184

Long chain alkane derivatives from Cinnamomum

obtusifolium, Elaeocarpus lanceaefolius and Bacaurea

sapida

185

4.1. Introduction

The North Eastern region of India, because of wet ecological conditions, good

solar radiation, temperature ranging from tropical to temperate and varied altitudinal

conditions is endowed with vast flora & fauna. It is estimated that about 50 % of the

total flora of India is found in this part of the country. Bestowed with a large number

of medicinal endemic plant species,1-16

this region is marked as one of the thirty four

mega-center of biodiversity. This region is inhabited by a large number of ethnic tribal

groups separated from each other as well as from the rest of the country for

generations due to geographical reasons. These aboriginals depend mostly on the local

herbs for their primary health care and thereby possess a vast knowledge base on

medicinal usage of relatively less known local flora. CSIR-North East Institute of

Science and Technology (Formerly Regional Research Laboratory), Jorhat, since its

inception has been putting emphasis on research on effective utilization of this unique

resource and has been working on chemical investigation of the medicinal plants in

search of bioactive molecules for developing drug or pest control agents. During the

last four decade a large number of such plants have been chemically examined at

NEIST, Jorhat (Formerly RRL, Jorhat) and isolated more than 100 new molecules of

different structural types. Most of these compounds have been evaluated against

different insects in order to find out their toxicity against insect pests and few of them

have also been evaluated against diseases like cancer, AIDS, etc.

186

This chapter involves the results of the investigation on a three of ethno-

botanically important medicinal plant species (Cinnamomum obtusifolium,

Elaeocarpus lanceifolius and Baccaurea Sapida) for their potential bioactive

phytochemical constituents active against pathogenic fungi Alternaria tenuissima and

Alternaria alternata. Phytochemical studies on these three species afforded isolation

very long chain alkane derivatives and results are presented here.

Fig. 4.1. Satellite map of North-East India (from Google)

187

4.2. Review of Literature on Cinnamomum obtusifolium, Elaeocarpus lanceifolius

and Baccaurea Sapida

4.2.1. Cinnamomum obtusifolium (Family Lauraceae)

Cinnamomum is a genus of evergreen aromatic trees and shrubs belonging to

the Laurel family, Lauraceae. The species of Cinnamomum have aromatic oils in their

leaves and bark. The genus contains over 300 species, distributed in tropical and

subtropical regions of North America, Central America, South America, Asia, Oceania

and Australasia. The genus Cinnamomum includes a great number of economically

important trees.1,2

Cinnamomum is acknowledged worldwide as an important genus because of its

beneficial uses of the essential oils produced by the barks. Essential oils, naturally

occurring substances, generally have a broad spectrum of bioactivity because of the

presence of several active ingredients that work through several modes of action.3

Out of 300 species Cinnamomum, C. cassia is the most famous one, which is

used for treating dyspepsia, gastritis, blood circulation disturbances, and inflammatory

diseases. The main constituents of C. Cassia bark oil are cinnamaldehyde and used as

antibacterial agent.4

Cinnamomum obtusifolium is a large tree. Its barks are grey or brownish white,

rough, up to 0.75 in. thick; blaze aromatic, yellowish or pale brown, turning darker

brown on exposure. Leaves 6-12 by 1.5-3.5 in., elliptic-oblong or elliptic, obtuse,

acute or acuminate, glabrous, sometimes glaucous beneath, very coriaceous; base 3-

nerved; nervules rather prominently reticulate beneath; petiole stout, 0.5-0.7 in. long.

Panicles large, long peduncled, sub terminal, usually exceeding the leaves, minutely

188

pubescent or puberulous, glabrate with age; branches more persistently pubescent;

pedicles short, up to 0.5 in. long usually hoary with silky pubescence parinath about

0.25 in across; lobes silky on both surfaces; of the inner 3 usually villous, all persistent

in fruit, elliptic or ovate. Stamens and ovary sharply pubescent. Fruit 0.3-0.5 in. long,

ellipsoid or sub globose, seated on the slightly enlarged perianth.17

Cinnamomum obtusifolium is such a species which is very less known in

literature but is used as a traditional medicine for treatment of digestive disorders,

rheumatism, and inflammation. Owing to the unique medicinal importance more

extensive studies need to be performed on this species. Recently Thantsin Khin and

coworkers have recently investigated the composition of semivolatile compounds of

Cinnamomum Obtusifolium along with nine other Cinnamomum species. Six

compounds namely borneol, cinnamaldehyde, -cubebene, eugenol, caryophyllene

oxide, and 4.4.8-trimethyltricyclo [6.3.1.0(1,5)] dodecane- 2,9-diol were identified by

GC-FID and GC-MS.5

Satoshi Morimoto and his coworkers6 described the isolation of three methyl

derivative (1, 2 and 3) (as shown in the following figure) of flavon-3-ols from the bark

of Cinnamomum obtusifolium.

189

In addition, from the bark of Cinnamomum obstusifolium, an acylated flavon-

3-ol glucoside 4 were isolated, together with the known proanthocyanidins 5, 6, 7 and

8.7

Chang and co-workers8 studied nine geographical provenances of indigenous

cinnamon (Cinnamomum osmophloeum Kaneh.) leaves and the essential oils isolated

were examined by GC–MS and their chemical constituents were compared. According

to GC–MS and cluster analyses the leaf essential oils of the nine provenances and their

relative contents were classified into six chemo types, cinnamaldehyde type,

cinnamaldehyde/cinnamyl acetate type, cinnamyl acetate type, linalool type, camphor

type and mixed type. In addition, the antifungal activities of leaf essential oils and

their constituents from six chemotypes of indigenous cinnamon were investigated in

this study. Results from the antifungal tests demonstrated that the leaf essential oils of

cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type had an excellent

O

OR1

R2O

OR4

OR3

OH

R1 R2 R3 R4

1 H H Me H

2 H H Me Me

3 Me Me Me H

O

O

HO

OH

OH

HO

OH

OH

OH

OH

OH

OH

6

O

O

HO

OH

OH

HO

OH

OH

OH

OH

OH

5

OH

8

O

O

HO

OH

OH

OH

OH

OOH

OH

HO

HO

O

OH

HO

OH

HO

OH

OH

OH

OHO

OH

OH

7

O

O

HO

OH

OH

OH

O

OH

OHO

OO

OH

OMe

O

HO

4

190

inhibitory effect against white-rot fungi, Trametes versicolor and Lenzites betulina and

brown-rot fungus Laetiporus sulphureus.

Leaves of five species of Cinnamomum, namely C. burmanni, C. cassia, C.

pauciflorum, C. tamala and C. zeylanica, were investigated by Prasad et al.9 for their

antioxidant activities. Among them C. zeylanica exhibited the highest total phenolic

content while C. burmanni had the highest flavonoid content among the five species.

They were then screened for their antioxidant potentials using various in-vitro models

such as total antioxidant capability, DPPH radical scavenging activity, reducing power

and superoxide anion scavenging activity at various concentrations. C.

zeylanica showed the highest DPPH radical scavenging activity, total antioxidant

activity and reducing power, while C. tamala exhibited the highest superoxide anion

scavenging activity. By the analysis of the high performance liquid chromatography

coupled to diode array detector (HPLC-DAD), three flavonoid compounds namely

quercetin, kaempferol and quercetrin were identified and quantified. The study

revealed that Cinnamomum leaf can be used potentially as a readily accessible source

of natural antioxidants.

Liao and co-workers10

isolated five novel glycosides Cinnacassides A–E (9–

13), with a unique geranylphenylacetate carbon skeleton from the stem bark

of Cinnamomum cassia. Each of the cinnacassides A–D (9–12) possesses one of the

four stereoisomers in the aglycone. Their structures were established by extensive

spectroscopic analysis and chemical and chiroptical methods.

191

Jayaprakasha et al.11

first reported the chemical composition of Cinnamomum

zeylanicum flowers. It was found to consists of 23% hydrocarbons and 74%

oxygenated compounds. (E)-Cinnamyl acetate, trans-γ-bergamotene and

caryophyllene oxide are found to be major compounds.

Park and co-workers12

studied the insecticidal and fumigant activities of

Cinnamomum cassia (Blume) bark-derived materials against the oak nut weevil

(Mechoris ursulus Roelofs) and compared to those of the commercially available

Cinnamomum bark-derived compounds (eugenol, salicylaldehyde, trans-cinnamic

acid, and cinnamyl alcohol). The biologically active constituent of

the Cinnamomum bark was characterized as trans-cinnamaldehyde by spectroscopic

analysis.

The antibacterial activity, minimum inhibitory concentration (MIC), and

minimum bactericidal concentration (MBC) of cinnamon stick extract against five

common foodborne pathogenic bacteria (Bacillus cereus, Listeria monocytogenes,

Staphylococcus aureus, Escherichia coli, and Salmonella anatum) were evaluated by

Shan and co-workers.13

Cinnamon stick extract exhibited significant antibacterial

192

properties. Major compounds in cinnamon stick were tentatively identified by GC-MS

and LC-MS as a predominant volatile oil component ((E)-cinnamaldehyde) and

several polyphenols (mainly proanthocyanidins and (epi)catechins). Both (E)-

cinnamaldehyde and proanthocyanidins significantly contributed to the antibacterial

properties.

Jayaprakasha and co-workers14

analyzed the steam-distilled volatile oil from

cinnamon fruit stalks (Cinnamomum zeylanicum Blume) with GC and GC-MS and

showed the presence of hydrocarbons (44.7%) and oxygenated compounds (52.6%).

Twenty-seven compounds constituting ca. 95.98% of the volatile oil were

characterized. (E)-Cinnamyl acetate (36.59%) and (E)-caryophyllene (22.36%) are

found to be major compounds. The volatile oil was screened for its potential as an

antioxidant by using in vitro models, such as the β-carotene-linoleate and

phosphomolybdenum complex method. The volatile oil showed 55.94% and 66.9%

antioxidant activity at 100 and 200 ppm concentration, respectively.

Chen et al.15

isolated five new compounds, kotolactone A (14), kotolactone B

(15), secokotomolide (16), kotodiol (17), and 2-acetyl-5-dodecylfuran (18), and 36

known compounds have been isolated from the stem wood of Cinnamomum kotoense.

The structures of these new compounds were determined by spectroscopic analysis.

The known butanolides, isoobtusilactone A and lincomolide B, showed in vitro

antitubercular activities with MIC values of 22.48 and 10.16 μM, respectively,

against Mycobacterium tuberculosis 90-221387.

193

Phytochemical analysis of the stems of Cinnamomum subavenium by Chen and

co-woekers16

resulted in the isolation of four novel compounds along with 17 known

compounds. The structures of 19−22 were determined by spectroscopic analysis.

Propidium iodide staining and flow cytometry were used to evaluate DNA damage of

the treated SW480 cells, and it was found that 19−22 caused DNA damage in a dose-

dependent manner after 24 h of treatment. These were analysed for their cytotoxicity

against human colorectal cancer cell line SW480 and it was found that 19−22 caused

DNA damage in a dose-dependent manner after 24 h of treatment.

O

O

O O

H

(CH2)11CH3H

H3C(H2C)11H

H3C

O

O

O O

H

(CH2)11CH3H

H3C(H2C)11H

H3C

H3CH

(CH2)8CH3

OHH

OH3CO O

H3COH

OH

24

O

CH3

O

H3C(H2C)11

14 15

16 17 18

O

H

O

HOH

H3CO O

H

O

HOH

H3CO

O

OCH3

O

H

O

HOH

OH3CO

1920

2122

194

Chen and co-workers18

investigated the anticancer effect of isoobtusilactone A

(IOA), 23, a constituent isolated from the leaves of Cinnamomum kotoense, on human

non-small cell lung cancer (NSCLC) A549 cells and found to induce the arrest of G2-

M phase, induce apoptosis, increase sub-G1, and inhibit the growth of these cells.

Further investigation revealed that IOA‘s blockade of the cell cycle was associated

with increased levels of p21/WAF1, p27kip1

, and p53.

23

Three new butanolides, tenuifolide A (24), isotenuifolide A (25), and

tenuifolide B (26), a new secobutanolide, secotenuifolide A (27), and one new

sesquiterpenoid, tenuifolin (28), along with 16 known compounds were isolated by

Lin et al.19

from the stems of Cinnamomum tenuifolium. The structures of these new

compounds were determined by spectroscopic analyses. Compound 27 was found to

induce apoptotic-related DNA damage, increase sub-G1 cells, and inhibit the growth

of human prostate cancer cells, DU145. In addition, treatment with 27 significantly

increased intracellular H2O2 and/or peroxide.

O O

HO H

C25H51

O O

HO

H C25H51

O O

H3CO C25H51

24 25 26

195

O

OCH3O

H

C23H47

HO

OO

OHH3CO

27 28

Chen and co-workers20

isolated three new butanolides, kotomolide A (29),

isokotomolide A (30), and kotomolide (31), and a new secobutanolide, secokomolide

A (32), along with 21 known compounds from the leaves of Cinnamomum ketoense.

Their strcutures were determined by spectroscopic analyses. Compound 32 was found

to induce significant cell death in the human HeLa cell line. Apoptotic-related DNA

damage can be positively related to the dose of compound 32.

OO

HOH

H

OO

HOH

H

OO

OCH3

H

H3CO

O

O

H OH

2930

31

32

196

Wang and co-workers21

isolated Subamolide E, 33 was isolated from

Cinnamomum subavenium and investigate the anticancer cytotoxic effects of natural

compound subamolide E on the human skin cancer melanoma A375.S2 cells. It was

found that Subamolide E demonstrated cytotoxicities in the cell-growth assay at

concentration ranges from 0 to 100 μM at 24 h.

OO

HOH

H

33

4.2.2. Elaeocarpus lanceifolius

Elaeocarpus lanceifolius (Family Elaeocarpaceae) is a medium sized or large

tree, up to 100 ft. high and 12 ft. girth with grayish brown fibrous bark, occurring in

eastern Himalayas and hills of Assam up to 8000 ft.22,23

Leaves lanceolate or

oblanceolate,coriaceous,turning red when old. Flowers small and white in axillary

racemes. Drupe ovoid greenish containing a longitudinally grooved, one seeded stone.

The wood (wt.36 Ib./cu.ft) is greyish white to brown, sometimes with darker

streaks, lustrous, moderately hard, close- and straight-grained. It is not difficult to

season, though it is somewhat liable to develop and splits. The wood is not durable in

exposed positions. It can be sawn and worked easily and can be finished to a lustrous

shiny surface. It is used for house building, boarding, tea boxes and for making

charcoal. It is suitable for match boxes and splints, and for general carpentry.

The fruits are edible. The stones are cleaned, polished, sometimes stained and

197

used as beads for rosaries, bracelets and other ornamental objects; they are frequently

set in gold.23

Elaeocarpus lanceifolius is also used as a traditional medicine, the unripe

fruits are crushed and used for treatment of blood dysentery.24

4/-Methylmyricetin (36) a flavonoid, has been isolated from the leaves of

Elaeocarpus lanceofolius together with the other flavonoid myricetin (34) and its 3-O-

rhamnoside (35).25

Like other flavonoids which are widely distributed in plant

kingdom, the flavonoids isolated from Elaeocarpus lanceofolius have been recognized

to show various biological activities including NO inhibitory activity.26

Volatile extract from the leaves of Elaeocarpus lanceifolius Roxb has the

antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Salmonella

typhi and Escherichia coli.26

Katavic and co-workers27

carried out the first phytochemical investigation of

the Papua New Guinean plant Elaeocarpush abbemensis which resulted in the

isolation of two new pyrrolidine alkaloids, habbemines A (38) and B (39), as a 1:1

mixture of inseparable diastereomers. The structures of these compounds and their

relative configurations were determined by spectroscopic means. An equimolar

ORO

OR O

OR2

OR

OR1

OR

(1) R = R1= R2 = H

(2) R =R1 =H, R2 = rhomnose

(3) R = R2 = H, R1 = Me(4) R = R2 =Et, R1 = Me

34, R = R1 = R2 = H

35, R = R1 = H, R2 = rhomnose

36, R = R2 = H, R1= Me

37, R = R2 = Et, R1 = Me

198

mixture of habbemines A and B showed human δ-opioid receptor binding affinity with

an IC50 of 32.1 μM.

38 39

Phytochemical analysis of the leaves of Elaeocarpus grandis by Carroll et al.28

resulted in two novel indolizidine alkaloids, grandisine A (40) and B (41), and the

known alkaloid (−) isoelaeocarpiline and their structures determined by 1D and 2D

NMR spectroscopy. The compounds showed affinity for the human δ-opioid receptor.

Grandisine A contains a unique tetracyclic skeleton, while grandisine B possesses the

unique combination of isoquinuclidinone and indolizidine groups in one molecule.

40 41

The first chemical investigation29

of the leaves of Papua New Guinean

plant Elaeocarpus fuscoides, resulted in one new indolizidine alkaloid, elaeocarpenine

(42), and three known alkaloids, isoelaeocarpicine, isoelaeocarpine, and elaeocarpine.

Their structures were determined by 1D and 2D NMR spectroscopy.

199

Compounds 34−37 demonstrated binding affinity for the human δ-opioid receptor with

IC50 values of 2.7, 35.1, 13.6, and 86.4 μM, respectively.

42

Katavic and co-workers30,31

isolated five new indolizidine alkaloids,

grandisines C, D, E, F, and G (43−47), and one known indolizidine alkaloid, (−)-

isoelaeocarpiline, from the leaves of Elaeocarpus grandis and their structures

determined by 1D and 2D NMR spectroscopy. Grandisines C, D, F, and G and (−)-

isoelaeocarpiline showed receptor binding affinity for the human δ-opioid receptor

with IC50 values of 14.6, 1.65, 1.55, 75.4, and 9.9 μM, respectively.

N

O

O

H

H

H

X

O

N

O

R

H

H

H

N

N

OCH3

O

H

H

43 44, R = OH, X = CH245, R = NH2, X = CH246, R = CH3, X = O

47

Bioassay-guided investigation of the bark of Elaeocarpus mastersii using KB

(human oral epidermoid carcinoma) cells as a monitor led to the isolation32

of two

cucurbitacins, cucurbitacin D and cucurbitacin F as cytotoxic principles, together with

two ellagic acid derivatives, 4′-O-methylellagic acid 3-(2″,3″-di-O-acetyl)-α-l-

rhamnoside (48) and 4,4′-O-dimethylellagic acid 3-(2″,3″-di-O-acetyl)-α-l-rhamnoside

200

(49). These compounds were evaluated against a panel of human tumor cell lines and

found display potent activity.

O

O

MeO

OH

O

ORO

OOH

O OOO

48, R = H49, R = Me

Elkhateeb and co-workers33

carried out bioassay-guided investigation of the

bark of Elaeocarpus parvifolius which led to the isolation of three new ellagic acid

derivatives, 4-O-methylellagic acid 3′-α-rhamnoside (50), 4-O-methylellagic acid 3′-

(3″-O-acetyl)-α-rhamnoside (51), and 4-O-methylellagic acid 3′-(4″-O-acetyl)-α-

rhamnoside (52) in addition to the known ellagic acid derivative, 4-O-methylellagic

acid 3′-(2″,3″-di-O-acetyl)-α-rhamnoside (53). Their structures were elucidated on the

basis of analysis of 1H NMR,

13C NMR, HMQC, HMBC and MS spectroscopic data.

All new compounds were evaluated for their growth-inhibitory effect on Babesia

gibsoni in-vitro. The compounds 50 and 52 showed very weak activity, while

compounds 51 and 53 showed moderate activity, with IC50 values of 28.5 and

52.1 μg/ml, respectively.

201

O

O

OCH3

OH

O

O

O

OHOR1O

R2O

R3O

50: R1 = H, R2 = OAc, R3 = OAc51: R1 = H, R2 = H, R3 = H52: R1 = H, R2 = OAc, R3 = H53: R1 = OAc, R2 = H, R3 = H

Piao et al.34

studied the antioxidant potential of 1,2,3,4,6-penta-O-galloyl-β-d-

glucose (PGG), isolated from Elaeocarpus sylvestris var.ellipticus, by using various

established systems based on cell-free and cell system experiments, such as radical

detection, antioxidant enzyme assay, lipid peroxidation detection, and cell viability

assay. PGG was found to quench the DPPH radical and intracellular reactive oxygen

species.

Miller and co-workers35

isolated a cyanogenic glycoside–6′-O-

galloylsambunigrin 54 from the foliage of the Australian tropical rainforest tree

species Elaeocarpus sericopetalus F. Muell. (Elaeocarpaceae). This is the first formal

characterisation of a cyanogenic constituent in the Elaeocarpaceae family, and only the

second in the order Malvales. 6′-O-galloylsambunigrin was identified as the principal

glycoside, accounting for 91% of total cyanogen in a leaf methanol extract.

202

O O

CN OH

OHHO

O

O

OH

OHHO54

4.2.3. Baccaurea Sapida

Baccaurea Sapida (Assamese Leteku fruit, Bengali-Latkan), is a semi wild

plant of Euphorbiaceae family, distributed over the sub-tropical and tropical regions of

Southeast Asia.36-39

These species have been taxonomically described by Burkill40

and

Heywood37

Medicinal properties of the species have also been discussed.41

Fruit of B. sapida have been found to contain sufficient sugars to make high

quality table wine.43,44

The fruits are palatable, bright in colour and are eaten fresh

during the summer season when they ripen. Commercial use of these fruits has not

been made, and the trees remain uncultivated and neglected. Kermasha et al.44

analyzed the B. sapida fruit for sugars, amino acids and minerals and found that the

fruit would be useful as supplements to a balanced diet.

Haq et al.45

performed alkaline extraction of pretreated fruits Peels of B.

Sapida which afforded a polysaccharide mixture which on hydrolysis gave (mainly)

D-Xylose and trace of D-Galactose, L-arabinose and an acidic substance.

Fractionation of polysaccharides gave D-Xylan. Methylation of xylan followed by

hydrolysis and chromatography examination gave the evidence for the presence of

2,3,4-tri-O-Me xylose, 2,3-di-O-Me xylose and 2-O-Me xylose.

203

Bordoloi et al.46

described the isolation of a novel anti-fungal

tetrahydrofurano-lactone meroisoprenoid (sapidolide A) from the medium polar

fraction of the crude extract of seed kernels of Baccaurea sapida. Sapidolide A has

exhibited strong inhibitory activity against pathogenic fungi such as

Helminthosporium oryzae, Phytophthera oryzae, Alternaria solani, Curvularia

eragrostidis, Collectotrichum gleosporioides.47

O

H

RO

OH

O

O

R= H, Ac

Sapidolide A

4.3. Results and Discussion

In continuation of our interest on the search for bioactive molecules from the

plants of the Sub-Himalayan region of North East India,48-54

we undertook the

chemical investigation of three plants (i) Leaves of Cinnamomum obtusfolium (family

Lauraceae) from Chessa, Arunachal Pradesh, (ii) Leaves of Elaeocarpus lanceofolius

(Family Elaeocarpaceae) were collected from Itanagar, Arunachal Pradesh and (iii)

fruit of Baccaurea sapida (family Euphorbiaeeae) growing widely in the

Brahmaputra valley, India.

204

4.3.1. Triacontanoic acid 1 from Cinnamomum obtusfolium

Silica gel column chromatography of the gummy mass obtained from a

petroleum ether (60-80 oC) extract of the dried powdered leaves of Cinnamomum

obtusfolium led to the isolation of a white powdered compound christened as

compound CO-1 for convenience of discussion.

The compound CO was analyzed as C30H60O2 by elemental analysis and EIMS

with [M]+ at 452. In the

1H NMR spectrum recorded at 300 MHz in CDCl3, a three-

proton triplet signal at δ 0.89 was assigned to a chain end methyl group. A broad

signal at δ 1.25 integrating for 52 protons indicated that the molecule contained 26

methylene groups in a nearly identical environment. One triplet at δ 2.40 with J = 6 Hz

and one multiplet at δ 1.55, each integrating for two protons were assigned to two CH2

group attached to a carbonylic acid group. In the IR spectrum, there was a strong band

at 1705.8 cm-1

indicating the presence of a carboxylic acid group in the molecule). Its

IR spectrum exhibited absorption bands at 3400 cm-1

for hydroxyl and 720 and 710

cm-1

for a long aliphatic chain. Based on these evidences, the structure of the molecule

was assigned as Triacontanoic acid, C29H59COOH 1. The structure of the molecule

was further confirmed by its 13

C NMR spectrum recorded at 75 MHz. The signal at δ

211.86 was assigned to the carboxylic acid carbon atom of the molecule. The signals

at δ 22.71, 23.91, 29.29, 29.38, 29.44, 29.50, 29.63, 29.68, 29.71, 31.94 and 42.84

indicated the presence of the methylenes in the molecule. The signal at δ 76.62 was

assigned to the carbon adjacent to the carboxylic acid function of the molecule. The

structure 1 of the molecule was further confirmed by the appearance of a number of

fragments with a systematic difference of 14 & 28 amus and the absence of a peak

205

corresponding to [M - 15]+ confirmed the straight chain nature of the compound

55 On

the basis of this evidence, this compound could be inferred to be triacontanoic acid 1.

Triacontanoic acid 1

4.3.2. Octatriacontan-1-ol 2 and dotriacontane 3 from Elaeocarpus lanseofolius

Silica gel column chromatography of the gummy mass obtained from a

petroleum ether (60-80 oC) extract of the dried powdered leaves of Elaeocarpus

lanseofolius led to the isolation of two white powdered compounds designated as

compound EL-1 and EL-2 for convenience of discussion.

The compound EL-1 was analyzed as C38H78O by elemental analysis and

EIMS with [M]+ at 550. In the 1H NMR spectrum recorded at 300 MHz in CDCl3, a

three-proton triplet signal at δ 0.89 was assigned to a chain end methyl group. A broad

signal at δ 1.25 integrating for 70 protons indicated that the molecule contained 35

methylene groups in a nearly identical environment. One triplet at δ 3.63 with J = 7 Hz

integrating for two protons was assigned to one CH2 group attached to a hydroxyl

group. One multiplet at δ 1.60, integrating for two protons was assigned to CH2 group

adjacent to a CH2OH group. In the IR spectrum, there was a strong band at 1705.8

cm-1

indicating the presence of a carboxylic acid group in the molecule. Its IR

spectrum exhibited absorption bands at 3419 cm-1

for hydroxyl and 730 and 719 cm-1

for a long aliphatic chain. Based on these evidences, the structure of the molecule was

206

assigned as Octatriacontan-1-ol, C38H78O 2. The structure of the molecule was further

confirmed by its 13

C NMR spectrum recorded at 75 MHz. The signal at δ 76.60 was

assigned to the carbon attached with primary alcoholic group. The signals at δ 14.71,

22.71, 29.71, 31.34 indicated the presence of the methylenes in the molecule. The

structure 2 of the molecule was further confirmed by the appearance of a number of

fragments with a systematic difference of 14 and 28 amus and the absence of a peak

corresponding to [M-15]+ confirmed the straight chain nature of the compound

55. On

the basis of these evidences, the structure of the compound could be inferred as a very

long chain saturated alcohol octatriacontan-1-ol 2.

Octatriacontan-1-ol 2

The compound EL-2 was analyzed as C32H66 by elemental analysis and EIMS

with [M]+ at 450. In the

1H NMR spectrum recorded at 300 MHz in CDCl3, a six-

proton triplet signal at δ 0.89 with J = 7 Hz was assigned to two chain end methyl

groups. A broad signal at δ1.25 integrating for 60 protons indicated that the molecule

contained 30 methylene groups in a nearly identical environment. There was no other

signals in the 1H NMR spectrum which indicated that the molecule did not contain

any functional group. In the IR spectrum, there was no strong band at near 1705.8

cm-1

indicating the absence of a carbonyl function in the molecule. The IR spectrum

exhibited absorption bands at 729 and 719 cm-1

for a long aliphatic chain. In the 13

C

NMR spectrum recorded at 75 MHz, the signals were observed only at δ 14.14, 22.71,

207

29.39, 29.72 and 31.35. This further indicated that the molecule does not contain any

functional group and it contains only methylenes and methyls. Based on these

evidences, the structure of the molecule was assigned as dotriacontane, C32H66 3. The

structure 1 of the molecule was further confirmed by the appearance of a number of

fragments with a systematic difference of 14 amu and the absence of a peak

corresponding to [M - 15]+ confirmed the straight chain nature of the compound

55.

On

the basis of this evidence, this compound could be inferred to be a long chain

hydrocarbon compound dotriacontane 3.

Dotriacontane 3

4.3.3. Oleic acid 4 and Palmitic acids 5 from Baccaurea sapida

Silica gel column chromatography of the gummy mass obtained from a

petroleum ether (60-80 oC) extract of the dried powdered seed kernals of Baccaurea

sapida led to the isolation of two major white fatty compounds and were designated

as compound A and B for convenience of discussion.

The compound A was analyzed as C18H34O2 by elemental analysis and EIMS

with [M]+ at 282. In the

1H NMR spectrum recorded at 300 MHz in CDCl3, a three-

proton triplet signal at δ 0.89 was assigned to a chain end methyl group. A broad

singlet at δ 1.25, integrating for 22 protons indicated that the molecule contained 11

methylene groups in a nearly identical environment. Multiplets at δ 2.04, 2.34 each

integrating for two protons are assigned as allylic CH2s. Multiplet at δ 5.36

208

integrating for two protons was assigned to two olefinic protons. In the IR spectrum,

there was a strong band at 1711 cm-1

indicating the presence of a carboxylic acid

group in the molecule. All these data indicate that the compound D is oleic acid 4. The

structure of the molecule was further confirmed by its 13

C NMR spectrum recorded at

75 MHz. The signal at δ 179.52 was assigned to the carboxylic acid carbon atom of

the molecule. The signals at δ 129.55 and 129.81 were assigned to the olefinic carbons

of the molecule. The signals at δ 13.99, 22.63, 24.94, 27.08, 27.13, 29.02, 29.12,

29.27, 29.29, 29.34, 29.43, 29.49, 29.63, 29.68, 31.88 and 34.23 indicated the presence

of the methylenes in the molecule. The structure 4 of the molecule was further

confirmed by the appearance of a number of fragments with a systematic difference of

14 amu. On the basis of this evidence, this compound could be inferred to be an oleic

acid 4.

Oleic Acid 4

The compound B was analyzed as C16H32O2 by elemental analysis and EIMS

with [M]+ at 256. In the 1H NMR spectrum recorded at 300 MHz in CDCl3, a three-

proton triplet signal at δ 0.89 was assigned to a chain end methyl group. A broad

signal at δ 1.25 integrating for 28 protons indicated that the molecule contained 14

methylene groups in a nearly identical environment. Two multiplet at δ 2.40 and 1.60,

each integrating for two protons were assigned to two CH2 groups attached to a

209

carbonylic acid group. In the IR spectrum, there was a strong band at 1694 cm-1

indicating the presence of a carboxylic acid group in the molecule. Its IR spectrum

exhibited absorption bands at 3400 cm-1

for hydroxyl and 727 and 720 cm-1

for a long

aliphatic chain. Based on these evidences, the structure of the molecule was confirmed

as palmitic acid 5. The structure of the molecule was further confirmed by its 13

C

NMR spectrum recorded at 75 MHz.

Palmitic acid 5

Palmitic acid is the most common fatty acid found in animals, plants and

microorganisms56

. Palmitic acid mainly occurs as its ester in triglycerides (fats),

especially palm oil, butter, cheese, milk and meat also contain this fatty acid. Palmitic

acid has been shown to alter the β cells in the pancreas that are responsible for the

secretion of insulin, and to suppress the body's natural appetite-suppressing signals.

Recently, a long-acting antipsychotic medication, paliperidone palmitate (marketed as

INVEGA Sustenna), used in the treatment of schizophrenia, has been synthesized

using the oily palmitate ester as a long-acting release carrier medium when injected

intramuscularly.

Oleic acid is a fatty acid that occurs naturally in various animal and vegetable

fats and oils. Oleic acid is the most abundant fatty acid in human adipose tissue57

.

Oleic acid may hinder the progression of adrenoleukodystrophy (ALD), a fatal disease

that affects the brain and adrenal glands. Oleic acid may be responsible for the

210

hypotensive (blood pressure reducing) effects of olive oil58

. Oleic acid also keeps cell

membranes soft and fluid, allowing helpful anti-inflammatory substances like omega-3

fatty acid to penetrate the cell membrane more easily and preventing the negative

effects of bad cholesterol59

. Adverse effects also have been documented, since both

oleic and monounsaturated fatty acid levels in the membranes of red blood cells have

been associated with increased risk of breast cancer60

.

4.3.4. Biological Activity of compounds Octatriacontan-1-ol (EL-1), dotriacontane

(EL-2), Triacontanoic acid (CO):

The test compound EL-1 and EL-2 showed 96.43 and 95.30 percent inhibition

respectively at 200 ppm, while standard fungicide (Captan) exhibited 100 percent

inhibition at 200 ppm. However, both of the compounds showed 100 percent

inhibition at 250 ppm of concentration. All the test compounds exhibited > 80 percent

inhibition at 200 ppm. Which indicates that the compounds posses good antifungal

activity. However the activity showed by EL-1 and EL-2 is comparable to the

Standard Zineb although the MIC of these two compounds is 250 ppm. The untreated

control sets of the experiment showed no inhibition of growth of the fungal pathogen.

Fatty acids (FAs) are an ever-present constituent of each genus and species of

living matter. Usually, they are bound in lipids in cells of most organisms with the

exception of oldest bacteria (Archaebacteriae spp.) that contain in cell walls isoprenoid

chains bound to glycerol by the etheric bond. Very long chain fatty acids (FAs) are

important components of different classes of lipids in all organisms from bacteria to

man. They include also, usually as minor components, odd-numbered FAs. These have

so far been given little attention because of technical difficulties inherent in their

211

detection and identification. Current modern analytical methods such as GC–MS

and/or LC–MS make this detection and identification possible, and should promote a

study of their properties61

.

It may be noted that five long chain aliphatic alcohols and 11 long chain

aliphatic saturated carboxylic acids were isolated from the heartwood of Rhizophora

apiculata62

. Teponno et al has isolated tetracosanoic acid from the tubers of Dioscorea

bulbifera L. var sativa63

.

A new long chain alcohol have been isolated from the shoots of Achyranthes

aspera and it was identified as 17-pentatriacontanol64

.

S Bauer et al have detected a number of long chain fatty acids from the skins of

tomatoes (Lycopersicon esculentum) by GC-MS studies of the surface wax65

.

However, to the best of our knowledge, this is the first report of isolation of

compound 1 to 3 from a living natural sources having inhibitory effect against

pathogenic fungi Alternaria tenuissima and Alternaria alternata.

4.4. Experimental

4.4.1. General Experimental Procedures

IR spectra were recorded on a Perkin Elmer System2000 FTIR spectrometer.

1H NMR (300 MHz) and

13C NMR (75 MHz) spectra were recorded on a Bruker

AVANCE DPX 300 NMR spectrometer in CDCl3 using TMS as the internal standard

and mass spectra were recorded on Mass spectrometer (Model: Trace DSQ GCMS

Instrument, Make: Thermo Fisher Scientific, Austria) system. Silica gel G was used

for TLC. All solvents used were distilled prior to use.

212

4.4.2. Plant Material

Isolation of Triacontanoic acid from Cinnamomum obtusfolium

Cinnamomum obtusfolium (Family Lauraceae) leaves were collected from

Chessa, arunachal Pradesh during June, 2007. Approximately 850 g of dried &

powdered leaves of Cinnamomum obtusfolium were extracted with petroleum ether

(60-80 oC) in a soxhlet apperatus and the residue, obtained after distillation of the

extract, was chromatographed over alumina (400g, neutral) column.

(i) Fractions 3 to 8 (100ml each) of pet. ether eluate were combined,

evaporated and crystallized from pet. ether to get 375 mg of white crystals of CO-2

triacontanoic acid 1. TLC, Rf 0.3 in Pet. ether – benzene (9:1), visualized with iodine.

(ii) Fractions (100 ml each) 18 to 36 of benzene eluate furnished 145 mg of -

sitisterol, identified by co-TLC & mixed m.p.

Spectral Data Triacontanoic acid:

1H

NMR (CDCl3): δ 0.89t (J = 7Hz, 3H),1.25brs (52H),1.55m (2H), 2.40t (J =

7Hz, 2H); 13

C NMR (CDCl3): δ 14.14, 22.71, 23.91, 29.29, 29.38, 29.44, 29.50, 29.60,

29.68, 29.71, 31.94, 42.84, 76.605, 211.86; IR: cm-1

(KBr): 3446, 2955, 2917, 2849,

1705, 1472, 1463, 1380, 1079, 729, 719; MS m/z at 452 [M]+, 451, 407, 365, 323,

281, 256, 255, 239, 235, 194, 127, 109, 96, 85, 71; Elemental Analysis, found C

79.61, H 13.39 % C30H60O2 requires C 79.58, H 13.36 %.

Isolation of octatriacontan-1-ol and dotriacontane from Elaeocarpus lanceofolius

Leaves of E. lanceofolius (Family Elaeocarpaceae) were collected from

Itanagar, Arunachal Pradesh during May, 2007. Approximately 450 g of dried &

powdered leaves of Elaeocarpus lanceofolius were extracted with pet. ether (60-80 oC)

213

in a soxhlet apperatus and the residue, obtained after distillation of the extract, was

chromatographed over alumina (150g, neutral) column. The column was successively

eluted with pet. ether (60-80 oC) and mixture of pet. ether – benzene and the eluates

were collected in 100 ml flasks.

i) Fraction-10-15 (100 ml each) of pet. ether eluate was evaporated and

crystallized from Pet.ether to get 120 mg of white crystals of octatriacontan-1-ol, m.p.

70 oC, TLC (Alumina plate), Rf 0.8 in Pet. ether, visualized with iodine.

ii) Fractions 19-21 (100 ml each) of Pet. ether – benzene (1:1) furnished 75 mg

of white crystal (ether-chloroform mixture) of dotriacontane, m.p. 89-90 oC. TLC

(Silica gel plate), Rf 0.5 in pure benzene; visualized in Sulphuric acid + heat.

iii) Fractions 24-28 (100 ml each) of pure benzene eluate, on evaporation and

crystallization from alcohol furnished 150 mg of beta-sitosterol identified by co-TLC

and mixed m.p.

Spectral Data of octatriacontan-1-ol:

1H NMR (CDCl3) : δ 0.89 t (J = 7Hz, 3H), 1.25 brs (70H), 1.60 m (2H), 3.63 t

(J = 7Hz, 2H); 13

C NMR (CDCl3) : δ 14.71, 22.71, 29.71, 31.34 and 76.60, IR: cm-1

(KBr) 3419.7, 2917, 2849, 1473, 1462, 1380, 1122, 1060, 730, 719; EIMS m/z at 550

[M]+, 532.5, 508, 479.5, 465.5, 464.5, 437.4, 436.4, 407.4, 393.4, 379.4, 351.3, 337.3,

309.3, 295.3, 267.2, 225.2, 211.1, 197.1, 169.1, 155.1, 141.1, 127.0, 113.0, 99.0, 97.0,

85.0; Elemental Analysis found, C 82.89, H 14.20 C38H78O requires C 82.83, H 14.27

Spectral Data of Dotriacontane:

1H NMR (CDCl3) : δ 0.89t (J = 7Hz,6H),1.25 brs (60H);

13C NMR (CDCl3) : δ

14.14,22.71,29.39,29.72.31.95; IR: cm-1

(KBr): 3446, 2956, 2917, 2849, 1473, 1463,

214

1378, 1071, 889, 719, 418; MS m/z at 450 [M]+, 506, 532, 470, 434, 378, 312, 285,

284, 264, 222, 213, 185, 157, 129, 97, 83, 72, 57; Elemental Analysis found, C 85.15,

H 14.81 %, C32H66 requires C 85.25, H 14.75 %.

Isolation of Oleic acid 4 and palmitic acid 5 from Baccaurea sapida:

Fruit of of B sapida were collected from Jorhat during July, 2007. Skin and

juicy part of fruit were removed and the seeds were dried under shades.

Approximately 300g of dried & powdered seed kernels of Elaeocarpus lanceofolius

were extracted with pet. ether (60-80 oC) in a soxhlet apperatus and the residue,

obtained after distillation of the extract, was chromatographed over 350g of silica gel

column. The column was successively eluted with pet. ether (60-80 oC) and mixture of

pet. ether – benzene and the eluates were collected in 100 ml flasks.

i) Fraction-12-18 (100 ml each) of pet. ether eluate was evaporated to get 100

mg of white crystalline substance palmitic acid 5 characterized as by 1H NMR,

13C

NMR, IR and mass spectral analysis

ii) Fractions 24-29 (100 ml each) of Pet.ether – benzene (1;1) furnished 65 mg

of white oily oleic acid 4 of oleic acid 4 characterized as oleic acid 4 by 1H NMR,

13C

NMR, IR and mass spectral analysis

Spectral Data of Oleic acid 4:

1H NMR (CDCl3): δ .89 (t, J = 7Hz, 3H), 1.30 (br s, 20H), 1.48 (m, 2H), 1.60

(m, 2H), 2.00 (m, 2H), 2.3 (m, 2H), 5.36 (m, 2H); 13

C NMR (CDCl3): δ 13.9, 22.24,

22.51, 22.6, 24.6, 24.8, 25.3, 27.08, 27.13, 29.02, 29.1, 29.27, 29.29, 29.34, 29.43,

29.63, 31.1, 31.47, 31.88, 33.06, 33.99, 34.23, 129.55, 129.81, 178.52; IR: cm-1

(Thin

flim): 3006, 2926, 2855, 1711, 1465, 1413, 1377, 1283, 1244, 1116, 941, 722; MS m/z

215

at 283.2 [M]+, 264.2, 263.1, 222.1, 171.0, 139.0, 125.0, 97.0, 68.9; Elemental Analysis

found, C 76.59, H12.17 % C18H34O2 requires C 76.54, H 12.13%

Spectral Data of palmitic acid 5:

1H NMR (CDCl3): δ 0.89 (T, J = 7Hz, 3H), 1.25 (m, 28H), 10.54 (br , 1H);

13C

NMR (CDCl3): δ 14.08, 22.7, 24.6, 29.07, 29.25, 29.36, 29.45, 29.61, 29.69, 29.70,

31.9, 34.1, 180.6; IR: cm-1

(Thin flim): 2916, 2848, 1694, 1471, 1463, 1430, 1410,

1249, 1296, 1227, 1205, 1187, 1098, 941, 781, 740, 727, 720; MS m/z at 256.1 [M]+,

213.0, 156.9, 128.9, 96.9, 72.0; Elemental Analysis found, C 74.97, H 12.62 %

C16H32O2 requires C 74.94, H 12.58%

4.4.3. Bioassay of the compound Triacontanoic acid, octatriacontan-1-ol and

dotriacontane:

Micro-organism preparation:

Stock culture of Alternaria tenuissima and Alternaria alternate were used

throughout the study. The fungus Alternaria tenuissima was isolated from Solanum

khasianum and was maintained on Potato Dextrose (PDA) having standard formula

consisting of infusion from potatoes 300 g dextrose 20 g and agar 15 g per litre (Hi

Media M096A). 41 g of standard PDA was suspended in 1000 ml distilled water,

boiled to dissolve the medium completely, sterilized by autoclaving at 15lbs pressure

(121 oC) for 15 minutes, mixed well before dispensing. Fungus was grown for 72 hrs.

on PDA at 28 oC. For use in experiment the fungus was grown separately on Potato

dextrose broth (PDB) containing infusion from potato 200 g and dextrose 20 g (Hi

Media, M 403). The broth culture was diluted to a concentration of 1x106

with sterile

distilled water for preparation of spore suspension of the test fungus.

216

Test sample concentration:

Stock solution of each sample was prepared having a concentration of 500

ppm. These varying concentrations of 250, 200, 150, 100, and 50 ppm were prepared

in hexane from the stock solution by standard broth dilution method. From each

concentration of the sample 0.1 ml was added to 10 ml of PDB Media containing

spore, shaked well, kept in incubator at 28 oC for growth and observed daily. Activity

was judged by measuring the dry weight of the test fungus after one week.

Minimum inhibitory concentration:

Antifungal activity was expressed as % Inhibition = Sample weight (treated)

/Control weight (untreated) x 100.66

Minimum Inhibitory Concentration (MIC) was

considered as the highest dilution at which 100% inhibition of growth of the test

fungus Alterneria tenuissima was observed.

Bioassay with Alternaria alternata was also done as above. Minimum

Inhibitory Concentration (MIC) was considered as the highest dilution at which 100%

inhibition of growth of the test fungus Alterneria alternata was observed.

217

Table 4.1. Antifungal activity of compound Triacontanoic acid, octatriacontan-1-ol

and dotriacontane against Alterneria tenuissima

Compounds

Inhibition % at different concentration (g/ml)

250 200 150 100 50

Triacontanoic

acid

90.52 ±

0.025166

80.43 ±

0.015275

72.85 ±

0.020817

65.71 ±

0.015275

50.20 ±

0.007506

Octatriacontan-

1-ol

100.00 ±

0.01

96.43 ±

0.032146

90.28 ±

0.015275

85.14 ±

0.026458

55.36 ±

0.030551

Dotriacontane 100.00 ±

0.012

95.30 ±

0.020817

88.16 ±

0.025166

82.40 ±

0.030551

56.12 ±

0.026458

Captan 100.00 ±

0.01

100.00 ±

0.01

96.10 ±

0.173205

94.20 ±

0.208167

70.12 ±

0.026458

Control 0 0 0 0 0

Table 4.2. Antifungal activity of compound Triacontanoic acid, octatriacontan-1-ol

and dotriacontane against Alternaria alternate

Compounds Inhibition % at different concentration (g/ml)

1700 1500 1200 1000 800 600

Triacontanoic

acid

100 ±

0.01

96.43 ±

0.020817

92.85 ±

0.011547

85.71 ±

0.01

82.15 ±

0.041633

75.00 ±

0.085049

Octatriacontan

-1-ol

100 ±

0.015

92.86 ±

0.011547

89.28 ±

0.061101

82.14 ±

0.496924

78.57 ±

0.119304

67.86 ±

0.174356

Dotriacontane 98.93 ±

0.108167

89.28 ±

0.02

82.14 ±

0.032146

71.43 ±

0.025166

67.86 ±

0.025166

64.28 ±

0.011547

218

4.5. Spectra of compounds

4.5.1. Triacontanoic acid

Fig. 4.1. IR spectrum of Triacontanoic acid

Fig. 4.2. 1H NMR spectrum of Triacontanoic acid

219

Fig. 4.3. 13C NMR spectrum of Triacontanoic acid

Fig. 4.4. Mass spectrum of Triacontanoic acid

220

4.5.2. Octatriacontan-1-ol

Fig. 4.5. IR spectrum of octatriacontan-1-ol

Fig. 4.6. 1H NMR spectrum of octatriacontan-1-ol

221

Fig. 4.7. 13C NMR spectrum of octatriacontan-1-ol

Fig. 4.8. Mass spectrum of octatriacontan-1-ol

222

4.5.3. Dotriacontane

Fig. 4.9. IR spectrum of Dotriacontane

Fig. 4.10. 1H NMR spectrum of Dotriacontane

223

Fig. 4.11. 13C NMR spectrum of Dotriacontane

Fig. 4.12. Mass spectrum of Dotriacontane

224

4.5.4. Oleic acid

Fig. 4.13. IR spectrum of Oleic acid

Fig. 4.14. 1H NMR spectrum of Oleic acid

225

4.5.5. Palmitic acid

Fig. 4.15. 13C NMR spectrum of Oleic acid

Fig. 4.16. IR spectrum of Palmitic acid

226

Fig. 4.17. 1H NMR spectrum of Palmitic acid

Fig. 4.18. 13C NMR spectrum of Palmitic acid

227

Fig. 4.19. Mass spectrum of Palmitic acid

228

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