STUDIES ON THE METABOLISM OF LINALOOL,

A NATURALLY OCCURRING FOOD FLAVOUR

December,

Being a Tiesis presented for the Award of a

Degree of Doctor of Philosophy

in the University of Surrey

by

KHANDAKER A.Q.M. QUDDUSUR RAHMAN,M.Sc. (Dacca), Grad.Dip. (New South Wales)

Department of Biochemist University of Surrey, Guildford,Surrey,England.

1974\

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Abstract

The metabolism of two terpenoid food flavours, linalool. and linalyl

acetate, has been studied in the rat.

14Linalool was labelled with C in the 1- and 2-positions and

administered intragastrically to rats; absorption from the gut was

rapid. Approximately 58% of the dose was recovered in the urine within

72 hours as linalool, dihydrolinalool and tetrahydrolinalool, mainly

conjugated with, glucuronic acid or sulphate. Some radioactivity in

the urine (101 of dose) was present as ^C-urea, and 25% of the label

appeared as ^ ’002 in expired air, indicating that linalool entered into

pathways of intermediary metabolism. A small proportion of the dose was

excreted in the faeces as free and conjugated linalool and as its

reduction products.

The reduction products of linalool were shown to arise by metabolism

occurring in the gut microflora. Both the liver and the intestinal

mucosa were shown to conjugate linalool with glucuronic acid in vitro

but the activity was much higher in the liver.

After intraperitoneal administration of linalool a substantial

proportion was excreted as conjugates in the bile and these were hydrolysed

in the gut with resultant enterohepatic circulation of linalool and its

metabolites.

Linalyl acetate was readily hydrolysed in the gut and was subsequently

metabolised similarly to linalool.

Studies of the effects of linalool on the hepatic drug metabolizing

enzymes showed that repeated daily intragastric doses (500 mg/kg body wt.)were

required before significant effects on these enzymes were observed. Dosing

(i)

for up to 64 days caused hepatomegaly with a transient increase of

DNA concentration suggestive of hyperplasia as well as cell hypertrophy.

Methylumbelliferone glucuronyltransferase activity was increased within

7 days whereas cytochromes P-450 and underwent a biphasic response

being initially inhibited and subsequently increased in concentration.

Linalool was shown to interact 'with cytochrome P-450 in vitro

producing a reverse type 1 binding spectrum (K = 1.9 x 10 ’M) and to_3inhibit biphenyl-4-hydroxylase (10 = 1.9 x 10 M). The observed changes

in vivo are indicative of an adaptive response to this mild inhibitor

but sensitivity to carcinogens may be increased during the period of

hyperplasia.

Studies of the metabolism of linalool by the intestinal microflora

of rat, mouse, guineapig, sheep, cow and human, revealed substantial

interspecies differences. Inter-individual differences in humans

appeared to depend on diet. Analysis by GC/MS, i.r. and n.m.r. showed

reduction products to be the most common metabolites in most species but

a novel intestinal reaction involving cyclisation of linalool to

a-terpineol and y”teipinolene was also observed in some species.

Acknowledgements

I wish to e qpress my deep sense of gratitude to Dr. Ronald

Walker and Professor Dennis V. Parke for their invaluable guidance

and unflagging encouragement throughout the course of this study.

I'would also like to express my sincere thanks to all members of

staff and colleagues within the Department of Biochemistry, and the

Animal Unit, for their helpful discussion, valuable suggestions

and assistance.

I gratefully acknowledge the financial support given by the

United Kingdom Overseas Student Affairs, for the period during the

war situation in Bangladesh, and to the British Council for their

fees award during my studies at the University of Surrey.

I am grateful to the Chairman of the Bangladesh Council of

Scientific and Industrial Research (B.C.S.I.R.) Dacca,, for granting

me leave of absence and for financial cover for a considerable

period.

To my brother, Alhaj Kh. A.A. Muhammad Habibur Rahman, my very

grateful thanks for financial support given at a very critical

period of my studies. Finally, I wish to thank the Secretarial

Staff of the Department of Biochemistry for their kindness in

typing this thesis for me.

(iii)

CONTENTSPage No.

Abstract (i)

Acknowledgements (ii)

Chapter I 1

Chapter I

General Introduction

Introduction 2

Composition of Essential Oils 3

Toxicology of Essential Oils and Terpenoid Compounds 4

Pharmacological Properties of Essential Oils * 5

Detoxication Mechanisms 7

Phase I Reactions ' 10Site of Reactions 10Microsomal Oxidation 12Components of the hepatic drug-metabolizing enzymes 12

A. Terminal Oxidase:cytochrome P-450 12Evidence for the involvement of cytochrome P-450

in drug metabolism 12Binding of P-450 to drugs 13

B. Cytochrome P^-450 14

C. Reductases 14NADPH Cytochrome c Reductase ~NADPH cytochrome P-450 Reductase 13

D. Non-haem Iron Protein 13

E. A Lipid Factor 13 [Microsomal Reduction!Phase II Reactions:Conjugation 16

Biological Consequences of Phase I and Phase II Metabolism 17 Factors affecting drug metabolism 17Stimulation of drug metabolism 18Inhibitors of drug metabolism 18Synergism 19

Effect of terpenes on the drug-metabolizing enzymes 19

The role of the gut microflora in the metabolism of foreign compounds(iv)

Aspects of the Gastrointestinal Metabolism of Foreign Compounds 21

A. Species Variation 21B. Effect of Diet 27

Toxicological implications of some of the reactions carried outby the gut microflora 27

a. Hydrolysis of glucuronides 27b. Hydrolysis of glycosides 29,c. Hydro lysis of sulphamates 30d." Nitro reduction 30e. Azo reduction 30f. Decarboxylation 31g. Aromatization 31h. Nitros amine formation 31

Metabolic adaptation of the gut microflora to foreign compounds 32Aims of the current work • . 33

Chapter 2Synthesis of |l,2-^C| Linalool

Introduction 35

Materials and Methods 37

Materials 37Methods 37. Syntheses 37

(a) Synthesis of vinyl bromide 37(b) Synthesis of linalool

Analysis and Purification 43

Determination of I.R. Spectrum 43

Determination of Radioactivity 43

Results 43(a) synthesis of vinyl bromide(b) synthesis of linalool

4446

Discussions 50

(v)

Chapter 5

In vitro metabolism of linalyl acetate and linalool by the intestinalmicroflora of rat and other species 52

Introduction 53

Materials 55

Methods 55

Rat caecal preparation 55Thin layer chromatography 56Separation and characterisation of metabolites by preparative gas chromatography, preparative thin-layer chromatography and mass spectrometry 56-59

Synthesis of 1,2 dihydrolinalool 59Purification ' 60Characterisation 60

(a) Thin-layer chromatography 60(b) Gas-liquid chromatography 60(c) Infra-red spectrometry 60(d) Nuclear magnetic resonance • 60(e) Mass spectrometry 60

Quantitative determination of metabolites produced from "^C-labelledlinalool by rat gut microflora 61

Species differences 61Thin-layer chromatography of metabolites 1Analysis with combined gas liquid chromatographyand mass spectrometry 63

Quantitation of the metabolites: from g.l.c. peak areas. % 63

Results 65Synthesis of dihydrolinalool 65Thin-layer chromatography 65Gas-liquid chromatography 65Infra-red spectrometry 65Nuclear magnetic resonance spectrometry 69

Metabolites produced by the rat caecal microflora:Thin-layer chromatography 70 •Preparative gas-liquid chromatography 70Infra-red spectrometry 70Mass spectrometry 70

Quantitative determination of metabolites of ^C-labelled linalool by rat gut microflora 77

Species differences 11

Discussion 84

Oi)

Chapter 4Absorption, distribution and excretion of ^C-labelled linalool in the rat

1. Introduction to absorption, distribution and excretion of xenobiotics

A. Absorption of foreign compounds 90(a) Gastrointestinal absorption 90(b) Factors affecting gastrointestinal absorption 92(c) Other routes of absorption 92

B. Distribution 95

C. Biotransformations 96

D. Excretion , 97

(a) biliar/ excretion . 97(b) enterohepatic circulation 98(c) excretion in urine 99(d) excretion in expired air 99.(e) excretion in gastrointestinal secretion 100

Materials and Methods 102

Materials 102

Experimental 102Administratioj^of "^C-labelled linalool and metabolic experiments: excretion of C in urine, faeces and expired air residual radio­activity, in tissues 72 hours after dosing; preparation of tissue homogenates 102

‘ “ B iliary excretion 103Enterohepatic circulation 104Determination of radioactivity : 106Radioactivity in urine and expired air 106Radioactivity in tissue 107Faecal radioactivity 107Biliary radioactivity 108Radioactivity in blood 108

Results 109

Discussion 116

(vii)

In vivo metabolic end products of linalool:identification and quantitativedetermination

Introduction 119

MATERIALS 120

' EXPERIMENTAL 121Thin-layer Chromatography: examination for glucuronides 121

Hydrolysis of polar conjugates in urine, faeces and bile 121

enzyme hydrolysis 121acid hydrolysis 121

Determination of urea 122

Thin-layer chromatography of the hydrolysates 122

Combined GC/MS 122

Quantitative evaluation of the metabolites 122

RESULTS 123Ether extractable radioactivity in urine and faeces 123

Polar substances in bile 123

Enzyme hydrolysates 123

Acid hydrolysates 124

Polar materials in urine 124Enzyme hydrolysates 124

Acid hydrolysates 128

Polar materials not hydrolysable with enzymes and acid 128

Polar substances in faeces: enzyme hydrolysates andacid hydrolysates 129

Bile from enterohepatic circulation: enzyme hydrolysate ^29DISCUSSION 131

(viii)

Chapter 6

Effect of linalool on some drug-metabolizing enzymes

Introduction 135

Materials and Methods 13?

Materials 137

Experimental 139

Animals and linalool administration 139Preparation of liver fractions 139Preparation of intestinal mucosal fraction 140Determination of biphenyl 4-hydroxylase activity 140Determination of 4-methylumbelliferone 240

glucuronyl transferase activity(a) in the liver 140(b) in the intestinal mucosa 140

Determination of alcohol dehydrogenase activity 141Determination of cytochrome P-450 141Determination of cytochrome b<- ' 142Determination of protein content 142Determination of DNA 142

Results 143Body weight 143liver weight and relative liver weight V ' 143Biphenyl hydroxylase activity 1434-methylumbelliferone glucuronyl transferase activity 143Alcohol dehydrogenase activity 144105,000 g supernatant protein content 144. cytochrome P-450 concentration 144cytochrome b^ concentration 157microsomal protein concentration 137DNA concentration ~ 137

Discussion 158

(ix)

Chapter 7

Studies of the effects of linalool on some microsomal enzymes

Introduction 164

Materials and Methods • - 165

Chemicals 165Animals 165in vitro inhibition studies ’ ' 165spectral dissociation constant (K ) of linalool 166in vitro conjugation studies s 166

Results 167

Inhibition studies (in vitro) 167Spectral dissociation constant (K ) of linalool 167Conjugation study s 168

Discussion 170

Chapter 8 .

General Discussion 177

References 186

Appendix 198

(x)

CHAPTER 1

General Introduction

1

INTRODUCTION

In order for food to be acceptable to the consumer it must possess

desirable organoleptic properties, namely, an attractive appearance, texture

and flavour. All of these properties are of considerable importance but

even if a food looks ’right* it will not be eaten if the consumer does not

find the flavour acceptable. There are, of course, no universally agreed

standards of what constitute an acceptable flavour and wide cultural

variation occurs. However, there is an increasing internationalization of

eating habits as instanced by the increasing acceptance of oriental cuisine

in Europe, and the substitution of bread for rice in the Orient.

Traditionally man has for many centuries used herbs and spices in food

both to add flavour interest and to mask unpleasant off-flavours. The

practice of using food flavours has been continued in industrialized food

production and is likely to be of increasing importance in imparting an

acceptable flavour to novel food materials such as spun or texturised

vegetable protein, leaf protein, fish meal or single cell protein. There is

a history of resistance to the acceptance of novel foods unless they can be

made to resemble traditional foods in appearance, texture and flavour and

the application of food additives of both natural and synthetic origin to

achieve this seems inevitable. While herbs and spices have been and are still

major sources of food flavours, they possess certain disadvantages to the

commercial food processors, viz:

(a) They have a weak flavour intensity and considerable quantities

need to be used.

(b) They are very variable in composition depending on the variety,

strain and growing conditions.

(c) They are periodically in short supply leading to wide price

fluctuations.

In order to overcome these disadvantages and to enable the food manufacturer

2

to produce a standardized product, use is widely made of extracted and

blended essential oils from traditional sources or of synthetic flavours

which may or may not have natural counterparts. At present essential oils,

or plant materials containing them, remain the most widely used food

flavours to which man is exposed and we are largely ignorant about the

consequences of this exposure. It is not only in foods and beverages that

man is exposed to essential oils, since they are also widely used in perfumes,

toiletries, cosmetics, aerosols ’air freshners', pharmaceuticals, tobacco,

furniture polishes and many other applications. They may be ingested, inhaled

or absorbed through the skin, and the fact that they are of natural origin

and have been used for centuries is no guarantee of their safety, of course.

Oil of sassafras and coumarin are examples of naturally occurring compounds

long used to flavour foods which have subsequently been shown to be

unacceptably toxic and which therefore no longer have GRAS status.

Further work on other natural flavours and aromatics would seem to be

of importance.

Composition of essential oils

The most important aromatic constituents of plant essential oils are

the monoterpenes and their oxygenated derivatives, terpenoid alcohols,

aldehydes and ketones. Alicyclic hydrocarbons, e.g. myrcene, ocimene and

cyclic hydrocarbons like limonene, a-pinene, etc. are frequently major

constituents of essential oils of citrus fruits, herbs and spices. Carbonyl

compounds are commonly encountered, e.g. - citral, citronellal,carvone andA

dihydrocarvone in caraway, cloves, spearmint, etc. Terpene alcohols are

important constituents of many oils, e.g. citronellol in citrus oils and

menthol in mint oils. Linalool and linalyl acetate, the compounds,studied in

this thesis, occur in a wide range of oils including cardamom, cinnamon,

coriander, laurel leaves, nutmeg, spearmint, sweet basil and thyme essential

oils. They are used in synthetic food flavours, in perfumary and other

non-food applications.

3

Toxicology of essential oils and terpenoid compounds

In view of the wide applications of essential oils and their components,

surprisingly little is known of their toxicology but the acute-toxicity

of any of them appears to be low (Jenner, et al, 1964). • There are a number

of reports, however, which indicate that one cannot feel conplacent about

the chronic toxicity and carcinogenicity of these compounds.

Over 30 years ago, Mackenzi, et al^(1941) reported that terpentine oil

and 1-pinene, its major constituent, were promoters of skin tumours in the

rabbit when applied topically.This report was substantiated by Salaman (1961)

who reported that application of citrus oils,, eucalyptus and linseed oil caused

epidermal hyperplasia in mice; activity in orange oil appeared to reside

mainly in the terpene hydrocarbon fraction. Tumour promoting activity was

reported for these terpene fractions but negative results were obtained with

terpeneless fractions. Roe, et al^(1960) and Roe and Field (1965) have

also reported that citrus oils are skin tumour promoters in mice but it

remains to be seen whether the terpenes can produce or promote tumours in other

tissue like the lung or gastrointestinal tract after chronic inhalation or

injestion.

While the non-oxygenated terpenes appear to be responsible for the tumour

promoting effects reported above, adverse reactions to a number of oxygenated

terpenes have also been reported. Thus, Leach, et al« (1956) reported that

citral caused lesions of the vascular endothelial cells when administered to

rabbits orally or subcutaneously at dose levels of 5yg/kg per day for 3 Weeks.

Similar lesions were observed in monkeys exposed to doses as low as 1 yg/kg/d

for the same period. Since this condition could be prevented by vitamin A,

it was suggested that citral might compete with retenene in the metabolism of

the endothelial cells and these observations raise the question whether

excessive consumption of citrus fruit beverages and marmalades might be

involved in the aetiology of the cardiovascular disease. In contrast to these

results, Benkos (1961) reported that terpenes inhibit the devebpment of

atherosclerotic plaques in rabbits receiving a diet rich in cholesterol.

The short-term toxicity of the terpenoid cyclic ketone, carvone was

studied in the rat by Hagan (1966) who reported growth depression in both

sexes at levels of 1000 ppm in the diet with testicular atrophy in males

receiving 10,000 ppm. The levels are of course, much higher than are likely

to be injected by man and problems of acceptability of diets containing such

high concentrations of flavours may have contributed to the observed growth-

depression. More serious-toxicity was reported for isobomeol acetate by

Gaunt, et alp(1971) who observed renal toxicity in rats receiving 90 mg/kg/d

for 13 weeks with liver damage at higher dose levels. The no effect level was

concluded to be 15 mg/kg/d which was stated to give a safety margin of over

one-hundred-fold the likely maximum daily intake in man but it is not clear

whether the calculation of this safety margin included likely exposure from

the many non-food applications mentioned earlier.

The fragmentary information on the toxicity of terpenes in experimental

• animals is supplemented by several reports of adverse reactions in humans.

Early work on skin sensitivity to terpenes (Hellstrom, et al«, 1953; Pirila

and Pirila, 1955, 1956, 1958, 1964) indicated that hydroperoxides of a number

of terpenes, particularly carene, are eczematogenic in man, which explained■ %

a number of adverse reactions to industrial exposure to terpenes, and

N u e y r 3d(1967) found that workers in a Shoe-polish factory inhaling (+)a-pinene

developed erythema, pruritis, neurological disorders, macrohaematuria

and methaemoglobinaemia.

It is not only industrial exposure to terpenes and terpenoids which has

been reported to cause a toxic response in man since heavy smoking of

mentholated cigarettes was reported to cause psychological disorders and

gastrointestinal symptoms (Luke, 1962).

Pharmacological properties of essential oils

Essential oil,herbs and spices have long been used in folk-medicine and

herbal remedies and examination has revealed a scientific basis for some of

5

these practices.

A number of essential oils,notably the oils of cloves and thyme , have

been reported to have fungicidal and bacteriostatic properties (Olcazki and

Oshima, 1952; Parry, 1969) and oil of caraway is used in the treatment of

scabies.

In a review of pharmacological properties of essential oils, Tucavon

(1960) noted that oils of mint, basil, lavender, caraway, fennel and

martricaria have an antispasmodic action on smooth muscle. Oils of cinnamon

and cassia are stated to have carminative and stimulatory properties (Pany,

1969). Camphor a constitutent of many oils, in low doses has a stimulatory

effect on the central nervous system while menthol in high doses appears to

be a CHS depressant (vide Supra) . Oil of matricaria is reported to have

antispasmodic and local anaesthetic properties in addition to antimicrobial

properties (Tucanov, 1960). Its ingestion increases the number of

circulating leucocytes up to three fold and its anti-inflammatory properties

have been claimed to be due to azulene content, principally chamazulene and'

guaiazulene.

While the observed pharmacological effects may suggest useful pharmaceutical

applications for essential oils, it is to be expected that at higher than

therapeutic dose levels adverse and possibly toxic side effects may occur.

Many essential oils are used as adjuvants in pharmaceutical preparations and

this adds to the total human exposure from the other sources mentioned earlier

but little is known about the effects of this exposure. Synergistic or

antagonistic effects on the pharmacological and/or toxic effects of drugs and

other environmental compounds might occur directly or due to effects on the

mammalian drug metabolizing enzymes. In view of this and the known hazardous

interactions between some drugs and dietary constituents (monoamine oxidase

inhibitors and tyramine) the consequences of increased environmental exposure

to essential oils and their use in pharmaceutical preparations along with

other drugs warrants closer investigation.

6

Detoxication mechanisms

Ingestion of low doses of potentially toxic compounds does not

necessarily result in a toxic response. Detoxication of such compounds may

occur by specific enzymic mechanisms and it would be expected that these

might have evolved, at least in herbivores and omnivores, to detoxicate

dietary terpenoid compounds.

The metabolism of foreign compounds may be considered as occuring in two

phases based on the nature of the reactions talcing place (Williams, 1970). In

both phases the compound is made more polar to facilitate excretion. In the

first phase the compound undergoes reactions classified as oxidations,

reductions and hydrolysis by which the compound requires suitable polar groups

for subsequent phase II conjugation reactions, which are essentially synthetic

in nature. For example the major pathways of metabolism of lipid soluble

benzene consists firstly of its oxidation to phenol and in the next step the

phenol is conjugated with glucuronic acid via its hydroxyl group to foim

phenyl glucuronide which is a fairly strong acid, soluble at physiological pH

values, and is readily excreted by the kidney.

C6H6 Phay 1 -----> C6H50H phase II C6H50 . C6H906b oxidation b conjugation '■ b

Typical Phase I reactions are classified in table 1.1 and the major routes of

conjugation are listed in table 1.2.

7

*Table 1.1 Typical Phase I Reactions

Chemicalreaction

Microsomal drug metabolising enzymes

Non-micros omal mammalian enzymes

Oxidation(oxygenation)

Aromatic hydroxylation

Acyclic hydroxylation

Alcohol oxidation(cytoplasm)

Aldehyde oxidation (cytoplasm)

Alicyclic hydroxylation Alicyclic Aromatization

Epoxidation

N-oxidation

S-oxidation

Desulphurat ion

Dealkylation

Deamination Deamination(Mitochondria & plasma)

Reduction Nitroreduction Reduction of sulphoxides and N-oxides (cytoplasm)

Azoreduction

Dehalogenation

Reduction of Disulphides

Hydrolysis Ester hydrolysis Hydrolysis of esters and amides (blood plasma)

Hydrolytic ring scission

Dehalogenation (cytoplasm

* From Parke (1968)

*Table 1-.2 Types of conjugation for animals

Conjugate Major target groups Deficient species

1. Glucuronic acidconjugation

2. Sulphate conjugation

3. Glycine conjugation

Ornithine conjugation'. IGlutamine conjugation

4. Acetylation

5. Mercapturic acidsynthesis

6. Methylation

-OH, -COOH, -NH2, >NH, -SH Cat, Gunn rat

aromatic-OH, aromat ic-NH2, some alcohols

aromtic-COOH,aromatic-alkyl-COOH

aromatic-COOH

aromatic-CH2-C0QH

aromatic~NH2 some aliphatic~NH2 hydrazides, S02NH2

aromatic hydrocarbons, -Cl, ~Br, -F, -N02

aromatic OH, NH2, >NH,-SH

Pig

Certain birds and reptiles

Most

Most

Dog

Guinea pig, Man

Certain birds and reptiles only.

Man and certain monkeys only.

* From Mandel (1971)

Phase I reactions

Site of reactions: The enzymes which carry out oxidations, reductions

and hydrolysis of foreign compounds, after absorption, are those which occur

predominantly in the liver,, although some activity is also found in kidney,

gastrointestinal tissue and to a lesser extent in the lungs, adrenals and

blood. Most of these reactions are carried out by the enzymes located in the

hepatic endoplasmic reticulum (enzyme of the microsomal mixed function oxidase).

The alcohol dehydrogenase in the soluble fraction and amine oxidase in the

mitochondria also catalyse phase I reactions to a certain extent.

Microsomal oxidation

Microsomal oxidation has a specific requirement for NADPH and O2 and is

catalysed by enzyme systems known as mixed function oxidases. During the

process one molecule of oxygen per molecule of substrate is consumed with

one atom of oxygen appearing in the product and the other undergoing an

equivalent reduction and appearing in water.

Mixed function oxidase activity appears to depend on the haemoprotein,

-cytochrome P-450, so-called because the reduced form of this enzyme binds

carbon monoxide with the formation of a peak in the absorption spectrum at

450 nm (Omura and Sato, 1964). The mechanism by which oxidation of the

substrate occurs is given in Fig.1.1

10

P-4503

P-4503Is cyt c k—

reductaseNADPH

0 2UsI

P- 4503V/.o|

cyt b5 ■cyt b5 reductasefNADH

Fig. 1.1 Scheme for cytochrome P-450 drug oxidation (Hildebrandt and

Estabroolc, 1971).

Components of the hepatic drug metabolizing enzymes

A. Terminal oxidase: cytochrome P-450

The terminal oxidase in the microsomal electron transport chain is a

haemoprotein having iron porphyrin as the prosthetic group. It is found in

hepatic microsomes and also in the microsomes and mitochondria of the adrenal

cortex (Estabrook, et al, 1963; Omura, et al, 1965a) and to a lesser extent

in the kidney and intestinal mucosa (Takesue, et al, 1965). Yohro and Horie

(1967) reported that mitochrondria of the corpus luteum also contains

this haemoprotein.

Evidence for the involvement of cytochrome P-450 in drug metabolism

Cooper, et alr3(1965) observed that carbon monoxide had an inhibitory

effect on the metabolism of certain drugs, e.g. codeine, monomethylaminopyrine

and acetanilide and reversal of this inhibition could be effected by irradiating

with light of .the same spectral characteristic as the carbon monoxide complex

of cytochrome P-450.

Cyanide does not bind with cytochrome P-450 and is not an inhibitor of

drug metabolism (Cooper and Brodie, 1955; Gillette, 1957) whereas carbon

monoxide form a complex with P-450 and inhibits drug metabolism.

Posner, et alp (1961) and Gillette, et ak^(1957) have shown that substances

such as deoxycholate , steapsin and snake venom which convert cytochrome P-450

to the inactive foim cytochrome P-420 also destroy the drug metabolizing

activity of the microsomes. Gillette (1966) and Silverman and Talalay (1967)

reported the detergents Triton N-101 and iso-octylphenoxypolyethoxy-ethanol

could partially solubilize lives*microsomes and have the ability to inhibit

the rate of drug metabolism in direct proportion to the conversion of

cytochrome P-450 to cytochrome P-420.

The parallel increase in the ratio of drug metabolism and cytochrome

P-450 levels during the administration of phenobarbital and 3-methylcholanthrene

and their parallel decrease ' to normal values after withdrawal of the drugs

led Sladek and Mannering (1968) to conclude that cytochrome P-450 is the

necessary component for drug metabolism. The implications of cytochrome P-450

‘ in drug metabolism, was also demonstrated by employing the inhibitors like

SKF 525A. Imai and Sato (1966) reported that the kinetics of binding of

aniline with oxidized cytochrome P-450 was similar to the kinetics of

hydroxylation of aniline by the same microsome (K = K = 2-3 nM in rabbit

liver microsomes). These workers therefore suggested that cytochrome P-450

is involved as the oxygen activating enzyme in the drug hydroxylation.

Binding to drugs of P-450

Remmer, et al (1966) and Schenkman, et ahj(1967) reported that there

are at least two types of P-450-drug complexes. The drugs ethylmorphine,

amidopyrine, hexobarbitone and most other drugs interact with cytochrome P-450

and cause a spectral change characterised by the appearance in the difference

spectrum of an absorption peak at 388 nm and a trough at 420-2 nm. The

second group of compounds, basic amines such as aniline, antipyrine,

nicotinamide, are known to cause type II interactions characterized by the

appearance of a peak at 430 and trough at 390.

A third type of spectral change known as Reverse Type I (RI) characterised

by a trough at 390 nm and a peak at 420 nm has been reported by Schenkman et ah,' %

(1972). The substrates which cause RI type interactions are usually the

short chain alcohols (methanol, ethanol, butanol ) and phenacetin.

Schenkman and Sato (1968) reported that type I binding spectra are

caused by interaction of the substrates with the haem moiety of the haemoprotein

with the modification of an existing ligand of the haem to the protein. A

wide range of compounds including drugs, mentioned above, fatty acids and

steroids show type I interactions with cytochrome P-450 and all these drugs

appear to be substrates of this enzyme (Ellin, et al.',' 1971).

The compounds causing type II spectral changes react with both the

ferricytochrome P-450 (absorption spectrum 419 nm) and substrates bound

haemoprotein (394 nm peak) as determined in the absolute spectrum, by

13

Remmer, et al i(1969) and in the difference spectrum by Schenkman, et al^

(1970). Omura and Sato (1964) reported that type II compounds interact at

the haem iron from the evidence that type II compounds, aniline and isocyanide

displace carbon monoxide from the reduced cytochrome P-450.

Schenkman, et ah, (1972) have suggested that reverse type I spectral

change is due to interaction of lipid soluble compounds with the substrate

bound form of cytochrome P-450 at a site other than type I. Both free and

endogenous substrate bound formsof P-450 exist in the liver microsomes.

Schenkman, et ah3(1969) also suggested that type I spectral change in both

absolute and difference spectra was .due to displacement. of some pre-existing

substance, bound in vivo, from the enzyme.

B. Cytochrome Pi-450

An observation that phenobarbital induced the metabolism of a much

larger number of drugs and other foreign compounds than did 3-methylcholanthrene

or 3,4 benzpyrene, or other polycyclic hydrocarbons (Mannering, 1968) led to

the assumption that two different inductive processes are involved (Mannering,

1968). It was concluded that polycyclic hydrocarbons cause synthesis of a

modified P-450. Imai and Siekevitz (1971) suggested that cytochrome P-450

and Pi"450 are interconvertible and are the physical modification of the

same enzyme system although Comi and Gaylor (1973) have reported these to be

different entities based on the evidence of the analysis of the soluble

cytochrome P-450 with ion exchange resin chromatography.C. Reductases

NADPH Cytochrome c ReductaseGillette (1966) suggested that NADPH cytochrome c reductase might be

a component of the microsomal electron transport system. His assumption was

based on the fact that the electron receptor cytochrome c and other electron

acceptors inhibited microsomal drug metabolizing enzymes. As there is no

cyctochrome c present in the microsomes and since there is no other known

natural substrate for the reductase in these organelles, NADPH cytochrome c

reductase is considered to play a role in the transfer of electrons from

14

NADPH to cytochrome P-450 across the microsomal electron transport chain

(see Fig.l).

NADPH Cytochrome P-450 Reductase

The implication of this enzyme in the microsomal electron transport

chain was demonstrated by Gigon, et al (1969). . They demonstrated that carbon

monoxide forms a complex with cytochrome P-450 in a microsomal suspension

that contained NADP and an electron generating system but not with oxidized

cytochrome P-450. Gillette and Gram (1967) and Gillette (1969) in a variety

of experiments have shown that the rate of drug metabolism more closely

related to the rate of reduction of cytochrome P-450 than the total amount

of cytochrome P-450. Gillette’s group thus concluded that NADP-cytochrome

P-450 reductase is rate-limiting in the overall reaction involving microsomal

drug metabolism.

D, Non-haem iron protein

Adrenodoxin, a non-haem iron protein, in presence of adrenodoxin reductase

and NADPH caused reduction of cytochrome P-450 in submicrosomal liver particles.

Omura, et al (1966); Kimura and Suzuki (1967) have shown that adrenodoxin is

a component of the electron transport system for the hydroxylation of steroids

by the adrenal microsomes and mitochondria and because of the similarity of

the microsomal hydroxylase systems of the adrenal and liver, it has been

assumed that adrenodoxin or some other non-haem iron protein is involved in

carrying electron between flavin enzyme (adrenodoxin reductase) and cytochrome

P-450 (Kimura and Suzuki, 1967).

E« A Lipid factor '

It has been shown by Lu and Coon (1968) that all three fractions

(a) cytochrome P-450; (b) an NADPH dependent reductase and (c) a heat stable

lipid factor (unaffected by heating at 100° C for 2 hours) separated from

solubilized microsomes were required for maximal drug-metabolizing activity.

Mannering (1972) has suggested that the lipid factor may act physically to

provide drugs access to cytochrome P-450.

15

Microsomal reduction

Microsomal enzyme systems can also catalyse reductive reactions.

Gillette (1966) demonstrated the reductions of azo and nitro compounds

RN = N-R' azo reductase— RNn2 + r' - nh2

rN02 nitro reductase >

There appear to be two pathways for these reactions one inhibited by carbon-

monoxide and the other not inhibited by carbonmonoxide. The first pathway .

involves cytochrome P-450 whereas the other pathway is catalysed by

NADPH-cytochrome c reductase alone. Oxygen inhibits the nitroreductase

activity and therefore occurs only in anaerobic conditions. Azoreductase

activity occurs in both aerobic and anaerobic conditions.

Phase II reactions: Conjugations

Foreign compounds or their phase I metabolites having suitable functional

groups for conjugation can couple with endogenous glucuronic acid, glucose,

cysteine via glutathione, glycine, ornithine, glutamine, sulphate, sulphur

from thiosulphate, methyl group from methionine and acetic acid (see table 1).

The route of conjugation varies with the foreign compounds, the species

and strain of animal and with dose, however, with few exceptions conjugation‘ %

with glucuronic acid is the most common. In some cases other glycosides,

e.g. glucosides are formed instead of glucuronides. Conjugations with

amino acids are also common, e.g. the conversion of benzoic acid to hippuric

acid in man, but the amino acid involved is species dependent (see table 1).

Hydroxyl compounds are frequently conjugated with sulphate while amino

compounds commonly form acetyl derivatives. Other routes of conjugation, e.g.

conjugation of cyanide with sulphur to form thiocyanate, usually represent a

detoxication mechanism specific to a particular compound or relatively small

number of similar compounds.

16

Biological consequences of phase I and phase II metabolism

The metabolic transformations in phase I reactions may alter the

biological activity of a foreign compound. The product may be less active

or more compared to the parent substance. However, in the phase II reactions

most of the compounds are made water soluble for ready excretion in urine

or bile to eliminate the toxic effect of a drug.

An example of detoxication by phase I reactions occurs with the short

acting hypnotic, hexobarbitone. This is metabolized by oxidation to

hydroxyhexobarbitone and other products which have little or no biological

activity (Tsukamoto, et ah, 1956). When the foreign compound is converted

to a toxic metabolite the process is known as intoxication or lethal synthesis.

For example the inactive insecticide parathion is metabolized by oxidation

to paraoxon which possesses a powerful anticholinesterase activity (Gage, 1953).

Parke (1968) and Peters (1963) describe the process of lethal synthesis where

some foreign compounds by virtue of their sufficient similarity to normal

endogenous substances enter the pathways of intermediary metabolism and as a

result become incorporated into the tissues. Fluoroethanol is not intrinsically

toxic but is oxidized in the body to fluoroacetic acid and this is converted

to fluorocitric acid which is highly toxic because of its inhibitory activity on

aconitase of the Tricarboxylic acid cycle,the energy yielding mechanism of

the body.

Although, as stated above, conjugation reactions usually lead to

detoxication a few examples are known where the conjugate may be more toxic

than the parent compound. For example, the glucuronide of N-hydroxy

fluorenylacetamide is a more potent carcinogen than the original compound

as is the acetyl derivative of I’-hydroxysafrole (Borchert, et aly 1973a,b).

Factors affecting drug metabolism

The metabolism of drugs is affected by a variety of factors (a) genetic

factors (species and strain differences; (b) physiological factors (age, sex,

nutritional status, pregnancy and disease) and (c) environmental factors

(stress due to adverse conditions, exposure to ionizing radiations and

ingestion of other foreign compounds that lead to inductive, inhibitory or\

synergistic effects), Parke (1968) recently reviewed the subject. The

phenomena of inductive, inhibitory and synergistic effects of drugs will be

discussed here briefly.

Stimulation of foreign compound metabolism

It has been established that drugs, pesticides and polycyclic hydrocarbons

activate the metabolism of other foreign compounds leading to the phenomenon

of drug synergism and tolerance, enzyme induction and carcinogenesis (Parke,

1968). Stimulation of the microsomal enzyme systems have been demonstrated

in various species and internal tissues. Most of the studies on mechanisms of

induction have been carried out with phenobarbitone and 3-methylcholanthrene

or 3,4-benzpyrene.

Phenobarbitone enhances almost all the drug metabolizing enzymes and

also elicits an increase in the magnitude of the type I and type II spectral

shifts. Chronic oral administration of phenobarbitone increases the size of

the liver and causes proliferation of the endoplasmic reticulum (Conney, 1967).

The stimulatory effect by 3-methyltholanthrene is much more rapid and

selective in contrast to phenobarbitone (Conney, 1967). Schuster (1964);

Emster and Orrenius (1965); Parke (1968) reported that the increase in the

microsomal enzymes caused by the foreign compound may result from an increased

rate of synthesis of these enzymes and/or decreased rate of breakdown.

Inhibition of drug metabolism

The hypnotic effect of hexobarbitone was prolonged by the administration

of the drugs SKF 525 A and Lilly 18947 and chlorpromazine. This evidence led

Gillette (1966) to suggest that co-administration of foreign compounds may

inhibit the metabolism of one another. In view of the spectral changes

exhibited by the foreign compounds in the liver microsomes it is expected that

the type I blocking agents should competitively inhibit the type I substrates

and type II substrates will be inhibited competitively by type II blocking18

agents. This has been found to be true with SKF 525, a type 1 inhibitor, and

hexobarbitone, amidopyrine and ethylmorphine, type 1 substrates. . However,

’the phenomenon of inhibition by type II inhibitors and its relation to spectral

interaction is not very clear, e.g. nicotinamide, a type II compound inhibits

the hydroxylation of aniline (a type II substrate) and also the demethylation

of ethylmorphine and amidopyrine, the type I substrates.

Synergism

The changes in toxicity and in the intensity of pharmacological activity

of foreign compounds could result due to inhibitory or stimulatory effects

by other foreign compounds. Philleo, et al«t(1965) have demonstrated the

synergistic effect of piperonyl butoxide, the inhibitor of microsomal

hydroxylation^on the toxic action of insecticides. In determining doses in

multiple prescribing,synergism of drug action plays an important role (Parke,

1968). In the treatment of epilepsy the combination of phenobarbitone and

diphenylhydantoin is used. As a result of chronic administration of

phenobarbitone the metabolism of diphenylhydantoin is accelerated and thus the

anticonvulsant effect is reduced. Sulser, et al->(1966) reported that the metabol'

of amphetamine was inhibited by desmethylimipramine and thus potentiated and

prolonged the effect of the drug.%

Effects of terpenes on the drug metabolizing enzymes

Since the influence of foreign compounds on the drug metabolizing enzymes

can have the profound consequences discussed above, the effects on these

enzymes of chronic exposure to dietary terpenes is of considerable importance

and a number of terpenes have been shown to affect the activity of enzymes

involved in both Phase I and Phase II detoxication reactions.

Jori, et ab, (1969) studied the effects of menthol, a- and $-pinene and

eucalyptol (1,8 cineole) on the metabolism of other xenobiotics in the rat.

Eucalyptol, administered subcutaneously or by aerosol spray was found to

increase the in vitro (9000 g fraction) liver metabolism of aminopyr jne,,

p-nitroanisole and aniline and jn -vivo metabolism of pentobarbital. This

19

demonstrated that the increased tolerance of barbiturates shown by mice or

rats housed on soft wood beddings may be dependant on an increased microsomal

enzyme activity displayed by these animals compared with animals kept on

hardwood beddings (Vessel, 1967). Recently Wade, et al',(1968) showed that

volatile hydrocarbon constituents of cedarwood such as the sesquiterpenes

cedrol and cedrene, are effective inducers of microsomal enzymes via the

inhalation route of administration.

Parke and Rahman (1969) studied the ability of the terpenes bomeol,

citral, terpineol, limonene, nerolidol,carvone ? and squalene to effect the

induction of the hepatic drug metabolizing enzymes after oral and parenteral

administration for 3 days (185 mg/kg/day). They reported that 3"ionone

increased the activities of biphenyl 4-hydroxylase, glucuronyl transferase and

4-nitrobenzoate reductase and cytochrome P-450 of about 50-751. Bomeol,

citral, limonene and terpineol caused smaller increasesof the order of 251.

Hie levels of biphenyl 4-hydroxylase, glucuronyl transferase or cytochrome P-450

were unaffected by linalool, nerolidol or squalene but these terpenes effected

the increase of the activity of 4-nitroreductase by 20-25%. They also

observed that 3-ionone which exhibited maximum inductive effect amongst the

terpenoids studied was the only one to be metabolized by hydroxylation. The

other terpenoids were metabolized by reduction and conjugation with glucuronic

acid. They concluded that amongst these classes of substances it was likely

that a compound could induce those enzymes which were involved in its: own

metabolism.

It seems that, if continued use of essential oils is to be made in food,

drinks and other applications leading to human exposure, both domestically and

industrially, it is highly desirable that more information should be obtained

about the pharmacology and chronic toxicity of essential oils and their

constituents.

In view of continued use of linalool and its esters, such as linalyl

acetate, linalyl cinnamate and linalyl isobutyrate attempts have been made

20

in the current work to study the effect of linalool, on the drug metabolizing

enzymes in the liver and in the intestinal mucosa.

The role of the gut microflora in the metabolism of foreign compounds

Attention has so far been directed at the metabolism of foreign compounds

by mammalian enzyme systems but in the case of orally ingested compounds,

or compounds which are secreted into the gut in bile, the gut microflora may

play a major role in the overall metabolism (Scheline, 1973).

In contrast to the mammalian detoxication reaction, the intestinal

reactions generally have a common characteristic .in that they involve

degradative processes so that various compounds undergo reduction in molecular

■size. It is of great significance that the altered compound may have increased

biological activity or toxicity. The major classes of reactions brought about

by the intestinal microflora are summarised in table l.*3.

Aspects of the Gastrointestinal metabolism of foreign compounds

A. Species variation

The 8-methyl ether of xanthurenic acid is known to be metabolized by

the gut microflora of rabbits by O-demethylation and dehydroxylation (Lower

and Bryan, 1969). The former reaction was found to account for about 20%

of the dose whereas values from about 2 to 9% were found for the dehydroxylation

reaction. When mice were given the same dose 99% was excreted unchanged.

Studies on the metabolism of homoprotocatechuic acid in rats and rabbits

have shown that dehydroxylation by the gut microflora occurs with part of

orally administered dose. The proportion of the dose dehydroxylated in

rabbits was about twice that obsewed in rats when the same dose was employed.

The aromatization of quinic acid is carried out by the gut microflora of

man and old world monkeys whereas new world monkeys and many species of lower

animals showed little or no conversion (Adamson, et al, 1970). These

variations may be related to the difference in the composition of the microbial

population in different species. The variation of microbial population of

different species along the gut from stomach to rectum and faeces has been

compared in tables 1.4, 1.5, 1.6, 1.7, 1*8.

Table 1.3 Reactions of foreign compounds by the gastrointestinal

microflora

1. Hydrolysis of glucuronides 10. Dehalogenation

2. Hydrolysis of glycosides 11. Heterocyclic ring fission

3. Hydrolysis of etheral sulphates 12. Reduction of double bonds

4. Hydrolysis of -CO-NH- compounds 13. Reduction of nitro groupsa. amides -i* r» j .r14. Reduction of azo groupsb. glycine conjugates' c. N-acetyl compounds 15‘ Red«ction of aldehydes

16. Reduction of ketones

17. Reduction of alcohols

18. Reduction of N-oxides

a. C-dehydroxylation 19. Reduction of arsonic acid

5. Hydrolysis of esters

6. Hydrolysis of sulphamates

7. Dehydroxylation

b. N-dehydroxylation 2Q_ Aromatization

8. Decarboxylation Deajnination

9. Dealkylation 22. Nitrosamine formationa. Odemethylationb. N-demethylation 23 • Acetylation

24. Esterification %After Scheline (1971, 1973).

The Stomachvirtually

The stomach contents from humans are/sterile (Sternberg, 1896, Dick, 1941

and Drasar, 1970). The bacterial count per gram of stomach contents from man

and common laboratory animals have been compared in table 1.4. The human

stomach contains 100-fold fewer bacteria per gram contents in contrast to

guinea pig, rat or mouse, and is only comparable to that of rabbit (like man,

a good producer of gastric acid).

22

Table 1.4 The flora of the stomach

Logio no. of viable organisms’, per gram of sample wet weight.

Man Rabbit Guinea pig Rat Mouse

No. of individuals examined 55 3 3 3 3

Enterobacteria - - 0-2 3-6 2-4

’Viridans' streptococci 0-5 - - - 5-6

Staphylococci - - - 0-5

Yeasts - - -- 4-5 0-7

Lactobacilli 0-4 - 2-4 7-9 8-9

Bacteroides 0-4 0-6 - 6-7 7-9

Bifidobacteria 0-3 0-6 4-6 7-8 7-9

Clostridia - - 0-4 2-3 0-3

Veillonella 5-7 6-7

- indicates the organism was not isolated.

The proximal small intestine

Cregan and Hayward (1953) and Bomside and Cohn (1965) have demonstrated

that most of the samples from the upper small intestine of fasting people are

bacteriologically sterile. Drasar, et alp (1969) isolated bacteroides from

3 out of 25 fasting samples. The microbial count of the upper intestinal

contents are summerized in Table 1.5.

•Table l.js* The flora of the proximal intestine

Logio no. of viable organisms per gram of sample wet weight.

Man Rabbit Guinea pig Rat .Mouse

No. of individuals examined 25 3 3 3 . 3

Enterobacteria - - - 3-5 3-6

Enterococci - - 0-2 3-5 3-5

Viridans' Streptococci 5 - . - 4-5 3-4

Staphylococci - - . 0-3 0-3 0-2

Yeasts. - - 0-3 3-5 0-7

Lactobacilli CMIO

— 2-5 6-7 7-8

23

Bacteroides 0-5 0-5 - 5-6 5-8

Bifidobacteria 0-4 0-5 4-5 5-7 6-8

Clostridia - - 0-3 - . 2-3

Veillonella - - 0-3 - 2-3

The distal small intestine

Although the stomach and upper small intestine of man are essentially

sterile, except immediately after a meal, the lower small intestine usually

is rich with permanent flora (Cregan and Hayward, 1953). Samples from various

levels of the lower small intestine of six subjects were examined by Drasar,

et al (1969) indicated that the numbers of microflora increase as the terminal

ileum i$ approached. From table (1.6 ) it can be observed that relatively little

difference exists in the microflora of the distal small intestine in man and

the common laboratory animals.

Table l.o The flora of the distal small intestine

Logio no. of viable organisms per gram of sample wet weight.

Man Rabbit Guinea pig Rat Mouse

No. of individuals examined 6 3 3 3 3

Enterobacteria 3-4 - 0-2 3-5 3-6

’Viridans’ Streptococci 3-4 0-2 - 2-6 4-7

Staphylococci - 0-2 0-2 0-2 -

Yeasts - - 0-6 3-5

Lactobacilli 3-6 - 2-3 6-7 7-8

Bacteroides 5-7 4-7 0-6 6-8 7-8

Bifidobacteria 5-7 5-7 4-6 6-8 7-8

Clortridia - 2-4 2-4 - 2-3

Veillonella - - - 4-5

- indicates the organism was not isolated.24 I

Hie large intestine, rectum and faeces

Although the microflora of the faeces may very closely reflect the

microbial population of the large intestine (Drasar, et al, 1970), the

microflora in these two areas were studied separately (Seeliger and Werner,

1963 and Drasar, et al, 1969).

Table 1.7 The flora of the large intestine

Logio no. of viable organisms per gram of sample wet weight.

Man Rabbit Guinea pig Rat Mouse

No. of individuals examined 11 3 3 3 3(appendices)

Enterobacteria 6-8 - - 4-7 3-6 •

Enterococci 5-9 - 2-3 3-6 3-7

Viridans Streptococci * -3 - - -

Yeasts * - - 5-7 4-7

Lactobacilli 4-9 - • 3-4 7-9 7-9

Bacteroides 6-10 8-9 6-8 7-8 8-9

Bifidobacteria 4-8 4-5 8-7 7-9 7-9

Clostridia * 0-4 . 2-3 iO 2-3

Veillonella 0-4 - 2-4 0-4 3-6

* Indicates the organism was not mentioned

- Indicates the organism was not isolated.

Table 1.3 The flora of the rectum and faeces

Logio no. of viable organisms per gram of sample wet weight.

Man Rabbit Guinea pi«g Rat Mouse

No. of individuals examined 25 3 3 3 • 3

Enterobacteria 4-8 0-2 0-3 5-7 4-6

Viridans Streptococci 2-6 2-4 1O 5-7 6-8

Staphyl ococci - - 0-4 - 0-2

Yeasts 0-4 - 0-4 5-6 0-6

Lactobacilli 2-7 3 3-4 8-9 8-9

Bacteroides 9-11 7-9 7-9 7-9 8-10

Bifidobacteria 9-11 7-9 8-9 8-9 8-9

Clostridia 0-5 0-3 0-3 . 0-6 0-3Veillonella 0-6 0-4 0-4 0-6

Human faeces predominantly contain non-snoring anaerobes; bacteroides

and bifidobacteria. Bacteroides are present in very large numbers and have

been shorn by Sanborn (1931) and Kaiser, et al^(1966) to be the dominant

organisms. However, Haenal (1961) considered bifidobacteria to be the dominant

microbes. Both of these groups of micro-organisms are dominant in the faeces

of all the animals studied (Drasar, 1970). The faeces of rats and mice

strikingly contains more lactobacilli and streptococci than in man and other

animals. There are notably fewer enterobacteria in rabbits and in guinea pigs.

It is therefore noted with interest that the composition of the gut

microflora of different species differ considerably from one another. Human

stomach and upper intestine is sterile whereas in rats and mouse is heavily

colonized. Although, in the guinea pig and rabbit, the flora are distributed

similarly to that of man, there are considerable qualitative differences. The

qualitative and quantitative differences in the gut microflora also influence

greatly the gut redox potential which influences the metabolism of a compound

in the gut. In view of the striking differences in the composition and nature

of the gastrointestinal micro-organisms amongst the species it is not

surprising to observe species variation in the metabolism of a compound by

their gut microflora.

Appreciable variations in the type of and numbers of micro-organisms have

been observed by Smith (1965) and Wilson and Miller (1964) in animals of

different ages with greatest changes occurring during early life. Fischer and

Weissinger (1972) reported that the deconjugation of diethylstilboestrol by

the bacterial 3-glucuronidase did not occur in newborn rats until they

reached the adult level of 25 days when enterohepatic circulation of this

synthetic hormone could be demonstrated (vide infra),

B. Effect of dietDiet may influence the nature of the gastrointestinal flora (Donaldson,

1964), yeasts are very-sensitive to the nature of the diet and are numerous

in animal fed mainly cereals but largely absent when meat and protein are

fed, (Smith, 1965). Lactobacilli also appear to be present in greater numbers

in animals fed on cereals. The effect of variation in the diet has been

observed in the same species. The conversion of cyclamate to cyclohexylamine

in the gut is carried out by enterococci. The gut microflora of a Japanese

individual contain large numbers of enterococci which, do not occur to a

significant extent in the gut of the British. It has been observed that all

the Japanese under investigation convert a cyclamate to cyclohexylamine

whereas the British could do so only after prolonged administration to allow

the gut microflora to adapt to the situation (Kojima and Ichibagase, 1966;

Renwick and Williams, 1969).

Toxicological implications of some of the reactions carried out by the gut

microflora

a. Hydrolysis of glucuronides

Less polar substances after absorption are conjugated in the liver to

render more polar and thus increase the water-solubility. These conjugates

are then excreted in the bile and/or urine. Those compounds which are excreted

in the bile when reaching the large intestine are hydrolysed by the bacterial

3-glucuronidase to liberate the aglycone which is reabsorbed and thus enters

the enterohepatic cycle. The glucuronide of stilboestrol^ a synthetic hormone,

is excreted extensively (Fischer, et ah, 1966; Clark, et ah, 1969) in bile.

Its excretion from the body through urine and faeces is delayed as its

glucuronide from the bile is hydrolysed by the gut microflora and the liberated

free stilboestrol is reabsorbed to establish enterohepatic circulation (see

Fig. 1.2 ). Toxicological implications of enterohepatic circulation may be

exemplified by the hydrolysis of the glucuronide conjugate of hydroxylated N-2

27

Fig.1.2 .. Enterohepatic .circulation and gut bacteria: 1^C-stilboesterol.

gut microflora (3-glucuronidase)

stilboestrolglucuronide stilboestrol

orother glucuronides

Gut

stilboestrol

injected vj/ stilboestrotLiver £ - - ~

stilboestrol glucuronide

fluorenylacetamide (FAA). The hydroxylated product is carcinogenic (Scheline,1971. It is reabsorbed and reconjugated with.glucuronic acid in the liver the thus

enters enterohepatic circulation. Formation of another typical toxic

metabolite by the gut microflora may be exemplified by the production of

thyrotoxic amino compounds from injected chloramphenicol, an antibiotic.

The antibiotic is excreted in large amounts in the bile as glucuronides

which are hydrolysed and reduced to aryl amines, the thyrotoxic compounds,

by the gut microflora.

28

b. Hydrolysis of Glycosides

Amygdalin, a glycoside present in almond, has LD^q (rat) 5000 mg/kg

when administered intraperitoneally whereas LD^q (rat) is 300 mg/kg

when administered orally. The toxicity lies in the fact that the glycoside

is hydrolysed by microflora in the gut to unstable mandelo nitrile which

readily breaks up to yield highly toxic cyanide (Williams, 1970).

CNiC -0. CgHioO^. 0 . CbHhOs IH in gut

vCNI vC - OH + 2 C6H, 206IH

CHO + HCN

A second example of glycoside toxicity appeared by the microfloral

hydrolysis in the gut of the gLycoside cycasin, a glucoside of

methylazoxymethanol, a constituent of cycad seeds which are used as food

in some parts of the Pacific Ocean region (Yand and Mickelsen, 1969).

The gut microfloral hydrolysis liberates the aglycone which in the lung

is believed to be metabolized to diazomethane which is a carcinogen.

However, eyas in itself is not active by injection and is inactive and

not absorbed when given orally to germ-free animals.

c. Hydrolysis of Sulphamates

The Sweetener cyclamate when administered orally for a long period of

time to animals or humans, cyclohexylamine was excreted as metabolite. These

amines are potentially toxic.

d. Nitro reduction

The reduction of nitro groups by the intestinal bacteria is well known

(Smith, et ah, 1949; 1950; Glazko, et al, 1952). It has been mentioned

earlier that chloramphenicol is reduced to amines in the gut ."which are

reabsorbed producing a thyrotoxic effect.

e. Azoreduction

Azodye food colours undergo reductive fission by the gut microflora. As

a rule, compounds having sulphonic acid groups on both sides of the azo

linkage are employed so that the fission products will be fairly highly

ionized compounds which do not possess appreciable biological activity and

are soluble in water so that these are easily eliminated from the body

(Scheline, 1971). One exception to this is the food colour Brown FK which

consists of sulphanilic acid connected through an azo linkage to several

diamino compounds lacking the sulphonic acid group. Walker, et al*5(1970)

have shown recently that the polyamino compounds formed by microbial azo

reduction are stabilized at the low redox potential in the large intestine

which does not favour their oxidative condensation. These polyamines are

absorbed and are myotoxic. This work illustrates an interspecies difference

in toxicity which results from differences in intestinal metabolism.

Thus, Grasso, et al«3(1968) reported that the azo dye Brown KF is toxic in

rat but not in mice under similar conditions whereas Walker, et al»j(1970)

showed that muscle lesions are produced in both cases when polyamino reduction

products of Brown FIC are given intravenously. These latter workers suggested

two reasons for the species difference in toxicity, (a) differences in the

composition of the gut flora, and (b) difference in the environment of the

large intestine of the two species, i.e. redox potential, which could

influence the stability of the fission products.

An example of pharmacological interest is the splitting of the azo

linkage of prontosil and neo-prontosil to liberate antibacterial sulphanilamide.

Sulphanilamide is absorbed and performs its pharmacological activity.

f. Decarboxylation

Hydroxylated benzoic, phenyl acetic and cinnamic acids are

decarboxylated by intestinal micro-organisms (Scheline, 1966, 1967, 1968;

Indeb and Sdieline, 1968). Hie phenols thus produced are toxic substances and

have been claimed by Bakke and Midtvet (1970) to play a significant role in

promoting tumours.

g. Aromatization

Indahl and Scheline (1973) and Goddard and Hill (1972, 1973) have pointed

out the ability of the gut microflora to aromatize cyclic compounds may lead

to the formation of polycyclic aromatic compounds from steroids with possible

carcinogenic activity.

h. Nitrosamine Formation

Nitrosamines are potent carcinogens which could possibly be formed

in the body from nitrite and secondary amines (Anon, 1970). Hawksworth and

Hill (1971) have demonstrated that bacterial formation of nitrosamines could

arise from two sites. The first is the intestine from nitrate in the diet

which after reduction to nitrite can react with those amines of intestinal

origin. The second possibility is in the urinary tract in individuals with

urinary infection. A majority of these infections are due to E.coli which is

one of the species which catalyses nitrosation of amines. Normal individuals

contain 5-10 times lower nitrate concentration than that which is apparently

required for nitros amine production, however, in areas with a high nitrite

content in the drinking water there is a danger of nitrosamine toxicity.

Metabolic adaptation of the gut microflora to foreign.compounds

Many of the intestinal reactions apparently occur readily without need

by the micro-organisms to adapt. A few examples are known, however, in which

prior exposure to the test compound is necessary before’appreciable

metabolism is observed and it seems likely that in these cases, adaptation

and selection may have occurred.

An excellent instance in this area has been reported by Kojima and

Ichibagase (1966) who showed that cyclamate could be metabolized to

cyclohexylamine. Wallace, et al‘y(1970) reported that no rat initially

converted cyclamate to cyclohexylamine while at the end of the year on diets

containing 0.4, 2.0 or 10% cyclamate, only 80% of the animals were

cyclohexylamine excretors and the amount excreted accounted for 35-40% of

dose. Drasar, et al (1971) reported that rat faecal micro-organisms coverted

more than five times as much cyclamate to cyclohexylamine when they were

incubated with cyclamate in protein containing media.

32

Aims of the current work

From the preceding review, it can be seen that dietary terpenes may

'be potentially toxic and that, after absorption or ingestion they may be

metabolized by mammalian or microbial systems with consequent changes in

biological activity. Linalool and its esters are common components in

essential oils, e.g. citrus oils, cardamom, coriander, etc., and may thus

have an influence on the consumer directly or by their effects on the

metabolism of other foreign compounds which may be encountered. In view of

this, it appeared that it would be of both academic and practical interest to

embark on the following programme:

i) to synthesize lt+C linalool to study the metabolic fate of linalool;

ii) to study the metabolism of linalool in vitro by the gut microflora of

rat and other species, and to identify the microbial metabolic end products;

iii) to study the metabolism of 1 linalool in vivo in male and female rats

to investigate the absorption, distribution, biotrans format ions and

excretion and to establish the sex differences, if any; and to

determine the kinetics of absorption and excretion;

iv) to identify the in vivo metabolic end products with a yiew to finding the

presence of any possible precursor of toxicity;

v) to study the effect of acute and chronic administration of linalool on

the drug metabolizing enzymes in the liver and the intestine, in rats and

to determine the mechanism of toxic response, if any, due to exposure of

the animal to linalool.

33

CHAPTER 2

Synthesis of []l, 2~1 ] Linalool

34

INTRODUCTION

Compounds labelled with radioactive tracers are commonly used

in studies of the metabolism of both endogenous and foreign compounds,

and tracer techniques are now routinely applied to investigating the

metabolism of drugs and other anutrients in laboratory animals

(R. ICuntzman, 1972). Hie radioactive isotopes most commonly employed

are the weak 8-emitters 3H and llfC which are common to almost all organic

compounds and present a minimal health hazard to the operator. Tritium

has the disadvantages that it frequently is labile and may undergo

exchange with protons of the solvent. Also, oxidations, conjugations etc.

at the site of labelling or adjacent to it can lead to displacement of

3H, often with loss' of activity. Consequently 1IfC is preferred as the

label for most purposes although the double labelling technique involving

3H and 14C at different sites can be extremely useful in some cases.

In view of the above comments, and to facilitate subsequent

quantitative studies in the metabolism of linalool, it was decided to

synthesise the llfC labelled compound. Noimant (1954) reported good yields

of linalool from the condensation of vinyl magnesium bromide with

2-methylhept-2-ene-6-one (methylheptenone) and this route was subsequently

used in a synthesis of [l,2-ltfC2] linalool from [^Cj ethylene by the

method of Filip and Moravek (1959). Details are shown in Figure 1.

ch2 CHj Br

Ethylene

■» CH2h CM? KDH

.Br Dibrome thane

* CHBr

Mo * * Ik--------- > a \2 = CH.Mg.BrT etraiiydrofuran Vinylmagnes ium

(THE) Bromide

CH2 = CH.Mg.Br +

Vinyl magnesium bromide

H3C ch3

Methylheptenone

HsC. OMgBr

h 3c

Figure 2.1

*CH;

CH,

[1,2-14 Linalool

The use of tetrahydrofuran in the Grignard reaction leading to the

formation of vinyl magnesium bromide is essential since Krestinskiy (1920)

showed that ethylene and acetylene were formed when diethyl ether was

used as the solvent. Yields of Grignard reagent from vinyl iodide were

also very low (Krestinskiy 1920). Nomant (1954) demonstrated that

improved yields of Grignard reagent from vinylhalides were obtained when

anhydrous tetrahydrofuran was used as solvent, apparently due to

stabilization as a result of formation of oxonium salts.

Consequently the method of Filip and Moravek (1959) was taken as the

basis of the synthesis finally employed, using commercially available

l,2-lltC labelled dibromoethane as the starting point. Since Filip and

Moravek (1959) did not report optimal conditions for the synthesis of

linalool several syntheses using unlabelled dibromoethane were carried

out first in which yields were checked at the stages of production of • vinylbromide and of linalool and an attempt was made to maximize the

yields at each stage.

36

MATERIALS AND METHODS

MATERIALS

(jL, 2 -14 C]Dibromoethane, 500 yCi (The Radiochemical Centre, Amersham) ,

unlabelled dibromoethane (BDH, minimum assay by g.l.c. was 98.51),

potassium hydroxide (BDH, Analar), 2-methylhept-2-ene-6-one (Ralph N.

Emanuel, g.l.c. assay was 98.5%), ammonium chloride (BDH, analar).

Carbowax 2QM (Phase Separations Ltd.), Celite 545 acid washed and treated

with dimethyldichlorosilane (Phase Separations Ltd.), magnesium metal

turnings for Grignard reaction (BDH).

METHODSSyntheses

The overall synthesis of linalool involved (a) synthesis of vinyl

bromide from dibromoethane and (b) synthesis of linalool from vinyl bromide

by condensing this Grignard reagent with 2-methylhept-2-ene-6-one

(methylheptenone) and subsequently hydrolysing the reaction mixture.

a) Synthesis of Vinyl bromide

The assembly for the synthesis of vinyl bromide has been shown in

fig.2.2. It consisted of a three-necked ground-joint flask (flask A, 100 ml)

fitted with a reflux condenser I (E) provided with a stillhead (L). The still-

head (L) was connected to a condenser (F) (condenser II) which in turn was joins

to a second three-necked flask (flask B, 250 ml) via a receiver adaptor (s)

which dipped into anhydrous tetrahydrofuran (120 ml) in the flask B resting

in a bath containing freezing mixture (acetone and solid CO 2). Freezing

mixture was also circulated by a pump through condenser II and a second

reflux condenser (G) (condenser III) on flask B. Unlabelled dibromoethane

11.5 g (0.063 mole ) was introduced from a dropping funnel into flask A

which contained 60 ml of 3.5M alcoholic KOH. Hie contents of the flask

37

Fig.

2.2

Appa

ratu

s for

Sy

nthe

sis

of C

Labe

lled

Lin

aloo

l

were heated at 45-48°C under reflux. Vinyl bromide (b.p. 16.5°C)

was produced and swept by dry nitrogen introduced into flask A through

a tube dipped into the liquid. The Nitrogen flow rate was regulated at

6 ml per minute. Hie vinyl bromide thus produced passed through the

condenser I and was condensed in condenser II (-15°C to -20°C) into the

tetrahydrofuran in flask B. Hie heating of flask A was continued for

2\ hours until there was no sign of further droplets of vinyl bromide in

condenser II. Flask B with 120 ml of THF was weighed before distillation

of vinyl bromide and weighed again after distillation to calculate the

vinyl halide produced.

To find the optimum concentration of alcoholic KOH to dehydrobrominate

the dibromoethane Investigations were carried out with four different

strengths of alcoholic KOH viz. (a) 2M KOH in 95% alcohol (b) 2M KOH in

absolute alcohol (99.9%),(c) 5.5M KOH in absolute alcohol (d) 4.5M KOH in

absolute alcohol. The reaction was carried out over a period of 2\ hours.

(b) Synthesis of linalool

Since the preparation of the Grignard reagent and its condensation

with methylheptenone necessitated the absence of moisture and oxygen, and

also in view of health hazard due to radioactive gaseous llM2 vinyl bromide

linalool was synthesised in a closed system. The same assembly (Fig. 2.2)

as in the synthesis of vinyl bromide was used but included some additional

operations for keeping the system free from moisture and air. Hie required

amount of magnesium metal (0.063 mole) was placed in the flask B. Dry

nitrogen, passed through two U-*tubes containing fused CaC^, was introduced

into flask A to remove air from the apparatus. When the funnel in vessel

B was closed a slight positive pressure was maintained at the nitrogen

tank as indicated by the level of mercury in the escape valve connected

Fig.

2.

3 A

ppar

atus

for

Sy

nthe

sis

of G

rigna

rd

Rea

gent

an

d C

onde

nsat

ion

with

M

ethy

l H

epte

none

PtfHo

PrZl.5 oiP-Ibl) A* S Eh•H Sw/g r•rH

bQG

ccJGOQCQ

ffiEhG• r-<

0 r—( Q) *1-1 CQ g CQ o

cvJ-{->GOOrm ^G S G O5 £ S ®a> §*.S ^2 >• pa> C/2 G

OoOCS3IO-MoOLO b£G•pHG• rP

"3o

(M «—«ocdU'CS0CQcSbi)G• pHG•pH-p->GOo o

0 3 0'S . C2 rg LtYoj VP S P

G0)GG•i-i

$2-4->0Go o•pH *»H

0GbD bJD re* oSs §

CQpHd•4H 0

.3CQ0Gbfia

G0)-}->0)

G0£ 1Eh

CO£CQCQCCJp HO

CQ Q O P-i W O O ’ H tD

jujijf! mlife-

40

through a rubber tubing to condenser III. When all the air had been

displaced, both the flasks (A and B) ivere heated gently 70°C for half

an hour under a continuous flow of nitrogen to ensure elimination of

any moisture adhering onto the surface of the glass or to the magnesium

metals. When the flasks were cooled the nitrogen flow was reduced to

a barely perceptible rate and 120 ml of tetrahydrofuran (dried over

metallic sodium) was introduced into flask B. Unlabelled dibromoethane

11.75 g (0.0625 mole) was introduced into the flask A and 60 ml of

3.5 N KOH in absolute alcohol was added from the dropping funnel. Dry

nitrogen was introduced at the rate of 6 ml/min through the tube (N)

dipped into the liquid of flask A. All the joints were checked to

maintain the airtight conditions. The contents of the flask were then

refluxed in an oil bath to avoid excess environmental moisture at a

temperature of 45-48°C, as before, and the vinyl bromide was- distilled

into tetrahydrofuran in flask B. After 2\ hours the heating of flask A

was discontinued and nitrogen flow rate was reduced. '

In the next stage, the Grignard reagent, vinylmagnesium bromide,

was prepared from vinyl bromide and this necessitated an alteration in

the assembly shown in Fig. 2.2. The condenser II was detached from the

stillhead (L) and traces of vinyl bromide washed into vessel B with

tetrahydrofuran. The condenser II was then detached from the vessel B

and thermometer (T) was inserted into the free neck of the vessel. In

the new assembly (Fig.2.3)which was essentially the second half of the

Fig.2.2,nitrogen was introduced at the top of the reflux condenser III.

Thus any air inside the flask was swept through the apparatus and escaped

at the mouth of the dropping funnel (p). One reason for the nitrogen

atmosphere was to prevent destruction of Grignard reagent by atmospheric

oxidation.

2RMgX + 02 — -----> 2R0MgX

41

The second reason was to eliminate moisture which could decompose the

Grignard reagent

RMgBr + H20 -------- — > RH + Mg(OH)Br

A small crystal of iodine was dropped onto the surface of magnesium

metal to initiate the reaction for the formation of vinyl magnesium bromide.

The freezing mixture surrounding the flask at this stage was replaced by

ice cold water (0-4°C). Five minutes after the addition of the iodine,

bubbles started to appear indicating the beginning of the formation of the

Grignard reagent. The vessel was kept undisturbed for half an hour then

stirring was started with a magnetic stirrer and continued for 45 minutes.

During this period the temperature of the vessel was gradually brought to

room temperature. Methylheptenone (6.3 g or 0.05 mole) was then added

drop by drop within a period of 15-20 minutes with stirring. The

condensation of methylheptenone with vinylmagnesium bromide was exothermic,

and therefore, was indicative of the progress of the condensation reaction.

At this stage the temperature was raised slowly to 50°G and kept at this

temperature for half an hour - and then further raised to and kept at

60°C under reflux for 30 minutes. The flask was then cooled down to room

temperature and stirring continued for one hour. At the end of the reaction

the linalool magnesium bromide complex was decomposed by hydrolysis with

30 ml saturated aqueous ammonium chloride solution to liberate free linalool.

.Ammonium chloride hydrolysis was preferred to acid hydrolysis because of

possible acid catalysed isomerisation of linalool to other terpenols.

The hydro lysate was extracted four times with ether and concentrated to

30 ml at 20°C under reduced pressure in a rotary evaporator. It was then

kept over anhydrous sodium sulphate overnight.

Analysis and Purification

For analysis and purification a Pye 105 Mark 2 Automatic Preparative

Gas Chromatograph equipped with a flame ionization detector

was used. Separations were accomplished on a 7ft x §in glass column

packed with 20% carbowax 20M on acid-washed and dimethyldichlorosilane-

treated celite 545, 60-80 mesh size. The oven temperature was main- o

tained at 140 C and injection temperature at 190°C, nitrogen was used

as the carrier gas and was introduced in the system at a flow rate of

110 nd/minute. Six components were detected, separated and collected

preparatively (fig -Table The peak corresponding to linalool

was characterized by retention time (Fig. 2.4 and by comparison

of its IR spectrum (Fig. 2.5a). See below (Results Section).

Determination of IR Spectra

Infra-red. spectra of the reaction product and authentic linalool

were recorded on the SP 200 IR spectrophotometer using a film of the

liquid between potassium bromide windows.

Determination of Radioactivity

Radioactivity was determined in a Packard Tricarb Liquid Scintillation

spectrometer', model 3320 at 0°, using 70/30 scintillator; 70 ml toluene,

30 ml absolute alcohol containing 0.3% PPO and 0.01% POPOP. The labelled

compound (lyl) was added directly to 15 ml of the scintillant contained

in the counting vial. Radioactivity thus measured was corrected for

quenching and counting efficiencies by internal standardization using

25 pi 1 C toluene, (specific activity 5.25 x 10^ d.p.m./g).

43

RESULTS

(a) Synthesis of vinyl bromide

The results are shown in Table 21 from which it can be seen that

maximum yields were obtained using 3.5M KOH in absolute alcohol in

otherwise similar conditions.

Table 2.1: Percent yield of vinyl bromide on the basis of equimolarquantities from dibromoethane.

Concentrations of KOH Percent yield of vinyl bromide

2M KOH in 95% aqueousalcohol 49.2

2M KOH in absolutealcohol (99.91) 58.7

3.5M KOH in absolutealcohol (99.9%) 65.5

4.CM KOH in absolutealcohol (99.9%) 64.8

44

Table 2.2: Retention time of the labelled and unlabelled compoundsin the synthetic mixture.

Compounds Peaks labelled/unlabelled Retentiontimes (mins)

Ether A unlabelled 0.7

Tetrahydrofuran(THF)

B 1.6

Methylheptenone C ii 6.2

Unknown D it 11.7

1 C labelled linalool E labelled 20.4

Unknown F ii 28.8

Authentic linalool — unlabelled 20.4

(b) Synthesis of linalool

The gas chromatographic analysis eluted six well-defined

symmetrical peaks. Their retention times and the results of the

radiochemical analysis are shown in Table 2.2 Peaks A, B and C

corresponded to ether, tetrahydrofuran and methylheptenone

respectively. Their presence was expected because of thety use in

different stages of the synthesis. Peak D and F were unknown compounds,

of which the latter was radioactive. Preparative GC yielded 108 mg

from Peak D (inactive) and 212 mg from Peak F, with a total activity

of 3.7 yCi. Neither compound was identified. Peak E was due to

linalool and was characterised by comparison with the retention time

20.4 min (Table 2.2, fig. 2.4) and infrared spectrum of authentic linalool

(fig. 2.5a & 2.5b). Hie total activity of the ether extract of the

synthetic hydrolysate was 200 yCi which was due mainly to 1LfC linalool.

After purification by preparative gas chromatography the linalool

fraction weighed 3.28 g (0.0213 mole) with a total activity of 170yCi.

Initially 11.75 g (0.0625 mole) of dibromoethane was used as a

starting material. Therefore, on the basis of the weight the % yield of pure linalool is P»Q2.L^c 100 _ ^ activity of

(jl, 21IfC] dibromoethane was 500 yCi and the activity of the final

product, 1IfC labelled linalool, was 170 yCi. Therefore, the % yield

in terms of radio activity is = 34%. Hence the yield both

in terms of weight and radioactivity was 34%. The specific activity

of [l,214C| labelled linalool was 170 yCi/3280 mg = 0.052 yCi/mg.

46

Fig.

2.4

Gas

chro

mat

ogra

m

of C

labe

lled

synt

hetic

m

ixtu

re

osuodsaj JopjoooH

0)S•H

47

mm

Fig.

2.

5a IR

of C

synt

hetic

lin

aloo

l

o o o ooo o^ PL CO |o -CO 1l o * - 1 CM j

O ICM rj°° — I|CO J

oLOCM

OO

ooCO

•'Cf1vH OO

00CMtHOOoO —

o03 oo

CMt-H°-4CO

o

ooCO

o o ” oo

oooCM

ooLOCMO

OOOCO

OCO O

OLOCO

Ooo'CPoCO ''CP LOo tH

r-fIou0"sIg£

o o o o

oounqjosqy48

Fig.

2.

5b IR

of au

then

tic

linal

ool

ooireqjosqy

DISCUSSIONS

The use of 2M KOH in absolute alcohol (99.9%) for dehydrobromination

of dibromoethane produced a higher yield of vinyl bromide compared with

2M KOH in 951 alcohol over the same period of time. The decomposition of

dibromoethane with exclusive formation of vinyl bromide with alkali in

absolute alcohol is purely a bimolecular reaction (Bernoul 1 i, 1933) .

With increased amounts of water, alkaline hydrolysis predominantly

produces ethylene glycol, and the velocity of reaction is considerably

reduced (Bernoul 1 i, ' ,1933) .

The reaction velocity changes exponentially with temperature

(Bernoulli, 1933). The reaction in the vessel A should not be so high

as to sweep alcohol vapour into the reaction vessel B by the stream of

nitrogen. Alcohols react with Grignard reagent at a faster rate than

ketones -(Fincif.-1967). Hence if alcohol was carried over into vessel B

it would react faster with vinylmagnesim bromide than methylheptenone

to produce radioactive gaseous ethylene.

*CH2 =*CHMgBr + Q^G^OHVinylmagnesium Ethyl bromide alcohol

-> O-L = QL + CHXILOMgBr Z Z 0 | zRadioactive Iethylene

(gas)Hydrolysis

CH CH OH Ethyl, alcohol

The synthesis of linalool with .improved yield is associated with

the following points

a. Regulation of temperature at 45°C in reaction vessel A.

b. Control of temperature in the reflux condenser (I) so that

it would condense all the alcohol that may be vaporized under the

50

reflux condition at 45°C in vessel A.

. c. Dry and inert atmosphere should be maintained during the

course of synthesis.

d. The reaction mixture in vessel B should be refluxed for at

least half an hour at 60°C after addition of methylheptenone to

facilitate the condensation of the ketone with the vinyl magnesium

bromide.

The total activity of the [i>214C| labelled linalool, synthesised

by the method described, was 170 yCi. The activity was enough for

the requirement to study the metabolic fate of _ pL, 2llfC] labelled

linalool in the rat.

CHAPTER 3

In vitro metabolism of linalyl acetate and linalool

by the intestinal microflora of rat and other species.

INTRODUCTION

The gastrointestinal microflora are able to metabolize a wide range

of foreign compounds including some food additives (Scheline, 1973).

The contributors of the gut bacteria to the metabolism of compounds which

are administered orally or any substance secreted into the intestine has

been discussed in Chapter 1.

In safety evaluation it is important that the intestinal microflora

of the potential recipient be taken onto account and compared with that

of the animal used for testing, for assessing the relevance of results.

Several of the micro-organisms, although common to many species, have been

found to vary in numbers and location in the intestine of different species *

and they could vary in the same species with nature of the diet (Miliams'p

1971). Tie composition of the microflora of different species at different

sections of the gut has been discussed in Chapter 1 (pages 22-26 ).

A striking example of species variation in the metabolism of quinic acid

(derived from chlorogenic acid, present in tea, coffee, fruits and

vegetables), by the gut microflora was observed by Adamson, et ah, (1970). It

was observed that only man and Old World monkeys converted quinic acid

extensively to benzoic acid (20-701) which was excreted entirely as hippuric

acid in the urine. In other species like rat, guinea pig, mouse, dog, cat,

ferret the excretion of hippuric acid was 0-10%. When Adamson et al,, (1970)

injected quinic acid intraperitoneally no quinic acid was found but oral

administration was followed by urinary excretion of hippuric acid. This thus

demonstrated that hippuric acid was formed by the action of microflora and

not by tissue metabolism.

Another example of a species variation influenced by the gut bacteria

is to be found in the metabolism of a food colour Brown FK. Brown FIC when

administered orally is toxic to rats but not to mice (Walker, et ah, 1970).

On further investigation by these workers it was found that gut microflora

of rat produced two myotoxic amines from Brown FK. When these two myotoxic

substances were injected to rat and mouse, cardiac and muscular lessions

were produced in both cases. It was therefore, suggested that the orally

administered Brown FK was myotoxic in rats but not in mouse due,probably,to

differences ,in the metabolism by intestinal microflora in the two species

(see Chapter 1).

In view of the ability of the microflora to introduce, remove or modify

the substituents and/or the structure of a compound thus altering its

toxicological and pharmacological properties the metabolism of linalylacetate

and of linalool by the gut microflora of rat and other species has been

investigated in relation to their safety in use as food additives.

54

MATERIALS AND METHODS

Materials

Linalool and linalyl acetate S6% g.l.c. pure grade (Koch-Light Laboratories

Ltd.), 1^C-labelled linalool (specific activity 0.52 yCi/mg, synthesised as

described in Chapter 2), silicagel G (E. Merck, Germany). Dimethyldichloro-

silane (British Drug House, England), celite 575 (60-80 mesh, Phase Separations

Ltd., England). Glucose-’Analar’ (British Drug House), peptone (Difco),

yeast extract (Difco). Tween 80 and anhydrous sodium sulphate-analar

(British Drug House).

Methods

Rat caecal preparation

Two Wistar albino rats, 10 weeks old kept on standard laboratory pellet

diet were sacrificed by decapitation and the caeca were removed. The caecal

contents were then suspended in 10 ml of sterilized broth media. The caecal

preparation was then made according to Scheline (1966). The composition of

the culture medium was 0.51 glucose, 0.5% peptone and 0.5% yeast extract in

0.1 M phosphate buffer, pH 7.4.

Incubations of linalool (3 mg/10 ml) and linalyl acetate were carried out

anaerobically for 48 hours in liquid broth medium according to Scheline (1966).

Tween 80 (3 mg/10 ml) was added to the medium to keep the lipophilic linalool

in suspension. At the end of the incubation period the medium was extracted

three times with 15 ml diethyl ether. The ether extracts were pooled, dried

over anhydrous sodium sulphate and concentrated to 2-3 ml in a rotary

evaporator at room temperature. For preparative scale studies 51 incubation

medium containing 1.5 g of linalool was incubated with preparations from the

contents of 6 rat caeca. The incubation was carried out as above. The ether

extract of the metabolites (4 x 600 ml) was dried over anhydrous sodium

sulphate and concentrated to 30 ml in a rotary evaporator.

Two controls were prepared similarly except in one case (a) the caecal

extract was omitted and in other (c) linalool was omitted.

Thin-layer chromatography

Hie ether extracts of linalool and linalyl acetate metabolites, and

two controls along with authentic samples were chromatographed on thin-layer

of silicagel G impregnated ivith 2.5% silver nitrate solution in water. The

solvent system used was chloroform + methylene chloride + n-propanol +

ethylacetate (10:10:1:1). The chromatogram was developed for 17 cm twice

in the same direction with the same solvent for better separation.

.Separation, and Characterisation of metabolites by preparative gas chromatography

(i) Preparation of acid washed and dimethyl dichlorosilane treated solid

support, Celite-545.

Acid washing is needed to remove traces of iron that may catalyse

decomposition and isomerisation of compounds (Liberti, 1S58). Celite

545 (60-80 mesh) was treated for sixteen hours with aquaregia. Hie acid

wash was then followed by several water washes. To remove traces of

residual acidic reaction which may interfere with the stationary phase

and also with the solute the solid support was treated with 2 M-NaOH.

It" was then followed by washing several times with water until neutral

and the Celite was dried at 120°C for eight hours.

(ii) Treatment with dimethyl dichlorosilane (DMCS). The solid supports tend

to absorb the solute and therefore, interfere to give trailing peaks and

consequently affect the retention volume. The absorption is particularly

evident in the case of polar compounds. Hydroxyl groups of silicious

compounds may be partly responsible for the absorption phenomena and can

be reduced by treatment with dimethyldichlorosilane (DMCS) followed by

a methanol (absolute) wash. Hie treatment with DMCS replaces the free

hydroxyl groups near the surface by silylgroups.

- Si - OH I0I

- Si - OH

Cl CH3\ /Si

/ \Cl ch3

CH;-2 HC1

- Si - 01 \ /0 Si1 / ~^ ch3

- SiI

0

Chlorosilyl ether is formed in case of one isolated hydroxyl group,

- Si - 0 HI0vl

- Si -

Cl ch3\ /Si

/ \Cl ch3

- HC1

- Si - 0 CH30I

- Si

Si/ X CH3

Cl

The SiCl group may give rise to OH-group after hydrolysis and therefore the

treatment with DMCS followed by washing with methanol gives rise to an inert

methoxy derivative as follows:

CH3ah- Si - o - s - Oh - HCl

0I

- Si -

[ c l +_HjO CH3

- Si - 0 - Si5 I 0

ch3 I0 - CH3

- Si I

Procedure: Acid washed Celite 545 (100 g) was treatedjvith gaseous nitrogen

bubbled through 40 ml of DMCS according to Prevot (196%), The DMCS treated

celite was washed with methanol (absolute) and dried at 100°C.

Coating of the solid support with stationary phase

Carbowax 20 M (10 g) was dissolved in 130 ml of methanol, the solution

was then mixed with 50 g of celite (AW-DMCS) in a 500 ml round bottom quick-fit

flask. The slurry was agitated gently to coat the solid support evenly with

the stationary phase. The solvent was evaporated at 40°C in a rotaiy

evaporator under vacuum.

Packing and purging of column

The packing material coated with stationary phase was seived to obtain a

60-80 mesh fraction. A 7ft.-x 1" column was packed and purged at

190°C under a slow stream of nitrogen for 16 hours.

Preparative chromatography

The Pye 105 Mark 2 Gas chromatograph equiped with flame ionization

detector was used for separation and collection of the metabolites in

milligram quantities, necessary for identification using physico-chemical

methods such as i.r., n.m.r. and also mass spectrometry. The column packed

with 20% Carbowax coated on celite 545 as decribed above was used. The

temperature was programmed from 75°C to 140°C at 2° increase per minute and

N2 (97 ml/min) was employed as carrier gas. The injection temperature was

maintained at 190°C. The separated metabolites were collected in U-shaped

glass traps kept cooled by immersing in liquid nitrogen in a Dewar flask.

Compounds corresponding to two major peaks A and B of retention time 41 min

and 28.4 min respectively were collected.

Checking of the homogeneity of the metabolites by thin-layer chromatography

The homogeneity for purity of the two compounds were checked by thin-

layer chromatography using silver nitrate impregnated silicagel G and the

same solvent system as above (Page 56)* Metabolite B was shown to be.homogene­

ous where as a metabolite A revealed the presence of two substances of values

0.34 (Ai)'and 0.52 (A2).

Preparative thin-layer chromatography for the separation of Ai and A2

Silver nitrate thin-layer chromatography and the same solvent system as

above was used for preparative collection of Ai and A2. Thin-layer silicagel G

(silver nitrate impregnated) plates (20 x 20 cm) of 0.75 mm thickness was used

for the purpose. After the application of the material, the plates were given

a double development. The separated components did not give any fluorescence

under u.v. light. The positions of the separated components were located by

spraying, with 7.5% phosphomolybdic acid in 95% ethanol, an exposed part

(1 cm) of the chromatographic plate whilst the major part was covered with a

(20 x 20 cm) glass plate. The .area of the plate corresponding to the separated

components was scraped off and extracted with ether.58

Mass spectrometry

The mass spectra of the extracted substances Ai and A2 were obtained

using the AEI MSI2, high resolution mass spectrometer.

Materials were introduced into the ion source by direct insertion probe

with inlet temperature 100°C. Source temperature 280°C, ionization voltage

70 EV and acceleration voltage 8 KV.

Synthesis of 1,2 dihydrolinalool

Dihydrolinalool was not available commercially and therefore, was

synthesised as follows: ethyl magnesium bromide was prepared from

bromoethane and metallic magnesium in anhydrous ether medium in presence of

iodine catalyst. The apparatus used for this purpose was similar to that in

figure 2.3 in Chapter 2. The ethylmagnesium bromide was then condensed with

methylheptenone in the same apparatus. The hydrolysis of the product formed

was carried out with saturated aqueous ammonium chloride, as described for the

synthesis of 11+C linalool in Chapter 2.

CH3-CH2-Br } CH3-CH2-MgBrBromoethane Ethylmagnesium bromide

CH3 ch3 ch3

Ethylmagnesium Methylheptenone bromide

1,2 dihydrolinalool

Purification

Hie purification was carried out with the Pye 105 Mark 2 Automatic

preparative Gas chromatograph equiped with flame ionization detector that was

used for the purification of 1 C-linalool (see Chapter 2). The same column

and similar conditions as linalool purification were used for the purification

of 1,2 dihydrolinalool and, strikingly, under the same conditions with 20 M

carbowax (on celite 545) column both had similar retention times.

Characterisation

Synthetic dihydrolinalool was characterised by physico-chemical methods

such as thin-layer chromatography, g.l.c., i.r., n.m.r. and mass spectrometry

(see below).

i. Thin-layer chromatography

The thin-layer chromatographic investigation was carried out for

examination of the presence of unsaturation in the synthetic- compound.

Silicagel G coated thin-layer plates were developed in chloroform and the

plates were sprayed with Fluorescein-Bromine reagent (Runge, 1963).

ii. Gas liquid chromatography

The gas liquid chromatographic analysis was achieved during the purification

process of the synthetic dihydrolinalool mentioned above. %

iii. Infra-red spectrometry

The infra-red spectrum of the liquid was taken between the two KBr plates

in the Perkin-Elmer 5133 spectrometer.

iv. Nuclear Magnetic Resonance Spectrometry

The nuclear magnetic resonance spectrum of the compound dissolved in

CDCI3 was determined with Perkin-Elmer R-10 60 MHz continuous wave n.m.r.

at magnet temperature of 33.8° with tetramethyl silane as internal standard.

v. Mass spectrometry

Materials were introduced into the ion source by direct insertion probe

in M.S. 12 mass spectrometry as previously described.

Quantitative determination of metabolites produced from 1^-labelled linalool

by rat gut microflora

The relative concentration of the metabolites were determined by t.l.c.

and scintillation counting. Hie ether extract was applied on silver nitrate

impregnated silicagel G plates and developed with chloroform + methylene

chloride + n-propanol + ethylacetate (10:10:1:1) as before.

For localization of radioactivity the t.l.c. plate was scanned on a

DUnnschicht-scanner II, (Model L.B. 2723, Counting range 10-101* cps,

Argon:methane gas (90:10) flow, 0.5 1/h, chart speed 2.5 cm/min). For

quantitative determination of metabolites parallel strips of silicagel (5 mm)

from the non-peak portion and 3 mm from the peak areas were scraped off from

the plate and were counted directly in a dioxane base scintillant (see

Chapter 4). Hie radiochromatogram has been shown in figure 3.1.

Species differences

In vitro metabolism of linalool by intestinal microflora from rat,

mouse and guinea pig, sheep, ox and two human individuals were carried out

according to Scheline (1966). Of the two human individuals one was accustomed

to food with spices whereas the other was used to spiceless food. The sources

of microflora from rat, mouse, guinea pig were caecal contents; from cow and

sheep were rumens and from human the source was faecal extract. The

metabolites along with unreacted linalool were extracted in ether as before.

The extract was examined for metabolites by t.l.c. and the LKB 2091 combined

GC/MS system.

Thin-layer chromatography

Silver nitrate impregnated silicagel G and the same solvent system as

used in earlier investigations were used for analysis of the ether extracts

from each species. The plates were given double deveopment for better

resolution of the components of the mixture.

61

Tet

rahy

drol

inal

ool

aoa

r— I rHCQ

COtH

OrHrH

•pH x — n

O LO 05O

i—< oo

\ — CO

aoaaa

*s0£oCQ

OOrHda• rHpH'd0rHrH03r*H1OtHm •0 d+ j ^1 ° d «+-h?H O4-> Sj><1 o0 -rHa a0rC 4->0s i

Oa 'd d 0 o o cq a o 'd o^ ada

CQQ30 h-h>>;zi.S o 'T £e £

£ 2H a

COobe•rHra

62

Analysis with combined gas liquid chromatrography and mass spectrometry system

A Pye 104 analytical g.l.c. linked with LIQ3 2091 Mass Spectrometer was

used in these studies. The column used was §n i.d. x 3 ft

and was packed with 20% 20 M carbowax on celite 545 (60-80 mesh size,

AW-DMCS). The column temperature was 140°C and injection temperature 190°C,

helium was used as carrier gas (30 ml/min).

All the components were introduced into the mass spectrometer through

a j et separator and the spectra were taken at acceleration voltage 3.5 KV

with a scan speed of 30 decade/sec;(slit setting of 0.05/0.05 mm; filter

200 Hz).

Quantitation of the metabolites: from the g.l.c. peak areas

The percent conversion was calculated by comparison of the area under

each peak of g.l.c. trace.

With differential detectors that respond to various concentrations of

solute in the carrier gas, the curves obtained are mainly Gaussian and the

areas they enclose are related to the quantity of eluted substance according

to the relation,

W. = K. A.l i iwhere W. is the weight of substance T’ passing through the detector, and K. the1 -v 1factor of proportionality, taking into account (amongst other factors) the

sensitivity of the particular detector to the substance being detected and

the sensitivity of the recording apparatus.

The area was determined in two different ways depending on shape of the

curve. For symmetrical peaks the area was determined according to Ball, jet al

(1968) considering the peak as a Gaussian curve.Where the peak was not

symmetrical the area was calculated according to..Kaiser (1965).___ —

Statistical MethodsIn all experiments at least eight animals (4 treated and 4 controls)

were used. Statistical evaluation of the results was determined according to Bailey (1959).

63

Fig.

3.2

IR Sp

ectr

um

of Sy

nthe

tic

1, 2

Dilw

drol

inal

ool.

maoJHo• pHs

LO<MCD

CDrH

oo00

o_ O O

OOO“'<MO r—t

00

O

O O ” CDrHO

CDOO00

o - o oCQo

o L oLO

CQO

oooCO

oo“ LO CO

CO

Oooo oo o

iHIo

Cl)rQagCL)c$>

o CO CD CQ

(%) oouu^imsuBJX

64

RESULTS

Synthesis of dihydrolinalool

Thin- layer cinematography

Mien the t.l.c. plate was treated with fluorescein-bromine reagent, the

spot corresponding to the synthetic dihydro-compound prevented eosin formation

by absorbing bromine. A similar quantity of authentic linalool prevented

eosin. formation of the larger area than the synthetic dihydro-compound whereas

tetrahydrolinalool gave no positive results. They have the comparable

polarities (linalool, value-44, synthetic compound 44, tetrahydrolinalool

46, using ordinary silicagel G thin-layer plates .and chloroform as solvent).

This therefore indicated that the synthetic compound possessed unsaturation

with similar polarity to linalool.

Gas-liquid chromatographic analysis

G.l.c. showed that the synthetic compound had a retention time of 19.6 min

compared to that of linalool of 20.1 min. This retention time was higher than

tetrahydrolinalool (14.3 minutes) under similar conditions.

Infra-red spectrometry

Hie i.r. spectral data has been tabulated in table 3.1. The i.r. spectrum

has been shown in figure 3.2.

Table 3.1 Infra-red spectral data of synthetic 1,2-dihydrolinalool

Compound Infra-red spectrum in KBr Cm 1

CH3 ch3

1,2 dihydrolinalool

y - OH - 3400 (broad)

CH H\ /CII

/ C\ch3 ch3

Y - CH2 -

( 2970 (( 1675 (( 835

1460

CH3i

Y - - C - 1375

Y - OHtertiary

Y - CH2 ~ CH31149

105565

Mass spectrometry

The mass spectrum showing the relative peak intensity against m/e ratio

has been shown in fig. 3.3 and the peaks due to possible fragments has been

worked out in table 3.2.

Table 3.2 Fragmentation pattern of 1,2 dihydrolinalool obtained in MS 12

Mass Spectrometry

Compound m/.e fragments

CH3rOH

CH3

ch3 ch3

A

109

73

OH i.e.i.e. A-(CH3-CH2j

A

CH3I

-J.~~ C -i l l.OH

CH3

69

H2C- H

ch3 ch3

55 HC\

H

ch3CHs

66

Compound m/e fragments

CH3

OH I- HC

CH3

+57

CH31ch3 ch3

"1

138

67

Fig.

3.3

Mas

s Sp

ectr

um

of Sy

nthe

tic

1, 2

Dih

ydro

linal

ool.

o_ CD

O LO rHO

CO _ CO

L. co

o L_ <M

05OOOO05

O CO

” ---------------------------------------- :------------------- L e05CD-------------------------------- — --- ~l50 t- L °~ I CD

io 10-------------5LOoLO

CO

05 f COCM ■hV J j whwuu i |,!arj3aiiw»im«

o o o o o o o o o o0 05 CO C— CD LO ^ CO CM rHrH

( % ) £[ISU8}UI 0AI}U-[0H

68

Nuclear magnetic resonance spectrometry

Hie n.m.r. spectrum of authentic linaloolttig 3.4)contains three quartets,

in the olefinic region, two of which are overlapping and one poorly resolved

triplet whereas in dihydrolinalool there is one triplet only. No absorption(fig.3.6)

peak was observable in tetrahydrolinalool in the olefinic region/and this is

expected because of absence of unsaturation. The three quartets in linalool

are due to the proton at 02- and two non-equivalent protons at C-l carbon

atoms leading to a typical ABX type spectrum (see fig. 4a).

10

CH3

OH2 / hx

S.Cl

H, HB9-y* 8 A

II!ch2

ch3 ch3

The quartet centered at 6 4.23 is due to the proton at 0 2 split by the

. protons and Hg (J^ =17 cps; Jg^ = 10 cps). The quartet centered at

6 4.88 and 5 5.0 are due to protons and proton Hg each being split by proton

HX JAX and by each other (J g or JgA - 2 cps). The

NMR spectrum of 1,2 dihydrolinalool shows only a triplet in the olefinic'v

region (6 4.7 - 5.2, fig. 3.5) which is also present in the spectrum of

linalool (6 5.35 - 5.54, fig. 3.4). This triplet in both cases is due to

the single me thine proton at C-6. The methine proton couples with the

methylene (-CH2-) proton at position 5 and the two methylene protons split

the methine proton absorption into a triplet. This confirms the fact that

in the synthetic 1,2-dihydrolinalool reduction of the double bond has

occurred. The downfield appearance of the chemical shift of the triplet in

the 1,2 dihydrolinalool compared to linalool is due to the dishielding effect

after the saturation of the tt bond at 1,2 position.

The strong absorption peaks of chemical shifts between 6 = 8.31 to 10.0,

the legion of the substituted ali.phatic compoundsjare due to tertiary CH3 groups

69

in linalool, dihydrolinalool and tetrahydrolinalool. Absorption by the

methylene groups at and Cg gives rise to multiplets in the same region,

overlapping in part the peaks due to the methyl groups at Cg and Cg.Metabolites produced by rat caecal microflora: thin-layer chromatography

Three compounds of values 0.42 (A), 0.50 (B) and 0.70 (C) were

separated by silver nitrate impregnated silicagel G thin-layer chromatography.

Linalool, dihydrolinalool (synthetic) and tetrahydrolinalool had R£ values 0.40,

0.50, 0.68 respectively and are comparable to compounds A, B and C. Thin-layer

chromatograms of the metabolites of linalyl acetate showed four spots of R^

values 0.41, 0.46, 0.50 and 0.70. The material of R^ value 0.46 was due to

unmetabolized linalyl acetate and the other spots appeared to be identical

with linalool (0.41) dihydrolinalool (0.50) and tetrahydrolinalool. It appeared

then, that the rat caecal microflora hydrolysed linalyl acetate prior to

reduction since acetates of dihydrolinalool and tetrahydrolinalool were not

detected.

Preparative gas liquid and thin-layer chromatography

Two samples corresponding to the major peaks of. retention times 21 min

(D) and 14.3 (E) were collected by gas liquid chromatography. The compound

(E) had a similar retention time to authentic tetrahydrolinalool. The

compound (D) was a mixture of two compounds (D and D2) as separated by

silver nitrate t.l.c. had the similar R£ value as linalool p.40) and ^

similar to dihydrolinalool p. 51).

Infra-red spectrometiy

The i.r. spectrum of B was similar to that of authentic tetrahydrolinalool

(Fig. 3.7) and this metabolite (B) was identified as tetrahydrolinalool.

Mass spectrometry

Compound and dihydrolinalool had the similar mass spectrum of m/ e 100,

73, 69, 43, 41, 55, 138, 67, 29, 57 (Fig. 3.3). The percent intensity of each

m/e peak was matchable to each other and therefore D2 was identified as

70

Fig.

3.4

N. m

. r.

Spec

trum

of

Aut

hent

ic

Lin

aloo

l.

§P4fto

03

00

CO

LO

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71

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CDrC■wSHO£O•r-lbnCDhi£«+H00r£-f->£•pHbn£•rHr-HQ*£O01£•rHwO pH“ Ir~1 *r—(03 i—<a <J o

d

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72

: ft

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o•rHH->CDA"S{sCOoau-MoCDftCO

incob/)•rHft

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73

Fig.

3.6

N.

m.

r. Sp

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of A

uthe

ntic

T

etra

hydr

olin

aloo

l.

pJ pIoT—I

00

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ID

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CO

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74

Fig.

3.7

ER

Spec

tra

of (1)

A

uthe

ntic

T

etra

hydr

olin

aloo

l(2)

M

etab

olite

B

CD ID

mS3ouo•rHs

CMCD

L oI oo

ooooo

I o_ oCQt—I©00

oor rHO

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OCD

Oooo

o _ o oCMoto

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O

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OCO OO

- LD CO

O~ ° oO "CfOCMO

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OO

iHIsou<DII>

£

(%) aoun^ixnsu'BJx

75

% R

adio

acti

vity

F ig . 3. 8 Chromatographic p rofiles of 14C -labelled linalool m etabolites produced by the rat gut m icroflora.

6 0 -

5 0 -

40-1

3 0 -

2 0 -

10 -

0 .4 1.00. 2 0.6 0.8Ry Values

A — Unidentified Metabolite

B - Unmetabolized 14C Linalool

C - 14C Dihydrolinalool

D - 14C Tetrahydrolinalool

E - Unidentified Metabolite

76

dihydrolinalool. Compound had superimposable m/e ratios with linalool

and therefore, was identified as unmetabolised linalool.

Quantitative determination of metabolites of ltfC-labelled linalool by rat

gut microflora

The relative proportions of the radioactivity present in the metabolites

have been' shown diagrammatically in fig. 3.8. The incubation for 48 hours has

produced 38% tetrahydrolinalool and 16% dihydrolinalool. Two other

unidentified metabolites of R£ values 0.24 and 0.9 containing 3.7% and 2.6%

of radioactivity respectively were also formed by the rat microfloral

metabolism.

Species differences

The thin-layer chromatographic examination of the products of metabolism

of linalool by the gut microflora of different species has been recorded in

table 3.3. It showed that incubation for 48 hours with rat caecal microflora

produced two metabolites of values 0.5 and 0.7 which corresponded to the

R£ values of dihydrolinalool and tetrahydrolinalool. The presence of

unreacted linalool was evident by the Revalue 0.42 of one of the components

of the earlier extract.

Incubation with intestinal micro flora of mice or sheep resulted in the

formation of a major metabolite of R£ value (0.50), that corresponded to

dihydrolinalool. A considerable quantity of unreacted linalool was also

present.

Incubation with rumen content of cattle gave rise to a metabolite of

R£ value 0.24 which was not produced by the other species except the human

individual who traditionally used spices in his food. Hie metabolic pattern

produced by the microflora of a second human individual who ate spiceless

food presented a different picture by producing a metabolite of Revalue 0.70

corresponding to tetrahydrolinalool.

Combined GC/MS revealed that tetrahydrolinalool was the major metabolite

77

Table 3.3 Thin layer chromatography of in vitro metabolism of linalool by

the gut microflora of different species. Silver nitrate

impregnated silicagel G thin layer (0.25 mm) plates were used

as stationery phase and chloroform + methylene chloride +

n-propanol + ethylacetate (10:10:1:1:1) as moAdng phase.

Authentic samples and metabolites

values Identified as/

Species

Linalool 0.41 -

Dihydrclinalool 0.52 - -

Tetrahydrolinalool 0.71 - -

A 0.24 not identified Man (spice user), cow

B 0.41 linalool (un­metabolised)

in all species

C 0.51 dihydrolinalool Rat, mouse, sheep

D 0.70 tetrahydrolinalool Rat, man (spice non-user)' %

78

and dihydrolinalool, a minor metabolite in rat caecal incubates with

considerable amounts of unreacted linalool. In mouse caecal incubates no

tetrahydrolinalool was present and dihydrolinalool was the only major

metabolite. Butyric acid was present as one of the metabolites and could

have been produced from the medium and not from linalool metabolism since the

branched carbon skeleton of linalool would be expected to give isobutyric acid.

However, butyric acid was not produced as a major metabolite in control

incubates in the absence of linalool.

In incubates with guinea pig caecal contents there was no sign of the

presence of metabolites thus suggesting that metabolism of linalool by the

guinea pig gut microflora was essentially non-significant.

Sheep produced two major metabolites one of which was identified as

dihydrolinalool the other metabolite F identified as a-terpineol (see below).

One minor metabolite (metabolite A, table 3.4) of retention time 5.0 min was

identified as y^terpinolene (peaks at m/1 93, 121, 39, 41, 79, 90, 27, 77,

53, 43).

Cattle rumen microflora produced a compound as major metabolite of

retention time (13.8) and having the same mass spectrum as that of the

metabolite F (table 3.4); its mass spectrum has been shown in figure 3.9.

Computer search revealed that the mass spectrum of metabolite F was a close

fit to (a) a-terpineol, (b) isopulegol, (c) nerol, (d) thuijyl alcohol;

however, the best fit was suggested to be the mass spectrum of a-terpineol.

The possible mechanism of formation of a-terpineol from linalool, under

anaerobic conditions has been proposed below.

+ OH

OH

•Linalool a-terpineol

Mechanism of foimation of a-terpineol from linalool

Rumen microflora of cattle also produced a metabolite (metabolite J,

- 26.2 min) which was common to that produced by faecal flora of the

spice-using human subject (see below).

Human (spice user) faecal flora produced a major metabolite of

retention time 13.6 minutes and possessed mass fragmentation pattern

as in metabolite F (fig.3.9), the same was the major metabolite both in.

sheep and cattle. Two other metabolites E and J of retention time 12.2

and 26.2 minutes were also produced. Their m/e ratio's have been recorded

in table 3.4. The mass spectrum of metabolite J showed the strongest

peak at m/e 60, the usual characteristic fragment of carboxylic acids.

Both these metabolites remained unidentified. % .

The faecal microflora of the second human subject (spice non-user)

produced tetrahydrolinalool (D, table 3.4) and compound F, (X-terpineol

as major metabolites. Metabolite G having.retention time 14.2 min. was present

as a minor metabolite, and was identified by mass spectrometry (m/e recorded

in table 3.4) as ethylhydroxy isobutyrate. The formation of ethylhydroxy

Fig

.3.9

M

ass

Spec

trum

of

Met

abol

ite

F.

LO­

COCO.

05

OCDOI- LO

OL rHOCO

oCS1rHO_ rH

Oo<D

CO05COCO

00

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( % ) i isua uj 0Ai^i9H

81

isobutyrate involves a branched form carbon atom fragment which may have

been derived from the branching at positions 3 and/or 7 in linalool.

The m/e ratios of the metabolites produced by the gut microflora from

linalool metabolism together with percent of linalool converted into each

metabolite have been recorded in table 3.4.

82

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unidentified

Cow

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12%)

acid

DISCUSSION

It has been observed that rat gut microflora after 48 hours of Incubation

converted linalool predominantly to tetrahydrolinalool (38%) and dihydrolinalool

(16%). The gut microflora under the anaerobic conditions can reduce

exogenous substances which may either be enzymic or chemical as demonstrated

by Scheline (1966) and Wilde (1966) (see below). Two unidentified minor

metabolites (3.7% and 2.6%) were also present in the rat caecal incubates.

•These may be artifacts that have originated under the experimental conditions.

The similar metabolic pattern of linalyl acetate to that of linalool is largely

due to the esterase activity of the gut microflora which could hydrolyse the

ester linkage to liberate linalool. The liberated linalool on further

metabolism produced metabolites similar to those produced in linalool

metabolism by the gut microflora. The ability of the intestinal microflora to

carry out hydrolysis of the ester linkage was reported previously by Scheline

(1973). Incubation of chlorogenic acid with rat caecal microflora resulted

in hydrolysis of the ester linkage and the further metabolism of the

ddsterified compound to m-hydroxyphenyl propionic acid.

It is interesting to note that the metabolism of linalool by the gut

microflora exhibited considerable species variation. Little metabolism was

observed as a result of the action of guinea pig caecal microflora whereas,

in the case of mouse caecal flora and sheep rumen flora, substantial reduction

to dihydrolinalool was observed In these cases reduction did not proceed

further whereas rat caecal content effected a second reduction to

tetrahydrolinalool which was the major metabolite. The rumen flora of the cow

were not seen to produce any reduction products of linalool, the major

metabolites being a-terpineol and an unidentified carboxylic acid.

a-Terpineol and y-terpinolene were also produced in substantial amounts by

sheep rumen microflora. Hie detection of dihydrolinalool and tetrahydrolinalool

as metabolites are not unexpected. Previously Scheline (1966) observed that

rat microflora reduced substituted cinnamic acid to the saturated phenyl

84

propionic acid and Wilde et al (1966) reported the reduction of double bonds

.of linoleic acid and oleic acid by the rumen microflora of sheep. The

phenomenon of species differences in relation to the metabolism of foreign

compounds is of great importance in evaluating their pharmacological and

toxicological implications. The cause of species variations in metabolism by

the intestinal microflora may be differences in the microbial species

inhabiting the gut of various animals (Scheline, 1968; Drasar, et al-, 1970,

see Chapter I). It has been reported that rabbit intestinal microflora have

been found to metabolize xanthurenic acid 8-methyl ether (Lower and Bryan,

1969) and (+) catechin (Scheline, 1970) differently from that which is seen

with the gut microflora of mouse and rat respectively. Again Walker, et al«5

(1970) have found differences in the metabolism of Brown FK by mouse and by

rat (see page 30 ), with consequent differences in toxicity. The difference

in metabolism may be due either to differences in the nature of the gut

microflora and/or differences in the environment in the large intestine of

these two species, e.g. redox potential. The differences reported here in the

metabolism of linalool between the gut microflora of mouse, sheep leading to

1,2 dihydrolinalool and rat leading further to tetrahydrolinalool may be due to

similar reasons. V

The studies using human faecal microflora revealed interindividual

differences which were as pronounced as the interspecies differences observed

above. Thus the individual who habitually used a highly spiced diet showed

many similarities to the cow in producing substantial quantities of

a-terpineol and the same unidentified acid metabolite. A second unidentified

metabolite E (table 3.4), which on the basis of mass spectral data appeared to

be structurally similar to a-terpineol, was unique to this individual, on the

other hand, the individual who regularly ate a bland diet more closely

resembled the rat in producing tetrahydrolinalool as the major metabolite.

Another interindividual difference was seen in the presence of ethylhydroxy

85

isobutyrate in incubates of faecal material from this individual. The

production of tetrahydrolinalool by the faecal flora of the', spice non-user

and not by the other may be due to the ability of the former to generate lower

redox-potential in the gastrointestinal environment.

The difference in the dietary factors may also have selective influence

in the composition of the gut microflora which may become adapted to metabolize

certain compounds thus leading to variation in metabolism of a compound. An

interesting example in this area is the conversion of cyclamate to

cyclohexylamine. Drasar, et al (1971) found that clostridia in rat faeces

were able to convert cyclamate to cyclohexylamine whereas in human and rabbit

enterococci were responsible for the same conversion. Hill, et al (1971)

showed that faecal enterococci are much higher in the Japanese than in the

British. Kojima and Ichibagase (1966) actually demonstrated that all

Japanese individuals investigated, excreted cyclohexylamine following oral

ingestion of cyclamate. Renwick and Williams (1969), in an experiment with

rat found that after a single dose of cyclamate no cyclohexylamine was produced.

However, rats receiving 0.50% cyclamate for 3 months all excreted

cyclohexylamine as a metabolite up to 351 of the dose.

Oriental foods are usually cooked with spices containing coriander seed

as an ingredient which contains essential oils, mostly linalool, and therefore,

it might have a selective influence on the gut microflora of the spice user

leading to an adaptive situation.

In the metabolism of linalool the observation that gut microflora of some

species carried out reductive reactions reported here, in general, illustrated

typical reactions previously reported by (Scheline, 1966 and 1973) and

Wilde, et al (1966). However, the identification of a-terpineol in the

incubates of microflora from two human individuals, sheep and cow; and

y-terpinolene in the incubate from sheep are evidence of a class of reaction

not previously reported, i.e. cyclisation. These metabolites are lipophilic

86

o /Ethylhydroxy Isobutyrate (man spice-nonuser ) /

SeveralSteps

Tetrahydrolinalool (rat, man spice-nonuser)

Dihydrolinalool (rat, mouse, sheep)

h 3c c h 3

o'-Terpineol Metabolite F (man spice-user, man spice- nonuser, sheep, cow)

Linalool

CHoOH

Geramol

Metabolite E(mass spectrum similar to o'-Terpineol but not identical in man spice-user)

Metabolite J (an acid - , in man spice-user, cow)

y-Terpinolene(sheep)

Fig. 3.10 The possible routes of metabolic transformation of linalool by the ‘gut-microflora of different species. The compounds in the square brackets have not been isolated. The species which can produce a metabolite has been put in parenthesis.

substances and therefore could be absorbed and metabolized in the liver.

In metabolic experiments in the rat in vivo it was observed (see Chapter 4)

that a considerable amount 1^C-labelled linalool was excreted as dihydro- and

tetrahydrolinalool glucuronides in urine and bile. It was also demonstrated

that glucuronides excreted in the bile were hydrolysed by the gut microflora

and linalool and its reduced products were reabsorbed to enter into the

enterohepatic cycle. As a result the excretion was delayed. The wide range

of compounds such as dihydrolinalool, tetrahydrolinalool, a-terpineol,

y-terpinolene, ethylhydroxyisobutyrate along with some unidentified metabolites

(see fig. 3.10) produced by the gut microflora of different species might have

pharmacological and toxicological implications regarding which little is

known. In view of the continued ingestion of linalool by humans and animals

the toxicology of its metabolites need to be evaluated in its safety testing

for use as a food additive. These observations clearly indicate the value of

intestinal metabolic studies, including man, in evaluating the hazard from

food additives since variations between species and individuals, and as

a result of dietary factors were observed. These factors have been discussed

in the literature previously (Scheline, 1971) but, are frequently ignored in

toxicity testing of food additives and other environmental qhemicals.

88

Chapter 4

Absorption, Distribution and Excretion of ^C-.labelled Linalool

in the Rat

89

Absorption, Distribution and Excretion of-^G-labelied Linalool in the Rat

1. Introduction to absorption, distribution and excretion of xenobiotics.

A. Absorption of foreign compounds

Absorption of foreign compounds may take place by oral, respiratory

or dermal routes and hence enter the blood stream. In the course of

absorption and distribution, the foreign compounds encounter membranes of

the gastrointestinal epithelium, the lining of the respiratory tract,

stratum comeum of skin the membranes enclosing the blood and other

body fluids, cell membranes and membranes within the cells, which control

the uptake into tissues and subcellular components. These membranes are

highly complex, dynamic lipoprotein structures consisting of several layers

of cells, e.g. skin and placenta, single layers of cells, e.g. intestinal

epithelium or less than one cell thickness, i.e. cell membrane. Substances

may cross the biological membranes one or more of the processes of (a) passive

diffusion, (b) filtration, (c) facilitated diffusion, (d) active transport,

(e) pinocytosis. Most foreign compounds cross the lipoidal membrane by

passive diffusion down a concentration gradient.

a) Gastrointestinal absorption. The mucosa of the gastrointestinal

tract is a semipermeable membrane across which various nutrients as well as

foreign compounds are transported into the blood by the above mentioned

mechanisms.

Passive diffusion is by far the most important process of absorption of

foreign compounds from gastrointestinal tract. No energy is required, it is

not saturable and the transport is directly proportional to the concentration

gradient and to the lipid/water partition coefficient of the drug. The higher

the concentration gradient and lipid/water partition coefficent the faster

is the rate of diffusion. Movement stops when the concentration on both the

sides of the membrane is the same but this equilibrium situation is very

seldom reached because transfer of the material by blood usually maintains a

90

concentration gradient across the membrane. The phenomenon of passive

diffusion becomes more complicated when the foreign compound is either a

weak base (e.g. Caffein) or a weak acid (e.g. Barbiturates). If these bases

and acids are fully ionized they are very slowly absorbed. Non-ionized drugs

diffuse across the membrane faster as they are readily lipid soluble. The

degree of ionization is related to pH and also to P "a. Acids with higher P^a

will diffuse faster whereas the reverse is true for a weakly basic drug

(Schedl, et al, 1961).

Studies of the gastric absorption of drugs in the rat and human have

shown that the epithelial lining of stomach is permeable to the lipid soluble

unionized form of drugs and relatively impermeable to the ionized foirni, unless

these too are highly lipid soluble. Weak acids namely, salicylates and

barbiturates, which are mainly unionized at the low pH of the stomach contents,

are readily absorbed. Conversely weak bases such as quinine and amidopyrine

are hardly absorbed at all. In gastric juice aniline is largely ionized but

.in the pH of the plasma remains mostly unionized. Therefore, according to

the pH partition hypothesis aniline will diffuse from plasma to the stomach

depending on the maintenance of a concentration gradient of the unionized

molecule.

The intestinal epithelium, like gastric mucosa allows ready penetration

of lipid soluble, unionized molecules and generally retards that of ionized

(Parke, 1968; Schanker, 1962). In the small intestine the contents have pH

of about 6.5 but true pH of the intestinal lumen is 5.3 (due to secretion of

hydrogen ions, Hogben, et al, 1959) where aniline will be less extensively

ionized than rat stomach pH and hence will be absorbed in the intestine.

The absorption in rat colon follows the similar pattern as that of

small intestine (Parke, 1968). The pH increases in the contents of colon

(pH 7-7.5). Stronger acids that are more highly ionized at this pH are poorly

lipid soluble and thus do not cross lipoidal membranes readily. Weak acids

and bases are generally readily absorbed.

The large intestine may be a site of absorption of compounds poorly

absorbed by small intestine, and particularly of their metabolites produced

by the micro flora of the caecum and colon. The main site ofkclabsolution of an ionized substance is largely dependent on itsp , and the

lipid solubility of its ionized and non-ionized forms.

b) Factors affecting gastrointestinal absorption

(i) Absorptive surface - Hie small intestine has particularly a huge

surface area. The absorptive surface is increased about thirty to forty times

by microvilli which are covered with lipid protein membrane and thus provides

one condition for rapid passive drug absorption.

(ii) Gastrointestinal motility - Accelerated gastric emptying reduces

gastric absorption but increases intestinal absorption. The extent of mixing

of the intestinal content depends on the rate of intestinal peristalsis which

to some extent influences the absorption (Parke, 1968). It has been reported

that anticholinergic drugs interfere with absorption by diminishing the

intestinal motility (Binns, 1971).

(iii) Circulation in the gastrointestinal tract - The increased

intestinal blood circulation will maintain the higher concentration gradient

and would thus accelerate the absorption of drug.

(iv) Alimentary secretions - The pH of the alimentary secretions varies

from pH 1-3 in the stomach to pH 6-6.5 of the intestinal and pH 7-7.5 in the

colon (Binns, 1971; Chasseaud, 1970). The effective pH of the intestinalICct lCclsurface is 5.3 and at this pH the acids with P 3 and bases with P > 8 are

poorly absorbed (Brodie, 1964). The intestinal juices contain numerous

hydrolytic enzymes such as esterases, amidases and hydrolysis in the gut of

conjugates excreted in the bile may lead to enterohepatic circulation due to

reabsorption of the deconjugated compound.

(v) Drug interaction within the lumen - Intraluminal interactions

affecting absorption were demonstrated by administering activated charcoal,

which decreased the absorption of promazine (Sorby, 1965), while the increased

absorption of fluoride caused by iron, phosphate and sulphate in the rat is

probably by the ionic interaction within the lumen (Stookey, et al, 1964).

The absorption of tetracycline is impaired by formation of chelates with

calcium JLron and other metals (Parke, 1968). Changes in intestinal absorption

due to change in the composition of diet have been reported (Underwood, et al,

1951; Reynolds, 1965). Underwood (1951) demonstrated that increased

absorption of Ca is effected by vitamin D. The absorption of glycine, fat

and xylase is depressed by the deficiency of vitamin Bi2. Little is so far

known about the effects of dietary changes on the extent of absorption of

non-nutrients but Booth, et ab*5(1958) demonstrated that different basal diets

fed to rabbits affected the absorption of hespiridin.

(vi) Other factors:

Species differences - The different sensitivities of experimental animals

to foreign compounds is well known. This may be exemplified by the difference

in absorption of coumarin between man and rat (Crampton, 1970). Daniel, et al.5

(1968) demonstrated that rat and man differ in their metabolism of 3,-5,di-

tert-butyl 4-hydroxytoluene (BBT). Such differences could be due to different

lumen pH affecting ionization (Crampton, 1970).

Sex - Crane (1960) has reported some sex differences in the absorption of

glucose in male and non-pregnant female rats.

Age - In the elderly decreased absorption of vitamin A and thiamin,

vitamin Bi2 and amino acids have been reported (Rafsky, et al, 1943; 1948;

Ellenbogen, et al, 1958; Fowler, et al*, 1960). The.primary causes have not

been established.

Gut microflora - Compounds which are themselves not readily absorbed may

be bio transformed by the gut microflora (See section C, Bio transformation) and

the intestinal metabolites subsequently absorbed. In view of the qualitative

and quantitative differences in the composition of the intestinal microflora

93

of different species (Drasar, et al*, 1970) the variation in the nature of the

metabolism carried out by the gut microflora may lead to increase or decrease

in absorption of the foreign compounds and their metabolites. Moreover, the

nature of the gut flora, and hence their metabolism, may also be affected by

diet.

Disease of the gastrointestinal tract

Removal of part of the gastrointestinal tract in diseases like peptic

ulcer and malignant tumours leads to the impairment of absorption. Tne removal

of the stomach, is followed by loss of gastric secretions, increased motility

of the small intestine and shorter transit times and therefore, a diminished

absorption is expected (Stammers and Williams, 1967). Underhill (1910-11)

suggested removal of appreciable segments of small intestine has surprisingly

little effect on change of absorption, however, diminished absorption of fat,

calcium, potassium and magnesium, vitamin A and other nutrients have been

observed when greater length of small intestine is removed (Trafford, 1956-7;

Croot, 1952; Althausen, et al, 1950).

c) Other routes of absorption

Absorption through skin, from trachea and lungs are of little importance

to nutrition but may be of considerable toxicological importance. The

epidermis of skin is a lipoidal barrier and is therefore more readily permeable

to lipid soluble substances. The topmost homy keratinous layer stratum

corneum gives the barrier property to the skin (Vinson, 1965).

Inhaled gases and vapours are rapidly absorbed through the alveoli which

have thin, highly vascular membrane of large surface area. Unlike the body

membranes, the pulmonary epithelium appeares to be highly permeable to lipid-

soluble molecules and also to lipid-insoluble molecules and ions (Enna, et aL,

1969). Absorption through skin and lung becomes important for volatile solvents

such as aromatic and chlorinated hydrocarbons. The antileprosy drug

diethyl dithiolisophthalate is administered percutaneously because of the

nature of the disease and because other usual routes of administration are

94

unsatisfactory (Ellard, et ai, 1963).

Administration through lungs becomes important for lipid soluble gases,

for example, anaesthetic gases. This route is also of some importance when

drugs are administered as aerosols, e.g. Intal in the treatment of asthma.

B. Distribution

Foreign compounds or their metabolites which are absorbed from the

gastrointestinal tract enter the liver via the portal vein.Absorption may also

take place via the lymphatic system and transport to the superior vena-cava.

Mien the substance reaches the liver it .may be metabolized and/or conjugated and

transported via the hepatic vein to the kidney for excretion in the urine.

Xenobiotics or their metabolites may be carried in blood stream (Schanker, 1964)

in one or more of the following ways.

1. As free-diffusible molecules dissolved in plasma.

2. As molecules reversibly bound to protein and other constituents of

serum.

3. As free or bound molecules contained within the erythrocytes.

These are then distributed by circulation in blood between various organs and

tissues as well as intracellularly. The concentration of the drug level in

the blood thus gradually decreases with tissue uptake. Bio transformation and

excretion further decreases the concentration of the compound in the blood. .

Some of the compound returns from the tissues to the blood, and the process.,

one of mobile equilibrium, continues. Mobile equilibrium involves interrelated,

interdependent and continuous processes in which the compound is circulated,

taken in, released and circulated. Finally, the compound is eliminated from

the body, usually partly bio transformed (Chasseaud, 1970). .The compound may

not be excreted quickly if it is localized in tissues (including adipose

tissue), erythrocytes or bound to plasma proteins. Tissue storage and plasma

binding of foreign compounds increase the residence times of the drug thus

prolonging its physiological action. Protein binding on one side of the

membrane increases the concentration gradient and thus facilitates absorption

by passive diffusion. Foreign compounds may displace endogenous substances

e.g. steroids that are bound to plasma proteins and thus unbalance the

bound:unbound ratio in the plasma causing altered physiological effects

(Westphal, 1969; Brodie, 1965).

C. Biotrans formation

The ability of the intestinal microflora to metabolize widely different

chemical structure has been reported (Scheline, 1973; Shrier & Noller , 1957).

Water soluble foreign compounds could reach the heavily populated caecum where

the gut flora may metabolize them before absorption. For example, Radomski

and Mellinger (1962) demonstrated that a number of water-soluble azo dyes were

poorly absorbed after oral administration and were metabolized to a large

extent by the gut microflora. Significant amounts of the metabolites

(reduction products) were then absorbed.

The metabolites that are produced by the gut microflora may be less or

•more toxic than the parent compound. Thus, the glycoside amygdalin when

administered orally to mice is toxic due to the liberation of cyanide after

hydrolysis of the glycoside by the gut microflora. Parenteral administration

of amygdalin is practically non-toxic. Similarly, the carcinogenic effect of

cycasin is due to the production, by the intestinal microflora, of methyl

azoxymethylglucoside which is believed to be metabolized to the proximate

carcinogen diazomethane. Cycasin is not carcinogenic when injected.

Bacterial glucuronidase may hydrolyse polar glucuronides of foreign

compounds or of endogenous substances which are excreted in the bile. The

parent compound is usually more toxic than the glucuronide and is also less

polar and thus can be reabsorbed to establish an enterohepatic cycle. The

effects of the compound would thus be prolonged.

Detoxication which is not common in the gut can be exemplified by the

deconjugation of biliary excreted carcinogenic N-hydroxy-N-2-fluorenylacetamide

96

glucuronide by the gut microflora to the parent compound which has lower

carcinogenic activity. The parent compound is further metabolized by the

gut flora to the dehydroxylated compound which in turn is less carcinogenic

(Walker, 1973; Grantham, et aL, 1970). The potential of the metabolic

activity of the gut microflora could be comparable to that of liver

(Drasar, et al, 1970). In addition microbial composition can change due to

various selection pressures. So that the adaptive capacity of the gut flora

is greater than that of the liver. For example prolonged exposure to

cyclamate can lead to increased conversion to cyclohexylamine by the gut

microflora both in experimental animals and in man (Williams, 1970). Hie

overall' metabolic transformation in the liver is commonly synthetic whereas

in the gut it is more usually degradative in nature.

However, the foreign compounds or their metabolites produced by the

gut microflora enter the portal blood circulation after being absorbed from

the gastrointestinal tract, and are transformed by the tissues principally by

the liver but in some cases by other organs like kidney, lung, placenta. The

nature of reactions and biological properties of metabolites indicate that the

general pattern of biotransformation is biphasic (Williams, 1960). In the

phase I reaction foreign compounds undergo oxidation, reduction and/or hydrolysis

to introduce -OH, -C00H, -NH2 groups. Phase II reactions involve conjugation of

the xenobiotic or its phase I metabolites with endogenous compounds to produce

more polar compounds and facilitate urinary and/or biliary excretion. In the

course of phase I reactions foreign compounds closely resembling endogenous

substrates may enter pathways of intermediary metabolism (Chasseaud, 1971).

In coprophagic animals, which includes most rodents, the regular ingestion •

of faeces results in an active microflora throughout the whole length of the

gastrointestinal tract and microbial metabolism may occur even in the stomach.

D. Excretion

a) Biliary Excretion - As a result of high permeability of the hepatic

parenchymal cells the boundary between the blood and bile is extremely porous

so that molecules and ions smaller than proteins can pass through. Many

substances therefore appear in bile in concentration similar to those of

plasma (Parke, 1968). Highly polar compounds such as bile salts, bilirubin

glucuronides and conjugates of foreign compounds are excreted into the bile

in much higher concentration by a process of active transport. Active

secretion has also been reported to occur with compounds which are present

in the blood as anions, and have molecular weight greater than 300 and are

bound to plasma proteins (Brauer, 1959). Food additives having suitable

conditions for conjugation may-form glucuronides, e.g. erythrosine (Webb,

et aL, 1962) and less frequently as sulphates, e.g. indocyl sulphate and

mercapturic acid, e.g. BHT. Compounds may appear in the bile in the original

form, e.g. Ionox 220 (Wrigert, 1966). DDT and its metabolites are also

excreted in bile (Bums, 1967).

Species variation has been observed in the excretion of foreign compounds

in bile. Highest biliary excretion was demonstrated in dog and rat

(Abou-el-Makarem, 1966). Increasing number of hydroxyl groups increases biliary

excretion since 4-4’ dihydroxybiphenyl monoglucuronide is more readily excreted

than 4 hydroxybiphenyl glucuronide (Parke, 1968). Decreased urinary excretion

may occur due to increased excretion in bile. This was observed for several

fluorescein dyes in rat (Eakins, et al, 1969).

b) Enterohepatic circulation - The conjugates of foreign compounds

excreted in the bile may partly undergo hydrolysis by the enzymes

3~glucuronidases,sulphatases, etc., present in the bile or intestinal secretion

or enzymes of the gut microflora. The hydrolysed drug may be reabsorbed

unchanged or metabolized further by the gut microflora before reabsorption.

In the liver the absorbed substances are conjugated and excreted in the bile

and/or urine. This cyclic process of recirculation of drugs or their

metabolites through liver-bile-liver is known as enterohepatic circulation.

The existence of enterohepatic circulation with stilboestrol and

98

chloramphenicol has been demonstrated (Parke, 1968). The residence time of

drug in the body is increased due to enterohepatic circulation and thus

physiological action is also prolonged. .

c) Excretion in Urine - Foreign compounds and their metabolites are

mostly excreted in the kidney by three distinct processes, glomerular filtration,

active tubular secretion of ionized substances and passive tubular absorption

of unionized substances (Goldstein, et al., 1968). Although different, these

mechanisms are complementary to one another. Glomeruli are concerned with

the ultrafiltration of the blood plasma. The lipid soluble unionized substances

in the ultrafiltrate passes into the kidney tubules and are reabsorbed into the

blood stream by passive diffusion across the distal tubular epithelium.

Active transport of certain anions and cations takes place from the blood

plasma to the glomerular filtrate across the tubular epithelium. Plasma

protein binding of foreign compounds and rate of renal clearance largely limit

the rate of urinary excretion of foreign compounds. The rate of renal

clearance in turn is largely dependant on the rate of glomerular filtration,

active tubular secretion and passive tubular absorption. Glucuronides,

sulphates, mercapturates are polar lipid insoluble molecules and are therefore

readily excreted. Excretion of weak acids and weak bases ai;e pH dependent

therefore at alkaline pH (larger proportion is ionized) of the tubular urine

the weak acids will be readily excreted and less is absorbed. The reverse is

true for the weak bases. Amphetamines when orally administered to humans is

readily excreted demonstrating the permeability of tubular epithelium to

unionized molecules (Beckett, 1966).

d) Excretion in Expired Air - Gaseous anaesthetics containing low

molecular weight compounds having sufficiently high vapour pressure are

excreted in the expired air. Rabbits when given oral doses (0.5 g/kg)

excreted 39% of benzene, 44% fluorobenzene, 27% chlorobenzene and 6%

bromobenzene in expired air (Parke, 1968).

99

The enzymes of intermediary metabolism may degrade relatively simple

foreign molecules or analogues of endogenous substrates wholly or partly

which may be eliminated in the expired air as CO2 and/or H2O.

The degraded moiety after passing through intermediary metabolism may be

incorporated into the tissue and therefore its complete excretion is delayed.

When Maleic acid was administered intraperitoneally to rats, 40% of the dose

was excreted as CO2 in five hours. Administration of ethylmethane sulphonate

by the same route in rats resulted in the excretion of 34% in the expired air

over twenty four hours, (Roberts, et al., 1958).

e) Excretion in gastro-intestinal secretion - Under favourable conditions

of concentration gradient according to pH partition hypothesis, ionizable

foreign compounds in circulating blood may be excreted into the gut. Aniline

base was secreted into stomach (pH 1) of rats from plasma pH'7.4 (Shore, et al.,

1957). As the pH down the intestine increases these bases are reabsorbed

intestinally.

The essential oils found in nature are mixtures of a number of

monoterpenes of which the oxygenated monoterpenes are the bearers of specific

odoures and are important constituents of many essential oils extensively used

in flavouring foods. Some have medicinal importance as many of them are

pharmacologically active.

In view of the wide range of use of essential oils the study of

metabolism and toxicological implications of this class of naturally occurring

compounds and their metabolites is of considerable significance to their

safety evaluation.

Hildebrandt (1901), in the course of his study of the phamacological

activity of many aliphatic terpenes, isolated Hildebrandt acid, and dibasic

acid, from the urine of rabbit given citral. Kuhn, et al.r(1935, 1936) studied

the fate in rabbit of a series of open chain terpene derivatives including

geraniol, citral and geranic acid. All of these were partially metabolized to

Hildebrandt acid and excreted into the urine. The total metabolic fate of

these terpenes was not determined.

Linalool, a tertiary unsaturated alcohol, isomeric with geraniol, is

the chief constituent of linaloe oil and is known as coriandrol in coriander

seed oil. It also occurs in oil of cinnamon, orange and other citrus fruits.

As a result of its wide distribution in citrus fruits, herbs and spices

linalool along with other oxygenated monoterpenes is being ingested by animals

and humans in significant quantities. Therefore, the study of metabolism of

linalool and its toxicological implications are of significant importance.

It has been widely accepted (Dacre, 1970) that toxicological studies of food

additives, whether intentional or unintentional, must include an investigation

of their metabolic fate. In this regard 1^C-labelled linalool was

administered and its absorption, distribution, blood levels, tissue storage,

metabolism and ultimate fate were determined in rat.

101

MATERIALS AND METHODS

Materials

^C-labelled linalool (specific activity 0.52 yCi/mg) was synthesised

in this laboratory as described in Chapter 2. 2-ethoxy ethanol, 99.51 pure

by g.l.c., anaesthetic agent ethylcarbamate, hyamine 10-X hydroxide solution

and all other chemicals were from BDH.

The cannula tubing used for the purpose of bile cannulation had i.d.

0.4 ram and o.d. 0.8 mm (Portex).

Scintillator chemicals such as 2,5-diphenyloxazole (PPO), l,4-bis-2

(4-methyl-5-phenyloxazolyl)benzene(dimethyl POPOP), "Cab-O-Sil", naphthalene

and toluene (5.65 x 105 dpm/g) internal standard were obtained from

Packard Instrument Company.

Experimental

Administration of linalool and metabolic experiments

Three male and 3 female rats of twelve weeks old weighing 355-370 g

(males) or 210-225 g (females) (Animal Unit of the Surrey University), were

housed individually in all glass Jencons metabolic cages (Metabowl Mark III,

Jencons Ltd., Hemel Hemptead, Hertfordshire, England) for at least 24 hours

for acclimatization prior to administration of linalool. The metabolic cages

were equipped with two Nilox columns (all glass) and a wash bottle containing

2-ethoxy ethanol:ethanol amine mixture (2:1) for absorption of CO2 from

expired air. Each column contained 500 ml of the absorbent liquid and the wash

bottle contain ed 200 ml of the same liquid. The airflow rate through the

metabowl was adjusted to between 380-400 ml/min. During experimentation, the

animals were allowed food and water ad libitum and were maintained on

Spratts laboratory pellet diet No.l. The animals were accommodated under

controlled conditions of temperature (20-22°C), and lighting (12 h light and

12 h dark). After the expiry ' of the period of acclimatization the animals

were given a single dose of 1^C-linalool (500 mg/kg; 5-10 yCi) intragastrically

102

as a-251 solution in propylene glycol. Samples of urine, faeces and

absorbent solution from the Nilox columns were collected at intervals of

6 h., 12 h., 24 h., 36 h., 48 h. and 72 h after dosing.

Residual radioactivity in tissues 72 hours after dosing: 'preparation of

tissue homogenates. ■ '

At the end of the excretion studies the animals were killed. To determine

the distribution of radioactivity in different tissues, homogenates from brain,

liver, heart, kidney and spleen were prepared using a Potter-Elvehjem

homogenizer to make a 251 homogenate in water. Skin and skeletal muscle we re

digested in 6 M NaOH by warning in a water bath at 40°C overnight. In samples

where dissolution was incomplete, the concentration was increased to 9 M NaOH

when a homogeneous solution was obtained. The lung was chopped with a scalpel

and dissolved in 3 M NaOH at 40°C to prepare a 25% homogenate. Tissue from

the gastrointestinal tract, which contains muscle and .connective tissue, was

cut into small pieces with a scalpel. Hie mass was then homogenized in water

containing glass beads in a Waring blender.

Biliary excretion.

Two twelve week old male rats weighing 330-360 g were anaesthesised

with urethane (1.75 g/kg as 25% solution in water) by subcutaneous administration

Complete anaesthesia was achieved one hour after urethane administration. The

ventral surfaces of the animals were shaved using clippers and the necessary

area was bathed with Wescodyne iodophor (Bengne & Co., Wembley, Middlesex). A

mid ventral line incision about two inches was made through skin and body wall

with a scalpel. After locating the common bile duct and clearing off the

connective tissues, the hepatic end of the common bile duct was ligated loosely

anterior and posterior to the site of incision. A ’V ’ shaped notch was made

with scissors on the bile duct between the two loose ligations and a 35 cm

polythene cannula (i.d. 0.4 mm, o.d. 0.8 mm) was inserted into the incision and

the anterior ligature tightened to hold the cannula firmly. Bile started

103

flowing through the cannula immediately. The duodenal end of the common

bile duct was then tied.off by tightening the posterior ligation. The

cannula was passed successively through the abdominal wall and the skin by

making two incisions with a hypodermic needle. To close the vertical midline

incision the abdominal wall was stitched by suture thread and the skin closed

with suture clips.

The bile was collected in preweighed tubes and when steady flow was

attained (3040 mins) 1 C-labelled linalool (1 yCi; 20 mg) was administered

interperitoneally as a 101 solution in prolyiene glycol. Bile was collected

at half hourly intervals over a period of 6 hours in preweighed 3 ml tubes.

The animals were covered with cotton wool and were kept warm by suitably

located Anglepoi.se lamps fitted with 60 W tungstan-filament bulbs; Saline 0.9

was administered interperitoneally at intervals to replace body fluid lost due

to collection of bile. '

Enterohepatic circulation.

Biliary excretion of 14C .and delayed excretion of radioactivity in the

faeces indicated the possibility of involvement of linalool in enterohepatic

circulation. This possibility was more pronounced when the study of tissue

distribution revealed that after the carcass the specific activity of 14C was'Vmostly concentrated in the liver and in the gut indicating the possible

existence of enterohepatic circulation of linalool.

To demonstrate the enterohepatic circulation two male rats of twelve

weeks old (358 g and 364 g) were anaesthesized with urethane ' (1.75 g/kg body wt.

as 251 solution in water) and their bile ducts cannulated as in Fig.l. The

hepatic end of the common bile duct of the first animal was cannulated as

described earlier. After passing the cannula through the two incisions

through the abdominal wall and skin and stitching the ventral midline incision

the cannula was introduced into the incision made in the duodenal end of the

common bile duct of the second animal. The hepatic end of the common bile

104

o\<=

Fig. 4 .1 C ross Over Experiment to dem onstrate Enterohepatic Circulation.

Com m on b ile duct

Cannula 2Cannula 1

Duodenum

Two m ale rats w ere anaesthesised and b ile ducts w ere cannulated. A cannula from the bile duct of the first animal w as inserted into the duodenal end of the b ile duct of the second animal while another cannula was inserted into the hepatic end of the bile duct of the second animal, the bile duct being ligated between the cannulae. The presence of radioactivity in the bile of the second animal afteri .p . adm inistration of 14 C -lab e lled linalool to the first anim al w as indicative of enterohepatic circulation.

105

duct of the second animal was cannulated and cannula 2 was introduced. The

bile duct was ligated at position (L), between two cannulae to ensure that

there was no diffusion of radioactivity either way. After making necessary

incision to pass the cannula through the abdominal wall and the skin the

'ventral midline incision of the second animal was stitched and bile was

collected in preweighed 3 ml tubes.

Determination of radioactivity.

The lhC content of the various samples was determined by the addition of

15-20 ml of scintillator solution to 0.1-0.5 ml of material and counting in a

Packard Tricarb Spectrometer Model 3320.

The following scintillators were used depending on the accommodative

capacity of water in the samples.

(a) 30/70 scintillator - 300 ml of absolute alcohol, 700 ml of toluene

(analar BDH), 0.1 g POPOP (1,4 bis-2 (4-methyl 5-phenyl oxazolyl-benzene) and

3 g PPO (2,5 diphenyl oxazole). This scintillator could accommodate 0.7% -

0.8% water. This scintillator was therefore used for counting radioactivity

in urine (0.1 ml) and bile (0.05 ml or 50 yl). This scintillator was also

used for determining the radioactivity in the 14C02 absorbed liquid that

contained expired air.

(b) Gel scintillator - Naphthalene.(60 g), PPO (4 g), POPOP (0.2 g),

1,2-ethanediol (20 ml) , methanol (100 ml) and 1,4 dioxane was added to make

the volume to 1 litre. This scintillator has a capacity of accommodating

water upto 10% and was used for counting samples of the tissue digests.

Radioactivity in urine and expired air.

Urine and expired air (in the liquid absorber) were collected at intervals

of 0-6, 6-12, 12-24, 24-36, 36-48 and 48-72 hours after intragastrie

administration of 1 kC-linalool. Aliquots of 0.1 ml urine were counted in 15 ml

of 30/70 scintillator while for expired air, 2 ml aliquots of the liquid

absorber were counted in 15 ml of the same scintillator. All the samples were

counted in duplicate ana corrected for counting efficiency by internal

standardization using luC-toluene (5 yl, 2381 dpm).*In order to confirm that the radioactivity in the expired air was

principally as ltfC02 the following experiment was performed. Aliquots of 5 ml

of the liquid absorber taken at the intervals 0-6 h, 6-12 h, 12-24 h, 24-36 h,

36-48 h and 48-72 h were pooled together. An equal volume of 2 M barium

chloride was added to precipitate ll*C02 as barium carbonate (Calvin, et al.,

1949), the mixture was then shaken for half an hour in a mechanical shaker and

allowed to stand over night. Inactive barium carbonate (500 mg) was added as

carrier, shaken for twenty minutes, allowed to stand for an hour and filtered

through a Gooch crucible. An aliquot of 2 ml of the filtrate was counted

for ndioactivity, if any, after precipitation of Balt+C03. The barium carbonate

precipitate was washed, dried and counted for llfC in 20 ml gel scintillator.

The radioactivity present in the barium carbonate precipitate was compared with

that of the known quantity of the pooled liquid absorber. This suggested that

radioactivity present in the expired air was principally due to 14C02 and not

■due to 14C labelled linalool or other volatile metabolites.

Radioactivity in tissues.

Tissue homogenates containing 100 mg of tissue were treated with 1 ml of

hyamine hydroxide 10 x 2, (BDH) and incubated at 37° for 12-16 hours in' %

scintillation vials. The samples were prepared in duplicate. Gel scintillator

(20 ml) was added to the vials and counted for 14C.

Faecal radioactivity.

The faeces were collected over five intervals 0-12, 12-24, 24-36, 36-48,

48-72 hours instead of six intervals as the animals did not defaecate during

0-6 hour period. Each faecal collection was treated in the cold with two

volumes of absolute methanol and allowed to stand overnight. The methanol

was decanted off and the faeces were homogenized in further methanol. After

addition of one more volume of absolute methanol the homogenized mass was

extracted thoroughly for one hour using a mechanical shaker. The methanol

layer was decanted after centrifugation. Extraction with fresh absolute alcohol

107

was repeated three times. The pooled methanol extract (0.5 ml) was then ♦

counted for radioactivity in 15 ml of 30/70 scintillator. The remaining

radioactivity in the extracted faecal residues was determined by digestion of

10-15 mg portion overnight at 37°C with 1.5 ml of hyamine-10 X hydroxide and

0.5 ml of water prior to the addition of gel scintillator (20 ml) (Crew, et al.,

1971). The counting efficiency was determined by using 1UC toluene as

internal standard. All samples were counted in duplicate.

Biliary radioactivity.

Bile (50 yl) was assayed for ltfC in 15 ml 70/30 scintillator. The samples

were counted in duplicate and corrected for counting efficiency as before.

Analysis of blood.

Heparinized (0.1 ml) blood was digested at 37°C for 24 hours with 1 ml

hyamine hydroxide in the scintillation vial. The digested blood was then

counted for llfC in 20 ml gel scintillator. lhC toluene, as before, was used

as the internal standard for the correction for counting efficiency.

108

RESULTS

‘The appearance of radioactivity (% dose) in urine, faeces and expired air

for the time intervals of 0-6 h, 6-12 h, 12-24 h, 24-36 h, 36-48 h and

48-72 h after oral administration of 1,2 14C-labelled linalool to the male

rats are presented graphically in figure 2. It was observed.that 57—58% of

dose of radioactivity was excreted in the urine in 72 h and most of it

(51-521) was excreted over the first 36 hours with no significant delay between

dosing and appearance of activity in the urine. After a delay of several hours,

substantial amounts of radioactivity (23% dose) appeared in the expired air.

In the experiment for the determination of extent of the radioactivity present

as llfC02 in the expired air no appreciable amount of radioactivity above

background count was detected in the filtrate after precipitation of lt*C0z> as

BaltfC03. The counts (dpm) in the aliquots were also similar to that from

BallfC03 obtained from a similar volume of the original absorbent solution. This

therefore, indicated that the activity in the expired air was principally as

1UC02 and not as 1 C-labelled linalool or any other volatile metabolites. The

excretion of radioactivity in the faeces was 14-15% of dose between 0-72 hours.

The shape of the curve signifies delayed excretion occurring mainly (ca. 7% of

dose) between 36-48 hour periods. The residual radioactivity in the tissues

72 h after dosing is shown in Table 4.2 from which it can be seen that the

activity was located principally in the liver, gut, skin and skeletal muscle

with insignificant amounts in the other tissues. Radioactivity in the blood

at this stage corresponded to 0.003% of the dose/ml.

The comparative rates of excretion of radioactivity through urine, faeces

and expired air after intragastric administration of 1 ,fC-linalool were also

studied in a group of three female rats. The results have been tabulated in

Table 4.1 from which it can be seen that there is no significant sex difference

in the amount of the dose excreted by the three routes studied.

The results of experiments with cannulated bile ducts have been shown in

figure 4.3. After intraperitoneal administration of 1^C-linalool, substantial

Rad

ioac

tivity

(%

dose

)

excr

eted

Fig. 4. 2 Cumulative Excretion of Radioactivity ( % dose) in Rat Urine, Faeces and Expired Air after single Intragastric Administration of 14C Labelled Linalool (10 p.Ci).

100Total

70 i

Urine

50 -i

so 4

Expired air

20 nFaeces

10 J

2010 30 50 70 7260

Time ( Hours )

Linalool was administered intragastrically to 12 week old male wistar rats as a 25% solution in propylene glycol at a dose level of 500 mg/kg body weight.

110

Exc

rete

d R

adio

activ

ity

( % do

se

) in

Bil

e

r ig. 4. 3 cum ulative ana nistogram representation 01 excretion of radioactivity ( %dose) in rat b ile after single intraperitoneal adm inistration of14C labelled linalool ( 1 pCi, 20 mg ).

25 i

21 30 4 5 6

Tim e After D ose ( hr )

Linalool w as adm inistered intraperitoneally to 12 week old m ale W istar rats as a 10 % solution in propylene glycol.

Ill

Exc

rete

d R

adio

activ

ity

(%do

se

) in

Bile

of

the

Seco

nd

Ani

mal

F ig. 4. 4 Cumulative and histogram representation of excretion of radioactivity ( % dose ) in b ile of the second animal after cro ss-o v er experim ent as shown in Fig. 4 .1

3 .0

2 .5

2. 0

1 .5

1.0

0 .5

1 2 3 4 5 6 7 8 9 10 11 12

T im e After Dose ( hr )

14C labelled linalool ( 1 pCi, 20 mg ) was adm inistered intraperitoneally as a 10% solution in propylene g lycol to the first rat and the b ile w as collected from the second animal.

112

biliary excretion took place, more than 25% of the dose appearing in the bile

-in 6 hours, mainly in first 4 hours. Hie radioactivity present in the bile

was exclusively in the form of polar conjugates, no free linalool being

detectable. Hie conjugates were hydrolysed to the extent of 68-691 by

3-glucuronidase and almost complete hydrolysis was achieved by the mixture of

3-glucuronidase and sulphatase with the formation of ether-extractable non­

polar substances. The characterization of the biliary metabolites is dealt

with in Chapter 5.

In the crossover experiment (fig. 4.4), 2.5% of the radioactivity

administered intraperitoneally to the first animal appeared in the bile of the

second animal indicating that linalool enters into enterohepatic circulation.

The peak of excretion occurred between 7 and 9 hours.

Comparative

rates

of' excretion of

radioactivity

{% dose)

in male and female rat

s after

intragastrie

administration of

1C-labelled linalool

(10-5

yCi)

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114

at a dos

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of 500 mg/kg

body

weight.

The figures

are expressed

as percent

of administered radioactivity,

results

are means

of two groups of

three

male or

three

female animals, w

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S.E.M. in

the parenthesis.

TABLE 4.2 Distribution of residual radioactivity in tissues of maleand female rats 3 days after intragastric administration of 1 C-labelled linalool (10-5 yCi)

Radioactivity present* 72 hours after dosing

TISSUES MALE% original dose

% residual activity

FEMUE% original % residual dose activity

Brain 0.01 0.30 - -

Lung 0.01 0.30 - -Liver 0.48 15.40 0.65 . 18.6Heart 0.01 0.30 - -Spleen 0.01 0.30 - -

Gut 0.55 . 17.70 0.6 17.2Kidney 0.05 1.60 - -Testes/Ovary 0.06 1.90 - -

Cornea + - -Skin 0.80 25.50 - -Skeletal muscle • 1.15 36.70 - -Carcass less liver and gut - - 2.25 64.2

Total (residual activity)

3.1 100.00 3.5 100.00

* Mean of two animals

+ Denotes no significant radioactivity

- Determinations were not made.

Linalool was administered intragastrically to 12 week old Wistar rats as a

25% solution in propylene glycol at a dose level of 500 mg/kg body weight.

DISCUSSION

From fig. 4,2 it can be seen that linalool appears to be rapidly absorbed

■from the gut as there was extensive and rapid urinary excretion of radioactivity

(ca. 52% of dose) over the first 36 hours with no significant delay between

dosing and appearance of radioactivity in the urine. Delayed appearance of

radioactivity was observed in the expired air that contained substantial amounts

of radioactivity (23% of dose) principally as 1UC02 and not as linalool or

other volatile metabolites. The appearance of ll|C02 and the delay in its

excretion suggests that linalool may be entering into pathways of intermediary

metabolism. This is supported by the observation that a substantial amount

of urinary radioactivity was present in the form of 14C-urea (see Chapter 5,

page 122).

Faecal excretion of radioactivity was delayed and occurred mainly between

• 36-48 h after dosing and ultimately 14-15% of the dose was excreted by this

route. Hie delayed excretion of radioactivity in the faeces after

.intragastric administration is not wholly explicable in terms of time for

gastrointestinal transit and therefore, suggests the possibility of biliary

excretion.

From figure 4.3 it can be seen that 25-26% of the dose of radioactivity%was excreted in bile, exclusively in the form of polar conjugates which, on

hydrolysis with 3-glucuronidase and sulphatase liberated a substantial amount

of non-polar ether-extractable radioactivity. Further evidence of hydrolysis

of biliary conjugates in the gut is afforded by the presence of non-polar

ether-extractable metabolites (about 5% of dose) in the faeces and these

non-polar metabolites (see Chapter 5?page 123)were absent from bile. In view

of the apparent rapid absorption of linalool from the gut,, it is probable that

the faecal metabolites originate from hydrolysis of biliary conjugates in the

lower colon. Hie nature of the faecal metabolites (Chapter 5) supports this

view since little unchanged linalool was present. Hiis therefore, suggests

that polar conjugates in the bile after entering the gut may be hydrolysed by

the 3"glucuronidase of the gastrointestinal tract or by the gut microflora

and the liberated aglycone may be reabsorbed to establish enterohepatic

circulation. Tissue distribution 72 h after dosing revealed that 16-18% of

the residual radioactivity was located in the liver and 18-19% in the gut.

The persistence of radioactivity in the liver and the gut 72 hours after dosing

along with the observation of delayed excretion of radioactivity in faeces also

suggests the possibility that linalool enters enterohepatic circulation after

oral administration.

In the crossover experiment 2.5% of the dose administered intraperitoneally

to the first rat was excreted (Fig. 4.4) in the bile of the second animal and

this therefore substantiates that linalool enters enterohepatic circulation,

at least after intraperitoneal administration.

Hie observation that 2.5% of the dose was excreted in the bile of the

second animal in the crossover experiment (Fig. 4.4) demonstrates the

occurrence of extensive enterohepatic circulation. Assuming, as previously

observed, that 25% of the original dose appeared in the bile of the first

animal over the period studied and also that 25% of the conjugates which are

hydrolysed and reabsorbed appeared in the bile of the second animal, it can be

calculated that 40% of the biliary conjugates are hydrolysed and reabsorbed on

the first pass. Thus extensive enterohepatic circulation is possible and may .

account, in part, for the delayed faecal excretion. Hie results in Table 4.1

reveal that there was no significant sex difference in the excretion of

radioactivity after intragastric administration. In view of this and the

insignificant amount of residual radioactivity present in the organs of the male

rat, other than liver and gut, the tissue distribution in the female rat was

carried out on the liver, gut and the carcass (less liver and gut). Hie total

residual radioactivity and the radioactivity in liver and gut in the female rats

72 hours after dosing were in all cases comparable to that in the male, Therefor.:

it can be argued in view of the continued existence of radioactivity in botli the

117

liver and the gut 72 hours after dosing that enterohepatic circulation is

also likely in the female rat.

This study therefore, suggests that large doses of linalool may be

metabolised in the'rat by conjugation and excretion in the urine, faeces and

bile while substantial portion of the dose may enter pathways of intermediary

metabolism. The rapid excretion of linalool and its metabolites suggests no

long term hazard from tissue accumulation on chronic exposure to levels

normally encountered in foods. However, at high dosage enterohepatic

circulation might have the effect of prolonging the metabolic load on the

liver over a relatively short period.

118

CHAPTER 5

In vivo Metabolic End Products of Linalool: Identification

and Quantitative Determination

119

INTRODUCTION

In the experiments described in Chapter 4 it was observed that most

of the orally administered linalool was excreted in the bile, urine and

faeces (28%, 59% and 60% respectively); only 20% was excreted in the

expired air. It was also observed that expired air contained mostly CO2 and

did not contain linalool or any of its volatile metabolites. It Is generally

considered that most foreign compounds are primarily metabolized in the liver

and the metabolizing enzymes can occur in the soluble, mitochondrial and

microsomal fractions principally the latter. However, before a food

additive or orally administered drug enters the portal circulation it is

exposed to the-metabolic activity of the gut microflora (see chapter l)and in

the complete study of the metabolism of foreign compounds it is essential

to study the contribution made from this source. In the liver the most

common route of metabolism of foreign compounds involve oxidations, reductions

and hydrolysis (phase I reactions) and conjugation (phase II reactions,

see chapter 1) to render the ingested substance more polar and water soluble

for elimination through bile, urine and faeces. In the previous chapter

it was noted that the biliary metabolites were exclusively in the form of

highly pol'ar compounds and in the urine only a . relatively small proportion

of the metabolites were ether extractable. In the faeces, significant amounts

of the excreted radioactivity was in the form of non-polar metabolites.

This suggests that the conjugation was occurring prior to excretion in the

urine and bile but that some hydrolysis of conjugates might have occurred

in the gut.

In this chapter the nature of the metabolites described in chapter 4 was

examined in detail to determine this qualitative and quantitative composition

and to elucidate the roles of mammalian and intestinal metabolism.

120

MATERIALS

3-Glucuronidase and 3-glucuronidase' sulphatase mixture (Sigma Chemicals);

urea, hydrochloric acid,analar (BDH); linalool and tetrahydrolinalool

(Koch-Light Ltd.); were purchased and purified. Dihydrolinalool was

synthesised as described in chapter 3.

EXPERIMENTAL

Thin-layer chromatography:examination for glucuronides

Bile, urine and faeces were examined by t.l.c. for glucuronide conjugates/

using silicagel G thin-layer plates and acetic acid butan-l-ol/ 1,2-dichloroethane

water (4:14:4:1 v/v) (Iveson, et al., 1971).. The glucuronides were detected

as blue spots by spraying with naphthoresorscinolreagent (0.2% w/v

naphthoresorscinolin acetone mixed with 9% H3P0if in water v/v in the proportion

of 5:1) and heating the plates for 10 minutes at 120°.

Hydrolysis of polar conjugates in urine, faeces and bile

Enzyme Hydrolysis

Samples of bile (3 ml), urine (10 ml) and aqueous faecal extract (5 ml)

were adjusted to pH 5 with 1 M MCI.

The enzymes 3~glucuronidase, or a mixture of 3-glucuron.idase and

sulphatase were added (1000 U/ml) separately in separate tubes (in duplicate).

Equal volume of 0.1 M acetate buffer, pH 5.0 was added and incubated for

16 hours at 37°C. The incubation was repeated for another 10 hours with fresh

enzymes before the termination of the experiment.

Controls of bile, urine and faeces were taken through the same procedure.

The enzymic hydrolysates were adjusted to pH 7 with 1 M NaOH and then

extracted four times with equal volumes of ether.

Acid Hydrolysis

The aqueous layers from the enzymic hydrolysates were further hydrolysed

with acid for conjugates which were unaffected by enzyme hydrolysis. The

acid hydrolysis was carried out by making the aqueous layer 6 M with respect

to HC1 by addition of conc. HC1. The hydrolysis was carried out in a boiling

water bath for 4 hours (Simmons, et al, 1973).

Determination of Urea

Urea forms crystalline compounds with certain acids and most important

of these are urea nitrate C0 (NH2)HN03 and urea oxalate (C0(NH2) ) H2C20u.2 2 2Urea (3 g) was dissolved in 15 ml of urine. Concentrated nitric acid (10 ml)

was added to precipitate urea as urea nitrate and filtered off. Hie urea

nitrate was dissolved in 20 ml of water and treated with 2 g of activated

charcoal in a boiling water bath for 2 minutes. After filtration ’the

filtrate was treated with conc. HN03 to reprecipitate urea nitrate. The

precipitate after filtering was dried and powdered. The powdered urea was

then counted for its lltC content in gel scintillator (as in Chapter 4)'.

Thin-layer chromatography of the hydrolysates

Silver nitrate impregnated silicagel G and methylene chloride:chloroform:

n-propanol:ethyl acetate = 10:10:1:1 system was used as in Chapter 3. After

double development the separated substances were located by spraying with

phosphomolybdic acid reagent (7.51 in 95% alcohol).

Combined Gas chromatography-mas spectrometry (GC/MS)

The same combined GC/MS system of LKB, under similar conditions as

described in Chapter 3, was used.

Quantitative evaluation of the metabolites

The quantitative estimations of metabolites were carried out by measuring

the peak areas of GC/MS total ion current trace and also by thin-layer and

scintillation spectrometry, as described in Chapter 3 (pages 61 and 63).

122

RESULTS

Ether extractable radioactivity in urine and faeces

Thin-layer chromatography of the ether extract of urine and bile showed

the presence of three major substances having values 0.38, 0.54, 0.71

corresponding to linalool, dihydrolinalool and tetrahydrolinalool respectively.

GC/MS examination confirmed the presence of these three terpenoids.

Hie ratio of linalool, dihydrolinalool and tetrahydrolinalool in the urine

extract was 1.0:1.2:1.9 and in the faecal extract was 1.0:1.0:1.6 as estimated

from the peak areas of gas chromatograms. Quantitative determination of the

radioactivity present in each of the component by using -thin-layer

chromatography and scintillation spectrometric technique revealed that linalool,

dihydrolinalool and tetrahydrolinalool were present as 1.2%, 1.11 and 2.2% of

the dose in the urine extract and 1.2%, 1.7% and 2.7% of the dose in the faecal

ether extract (see figures 5.1 and 5.3).

Polar substances in bile

Enzyme hydrolysates

Thin-layer chromatography for glucuroni.de conjugates indicated the

presence of glucuronides of R£ values 0.42, 0.53, 0.57, 0.64, 0.71. Out of

these the substances of R^ values 0.53 (A), 0.57 (B) and 0.64 (C) were

radioactive; A being the major and B and C were very minor metabolites.

Thin-layer chromatography of the ether extract of B-glucuronidase hydrolysate

of bile containing 17% of the dose of radioactivity separated three

radioactive components having R£ values 0.38, 0.49 and 0.68 corresponding to

linalool, dihydrolinalool and tetrahydrolinalool and the confirmatory evidence

of the presence of these was obtained by combined GC/MS. The quantitative

determination of the radioactivity (% dose) present in each of linalool (15.4%)

dihydrolinalool (0.6%) and tetrahydrolinalool (0.8%) was carried out by

thin-layer chromatography and scintillation spectrometry. Linalool was present

in large amounts compared with the small amounts of the other half saturated

123

and fully saturated derivatives.

Hie hydrolysis with 3-glucuronidase/sulphatase mixture increased the

radioactivity of the ether extractable materials by ca 1% of the dose thus

indicating the extent of sulphate conjugates (ca 7%) excreted in bile.

Hi in-layer, GC/MS confirmed the presence of linalool to a very large extent

compared to dihydro and tetrahydrolinalool, the ratio of which was

22:0.7:0.9 respectively as determined by peak areas. Quantitative determination

of the radioactivity present (% dose) in linalool (22.1%), dihydrolinalool (0.9%)

and tetrahydrolinalool (1.0%) was carried out by thin-layer and scintillation

spectrometry. The aqueous layer contained about 2.0% of the dose of

radioactivity.

Acid hydrolysate of bile

Hie ether extract of the acid hydrolysate of bile contained almost all

the radioactivity leaving little in the aqueous layer. Combined GC/MS

examination suggested the presence of linalool, dihydrolinalool, tetrahydro­

linalool in the proportion of 1.1:0.12:0.14 along wTith some (0.7%) unidentified

substances possibly as artifacts that might have arisen from acid catalysed

isomerisation of the terpene alcohols. Thin-layer chromatography indicated

the presence of a substance having value 0.55 which corresponded to

authentic geraniol, an isomer of linalool^' r. . . which is.known to

be produced by an acid catalysed allylic rearrangement.

Polar materials in urine

Hiin-layer chromatography showed the presence of a number of glucuronides

of R£ values 0.47, 0.52, 0.58, 0.63, 0.70, 0.76 of which three R^ values

0.52 (A), 0.58 (B) and 0.65 (C) were radioactive.

Enzyme hydrolysates

3-Glucuronidase hydrolysis showed that 31% of dose was excreted in the

urine as glucuronide. The combined GC/MS examination of the 3-glucuronidase

hydrolysis confirmed the presence of linalool,dihydrolinalool and

Fig.5.I»

Urine

Fractionation

Scheme

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Linalool was administered intragastrically to

12 weeks

old Wistar rats

as a 25°& solution in

propylene

glycol at

dose

level

of 500

mg/kg

body

weight.

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126

Linalool was administered intraperitoheally

to 12

wk old mal

e Wistar rats

as 101 solution in

propylene

glycol.

Fig.5.3

Fractionation

Scheme of

Faecal Metabolites

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127

tetrahydrolinalool in the proportion of 3.1:1:1.5 estimated from the area

under .their respective peaks. The quantitative determination of the

radioactivity present in linalool (18% dose), dihydrolinalool (5%) and

tetrallydrolinalool (8%) was carried out by thin-layer chromatography and

scintillation spectrometry. These proportions agreedwell with those obtained

by GC/MS. The glucuronidase-sulphatase hydrolysed urine sample showed an

increase of the radioactivity of 6% over glucuronidase hydrolysis alone

indicating the presence of sulphate conjugates of the metabolites. Thin-layer

GC/MS and thin-layer scintillation spectrometry examination revealed that

linalool, dihydrolinalool and tetrahydrolinalool were present to the extents

of 19.7%, 7.4% and 10.5% of dose.

Acid hydrolysates

The radioactivity in the aqueous layer not removed by ether extraction

after 3-glucuronidase-sulphatase hydrolysis was further recovered to the

extent of 4.0% of the dose of radioactivity by ether extraction after acid

•hydrolysis. Tnin-layer chromatography - GC/MS confirmed the presence of

linalool, dihydrolinalool and tetrahydrolinalool along with some small peaks

of unidentified minor components probably artifacts derived from the isomerisatioi

of the terpenealcohols produced by the action of aci d. Trie amount of'v

radioactivity associated with linaloof, dihydrolinalool and tetrahydrolinalool was

1.0%, 0.9% and 1.2% respectively (see figure

Polar materials not hydrolysable with enzymes and acid

. In the experiment for the determination of urea in the urine the

precipitated urea nitrate was found to contain 10.5% of dose radioactivity.

The filtrate after filtering urea nitrate contained 3.2% of radioactivity

(% dose). This fraction was not further investigated in view of the insufficient

material present.

Polar substances in faeces

'Thin-layer chromatography, GC/MS and thin-layer chromatography-

scintillation spectrometry examinations showed that the ether extract of the

3-glucuronidase hydrolysate contained linalool, dihydrolinalool and

tetrahydrolinalool respectively which, contained 1.1%, 1.7% and 2.3% of the

dose of radioactivity. The mixture of glucuronidase-sulphatase hydrolysis

produced linalool, dihydrolinalool and tetrahydrolinalool containing 1.3%.,

1.8% and 3.9% of the dose of radioactivity respectively. The sulphate

conjugate, therefore, contains 1.9% of the dose of radioactivity. The aqueous

layer after ether extraction contained about 2.3% of the dose of radioactivity.

Hydrolysis with acid and subsequent extraction with ether indicated little

residual activity in the aqueous layer thus pointing to the likelihood of

the presence in the faeces of acid hydrolysable conjugates other than

glucuronides and sulphates. The peak areas of the GC/MS total ionisation

current traces indicated the presence of linalool, dihydrolinalool and

tetrahydrolinalool in the proportion of 1:0.8:1. The percentage composition of

linalool (0.8%), dihydrolinalool (0.7%) and tetrahydrolinalool (1.2%), was

determined by thin-layer and scintillation spectrometric analysis (see

figure 5.3).

Bile from enterohepatic circulation ; 'Enzyme hydrolysate

The ether extract of 8-glucuronidase-sulphatase hydrolysate of bile from

the enterohepatic circulation experiment on examination by t.I.e.-GC/MS and

t.I.e.-scintillation spectrometric analysis showed that linalool, dihydrolinalool

and tetrahydrolinalool were present to the extents of 0.8%, 0.7% and 1.1% of

dose respectively.

129

130

DISCUSSION

From the results above it was observed that after oral administration,

linalool is metabolized primarily to dihydro and tetrahydrolinalool and

excreted mainly as conjugates in urine and faeces and the excretion in the

bile was effected exclusively as conjugates. The bile from the enterohepatic

circulation (and urine from animals dosed intragastrically with linalool)

contained conjugates of dihydro and tetrahydrolinalool as major metabolites

whereas these reduction products of linalool were present in insignificant

quantities in the 'first pass' bile of animals which had been dosed

parenterally. It may therefore be suggested that the reduction products

originated in the gut. . In the in vitro metabolism of linalool by the gut

microflora of rat (Chapter 3) it was observed that rat gut microflora

reduced linalool to dihydro- and tetrahydrolinalool. This observation,

therefore, further substantiates the assumption that gut was the origin

of the production of dihydro- and tetrahydrolinalool formed by the gut

microflora from linalool.

It was also observed that linalool and its metabolites were excreted

in the bile exclusively in the foim of polar conjugates whereas faeces

contained free linalool, dihydro and tetrahydrolinalool (4-51 of dose)

and it is possible that the polar conjugates of linalool were hydrolysed

and reduced in the gut. After being liberated from conjugation linalool,

dihydrolinalool and tetrahydrolinalool were reabsorbed and this was the

mechanism by which enterohepatic circulation was established as demonstrated

in Fig. 4.1 (see Chapter 4).

The appearance of conjugates of dihydrolinalool and tetrahydrolinalool

in urine suggests that substantial reduction in the gut occurred before

absorption of linalool or reabsorption of linalool released from biliary

conjugates.

In addition to the presence of conjugates of linalool, dihydrolinalool

and tetrahydrolinalool in the urine, 10-111 of radioactivity was present in

131

the urine as urea indicating the involvement of linalool in intermediary

metabolism. Radioactivity (20-231) was excreted in the expired air as llfC02 (see

Chapter. 4) suggesting the possibility that linalool was entering the pathways

of intermediary-metabolism and this assumption could therefore be

substantiated by the availability of radioactivity in the urea. On the

basis of the present findings the possible in vivo metabolic transformations

of linalool has been proposed in figure 5.4.

The percent dose excreted as metabolites through the three major routes

of excretion, viz. expired air, urine and faeces is shown as a balance sheet

in table 5.5.

Fig. 5.

5 Percent

dose

excreted as

metabolites

in expired

air, urine

and faeces

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The figures

are expressed

as % of

administered radioactivity, and the results

are means

of three

animals

with S.E.M.in parer

Chapter 6

Effect of Linalool on some Drug Metabolising Enzymes

\

134

Introduction

The potential toxicity of food additives is not an isolated problem

but must be considered in context with the exposure of man and animals to

other foreign compounds (Gillette and Brodie, 1970).

A substance may be harmless when administered alone but may be toxic

when taken with other substances. For example,if persons being treated with

tranylcypromine or trifluoperazine, the monoamine oxidase inhibitors, eat

cheese, they may suffer hypertensive attack due to build up of unmetabolized

amines, (Asatoor, et al, 1963). Benkos, et al.(1961) studied the effects of

essential oils on atherosclerosis of cholesterol-fed rabbits. They suggested

that the formation of a the ros cl eros is plaques was prevented by treatment

with terpenes, notwithstanding a high level of serum cholesterol. They

visualized that this effect of terpenes partly resulted from competitive

inhibition or perhaps they acted by blocking mediatory factors promoting the

penetration of lipids into the arterial wall. Keto and Gozsy (1958) found that

linalyl acetate, citronellal, bomeolacetate and 104 other volatile oils and

■ their chemical derivatives enhanced the therapeutic efficacy of small doses of

dihydrostreptomycin in the treatment of experimental tuberculosis of the

guineapig. Salaman (1961) reported that repeated application of oil of

orange, lemon, grapefruit, lime and bergamot to skin of mice produced and

maintained epidermal hyperplasia and this was regarded by Salaman as evidence

of tumour promoting action. Roe and Field (1965) demonstrated that rats

pretreated with subcarcinogenic doses of benzpyrene or urethane developed skin

tumours if treated subsequently with citrus oil.

Some foreign compounds are bio transformed by enzymes of intermediary

metabolism because of the resemblance of these chemicals to normal endogenous\

substrates but most foreign compounds, since they have no endogenous

counterpart, are metabolized by the drug metabolizing enzymes of the endoplasmic

reticulum. The liver is the main site for metabolism of foreign compounds;

other tissues including gut also participate in varying degrees. The enzymes

135

o£ the endoplasmic reticulum carry out the oxidative reactions and

conjugations such as glucuronide formation. The enzymes of the mitochondria

also catalyse some bio transformations. In view of the autooxidation of many

intermediary metabolites the conversion of azo and nitro compounds to amines

is of minor importance in well oxygenated tissues. The anaerobic conditions

in the gut is therefore important in the reduction of azo and nitrocompounds

by the enzymes of the gut and particularly by the gut microflora.

Jori, et al. (1969) studied the effect of menthol, a-pinene and eucalyptol

on the metabolism of other drugs in rat. Subcutaneous administration or aerosol

spray of eucalyptol was found to increase hepatic metabolism of aminopyrine,

p-nitroanisoles, aniline and pentobarbital in vitro. These workers, therefore,

suggested that' eucalyptol induces the microsomal enzymes and enhances the

metabolism of foreign compounds to protect against their toxic effects.

In this context Parke and Rahman (1969) studied the short term (after 3 days

repeated administration, 150 mg/kg body weight) effect of some terpenes.

They found that linalool, nerolidol and squalene had no effect on biphenyl

4-hydroxylase, glucuronyl transferase and cytochrome P-450 but 4-nitrobenzoate

reductase was increased by 25-50%. In view of the present widespread use of

linalool in natural (citrus fruits, spices and herbs) and synthetic food

additives (in soft drinks) the long term effects of linalool on the drug

metabolizing enzymes after repeated doses have been studied.

\

136

MATERIALS AND METHODS *

Materials

Buffers

Tris (2-amino-2-hydroxymethyl propane-1,3 diol) was obtained from

Sigma Chemical Co. After preparing solutions of requisite ionic strengths,

dilute hydrochloric acid was added to adjust to the required pH. Phosphate

buffers were prepared from Analar grade (BDH), potassium dihydrogen phosphate

and dipotassium hydrogen phosphate of the required ionic strength, and pH

as described by Dawson, et al.(1959).

. Acetate buffer (0.2 M, pH 4.6) was prepared by addition of 510 ml of 0.2 M

acetic acid (BDH) to 490 ml of 0.2 M sodium acetate (BDH).

Glycine buffer (0.5 M, pH 10.4) was prepared by dissolving 18.76 g of

glycine (BDH, Analar grade) in 450 ml of water and the pH 10.4 was adjusted

with dilute NaOH solution. The total volume was finally made upto 500 ml with

water. Glycine buffer (0.1 M, pH 9.6) was prepared similarly by dissolving

37 g of glycine, adjusting the pH to 9.6 with dilute NaOH solution and making

the volume to one litre.

Substrates

Biphenyl, m.p. 70°C (British Drug Houses, England) purified as described

by Bridges, et al (1965); 185 mg in Tween 80 (2.0 g) was diluted to 1Q0 ml

with hot 1.151 KC1 solution. Any crystals of biphenyl which precipitated

were redissolved by warming before use (Creaven, et al, 1965).

4-Methylumbelliferone, m.p. 182-184° (Koch-Light Ltd.), 32 mg recryst.

4-methvlumbelliferone fMead, et al, 1955) was dissolved in 100 ml hot 1.151

KC1, and aliquots of 0.3 ml (0.6 ymol) were used in 1 ml incubation mixture.

Coenzymes

Nicotinamide adenine dinucleotide phosphate monosodium salt was dissolved

in 1.15% KC1 (5.04 mg/ml). An aliquot of 0.25 ml contained 1.5 ymol per

incubation.

137

Uridine-5'-diphosphoglucuronic acid ammonium salt, (Sigma Chemicals Co.

Ltd.) was dissolved in 0.1M Tris buffer, pH 7.6 (5.8 mg/ml).

Nicotinamide adenine dinucleotide (Sigma Chemicals Co. Ltd.), 10 mg was

dissolved in 1 ml of 0.1 M glycine buffer (pH 7.6). An aliquot of 0.2 ml was

used in 2.4 ml incubation mixture for the determination of alcohol

dehydrogenase activity.

Standards

Protein standard was prepared by dissolving crystalline bovine serum albumin

(Koch-Light Ltd.) in water (20 mg/ml).

DNA standard (stock solution) was prepared by dissolving DNA (Sigma

Chemicals Co. Ltd.) in 5 mM sodium hydroxide solution (40 mg/100 ml). The

working standard (200 yg/ml) was prepared by mixing stock DNA solution with

10% perchloric acid solution (1 volume/1 volume).

4-Hydroxy biphenyl, m.p. 162° (British Drug HousesVas purified as described

by Bridges, et al (1965). The solution was made up as described by Creaven,

et al.(1965).

4-Methylumbelliferone stock solution (0.1 ymol/ml) was prepared by

dissolving recrystallized 4-methylumbellifercne in 1.15% KC1 solution

(17.6 mg/litre). The working standard 0.01 ymol/ml was prepared by diluting

10 ml of stock solution to 100 ml with 1.15% KC1 solution as required. . ;.

4-Methylumbelliferone in 1 litre 1.15% KC1 solution. The working

standard 0.01 ymol/ml was prepared by diluting 10 ml of stock solution to

100 ml with 1.15% KC1 solution as required.

Enzymes .

3-Glucuronidase (E.C. 3.2.1.3.1) (Sigma Chemical Co. Ltd.) was dissolved

in 0.2 M acetate buffer pH 4.6 to give an activity of 500 units/0.5 ml.

Other materials •

Tween 80 (polyoxyethylene Sorbitan monooleate) was obtained from Koch-Light.

Folin and CiocalteuTs reagent from BDH was used as supplied. Diphenyl amine

reagent was obtained from BDH. Sodium dithionite, copper sulphate, sodium

•carbonate, sodium-potassium tartarate, magnesium chloride and other chemicals

were obtained from British Drug House and other usual sources.

Experimental

Animals and linalool administration

Four week old 48 male Wistar albino rats were divided into 12 groups

each consisting of four animals, six groups for controls and six groups for

linalool fed. Each group of the animals was caged in polypropylene cages

(26” x 15gn x 8M) and were kept on laboratory pellet diet (Spillers Ltd.)

and'water ad-libitum. Linalool was administered to the rats by daily

intragastrie intubation at a dose level of 500 mg/kg/d as a 25% solution in '

propylene glycol used as vehicle. The controls were given a similar volume

of propylene glycol. The animals were accommodated under the controlled

conditions of relative humidity (50%), temperature (20-22°C) and lighting

(12 h light and 12 h dark).

Preparation of liver fractions

At intervals of 0, 3, 7, 14, 30 and 64 days from the commencement of

dosing, animals were weighed and killed by cervical dislocation in the morning

between 9 a.m. to 10 a.m. to minimise diurnal variation (Ractzialowski and

Bousquet, 1968). Immediately after killing the livers were rapidly excised,

freed from connective tissues, blotted with filter paper (Whatman No.l) to

remove excess blood and weighed in a beaker containing 1.15% KC1 which helped

in keeping the temperature, down and washing off the blood which may interfere

in the determination of cytochrome P-450. Liver homogenate was prepared and

fractionated according to the method of Basu, et al.(1971). into the following

fractions (a) 10,000 g supernatant (microsomal fraction); (b) 105,000 g

supernatant (soluble fraction); (c) 105,000 g pellet, washed and resuspended

in the original volume of 1.15% KC1 (microsomal suspension).

Preparation of intestinal mucosal fraction

From duodenum (inclusive) a 30 cm section of the small intestine was

excised and immersed in ice cold 1.151 KC1 (4°C). The gut section was washed

free of chyme with ice cold 1.15% KC1 using a needleless syringe inserted into

the gut. section. The gut was then opened by a longitudinal incision. The

mucosa was removed by gently scraping the inner gut wall with a microscope

slide, using the blunt edge, and placed in a preweighed ice-cold beaker.

The undiluted mucosa was homogenized for one minute by repeated up and

down strokes in the .Potter Elvehjem homogenizer driven by a Black & Decker

electric drill at a speed 2950 r.p.m. The partially homogenized material was

diluted with three volumes of ice-cold 1.15% KC1 (W/v) and further homogenized

for one minute. The mucosal suspension (1 in 4) was diluted five fold with

ice-cold 1.15% KC1 to make a 5% (W/v) mucosal homogenate. The homogenate was

centrifuged at 0-4°C at 10,000 g in M.S.E. High Speed centrifuge in 50 ml

.stoppered polypropylene centrifuge tube in 80 x 50 ml angle head rotor.

Determination of biphenyl 4-hydroxylase (E.C. No.1.99.1.1) activity

Biphenyl 4-hydroxylase activity was determined fluorimetrically in the

10,000 g supernatant by the method of Creaven, et al. (1965) as modified by' N • '

Neale (1971). All the samples were run in duplicate. Standards of

4-hydroxy biphenyl were prepared by the same procedure (30 yg/ml). The activity

of the enzyme was expressed as ymoles of product formed per gram liver per

hour. .

Determination of 4-rnethylumbelliferone glucuronyl transferase (E.C. No.2.4.1.17)

activity (a) in the liver and (b) in the intestinal mucosa

(a) Hepatic 4-methylumbelliferone glucuronyl transferase

The activity of 4-methylumbelliferone glucuronyl transferase was determined

in the 10,000 g supernatant previously diluted twenty fold with 1.15% KC1.

The assay was carried out fluorimetrically according to Bollet, et al. (1959) .

140

All the samples were run in duplicate. A standard curve of 4-methylumbelliferone

was prepared by measuring the fluorescence of varying amount (0.001 - 0.01 ymol)

of 4-methylumbelliferone in acetate buffer (0.1 M) pH 4.6. After measuring the

fluorescence in the samples of liver preparations the concentration of

4-methylumbelliferone was calculated from the standard curve. The enzyme

activity was expressed as pinole of product formed per gram liver per hour.

(b) 4-Methylumbelliferone glucuronyl transferase (E.C. No. 1.99.1.1) in

the intestinal mucosa.

The activity of the enzyme (pmole/g liv./hr) in 10,000 g mucosal fraction

(5%, 1 in 20) was determined fluorimetrically following the same procedure as

in the liver fraction (Bollet, et al., 1959).

Determination of alcohol dehydrogenase activity (E.C. No. 1.1.1.1)♦

Hepatic alcohol dehydrogenase is remarkably unspecific and catalyses

reversibly the interconversion of broad groups of alcohols and their

respective aldhydes or ketones (Theorell, 1965).

CH3CH20H + NAD+ * CH3CHO + NADH+ + H*'

The assay of liver alcohol dehydrogenase activity (ymol/100 mg/protein/hr)

was based on spectrophotometric measurements of the reduction of NAD+ to + +NADH + H by excess ethanol as substrate at alkaline pH (9.6) as developed

by Theorell and Bunnichsen (1955) and subsequently modified by Dalziel (1957).

The change in absorption at 340 nm due to formation of NADH + H* per minute

was the measure of enzyme activity and was expressed as ymol/1,000 mg protein

(soluble fraction) /hr as calculated using the coefficient of NADH + H+ at

340 nm (£340 = 6.22 x 106 cm2/mole).

Determination of cytochrome P-450

Cytochrome P-450 was determined in the microsomal suspension. The

microsomal suspension was diluted three fold with 0.2 M potassium phosphate

buffer, pH 7.4. The cytochrome P-450 in the hepatic microsomal suspension was

determined according to Sladek and Mannering (1966). Hie difference in

141

absorption between the reduced and CO-complex of the reduced haemoprotein

within the range 450 nm to 490 nm was the measure of cytochrome P-450

concentration (nmole/g liver) calculated according to Omura and Sato (1964)

using a molar extinction coefficient of AE450-490 = 91 cm 1 mM 1.

Determination of cytochrome b5

Hie concentration of cytochrome b5 was determined in the diluted

microsomal suspension, prepared as for the determination of cytochrome P-450,

by the method of Schehkman, et al. (1965). Hie difference in absorption between

the reduced and oxidized microsomal suspension within the range 410 nm to

424 nm was the measure of cytochrome b5 concentration (nmol/g liver) and was

calculated using the molar extinction coefficient AE424— 410 = 185 mM 1 cm 1.

Determination of Protein Content

The concentration (mg/g liver) of protein in the microsomal suspension

and -in-the soluble fraction was determined according to Lowry, et al.(1951).

The microsomal suspension was diluted to 1 in 200 with 1.151 KC1 and the

soluble fraction was diluted to 1 in 400 and protein determinations carried

out in 1 ml of the diluted suspensions. A standard curve was prepared by

plotting the optical density against the corresponding concentrations of

bovine serum albumin (20 yg - 200 yg). The concentrations of protein content

in the samples were calculated by plotting the optical densities onto the ' •

standard curve.

Determination of DMA

DMA from the liver homogenate was extracted according to the method of

Schreider (1945) as modified by Burton (1955). The determination of DNA

content based on the colour reaction with diphenylamine reagent was described

by Dische (1930) and partly modified by Burton (1955). All the samples were

run in triplicate. Standard curves were prepared with known amounts of DNA

(20 yg - 200 yg) by running through the same procedure for colour development

with diphenylamine reagent. Hie DNA content (mg/g liver) in the liver

142

homogenate was calculated from the standard curve by plotting the measured

optical densities at 600 nm.

Results

The results of 0-64 days pretreatment with linalool are shown in

figures 6-1 to 6-10. Tabulated results are shown in the Appendix, tables

6-i to xi.e -

Body weight

From Figure 6-1 it can be seen that there was a slight but statistically

not significant decrease in body weight gain in animals treated with linalool

relative to their controls.

Liver weight and relative liver weight

Figures 6.2 and 6.3 show the effect of linalool on pretreated rats

compared to controls with respect to liver weight and relative liver weight.

It was observed that there was no significant difference between the tests and

controls in these two parameters upto the 30th day of exposure. After

continued exposure for 64 days, significant, although slight, increases were

observed (p < 0.05) in both these parameters.

Biphenyl 4-hydroxylase

Figure 6-4 compares the activity of biphenyl 4-hydroxylase in the controls

and linalool treated rats. There was no significant change in the activity of,

the enzyme in linalool treated rats compared to untreated animals. However,

one noticeable observation was that the level of this enzyme was gradually

dropping in developing rats compared to 28 days old animals. To begin with

28 day animals the enzyme level was 4.7 ymol/g liver/h and after 64 days at

the age of 13 weeks the level had dropped to 3.5 ymol/g liver/h amounting to

a 26% decrease.

4-Methylumbelliferone glucuronyltransferase

Figure 6.5 shows the 4-methylumbelliferone glucuronyltransf erase activity

in the liver of controls and linalool treated animals. It was observed that

the activity of this enzyme was dramatically increased by chronic exposure

to linalool. Hie activity increased steadily by 17% (p < 0.02); 29% (p < 0.01);

81% (p < 0.001); 98% (p < 0.001) and 150% (p « 0.001) after exposure to

linalool for 3, 7, 14, 30 and 64 days respectively.

The intestinal 4-methylumbelliferone glucuronyltransferase, unlike liver,

showed a biphasic response to linalool (Fig. 6.6). After an initial depression

of 33% in the activity there was steady increase reaching the control value

on 14th day. By the 30th day the activity was increased by 45% (p < 0.01)

relative to controls and by the 64th day the increase in activity was 8.3%

(p ■< 0.001).

Alcohol dehydrogenase .

Alcohol dehydrogenase in the soluble fraction (105,000 g supernatant) of

the linalool treated rats showed a biphasic response (Figure 6.7). It was

initially depressed by 33% (p < 0.02) on the three days pretreatment followed

by sharp dramatic increase of 36% (p < 0.001) on the 7th day. After 14 days

exposure in the treated rats the enzyme activity was 11% higher (p < 0.05)

than the control, thereafter, no significant change was observed between the

test and the controls of 30 days and 64 days.

105,000 g Supernatant (soluble fraction) protein concentration

Figure 6.8 shows the protein content (mg/g liver) in the soluble fraction,

of untreated and linalool treated rats. The protein concentration in the

soluble fraction remained unchanged for the entire period of 0-64 days

treatment. The significant change in alcohol dehydrogenase in the treated

animals has therefore shown no noticeable effect on the protein concentration

of the soluble fraction. ~ .

Cytochrome P-450 concentration

The progressive change in the concentration of cytochrome P-450

(n mol/g liver) in the linalool treated rats has been shown in Fig. 6.9;

a biphasic response was observed. The enzyme was inhibited to the extent of

144

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20% (p < 0.02) in the test animals after 7 days pretreatment compared to

controls. It regained control value on 14th day with a sharp increase

(50%, p < 0.01) on the 30th day. The concentration of this cytochrome

continued to remain at this elevated level after 64 days exposure to linalool.

Cytochrome b5 concentration

The concentrations of hepatic cytochrome b5 (n mol/g liv.) at the

different stage of treatment in the linalool treated rats compared to controls

have been shown in Figure 6.10. It resembles cytochrome P-450 in showing a

biphasic response with 29% inhibition after seven days exposure with

non-significant change on three days pre treatment. The level of cytochrome

b5 reached the level of the control value on the 14th day with a sharp increase

(50%, p < 0.01) after 30 days pretreatment. The 64 day exposure led to a 75%

increase in the test compared to the control.

Microsomal protein concentration

The change in microsomal protein concentration (mg/g liver) is shorn in

Figure 6.11. Hie microsomal protein concentration was unchanged in the test

animal compared to controls up to the 14th day of exposure. However, after

continued exposure for 30 days a significant increase (20%, p < 0.02) was

observed in the pretreated rats and the microsomal protein concentration

remained at this elevated level after 64 days pretreatment. ' • ■

DMA Concentration

The change in DNA concentration (mg/g liver) is shown in Figure 6.12.

The DNA concentration in the tests was unchanged, compared to controls after

3 days pre treatment. Seven days pre treatment caused a sharp increase of 32%

in DNA concentration but further exposure for 14 to 64 days did not cause a

significant difference in DNA concentration between tests and controls.

Discussion

In an earlier study involving a number of terpenoids, Parke and Rahman .

(1969) administered linalool intraperitoneally to rats at a dose level of

150 mg/kg for three days and found no significant effect on liver weight,

cytochrome P-450 concentration, biphenyl-4-hydroxylase and glucuronyl

transferase activities. The current study using high dose levels by the

oral route shows that, with most of the parameters studied, prolonged

administration was necessary for significant effects to be observed.

The liver weight and relative liver weight increased over control values

slowly but consistently after exposure to linalool for 14 days but the

differences only became statistically significant after 64 days. This increase

in liver weight was preceeded by a peak in the DNA concentration at 7 days

(p < 0.001) which subsequently returned to control levels. This suggests

increased mitotic activity at this stage indicating that the increased

liver weight involved some hyperplasia in addition to cell hypertrophy.

Microsomal (but not soluble) protein concentration also increased in the

later stages of the study, being significantly higher than control values

after 30 and 64 days of dosing and it appears that the earlier hyperplasia

was followed by a proliferation of endoplasmic reticulum with associated cell

hypertrophy. .The latter effect appears to be the major contributor to the

observed hepatomegaly. The time course of the changes in the parameters

studied is of particular interest (see Fig. 6.13).

The earliest observable effects of administration of linalool were in the

activity of 4rmethylumbelliferone glucuronyl transferase and alcohol

dehydrogenase. The activity of the former enzyme was significantly increased

after only 3 days which, together with the observations (Chapter 4) that

linalool and its metabolites are largely excreted in urine and bile as

conjugates with glucuronic acid, suggests an adaptive response to the increased

metabolic demand. The up-surge in glucuronyl transferase activity between

% C

ontr

ol

Val

ue

F ig. 6 .13 T im e Course of the Effect of Linalool Adm inistration on som e R at-L iver Param eters.

1.50P < 0.001

140 “I

130“ i

110P < 0 .001

100 i

90 “ !

8 0 - P < 0 .001

7 0 -P < 0 .002

P < 0 .0 1P < 0 . 0150 H

40 J/PC0.01

R < 0.001P < 0. 02

P < 0. 02

\n. si10 - n. s n. s .>e • • —jsg

-----------o

- 1 0 - Days of Pretreatm ent

4-M ethylum belliferoneG lucuronyltransferase

o Alcohol Dehydrogenase©.......© Cytochrome P-450a— .—a Cytochrome b 5

2 0 -

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A A M icrosom al ProteinD N A

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Linalool (500 mg/kg) w as adm inistered daily by intragastric intubation as a 25% W /V solution in propylene glycol.

159

7th and 14th day may result from gradual tissue accumulation since it was

'observed (Chapter 4) that linalool may enter into enterohepatic circulation,

delaying complete excretion. Subsequently the higher level of glucuronyl

transferase activity was maintained and it is to be expected that in the

later stages of the experiment it was sufficient to maintain acceptable tissue

levelsof linalool despite the repeated dosing.

The up-surge in glucuronyl transferase activity between the 7th and 14th

day coincided with anobservable increase in microsomal protein concentration

which had appeared normal up to that point. This suggests that the initial

increase in glucuronyl-transferase activity resulted from activation of the

enzyme already present in the endoplasmic reticulum while the second up-surge

in activity resulted from proliferation of the endoplasmic reticulum. This

agrees with the observations of cytochrome P-450 and b5 concentrations where

some inhibition is evident after seven.days but subsequently, as microsomal

protein concentration increased, so did the concentration of the cytochromes.

Since linalool is already oxygenated and readily conjugated via the OH group,

it would not be expected to act as a substrate for cytochrome P-450 and hence

the increase in concentration of this cytochrome might reflect a non-specific

proliferation of endoplasmic reticulum associated with the increased synthesis

of glucuronyl-transferase. However, it has been shown (Chapter 7) that

linalool binds to cytochrome P-450 to produce a reverse type I binding spectrum

and is a mild inhibitor of its monooxygenase function. Thus the increase in

cytochrome P-450 concentration might,, at least in part, reflect adaptation to

this inhibition.

The early biphasic effect on alcohol dehydrogenase activity may also

relate to initial tissue accumulation of linalool which is observable first

as an inhibition of alcohol dehydrogenase activity after three days. The

subsequent increase in activity after seven days then probably results from

160

the combined effect of an adaptive induction of alcohol dehydrogenase together

with an increased capacity for glucuronidation (vide Supra) which would lower

the inhibitory effect. Subsequently, when the increased activity of

glucuronyl-transferase was fully able to cope with the increased metabolic

demand, alcohol dehydrogenase levels returned to, and remained, normal.

It is interesting to note that, despite the biphasic fluctuation in the

cytochrome concentrations, the activity of biphenyl 4-hydroxylase was

unaffected throughout the course of the study. This might seem surprising

at first sight, but other workers (Rahim, Parke and Walker, 1974) have

reported selective induction of biphenyl 4-hydroxylase or ethylmorphine-N-

demethylase with antioxidants in which there was no correlation between the

function of the enzyme and cytochrome P-450 levels with one or other substrate.

Hie increase of cytochrome P-450 and bs which are at the terminal end of

the microsomal electron transport system may be involved in the oxidation of

NAIH2 to keep up the level of oxidized NAD+ for the synthesis of UDPGA for

continued conjugation to dispose off the load of linalool,

UDPG + 2NAD ^ UDPGA + 2NADH2. . - — . . -> *-UDPG dehydrogenase

UDPGA' + ROH R0C6H906 + UDP. . — .1 ■ ,1,1 ».|„||| ■ .« — , - Ml.

UDP transglucuronylase +In mobilizing'the reserve of NAD the cytochrome P-450 and b5 may therefore

enhance the metabolism of other exogenous and endogenous compounds including

cholesterol (steroid) metabolism as was observed earlier by Benkos, et al.

(1961) who suggested that formation of atherosclerosis plaques was prevented .

by terpene treatment.

The intestinal 4-methylumbelliferone glucuronyl-transferase, unlike the

liver enzyme showed a biphasic response. The initial inhibition at 3 days

indicates that the intestinal 4-methylumbelliferone glucuronyltransferase is

first inhibited and then more rapidly synthesized to give the adaptive

161

response to the increasing load of linalool. In this regard the steady

increase of liver 4-methylumbelliferone glucuronyltransf erase may be

indicative of a more efficient adaptability of the liver enzyme than the

intestinal one.

The results of the present investigation therefore show that with the

exception of alcohol dehydrogenase and glucuronyltransferase prolonged

exposure to linalool is necessary before highly significant effects are

observed on the other drug metabolizing enzymes. • It appears that the

observed effects represent a physiological adaptation to the continued

exposure of linalool and not a pathological condition. However, the increase

in DNA concentration after the 7 day period, indicative of increased mitosis,

suggests that an increased vulnerability to carcinogens might occur at this (c.f. Peraino et al.,1971)

stagq^* Experiments to investigate this possibility and to study the possible

synergistic effect of linalool on chemical carcinogenesis seem warranted.

This is particularly important since a number of essential oils have been

reported to possess carcinogenic and co-carcinogenic activity (Roe and Field,

1965).

Chapter 7

Studies of the Effects of Linalool on some

. Microsomal Enzymes

INTRODUCTION

In Chapter 6 the increase in 4-methyl umbelliferone glucuronyltransferase

activity was described and was considered to be an adaptive response of

this enzyme to the requirement for glucuronidation of linalool. It was

therefore decided to investigate the ability of microsomal glucuronyltransferase

to conjugate linalool directly both in normal rats and rats which have been

exposed to linalool. It was further suggested that delayed induction of

cytochrome P-450 may have been due to linalool acting as an inhibitor of

this enzyme. It seems unlikely that the already oxygenated linalool would

be a substrate for cytochrome P-450 in vivo but experiments to investigate

whether linalool might bind to cytochrome P-450 and act as an inhibitor of

cytochrome P-450 were indicated.

164

MATERIALS AND METHODS

Chemicals

Linalool, 99% was obtained from Ralph N. Emanuel Ltd., ltfC linalool

was synthesised in our laboratory (see Chapter 2) and Uridine 5' -diphospho-

glucuronic acid (UDPGA) ammonium salt was procured from Sigma, London.

The other' chemicals have been mentioned elsewhere in the text.

Animals

Male Wistar rats were used and accommodated under controlled conditions

of temperature (20°-22°C), relative humidity 52% and lighting (12 h light,

12 h dark).

In vitro inhibition studies

Four 28 day old normal rats (untreated) were killed by cervical

dislocation, their livers pooled and a double washed microsomal suspension

prepared according to the method of Basu, et al. (1971). The biphenyl 4-

hydroxylase activity of this microsomal preparation was measured over a range

of substrate concentrations and in the absence or presence of various

concentrations of linalool.

Linalool was added as a 250 mM solution in 95% aqueous ethanol at a

maximum volume of 6 yl per incubation mixture and a similar volume of 95%

ethanol was added to controls without linalool.

Biphenyl activity was assayed by the method of Creaven, et al. (1965)

as modified by Neale and Parke (1970), except that the substrate concentration

was varied between 0.6 - 3.0 pM and linalool was added at a concentration of

0 - 6.25 mM.

Spectral dissociation constant (Ks) of linalool

A double-washed hepatic microsomal suspension (2 mg protein per ml in

0.1 M phosphate buffer pH 7.6) was prepared according to the method of

Schenkman, et al. (1967) from the liver of 28 days old rats. The Ks was

determined according to the method of Schenkman, et al.modified as follows:

165

Microsomal suspension (2.5 ml) containing 2 mg of protein per ml was

pipetted into both of the two glass cuvettes. These were sealed with

.subaseal .stoppers (Gallenkamp Ltd.', London) and placed in the reference and

sample beams of the SP 1800 spectrophotometer. Serial additions of microlitre

quantities of linalool- in'alcohol (0.1 to 0.6 mM) were made to 2.5 ml

microsomal suspension in the. sample cuvette and equal quantities of alcohol

(95%) in the reference cuvette. The difference spectrum at 380 nm to 425 ran

was measured.

In vitro conjugation study . . .

Linalool was administered intragastrically (500 mg/kg in 25% propylene

glycol) to 28 day old rats, for four weeks. Three paired fed controls

received the same quantity of propylene glycol and three controls had access

to food and water ad' libitum. The rats were killed on the 29th day. Double

washed microsomes were prepared from the liver and intestinal mucosa of

these three groups of animals each separately.

The glucuronide formation in the microsomal suspension was determined

according to Axelrod and Inscoe (1960) as modified by Yeh and Mitchell (1972)

except that lkC labelled linalool (6.0 pinole containing 111,500 dpm) was used

as substrate and microsomal suspension of 12.5% from liver microsomes and' >25% from the intestinal mucosal microsomes were used as enzyme sources.

Hie incubation mixture contained O'. 2 ml of double-washed microsomes (liver

or intestinal mucosa), 6.7 ymoles of UDPGA (0.3 ml), 42.5 yl of 1 C-linalool

solution in alcohol (1.4 x 10 1 M 1 C-linalool in alcohol) that contained

6 ymole 1 C-linalool (lli,000 dpm), 0.3 ml Tris buffer (0.05 M Tris buffer,

pH 8.0), 0.2 ml MgCl2 (1 mmole) solution in 1.15% ICC1 and the final volume

of the mixture was 1.0 ml. Incubations were carried out in duplicate in a

shaking water bath at 37°C for 30 minutes under atmospheric air. The reaction

was terminated by placing the tubes in ice cold water and the excess

unconjugated 1IfC linalool was extracted three times with ether. The water

layer after being freed of residual ether by a slow stream of nitrogen at

166

room temperature, was hydrolysed with 500 units of 3-glucuronidase in

acetate buffer (0.1 M) of pH 4.6 according to Arias (1962). After hydrolysis

the liberated llfC-linalool was extracted with ether. The amount of linalool

in the ether extract was determined by counting for 1 in 30/70 scintillator

in a Packard Tricarb Spectrometer model 3320.

The enzyme activity was expressed as ymole of linalool conjugated per

gram liver per hour.

RESULTS

Inhibition Studies

The inhibiting"effects of varying concentration of linalool on biphenyl

hydroxylase activity is shown as Dixon plots' in figure 7.1. The straight

line curves intersect one another on a point on the base line where Vv= 0t

and linalool concentration is 1.9 x 10 3 M. This, therefore, characterises

linalool as a non-competitive inhibitor with a Ki of 1.9 x 10 3 M.

Figure 7.2 is the Lineweaver-Burk plot of velocity over a range of

substrate concentration and extrapolation of the plot to /v= 0 cuts the absciss

at -(Vs) - - "2TTT x 10 3 M which is equivalent to - hence the value of— 1 ^Km = 3.45 x 10 k M. The intercept at the y-axis ( /v) correlates with the

theoretical value of (1 + -) x thus showing a characteristic of non­

competitive inhibition.

Spectral dissociation constant (Kg)

The spectral interaction of linalool with microsomal suspension exhibited

a difference spectrum with trough at-390 nm and peak at 420 nm. This spectral

change is associated with the reverse type I compounds (Orrenius, 1970;

Schenkman, 1972) and thereby, linalool is characterised as reverse type I

compound. The extent of the binding of linalool with cytochrome P-450 was

represented by the magnitude of the difference (AE) between absorbance at

420 nm and 390 nm at various linalool concentration. The double reciprocal

plot of AE against linalool concentrations is shown in Fig. 7.3. The plot

was extrapolated to AE = 0, where it cut the abscissa- at a substrate

concentration -1.9 x 10 4 M ( " ^ = “5.3 x 10 3 M) which by definition is

- the negative equivalent of the substrate concentrates at half maximal

spectral change. The value of Kg = 1.9 x 10 ** M indicates considerable affinity

of linalool for cytochrome P-450.

Conjugation study

The results have been tabulated in table 7.1. No appreciable variation

was observed between‘the activity in the control and the pair-fed animals

whether in'the liver or gut mucosa. However the liver microsomes from

linalool-treated animals showed a 711 increased rate of conjugation whereas

with the intestinal mucosal microsomes the rate of conjugation was increased

by 33% compared with their respective control values.

168

Table

7.1

Conjugation

Study.

Increased

rate

of glucuronide

conjugation

of C-linalool by

microsomal to

S'0oto0P0COo&<L>O•Hsp•6P0+-*ojajto0u11—I 03 0 •H•Pto0■P

■0§P0>•H■Pa3P<4-tOto0to03P.0mto

pi—iIp0B O•P

LOrH UOt—I•HrHOO rH

to

VO mrH

LOrH rH

to

rHrHrH

rHCN1

rHrHrH

LO 01rHrH

LOCO

rH rH

rHOOrH

rH

rH

+ I<P00to0Pft0Pa3•Po300£

m0to•pto•H

1 u'6030

SopbO

■P•HtSrH'010 3 1

-P0•H000mop■p0OU•0

&ofeoop■p

g-u•00mp•H0ft

ov

ft

tSrH*01 rt I0,0

o13goo•p4-Jo0ftto0p

■5•H£•0

toi—io p ••p0OU•0.0mp•H05fto+->•po0ftto0p

• H£

to•po0mm0

u•Hm•Ha•HC/D

LO8o

ft**

169

DISCUSSION

The in vitro studies indicate that linalool binds to cytochrome P-450,

‘exhibiting a reverse type I type of reaction difference spectrum and is a

non-competitive inhibitor of the hepatic microsomal drug metabolizing enzymes.

Biphenyl is a substrate of cytochrome P-450; hence during the process of

biotransformation linalool and biphenyl may inhibit each other competitively

if they bind on the same site of the cytochrome P-450. Our present study

revealed that linalool binds to the reverse type I "site" of the cytochrome

P-450 and that linalool inhibits biphenyl 4-hydroxylase non-competitively.

Biphenyl is a type I compound (Tredger ,19 74) . Inhibition of biphenyl 4-hydroxylase

by linalool is, therefore, non-competitive because of interaction of biphenyl

and linalool at different site of cytochrome P-450. The apparent interaction

of linalool at the reverse type I "site" may be due to a conformational change

of the cytochrome. It is therefore unlikely that linalool. is a substrate for

cytochrome P-450, which could be anticipated as linalool is an oxygenated

compound with a potential hydroxyl group for ready elimination from the

body as polar conjugates.

Despite the fact that linalool was found to be an inhibtor of biphenyl

4-hydroxylase in this study, it is not clear why it was without effect on/ N

biphenyl 4-hydroxylase activity after administration in vivo.

The initial inhibition of cytochrome P-450 and subsequent sharp

induction (see Chapter 6) are likely to be caused by tissue build up of

linalool due to repeated doses and enterohepatic circulation which inhibit

the mixed function oxidases at early stages (7 days pretreatment) and then

rapid synthesis of these enzymes is triggered off amounting to sharp induction

to meet the requirement for normal metabolism of endogenous and exogenous

substances. .

170

Table 7.2 Comparison of spectral dissociation constants (K ). and

dissociation constants (X) of enzyme-inhibitor complex.

Compound Type of Binding

Species K.l Reference

Linalool RI R 1.9x10"3M 1. 9x10"*1

Dexamethasone RI R 1.21x10"1I H 0.9xl0_1 ) '

CorticosteronLe I R 2.Ixl0~1I H 0.7X10"1 )

Prednisolone II ? R 0.7x10"1 )Tredger (1974)I H 0.7x10"1 )

Betamethazone II? R 0.7X10"1I H 0.3X10"1

J

Cortisol RI R - ))

I H 1.2x10"1

Phenobarbita] 2.4x10"1 Kutt,et al. (1971)

Ethoxyquin I R 4.3x10~6M 1.1x10"2 Parke,et al.(1974)

SKF 525 A I R 6.0x10"eM 0.7x10"2 )Manneririg (1969)) and

SKF 8742 A I . R 3.6xl0~6M - )Anders (1971)

Aniline II R — 12.5x10“1 Sweat,et al.(1970)

RI = Reverse type I ? The type of binding of prednisolone and betamethasone

R = rat could not be categorised. Prednisolone gave max at

H = hamster 425 nm min 380 nm and betamethasone max at 415 nm

and min 375 nm.

F ig . 7 .1 Dixon plot showing non-com petitive inhibition of biphenyl 4 -hydroxylase activity by linalool.

1 .4 -1

05

-2 -1 1 2 3 4 5 6 7 8 9

Concentration of linalool x 10"3 M

Biphenyl 4-hydroxylase activity (ju m o le /g liv er /h ) was determ ined by the method of Creaven et a l (1965) a s m odified by Neale (1970), at substrate concentrations of 0 .6 mM, 1, 8 mM, 3 .0 mM and linalool concentrations 1 .2 5 mM to 7. 5 mM.

Fig. 7.2 Lineweaver - Burk reciprocal plot for the determination of theapparent Michaelis constant ( Km ) of biphenyl 4 -hydroxylase.

Km = 3.4-5 x 10 M

1.2“

1.0H

r—I

o S §.g 0.6"si ft a —i•8* 123 “

0 .4 -Ph ^ rH /

75 61 3 42124 3 1________Biphenyl x 10 3 M

Biphenyl 4-hydroxylase activity (jumole/g liv er /h ) w as determ ined by the method of Creaven et al.(1965) as modified by Neale (1970) at substrate concentrations of 0 .1 5 mM to 1. 5 mM.

Fig. 7. 3 Lineweaver - Burk reciprocal plot for the determinationof the Spectral dissociation constant (Ks) of linalool.

o<M

o

10-4

5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 12

r ; - .---------- — r:— (M) x 10Linalool concentration

Spectral d issociation (Ks) of linalool w as determ ined by the method of Schenkman et al.(1967). Increasing concentrations of linalool (0.1 mM - 0 .6 mM) w ere added to 2. 5 ml of double washed m icrosom al suspension (2 mg protein /m l) made from rat liver m icrosom e preparation in 0 .1 M phosphate buffer,. P H - 7. 6. Maximum volum e of 10 p i of ethanolic solution of linalool (250 mM) w as added to the 2. 5 ml of the above m icro ­som al preparation in cuvettes of 1 cm pathlength.

The K and of linalool have been compared in table 7.1 with those

of some known inducers and inhibitors of drug metabolizing enzymes. Tredger

(1974) studied the binding characteristics of some of the glucocorticoids.

Cortisol and dexamethasone were reported to bind to the reverse type I

"site" of the rat liver microsomal P-450 but exhibited characteristics of a

type I substrate with hamster liver cytochrome P-450. These steroids and

others such as corticosterone, prednisolone, betamethazone were shown to

possess Kg values of similar order to that of linalool. Since cortisol and

dexamethasone have similar ’'reverse type I” binding characteristics to linalool

they may compete with one another for metabolism or may be synergistic. In

this regard the work of Benkos, et al. (1961) may be worth mentioning. He

reported that the formation of atherosclerosis plaques was prevented by terpene

treatment, nowithstanding a-high level of serum cholesterol in the cholesterol

fed rabbit. The K value of linalool was also comnared to other inducers andsinhibitors. Phenobarbital, an inducer of drug metabolism has Kg 2.44 x 10 1 mM

of similar order to linalool 1.9 x 10 1 mM. In comparing the JL of linalool

(1.9 x 10 3 M) with other potent inhibitors like SKF 525 A (6.0 x 10 6 M),

SKF 8742 A (3.6 x 10 6 M) and ethoxyquin (4.3 x 10 G M) it is plausible that

linalool is a mild inhibitor.

However, the ability of a compound to form a binding spectra with

cytochrome P-450 does not guarantee its metabolism. Imai and Sato (1967) have

pointed that barbital and benzene are hydroxylated by microsomes although they

do not produce binding spectra. Conversely, many alkylamines combine avidly

with cytochrome P-450 to produce type II binding spectra but are not metabolized

(Jefcoate, et al, 1969). Linalool having a potential -OH group could undergo

phase II reaction to be eliminated from the system and phase I oxidations

would appear to be unnecessary for detoxication.

In the light of the earlier metabolic studies (see Chapter 6) increased

activity of the glucuronyltransferase in the hepatic and intestinal mucosal

microsomes may also be considered as an adaptive response to the increasing

load of linalool so that the latter could be eliminated from the body after

.conjugation with endogenous substances.

176

General Discussion

As the experimental results have been discussed in the individual

chapters, this general discussion will be confined to broader implications.

The in vitro studies on the metabolism of linalool by caecal

microflora indicated the potential of these organisms to effect reduction

to dihydrolinalool and tetrahydrolinalool, and these metabolites were

also identified , both free and conjugated with glucuronic acid or

sulphate, after in vivo metabolism. The fact that, after intraperitoneal

administration, very small amounts of these reduction products appeared

in bile after the first pass (linaloolidihydrolinalool + tetrahydrolinalool,

9:1) whereas after the second pass the reduction products predominated

(linalool:dihydrolinalool + tetrahydrolinalool, 1:3) confirms that the

major site of reduction of linalool is in the intestine. The in vitro

studies are therefore a fair indication of intestinal microbial metabolism

in vivo.

Reduction of - C O double bonds by intestinal micro-organisms has

been reported previously for dietary unsaturated fatty acids (Polan et- al.

1964, Watson, 1965; Wilde & Dawson, 1966) and for a number of xenobiotics

including coumarin, cinnamic acid and its derivatives (Hansen &

Crawford, 1968; Scheline, 1968; Peppercorn & Goldman,. 1972, Booth &

Williams, 1963). This, therefore, appears to be a common route of

microbial metabolism of unsaturated compounds and may result from

specific reduction mechanisms or merely as a result of the low redox

potential generated in the gut by the microbial population.

Further, since linalool was excreted in bile as conjugates on the

first pass and appeared as conjugates of the reduction products on the

second pass, it seems likely that hydrolysis of the glucuronides

occurred in the gut'with subsequent reduction of the aglycone prior to

177

reabsorption. There are two important consequences of this. First,

linalool or its reduction products, enter into enterohepatic

circulation which delays their excretion so that any biological effects

are more prolonged and second, whether the compound is'administered

orally or parenterally, similar metabolites may ultimately.be produced

in the gut. These consequences are not confined to linalool since it

is well known that drugs such as stilboestrol enter into enterohepatic

circulation which delays excretion and Tyrolones pharmacological activityparenterally administered ;

(Clark et al., 1969) while conjugates of]\chloramphenicol may be

hydrolysed in the gut and further, metabolised to toxic compounds which{£arke,1968) j

are reabsorbed causing blood dyscrasias^. It would appear, then, that

in conventional animals, biliary excretion is a less effective and less

reliable detoxication mechanism than is urinary excretion, though this

may not be the case in axenic (germ-free) animals.

The extent to which a compound is excreted in bile is dependent

inter alia on a number of molecular parameters, principally polarity

and molecular weight (Williams et aL, 1965). There appears to be a

species-dependent threshold molecular weight above which compounds

may be excreted in bile and hence there will be species differences

in the extent to which parentally administered compounds reach the

gut for metabolism by gut microflora. There may thus be consequent

species differences in pharmacological activity or toxicity.

The wide species differences observed in the metabolism by the

gut microflora (Chapter 3) raise questions of species selection in

toxicity testing. In many cases, the intestinal microflora are

responsible for the production of toxic metabolites (Scheline, 1973,

Section VII) and species differences in toxic response to a test

compound may sometimes arise from differences in intestinal metabolism.

178

Thus Walker et al. (1970) demonstrated that the species differences

between rat and mouse in the toxicity of orally adminstered Brown FK

resulted from differences in the extent of intestinal reduction to myotoxic

amines.

A number, but not all, of the species examined produced a-terpineol

and y-terpinolene by a cyclisation process not previously reported to

occur with intestinal micro-organisms. These cyclic compounds may then

undergo aromatization, as occurs with quinic acid (Scheline, 1968) and

some steroids (Goddard & Hill, 1973), although aromatized metabolites

were not detected in the current study. Little is known about the

toxicity of a-terpineol and y-terpinolene but the presence of the latter

compound is of concern because of the tumour promoting effects of a

number of essential oils appears to reside in the hydrocarbon fraction

(see Chapter 1).

It seems prudent to consider the consequences of intestinal

metabolism in the selection of species for toxicity testing and

extrapolation of the results obtained to man. However, a further

difficulty arises in this area since there is obviously no archetypal

man in respect of intestinal metabolism. In the current study, the

faecal microflora of a habitual spice-user metabolised linalool in a

substantially different way from the flora of a subject who regularly

ate a bland diet and similar differences have been observed in the

metabolism of cyclamate by Japanese and Europeans. (Kojima &

Ichibagase, 1966; Hill et al, 1971). In the latter case, the difference

was explained by large qualitative and quantitative differences in

intestinal microbial populations which were believed to arise from dietary

differences. It has been reported that individuals on a high cereal

diet consistently harbour more yeasts in the intestine than normal

( Schelinq 1968) while considerable changes in the intestinal microflora

179

accompany weaning (Lee & Gemmell, 1972) with a reduction in the

number of aerobes and the establishment of dominant population of

strict anaerobes. It seems likely, therefore, that the inter-

individual differences observed in the current study arose from the

differences in diet. The microflora of the spice-user would have been

repeatedly exposed to essential oils containing terpenoid compounds

and may consequently have adapted by selection of organisms which

were capable of metabolising these compounds and/or which were

relatively resistant to their bactericidal effects (Lee & Gemmell, 1972).

In this context,coriander is regularly used in oriental cuisine and is

particularly rich in linalool. Adaptation on chronic exposure with

subsequent enhanced ability to metabolise cyclamate to cyclohexylamine

has been reported (Kojima and Ichibagase, 1966) and the interindividual

differences in linalool metabolism may be a manifestation of a

similar phenomenon.

It is noteworthy that, while rat caecal microflora and the

faecal microflora of the human non-user of spices produced mainly

reduction products with tetrahydrolinalool predominating, mouse and

sheep only effected reduction as far as dihydrolinalool while the cow

and the spice-user produced no detectable reduction products. Mice

have previously been reported to be less effective than rats in reducing

azo compounds (Walker et al., 1970) and it was thought that this may

result from differences in the redox potential generated by the gut

flora. From the current study, ruminants,mouse and the spice-user

were only poorly able to reduce linalool and this probably reflects

differences in gut populations. It was noted above that weaning

caused a change from essentially aerobic or facultative populations to

anaerobic populations in which Bacteroides, Bifidibacteria and

Fusobacteria predominate and it is possible that the ingestion of

180

essential oils by the spice-user was having a selective bactericidal

effect on these fastidious anaerobes. As a consequence, the symbiotic

equilibrium would be shifted towards the facultative and aerobic

organisms with a raising of the redox potential in the lumen. In this

context it is noteworthy that only in those species in which reduction

products of linalool were not detected did the unidentified carboxylic

acid (metabolite J),produced by an oxidative route, appear. Further

studies on the effects of essential oils on intestinal microflora

and vice versa, are obviously indicated.

The fact that radioactivity from llfC-linalool was excreted to the

extent of about 97% in 72h, mainly in the first 48h, suggests that there

is unlikely to be a problem of tissue accummulation at the normal levels

ingested in foods. Since linalool was observed to enter pathways of

intermediary-metabolism with the formation of 14C02 and ll|C-urea, the

residual activity in the tissues at the end of 72h may well be present

as normal tissue components. However, when one considers the weight

of individual organs and tissues, the residual activity was concentrated

to some extent in the gut, liver and kidney and this may arise from the

observed enterohepatic circulation of linalool, and its metabolites,

delaying excretion with some activity entering the urine on each turn

of the enterohepatic cycle.

The dose levels used in the in vivo studies (500 mg/kg) were very

much higher than are likely to be ingested in foods and consequently

the demands on the detoxicating pathways vis a vis pathways of

intermediary metabolism may have been increased. At lower dose levels

comparable to those ingested in foods, intermediary metabolism may

predominate and it would be of interest to study the effects of dose

level on the relative proportions of the metabolites produced.

Since most of the urinary and biliary metabolites of linalool

were.conjugated with glucuronic acid, the importance of this Phase II

detoxication mechanism is emphasized. It was perhaps not surprising,

therefore, to find that repeated dosing with linalool had a more

rapid and marked effect in increasing the activity of glucuronyl

transferase than on the other enzymes studied. This increase in

hepatic glucuronyl transferase activity with, later, an increase also

in intestinal mucosal glucuronyl transferase activity, might therefore

be considered as an adaptive response. In the normal individual receiving

linalool in the diet at customary levels, this detoxication mechanism

is. probably quite adequate but it is well known that in neonatal

animals and in certain strains and species (e.g.Gunn rat; cat)

glucuronyl transferase activity is depressed (Parke, 1968). In these

cases, toxic effects might occur with linalool due to competition

with endogenous substrates like bilirubin resulting in hyperbilirubinaemia

and icterus. Similar considerations apply to individuals with impaired

liver function due to disease, alcoholism etc., and further work on the

potential of linalool to cause injury in these cases would be justified.

The Gunn rat might be a useful experimental model in this sort of work.

The relative activities of hepatic and intestinal glucuronyl transferase

suggest that conjugation in the intestine is unlikely to be adequate

fo.r the complete detoxication of linalool when hepatic activity is

depressed.

While the involvement of glucuronyl transferase in conjugating

linalool and an adaptive increase in this enzyme was not unexpected,

it was perhaps more surprising to find that linalool also had an effect

on the Phase 1 microsomal mixed function oxidase system. As linalool

is already oxygenated it would not be expected that further oxidative

detoxication mechanisms need operate. This apparent anomaly was

182

explained by the observation that linalool interacts with cytochrome

P450 to produce a reverse Type 1 binding spectrum and is a mild

inhibitor of the activity of this enzyme with respect-to hydroxylation

of biphenyl. A number of compounds displaying this type of spectral inter­

action are not substrates for cytochrome P450 (Schenkman et al., 1972)9

but apparently cause conformational changes and non-competitive inhibition.

Linalool has been shown to be a non-competitive inhibitor of biphenyl 4-hydroxy­

lase in the current study.

The biphasic response of cytochrome P450 and cytochrome b5 to

repeated dosing with linalool was explained (Chapter 6) as initial

inhibition with a somewhat delayed adaptive response. 'While the increase

in these cytochromes may have little toxicological significance per se,

a number of other considerations arise.

During the inhibitory phase when cytochrome P450 was depressed

(days 3-7) the detoxicating activity with respect to other xenobiotics

would be likely to be impaired, modifying their pharmacological and

toxic effects. Thus Parke, et al. (1974) observed that single doses

of ethoxyquin had an inhibitory effect on hexobarbital metabolism in

rats with a dramatic increase in the sleeping time from 15 min to over

8 h. whereas repeated dosing caused induction of hepatic enzymes and a

slight reduction in sleeping time. Prior to the adaptation, then,

the pharmacological activity of hexobarbital was enhanced and similar

considerations apply to the inhibitory phase of the linalool induced

changes. This raises questions concerning the not uncommon practice

of using essential oils as pharmaceutical adjuvants where initially

they may increase the pharmacological activity and toxicity of a drug

and subsequently, in the adaptive phase, render it less effective.

Furthermore, some enzyme inducers like phenobarbital are claimed

183

to potentiate the carcinogenicity of other compounds.such as

N-fluorenylacetamide (Peranio et aL, 1971). This may result from

an increased sensitivity to the carcinogenic effects when the liver is

enlarging as a consequence of exposure to the enzyme inducer. In this

context, prolonged exposure to linalool caused hepatomegaly with

a transient increase in DNA on day 7. Hepatomegaly therefore appeared

to result both from hyperplasia and cell hypertrophy and during the

stage of increased mitotic activity, sensitivity to carcinogens may be

enhanced. One cannot, therefore, merely discuss the observed changes

as an adaptive response with no toxicological significance; consideration

needs to be given to these other possible consequences. A study of the

effects of co-administration of linalool and drugs or carcinogens might

well be justified to determine the validity of these speculations.

One final point to emerge from this study is the possible effects

of linalool in neonatal animals and man. It has already been mentioned

that, due to the lower activity of the detoxicating enzymes in neonates,

the toxicity of environmental chemicals may be increased. Conversely,

the increase in the activity of the drug metabolising enzymes which

accompanies weaning (Parke & Rahman, 1969) may result from exposure to

dietary anutrients including essential oils. Weaning is also accompanied '

by dramatic changes in intestinal microflora so that at this stage of

development considerable changes in patterns of metabolism both by

mammalian and microbial mechanisms is to be expected. While changes in

the former would usually result in improved detoxicating capacity, changes

in the latter might lead to the production of different, more toxic,

metabolites. In addition, the complication of enterohepatic circulation

is likely to increase when conjugating activity in the liver and

hydrolytic activity in the gut are increasing simultaneously.

184

As far' as the toxicity studies of essential oils are concerned

only the acute toxicity lias so far been reported in most cases. .The

toxicologiccil implications including the biochemical and biological

effects of the essential oils and their synthetic constituents in the

GRAS list need to be investigated in view of thei? continued use in

every day life, particularly in food.

References

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197

APPENDIX

Table 6-i Effect of chronic administration of Linalool on the

body weight of developing rats.

Days Body Weight (g) in Rats ’p' valuesControls Pretreated

0 78 + 1 75 + 2 ns

3 96 + i 88 + 2 ns

7 124 + 2 . 113+6 ns

14 190 + 4 187 + 1 ns

30 266 + 10 * 250 + 10 ns

64 324 + 10 369 jh 12 ns

Linalool was administered intragastrically to 28 days old. Wistar male rats

as a 25% ( /v) solution in propane 1,2-diol at a dose level of 500 mg/kg

body weight. Controls and pretreated each consisted of four animals. Values

given are the means _+ SEM.

"’ns1denotes 'p1 values are statistically non-significant.

199

Table 6-ii Effect of chronic administration of Linalool of the

liver weight of developing rats.

Days Liver weight Controls

(g) in ratsPretreated

*p’ values

0 2.9 +0.4 2.9 +0.4 ns

3 4.2 +0.4 3.7 +0.2 ns

7 5.2 + 0.7 5.0 +0.4 ns

. 14 8.1 + 0.4 9.0 + 0.3 ns

30 11.0+0.5 11.7 +0.8 ns

64 12.8 + 0.5 15.5 + 0.7 0.05

Linalool was administered intragastrically to 28 days .old Wistar male rats

as a 251 (W/v) solution in propane 1,2-diol at a dose level of 500 mg/kg

body weight. Controls and pretreated each consisted of four animals. Values

given are the means _+ SEM.

ns'1 denotes ’p’ values are statistically non-significant.

200

Table 6-iii Effect of chronic administration of Linalool on the

relative liver weight of developing rats.

Days

.

Relative Liver weight (g) in ratT p 1 values

Controls Preteated

0 4.11+0.7 4.11+0.7 ns

3 4.4 +0.3 4.22+0.3 ns

7 4.2 +0.4 4.4 +0.3 ns

14 4.3 +0.1 4.7 +0.2 ns

30 4.22+0.1 4.7 +0.0 ns

64 3.2 +0.2 4.1 + 0.0 0.05

Linalool was administered, intragastrically to 28 days old Wistar male rats

as a 25% (w/v) solution in propane- l,2,*-diol at a dose level of 500 mg/kg

body weight.

Controls and pretreated animals consisted for four animals.^

Values given are the means _+ SEM.

’ns’denotes 'p1 values are statistically non-significant.

Table 6-iv. Effects of chronic administration of Linalool on the

Biphenyl 4-hydroxylase activity of the developing

rats

Days

Biphenyl 4-hydroxylase activity (ymol/g liver/h) in rats ’p 1 values

Controls Pretreated

0 4.9+0/3 4.9 + 0.3 ns

3 4.5 +0.3 3.9 + 0.3 ns

7 3.6 + 0.1 4.0 +0.1 ns

14 3.9 +0.2 4.1 + 0.2 ns

30 3.5 + 0.0 3.7 + 0.1 ns

64 3.5 + 0.3 3.4 + 0.2 ns•

Linalool was administered intragastrically to 28 days old Wistar male rats

as a 25% (W/v) solution in propane. 1,2-diol at a dose level of 500 mg/kg

body weight.

Controls and pretreated animals consisted of four animals.

Values given are the means _+ SEM.f '’’ns'denotes fp* values are statistically non-significant.

Table 6-v Effect of chronic administration of Linalool on hepatic

'4-methylumbelliferone glucuronyltransferase activityof developing rat

Days4-Methylumbel1iferone glucuronyltransferase

(pmol/g liv./hr) fp* values

Controls Preteated

0 34.7 + 3.2 34.2 + 3.1 ns

3 37 + 2 44 + 1.0 <0.02

7 35 + 2 • 45 + 2 < 0.01

14 35 + 2 64 + 3 < 0.001

30 37 +1 73 +1 . < 0.001

64 33 + 3 83 + 1 < 0.001

Linalool was administered intragastrically to 28 days old Wistar male rats

as a 25% solution (w/v) in propane 1,2-diol at a dose level of 500 mg/kg

body weight.

Controls and pretreated animals (tests) each- consisted of four animals.

Values given are the means _+ SEM.

’ns’denotes the fp1 values are statistically non-significant.

203

Table 6-vi Effect of chronic administration of Linalool on

4-methylumbellifcrone glucuronyl transferase activity

(ymol/g liver/li) in the intestinal mucosa of developing

rats.

Days

.

4-Methylumbelliferone glucuronyltransferase (ymole/g liver/h)

*pf valuesControls Pretreated

0 4.6 + 0.3 4.6 +0.3 ns

. 3 4.3 +0.4 2.9 + 0.3 < 0.02

7 3.9 + 0.6 3.7 +0.5 ns

14 4.0 + 0.2 5.9 + 0.2 < 0.01

30 3.5 + 0.3 6.2 +0.4 < 0.001

Linalool was administered intragastrically to 2.8 days old Wistar male rats

as a 25% solution (W/v) in propane 1,2-diol at a dose level of 500 mg/kg

body weight.

Controls and pretreated animals (tests) each consisted of four animals.

Values given are the means _+ SEM.

»ns»denotes the !p* values are statistically non-significant.

204

Table. 6-vii Effect of chronic administration of Linalool on

alcohol dehydrogenase (ymol/100 mg protein/hr)

of developing rats

■

Days

;Alcohol dehydrogenase (ymol/100 mg protein) fp 1 values

Controls Pretreated

0 33.1 + 2.2 33.8 + 2.2 ns

3 34 . + 1 20 + 2 < 0.002

7 35 +1 45 + 1 < 0.001

14 38 + 6 42 +2 ns

30 35 + 1 37 + 1 ns

64 22 +1 21 +1 ns

Linalool was administered intragastrically to 28 days old Wistar male rats

as a 25% solution (W/v) of propane 1,2-diol at a dose level of 500 mg/kg

body weight. .

Controls and tests each consisted of four animals.

Values given are the means _+ SEM.

’ns’denotes the ’p* values are statistically non-significant.

205

Table 6-viii Effect of chronic administration of Linalool on

105 jOQQ g Supernatant (soluble fraction) protein

content of developing rats

'

Days105,000 g Supernatant Protein,

(mg/'g liver) ‘pf valuesControls Pretreated

0 68 + 4 68 + 4 ns

3 70 + 1 73 + 2 ns

7 69 + 1 73 + 1 ns

14 70 + 2 71 1 1 ns

30 79 + 4 75 + 2 ns

64 74 + 3 73 + 4 ns

Linalool was administered intragastrically to 28 days old Wistar male rats

as a 25% solution (w/v) of propane 1,2-diol at a dose level of 500 mg/kg

body weight.

Controls and tests each consisted of four animals.

Values given are the means +_ SEM.

’ns’denotes the Tp * values are statistically non-significant.

206

Table 6-ix Effect of chronic administration of.Linalool on hepatic

cytochrome P-450 concentration of developing rats.

Cytochrome P-450 (nmole/g/liv.) in ratsDays Controls Pretreated ’p’ values

0 17.7+0.7 17.7 + 0.7 ns

3 20.8 + 0.9 25.6 + 1.4 ns

7 19.4 + 1.0 15.5 + 0.8 0.02

14 16.8+1.0 20.0+1.3 ns

30 22.2 + 2.1 34.0+1.5 0.01

64 19,8 + 1.1 29.5+1.0 0.01

Linalool was administered intragastrically to 28 days old Wistar male rats

as a 25% solution (W/v) of propane 1,2-dioi at a dose level of 500 mg/kg

body weight.

Controls and tests each consisted of four animals.

Values given are the means _+ SEM.

'ns’denotes the *p' values are statistically non-significant.

Table 6-x Effect of chronic administration of Linalool on

cytochrome bs content of developing rats.

Cytochrome b 5 (nmole/g liver) in ratsDays Control Pretreated 1p1 values

0 9.2 + 1.2 9.3 +1.0 ns

3 11.3+0.4 12.6 +0.1 ns

7 11.8 + 0.2 8.6 + 0.2 0.02 '

14 8.6 + 0.3 10.6 + 0.9 ns

30 11.2+1.2 16.5 + 0.3 0.01

64 9.44+ 0.6 16.1+0.8 0.002

Linalool was administered intragastrically to 28 days old Wistar male rats

as a 25% solution (w/v) of propane 1,2-diol at a dose level of 500 mg/kg

body weight.

Controls and tests each consisted of four animals.

Values given are the means +_ SEM.

ns’denotes the fpf values are statistically non-significant.

Table 6-xi Effect of chronic administration of Linalool on the

liver microsomal protein content of. developing rats.

Microsomal protein (mg/g liver) in rats.Days Controls Pretreated Tp 1 values

0 22.2+1.3 22.2+1.3 ns

3 24.5+1.5 26.5 + 0.3 ns

7 23.2+1.5 21.4 + 0.5 ns

14 19.8 + 0.3 20.8 + 2.3 ns

30 27.6 + 2.3 32.8 + 1.1 < 0.02

64.

26.9 + 0.6 32.1 + 0.6 < 0.02

Linalool was administered intragastrically to 28 days old Wistar male rats

as a 25% solution (w/v) of propane 1,2-diol at a dose level of 500 mg/kg

body weight.

Controls and tests each consisted of four animals.

Values given are the means _+ SEM.

’ns’denotes the ’p’ values are statistically non-significant.