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Pak. J. Chem., Vol. 3 (1), 2013

In the Honor of International Year of Chemistry

Issue: March, 2013

ISSN (Print): 2220-2625

ISSN (Online): 2222-307X

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ISSN (Print): 2220-2625 ISSN (Online): 2222-307X

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Pak. J. Chem Table of Contents


Author(s) - Title Page #

N. M. Tariq, S. U. Wisam, H. M. Faik and T. H. Mayson - The Antioxidative Activity of Aqueous and

Ethanolic Extracts of Rosemary and Green Tea Leaves: A Comparative Study 1

S. K. Bariyah - An Extensive Survey of the Phytochemistry and Therapeutic Potency of Ocimum sanctum

(Queen of Herbs) 8

A. H. Banday - Piperidine Promoted Regioselective Synthesis of α, β-unsaturated Aldehydes 19

N. Fatima, S. Z. A. Zaidi, S. Nisar and M. Qadri - pH Effect on Stoichiometry and Stability of Ferrous

Complexes of (-)-3-(3,4-dihydroxyphenyl)-L-alanine 23

R. Parveen, M. Ashfaq, J. Qureshi, S. M. M. Ali and M. Qadri - Estimation of Chromium in Effluents

from Tanneries of Korangi Industrial Area 29

H. N. Majeed and A. K. Hasan - Determination of 106Ru,134/137Cs, and 241Am concentrations and action

level in the foodstuffs consumed by inhabitants of Iraq 34

N. M. Salih AL-Janabi, A. M. Mahmed AL-Samraee and W. S. Ulaiwi - The Effects of Squeezed

Grapes Residue on the Preservation of Stored Fried Potato Chips 41

R. Azmat - A Visual Demonstration of Solvent Effect in Chemical Kinetics through Blue Bottle

Experiment 45

Pak. J. Chem. 3(1): 1-7, 2013 Full Paper

ISSN (Print): 2220-2625


*Corresponding Author Received 29th

September 2012, Accepted 14th January 2013

The Antioxidative Activity of Aqueous and Ethanolic Extracts of Rosemary and Green

Tea Leaves: A Comparative Study

*N. M. Tariq, S. U. Wisam, H. M. Faik and T. H. Mayson

Basic Science Section, College of Agriculture, University of Baghdad, Baghdad-Iraq

E-mail: *[email protected]

ABSTRACT The antioxidant activities of rosemary and green tea leaves, aqueous and ethanolic extracts, have been studied by using two

different methods (reducing power and chelating ability). It was found that the total phenolic compounds in aqueous and ethanolic

extracts of rosemary and green tea leaves were 13.44, 18.75, 39.38 and 48.44 mg/ 100 mg dry extract respectively. The flavonoids

(which is a part of the phenolic compounds) were found to be 9.54, 12.65, 17.69 and 22.70 mg/ 100 mg dry extract in aqueous and

ethanolic extract of rosemary and green tea leaves respectively. The ethanolic extract shows high content of phenolic compounds

and in turn highly antioxidative activiy for both rosemary and green tea leaves as compared with aqueous extract.The aqueous and

ethanolic extracts of rosemary and green tea leaves show high reducing power ability comparing with their abilities as chelating

agents. Although, the phenolic compounds of green tea leave almost about 3-fold as compared with rosemary leave in both

aqueous and ethanolic extracts, their extracts show extremely the same mode of action in both methods of determination (the

reducing power and chelating ability). Therefore, we are fully recommended the rosemary leave extracts as a potent food

preservative.

Key words: Rosemary & green tea, leaves extracts, antioxidants

1. INTRODUCTION Oxygen free radicals induce damage due to peroxidation to biomembranes and also to DNA, which lead to tissue

damage. Antioxidants neutralise the effect of free radicals through different ways and may be prevent the body from

various diseases. Synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole

(BHA) have recently been reported to be dangerous for human health. Thus, the search for effective, non-toxic natural

compounds with antioxidative activity has been intensified in recent years 1. About 5% or more of the inhaled oxygen

(O2) is converted to reactive species (ROS) such as O2-, H2O2 and OH by univalent reduction of O2

2. Antioxidants can

act by scavenging reactive oxygen species, by anhibiting their formation (e.g. by blocking activation of phagocytes),

by binding transition metal ions and preventing formation of OH and /or decomposition of lipid hydroperoxides by

repairing damage (e.g. α-tocopherol repairing peroxyl radicals and so terminating the chain reaction of lipid

peroxidation) or by any combination of the above 3.

Rosemary (Rosamarinus officinalis), is a woody, perennial herb with fiagrant, evergreen, needle-like leaves

and white, pink, purple or blue flowers, native to Mediterranean and Asia regions. It is a member of the mint family

(Lamiaceae). The name rosemary drives from the latin (Rosamarinus), which means dew of sea 4. Rosemary extracts

contain several compounds which have been proven to exert antioxidative functions. These compounds belong mainly

to the classes of phenolic acids, flavonoids, diterpenoids and triterpenes 5. The principal antioxidative components of

rosemary extracts are the phenolic diterpenes carnosol, carnosic acid (the most abundant) and rosamaric acid (Fig.

1).carnosol and carnosic acid exert potent anti-inflammatory and anti-carcinogenic properties 6. They impair the

proliferation of several cancer cell lines and induce apoptosis 7-12

.

Carnosic Acid Carnosol Rosamarican Acid

Fig-1: Chemical structure of the three major antioxidative compounds in rosemary extracts

Tea (Camellia sinensis) refers to the aromatic beverage prepared from cured leaves by hot or boiling water 13

. Tea is

the second most popular drink in the world 14

. The green tea is relevant in the terms of preventive effect on metastasis

of lung, breast cancer 15

, prevention of inflammation and thrombosis 16

, preventive effect on atherosclerosis and

decreasing cholesterol concentration in the blood 17

. The antioxidant activities have been established for the green tea

by the ability to bind and neutralize the free radicals 18

. Catechins which is a fraction of flavonoids are the basic

phenolic compounds in green tea (especially the main compound, epigallocatechin-3-gallate, Fig-2) are responsible for

antioxidant activities 19, 20

.

mailto:[email protected]

Pakistan Journal of Chemistry 2013

2

Fig-2: The epigallocatechin-3-gallate compound.

The present work is a comparative study between rosemary and green tea leaves throughout their abilities as

antioxidants by using two different methods, and find out, which of them could be recommended as a natural

preservative in foods.

2. MATERIALS AND METHODS The rosemary and green tea leaves were locally obtained, cleaned and ground. 20 gr of ground material was extracted

by 250 ml distilled water or ethanol 95% at boiling point, under reflux for 1 hr. The extractive wasfiltered and

evaporated at 50oC to the compete dryness.

2.1 Determination of total phenolic compounds A Folin-ciocalteu's colorimetric method was used as described by Ayoola et al. (2008)

21. To a 0.5 ml of (1 mg/ml)

extract a 2.5 ml of a ten-fold diluted Folin-ciocalteu's reagent and 2ml of 7.5%.sodium carbonate solution were added

before the reaction allowed standing for 30 min at room temperature. The absorbance was recorded at 760 nm by

using UV/VIS Spectroscan 80 D spectrophotometer. The total phenolic compounds were determined according to

gallic acid standard curve (0.01 to 1 mg/ml) (Fig. 3)

2.2 Determination of flavonoids The total flavonoids in aqueous and ethanolic extracts were determined according to Rao et al, (2012)

22. 1 ml extract

solution (1mg/ml) was placed in 10 ml volumetric flask. 5 ml of distilled water and 0.3 ml of 5% NaNo2 solution were

added. After 5 min 0.6 ml of 10% AlCl3 was added. 2 ml of 1M NaOH solution was added after another 5 min, and

the volume was made up to 10 ml with distilled water. The mixture was mixed thoroughly and the absorbance was

measured at 510 nm. The total flavonoids were expressed as µg catechin equivalents per gram dry matter according to

catechin standard curve (Fig-4).

Tariq et al, 2013

3

2.3 The assay of antioxidant activity

2.3.1 The reducing power The reducing power was estimated as described by Chou et al.( 2009)

23. 1ml extract of (2-10 mg/ml) was mixed with

2.5ml of 1% potassium ferric cyanide and 2.5ml of 0.2M (pH, 6.6) of sodium phosphate buffer, and incubated at 50co

for 20 min. To stop the reaction, 2.5ml of 1% trichloroaceticacide (TCA) was added to the mixture and centrifuge for

10 min at 3000 rpm. 0.5ml of the supernatant was mixed with 1ml of 1% ferric chloride and stand for 10min.The

absorbance was measured at 700nm. 0.02% of BHT used as reference.

2.3.2 The chelating ability Chelating ability was determined according to Su et al. (2008)

24 with some modification. 1ml of (2-10mg/ml) extract

was mixed with 0.2ml ferric chloride of 2mM and 0.2ml 8-Hydroxyquinoline (5mM). After 10min at room

temperature, the absorbance was determined at 562nm.The EDTA-Na2 was used as reference.

3. RESULTS AND DISCUSSION Polyphenols are widely appreciated for their potential beneficial health effects, like antioxidant activity

25. Table-1,

shows the percentages of total phenolic compounds and flavonoids which are represent the main antioxidant

compounds in aqueous and ethanolic extracts of rosemary and green tea leaves. The total phenolic compounds which

expressed as gallic acid and flavonoids as catechins were determined according to standard curves, phenols were

determined by Folin-Ciocalteu's colorimetric method and flavonoids by aluminum chloride colorimetric method. As

shown, the total phenolic compounds in both, aqueous and ethanolic extracts of green tea leaves are higher than

rosemary leaves which refer that, the antioxidative activity of the tea leaves will be more effective as compared with

rosemary leaves for the both extracts. The high percentages of the total phenolic and flavonoids in alcoholic extract

mean that, the ethanol as extracting solvent and according to the chemical composition of phenolic compounds are

more effective than water 26

.

Table-1: The total phenolic and flavonoid contents of rosemary and green tea leaves extracts (on dry-basis.

The plant Extract % Phenolic compounds % Flavonoids

Rosemary leaves

aqueous 13.44 9.54

ethanolic 18.75 12.65

Green tea leaves

aqueous 39.38 17.69

ethanolic 48.44 22.70

Free radicals are naturally formed in a wide range of biological as well as chemical systems. They are chemical stable

atoms and molecules, which have one (or rarely more) free electron / electrons in the electron envelope 27, 28

. The free

radicals are responsible for many pathological processes and cause important secondary damage to the biological

systems and cells 29-32

. The antioxidant activity of the compound (or mixture of compounds) to inhibit oxidative

reaction of various biomolecules (e.g. prevent the peroxidation of lipids). As shown in Figs 5 and 6, which are refer to

the reducing power method for the determination of the antioxidative abilities of aqueous and ethanolic extracts of

rosemary and green tea leaves (as compared with BHT as a reference), that there was a similarity in the way of how

they are acting, in spite of, the total phenolic compounds in ethanolic extracts in both plants are more than that of

aqueous extracts. This will depend on the kinds of phenols those were available in each extract at a certain

concentration.


4

Almost, the same mode of action is also associated with the second method of determination (the chelating ability,

Figs 7 and 8). As shown in Figs 7 and 8, the abilities of aqueous and ethanolic extracts of rosemary and green tea

leaves, as chelating agents (comparing with EDTA as a reference) are less than their abilities as reducing power.

Determination of the antioxidant activity is one of the ways how to biologically and nutritionally evaluate the quality

of the fruit. It has been proved that antioxidant activity depends on the type of phenolics present in the plant, as some

phenolic compounds exhibit higher antioxidant activity than others 33-38

.

Ch

ela

tin

g ab

ility

%

Concentration (mg/ml) Figure 7.Chelating ability of aqueous and ethanolic extracts of green tea

leaves as comparad with EDTA at the same concen tration …

et…

Ch

ela

tin

g ab

ility

%

Concentration (mg/ml)

Figure 8. Chelating ability of aqueous and ethanolic extraxts of rosmary

leaves as comparad with EDTA at the same concentration

et…

Tariq et al, 2013

5

As shown in Fig.9, which refer to the activity of ethanolic extracts for the both plant leaves (rosemary and green tea),

the ethanolic extract of green tea shows, to some extant, high reducing power ability as compared with rosemary,

especially for the concentrations above 4 mg/ml. In Fig. 10, which represents the ability of aqueous extracts for the

both plant leaves, the ability of aqueous extract of the rosemary leave shows high percentages of reducing power as

compared with green tea, especially for the concentrations from 4 mg/ml to 8 mg/ml.

Figs 11 and 12 show the chelating abilities for the aqueous and ethanolic extracts of rosemary and green tea leaves. It

is clear, that the active compounds in rosemary extracts (especially the ethanolic) having high ability as chelating

agent comparing with green tea.


6

Carnosic acid is the most abundant antioxidant substance found in the leaves of the rosemary plant and is the main

compound responsible for its antioxidant activity 39

. Its radical scavenging activity follows a mechanism which is

explained by the presence of two O-phenolic hydroxyl groups found at atoms C11 and C12 40

.

Rosemary can inhibit lipid oxidation, chelating metals and scavenge superoxide radicals. Nakatani (2003) 41

reported

that phenolic diterpenes from rosemary are particularly antioxidative. The antioxidant activity of carnosic acid is more

than twice that of any other phenolic diterpene. It has several times the antioxidative capacity of BHT and BHA 42

.

Furthermore, carnosic acid and carnosol chelate iron and scavenge peroxyl radicals, especially in lipid-based systems 43

.

Green tea has substantial antioxidative activity, much of which appears to be due to natural flavonoids. Antioxidant

activity of green tea infusions appears to be linearly related to phenol content 44

. Catechins, polyphenolic flavonoids in

green tea, are particularly effective free radical scavengers 45

. The primary catechin polyphenol constituent and major

peroxyl-radical-scavengeing compound is (-)-epigallocatechin-3-gallate 46, 47

.

On the average, 65-70% of population is excessively impacted by oxidation stress caused by free radicals.

Therefore, oxidative stress monitoring is an important part of reasonable health prevention 48-51

.

4. CONCLUSION In general, the ethanolic extracts of rosemary and green tea leaves are high in phenolic compounds as compared with

aqueous extracts. The green content of phenolic compounds of both aqueous and ethanolic extracts about 3-fold

comparing with rosemary. Although, there were differences in their phenolic content, rosemary and green tea leaves

extracts gave almost similar mode of action as antioxidants (May due to the type of phenolic compounds in each

plant). The phenolic compounds in rosemary leave (mainly, carnosic acid, carnosol and rosamaric acid) and green tea

leaves (catechins, mainly epigallocatechin-3-gallate) gave high reducing power ability rather than chelating agents. As

a result, we are fully recommended the extract of the both plant leaves, especially the rosemary, as a natural

preservative in the food systems.

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Pak. J. Chem. 3(1):8-18, 2013 Full Paper

ISSN (Print): 2220-2625


*Corresponding Author Received 30th July 2012, Accepted 31st December 2012

An Extensive Survey of the Phytochemistry and Therapeutic Potency of Ocimum

sanctum (Queen of Herbs)

*S. K. Bariyah

Department of Chemistry, Forman Christian College (A Chartered University), Lahore, Pakistan.


ABSTRACT Ocimum sanctum, known as Queen of Herbs, is an important member of the family Lamiaceae due to its use in herbal medication

centuries back, especially, in India and other parts of the sub-continent. It is still a subject of immense importance in modern

medical research and it is due to the chemical constituents present in it like flavonoids, terpenoids, alkaloids, saponins, vitamins,

minerals, proteins, carbohydrates and many others. It has shown a wide range of therapeutic potencies like antimicrobial,

anticataleptic, antitoxic, immunomodulatory, analgesic, antidiabetic and cardioprotective activities. The aim of the present review

is to present an extensive survey on the phytochemistry and pharmacological applications of the herb.

Keywords: Ocimum sanctum, Queen of Herbs, phytochemistry, pharmacological applications.

1. INTRODUCTION Medicinal plants have considerable importance as source of medicines in both rural and urban lives. Traditional

medical practitioners use these plants in their day to day practice1. The phytochemicals found in plants are preventive

against a number of diseases like diabetes, nervous disorders including nervous degenerative disorders and cancer2-4.

From ancient times till today, these plants are being investigated for their phytochemistry and medicinal potency.

Ocimum sanctum is different from pesto form of the basil, that is, Ocimum basilicum. It is the purest and most sublime

plant. It is found in tropical part of Asia and has been grown in India for more than 3,000 years. In India, O. sanctum

is considered as the most sacred plant by Hindus as it is used for religious purposes in addition to its medicinal value5.

It symbolizes Laksmi Devi, according to their religion, who is considered as the most important lady. Tulsi means

“The incomparable one” and is regarded as “the queen of herbs”. It is known as religiously and spiritually pious.

Tulsi, also known as Holy Basil, has got immense Ayurvedic medicinal importance especially in the Eastern areas.

2. BOTANICAL DESCRIPTION O. sanctum is an erect, medium sized perennial herb which is woody below. Leaves are 2.5-6.0 cm long, being oblong

and narrow at both ends. Petiole is thin and about 1.5-3 cm long. Floral leaves are sessile. Flowers are small and are

arranged on small stalks in 15-20 cm long bracteate racemes. Colour varies from white, pink to purplish. Calyx is

campanulate, membranous and enlarged. Corolla is extended beyond calyx. A group of four nutlets are enclosed in it.

They are round, smooth and pale reddish-brown in colour. Nutlets are sub-globose and elliptic. Odour is aromatic and

taste is pungent. Root is thin, soft, hairy and branched being blackish-brown externally and pale violet internally.Stem

is herbaceous, hairy, woody, branched and sub quadrangular. It is purplish-brown to black externally and cream

coloured internally. Odour of the stem is slightly aromatic. Seeds are oval to round and mucilaginous. They are

odorless and taste is slightly pungent6.

The soils best fit for cultivation of the herb are saline, rich loam to poor laterite and alkaline to slightly acidic.

At altitudes of 900m (tropical and sub-tropical regions) best cultivating climate is available for the herb. For good

vegetative growth, well-drained soil is best. Reasonable rainfall, humid conditions and long days with high

temperature are fit for its proper growth and good oil production. Low oil production results in case of partially shaded

habitat.

Ocimum sanctum plant with flowers

3. ETHNO MEDICINAL APPLICATIONS Leaves of O. sanctum are expectorant. Juice of leaves is used as stimulant, stomachic, diaphoretic, antiperiodic, used

in gastric disorders and hepatic infections. It is also used in earache, catarrh, bronchitis and bronchial asthma. Dried



9

leaves of the herb are used as snuff. In malarial fever, root decoction proves to be beneficial. Seeds of O. sanctum are

demulcent and effective in urinary disorder. The plant as a whole is a remedy for snake and mosquito bites. It is also

used for the treatment of arthritis, diarrhea, skin diseases, eye infections and chronic fever. The leaves of the herb

contain a bright yellow volatile oil which is effective against insects and bacteria. It inhibits the in vitro growth of M.

tuberculosis and M. pyogenes var. aureus. When externally applied, it helps in curing ringworms and other skin

diseases. Juice or paste of leaves taken twice a day on an empty stomach increases the resistance of the body and

reduces the chances of inviting swine flu. This potential of O. sanctum has been discovered recently. It helps in

fighting against Japanese Encephalitis7. The herb is also used to treat hemorrhage and dyspepsia It is also known to

have antimicrobial, antifungal, antifertility, anticancer, antidiabetic, hepatoprotective, cardioprotective, antiemetic,

analgesic, antispasmodic and adaptogenic potential1.

3.1 Taxonomical Classification 3.2 Vernacular Names

Kingdom: Plantae Sanskrit : Surasā, Krsnatulas i, Bana Tulas i

Phylum: Magnoliophyta Assamese Tulasi

Class: Magnoliopsida Bengali : Tulasi Order: Lamiales English : Holy Basil Family: Lamiacaea Gujrati : Tulasi, Tulsi Genus: Ocimum Hindi : Tulasi

Species: sanctum Kannada : Tulasi, ShreeTulasi, VishnuTulasi Kashmiri : --

3.3 Parts Used Malayalam Tulasi, Tulasa

Leaves, seeds, whole plant, oil Marathi : Tulas

Oriya : --

3.4 Production lead time Punjabi : Tulasi

Four weeks. Tamil : Tulasi, Thulasi, ThiruTheezai Telugu : Tulasi Urdu : Raihan, Tulsi6

4. PHYTOCHEMICAL STUDIES Whether the plant colour is green or purple, all O. sanctum plants have the same chemotype. High amounts of eugenol

or methyl eugenol or sesquiterpenes are found in O. sanctum belonging to different habitats. Eugenol is the major

constituent of essential oil (27–83%) along with methyl eugenol (3– 24%), methyl chavicol (10–15%) and

sesquiterpene hydrocarbons. Fifty one components, representing 94.2% of the whole oil, were detected in O. sanctum

(green) essential oil. The composition of the oil is eugenol / sesquiterpene-type: eugenol (41.7%), sesquiterpene

hydrocarbons (39.2%) and total sesquiterpene components (45.9%). α-caryophyllene and α-elemene were the main

sesquiterpene hydrocarbons. In one study, a solvent system for HPTLC analysis of quercetine in aqueous and

alcoholic extract of O. sanctum showed the amount to be 0.74 mg/ml8. In leaf of O. sanctum, volatile oil, phenols,

alkaloids, tannins, saponins, flavonoids, protein and carbohydrate were recorded to be 0.8, 1.02, 3.9, 0.42, 0.24, 1.10,

3.3 and 4.5 mg/100g, respectively. In stem and root, the quantities were found to be 0.7, 0.72, 1.9, 0.14, 0.22, 1.08,

2.82, 2 and 0.4, 0.99, 1.1, 0.10, 0.20, 0.96, 1.20 and 2.1 mg/100g, respectively. Thiamin (Vitamin Bl), riboflavin

(vitamin B12) and niacin were estimated to be 0.48, 0.24 and 0.27 mg/100g, respectively, whereas, magnesium,

calcium, potassium, phosphorus, sodium, iron, zinc and manganese concentrations were 0.48 to 0.18, 3.598 to

195.02,4.98 to 396.94, 196.05 to 100.54, 81.34 to 26.94, 16.18 to 0.22, 0.10 to 0.08 and 0.18 to 0.12 mg/100g,

respectively9. In crystal springs O. sanctum Local, Stoneville O. sanctum Local, Beaumonte O. sanctum Local and

Verona O. sanctum Local the concentrations of phytochemicals were: α-humulene (5.53 ± 0.662, 2.88 ± 0.281, 3.89 ±

1.11and 4.38 ± 0.710, respectively) eucalyptol (23.4 ± 0.770, 7.45 ± 3.00, 6.72 ± 1.09 and 13.6 ± 1.30, respectively)

humulene epoxide II (1.63 ± 0.135,2.49 ± 0.0886, 2.44 ± 0.328 and 2.21 ± 0.204, respectively) methyl chavicol (25.1

± 3.12, 11.9 ± 1.37,7.02 ± 0.814 and 17.3 ± 0.703, respectively) and (–)-trans-caryophyllene (1.35 ± 0.146, 0.884 ±

0.206, 1.04 ± 0.348 and 2.37 ± 1.61, respectively)10. The phytochemical estimation of O. sanctum showed the

presence of alkaloids, tannins, flavonoids, amino acids, steroids, carbohydrate, reducing sugar, triglycerides,

phospholipids, cholesterol, LDL – cholesterol, VLDL – cholesterol and HDL – cholesterol11. The qualitative analysis

of leaf extract of O. sanctum detected tannins, saponin, flavonoids, steroid, terpenoids and cardiac glycerides. GC-MS

analysis of hydroalcoholic extract showed the presence of ten compounds: caryophyllene (26.53%), eugenol (43.88%),

cyclopropylidene-(1.02%), 1, 2, 4-triethenyl (15.31%), 1, 1-dimethoxy-(2.04%), N, N, a, 4-tetramethyl-(2.04%),

benzene methanamine, cyclohexane, cyclopentane, octadecane12. Eugenol (80.94%) was extracted from the leaves of

O. sanctum by kinetic method in 90 min with the agitation speed being 1000 rpm13. Ursolic acid, apigenin, luteolin,

oleanolic acid, rosmarinic acid, carvacrol, linalool and β-caryophyllene have been identified in O. sanctum14.

Alkaloids, glycosides, gums mucilage, proteins, amino acids, tannins, phenolic compound, triterpenoids, steroids,

sterols, saponins, flavones and flavonoids were found to be present in O. sanctum located in South Eastern Odhisa15.

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In Thailand, α-thujene, camphene, sabinene, β-pinene, limonene, linalool, borneol, α-copaene, β-elemene, β-

caryophyllene, α-humulene, γ-murolene, α-bulnesene, eugenol and methyl eugenol were detected by GC-MS analysis

of the hydro distillate of aerial parts of O. sanctum16. In one study, alkaloids, flavonoids, tannin, saponins and

cyanogenic glycosides in stem and leaves of O. sanctum were estimated to be 7.50, 6.30, 0.45, 0.76, 32.18 mg/100g

and 11.80, 11.50, 3.55, 0.28, 25.66 mg/100g, respectively. Nutritional analysis showed protein, lipids, fiber,

carbohydrates, moisture content and ash in stem and leaves to be 9.25%, 2.75%, 18.30%, 68.05%, 88.30%, 20.15%

and 12.30%, 3.0%, 7.0%, 77.70%, 83.55%, 18.35%, respectively17.

5. PHARMACOLOGICAL STUDIES Vast research has been done regarding the therapeutic potential of O. sanctum. Some of the work is mentioned below.

5.1 Anti aflatoxin Potential Reduction of aflatoxin B1 (AFB1) in stored rice by extracts of S. aromaticum, C. longa, A. sativum and O. sanctum

was investigated. S. aromaticum (5 g/kg) showed complete inhibition of AFB1 production. C. longa, A. sativum and

O. sanctum also inhibited aflatoxin B1 production (72.2–85.7%) at the same concentration18.

5.2 Anti amnesia Potential Antiamnesic effect of the aqueous extract of O. sanctum was investigated on time induced amnesia and sodium nitrite-

induced amnesia in mice. Prior to experiment, for a period of three days, the extract was administered to mice at 100

and 300 mg/kg dosage intraperitoneally. The effect of the extract was noticed by object discrimination task and

elevated plus maze task. The standard drug used was Piracetam. When given before and after training trial,O. sanctum

was found to have a potential memory enhancing character19.

5.3 Analgesic Potential The ethanol extract of the leaves of O. sanctum was used to test the analgesic potential of the herb. In the hot-plate

test, at the doses of 250 and 500 mg/kg body weight, the extract showed a significant (p<0.05) dose dependent

increase in reaction time in mice. Significant (p<0.05) anti-inflammatory effect was also observed by inhibition of

paw volume by 43.33% at the dose of 500 mg/kg body weight at the fourth hour of study. The ethanol extract of O.

sanctum proved to have dose-dependent analgesic and anti-inflammatory activity20.

5.4 Anthelmintic Potential The hydro alcoholic extracts of W. sominifera and O. sanctum were investigated for anthelmintic potential against

earthworms. For both the plants, dose of 40 mg/mL possesses more wormicidal activity. For O. sanctum and W.

sominifera, the paralysis time was 2.5±0.6 and 2.8±0.8 and the death time was 6.5±0.7and 7.1±0.9, respectively. O.

sanctum proved to be a better anthelmintic cure21.

5.5 Immunomodulatory Potential Immunomodulatory potential of the aqueous extract of O. sanctum leaves in wistar rats was investigated by studying

the humoral antibody titre (HA), total leukocyte count (TLC) and differential leukocyte count (DLC). Levamisole was

the standard drug used.The data obtained proved the antianaphylactic activity of O. sanctum22. Comparison of

immunomodulatory activity of alcoholic and aqueous extracts of O. sanctum was undertaken by giving doses of 50,

100 and 200 mg/kg/day for 14 days to Swiss albino rats. Humoral (haemagglutination antibody titer model) and

cellular immunity (delayed type hypersensitivity reaction models) were used for testing the potential. Alcoholic

extract had more potential in producing immune stimulation than the aqueous extract23. Immunomodulatory effect of

aqueous extract of O. sanctum in rat was investigated by its administration orally at 100 and 200 mg/kg/day for 45

days. Increasing antibody production in dose-dependent manner was observed along with increased RBC, WBC and

haemoglobin production, thus, proving immunomodulatory and haematological activity of the herb24. Amelioration of

endosulfan induced immunotoxicity by O. sanctum was studied in male wistar rats by mixing in ground nut oil in the

concentrations of 6, 3 and 1.5 mg/kg body weight. The mice were divided in groups. To some groups, in addition to

endosulfan, O. sanctum was given. Immunity improved in the O. sanctum treated groups25.

5.6 Cardio protective Potential Cardio protective activity of aqueous extract of O. sanctum was tested in male albino rabbits. Adult male rabbits were

given 0.5mg/kg cholesterol with or without plant extract for 45 days. Normal control, cholesterol control and O.

sanctum groups (10mg, 25mg, and 50mg/kg body weight/day) were made. Biochemical evaluation of serum lipid was

done for 4 months then tissues were analyzed for cholesterol. O. sanctum lowered the blood and tissue cholesterol

levels, thus, proving to have anticholesterol property26. Treatment with C. mukul and O. sanctum showed decrease in

serum triglyceride, cholesterol and MDA. These indigenous drugs had significant positive effect on

hypercholesterolemic rabbit model27. Male albino rats were subjected to restraint stress 3h/day for 6 days. Aqueous

extract of O. sanctum was given (100 mg/kg for 6 consecutive days) following stress. In cerebrum, cerebellum and


11

brain stem, MDA, nucleic acids and proteins were estimated. Restraint stress increased the rate of lipid peroxidation

and decreased nucleic acids and proteins. Aqueous extract of O. sanctum prevented the stress related changes, hence,

proving the protective role of the herb28.

5.7 Wound Healing Potential To test wound healing potential of O. sanctum streptozotocin induced type 2 diabetic rats were given 2 gm fresh

leaves of the herb for 30 days. Serum glucose and lipids were estimated by using diagnostic kits. Incision and excision

models for observation of wound healing potential were generated. O. sanctum had pro-healing effect. DHR-S rats

treated with O. sanctum leaves, took less number of days for healing as compared to DHR and CR group29. In one

study, comparison of the oral and topical application of O. sanctum for wound healing potential was done. Excision

and incision methods were used for study in albino rats. Four groups of rats were made for oral test, control and

topical test with six rats per group. Oral and topical treatment showed better results as compared to control30. In wistar

albino rats, wound healing potential of cold aqueous extract of O. sanctum leaves along with its effects on tumor

necrosis factor –α was assessed. Topical application of O. sanctum extract in petroleum jelly resulted in faster healing

as compared to the application of petroleum jelly alone. Significant healing was noticed in animals, which in addition

to topical application of 10% O. sanctum extract, were given 250 mg/kg b. w of aqueous O. sanctum extract for 20

days. TNF-α level was found to be increased by O. sanctum treatment, thus, promoting wound healing potency31.

5.8 Anti diabetic Potential Anti-diabetic potential of O. sanctum was found by experimenting on male rats. After induction of diabetes by

alloxan, fall in blood glucose, blood urea, serum cholesterol and serum triglyceride was noticed by treating the

diabetic respondents sub-cutaneously with O. sanctum leaf extract, thus, proving anti hyperglycemic action of the

herb32. From PAU, Ludhiana, ninety male diabetic patients (40-60 years) who were non-insulin dependent were

selected to study the effect of O. sanctumand A. indica leaves on them. After a month control period, the patients were

divided into group 1, 2 and 3 (30 each). In capsule form, group 1 was given O. sanctum leaf powder, group 2 A. indica

leaf powder and group 3 was given mixture of both powders. Four capsules constituting 2 gm powder were given in

lunch and dinner for 3 months period. Diabetic symptoms lessened in all the three groups, but, the maximum

improvement was seen in group 3 patients. Hence, combination of the leaves proved to be beneficial for diabetes33.

The effect of aqueous extract of A. indica, A. sativum, A. cepa, A. indica, M. sapientum, M. indica, M. koenigii, O.

sanctum, P. amarus and T. cordifolia on type 2 diabetic patients was investigated. Four hundred out of 828 patients

were selected for the study. Ten experimental and ten control groups were made (20 diabetics/group). After 2 months

study, M. indica, M. koenigii, O. santum, P. amarus, A. cepaand A. indica proved to be anti-diabetic and

hypolipidemic. O. sanctum decreased total cholesterol (142±14 to 137±15 mg/dl, p<0.03), LDL (91±14 to 85±19

mg/dl, p<0.03) level and enhanced HDL (25±3 to 27±4 mg/dl, p<0.03) level34.

5.9 Hepatoprotective Potential The hepatoprotective potential of aqueous extract of O. sanctum in lead induced toxicity in wistar albino rats was

studied. Six groups comprising of six rats each were made. Oral administration of the herb for a period of 21 days was

made. After analysis of antioxidant status of the animals, lipid per oxidation (LPO) and hepatic serum markers (AST,

ALT, ALP, GGT), increase in the serum marker enzyme level and serum bilirubin content, lowered levels of serum

protein and tissue glycogen was noticed due to lead toxicity. In hepatic tissues lipid peroxides accumulation was

observed. Restoration to normal level of all the parameters was seen when treated with the aqueous extract of O.

sanctum, depictive of the hepatoprotective nature of O. sanctum in lead induced toxicity35

. In another study,

hepatorenal protective potential of aqueous filtrate of dried leaf powder of O. sanctum (100, 75 and 50 mg/kg./oral) in

distilled water was tested. In second experiment, lethal dose (1gm/kg./i.p) of acetaminophen was tried at its maximum

dose (1.5gm/kg/oral). Leaves of O. sanctum reduced hepatorenal toxicity of acetaminophen in mice36.

5.10 Anti toxic Potential The protective potential of the herb was tested in 35 white leghorn chicks (4 weeks old males). Five groups i.e. 1

(control), 2 (Pb 50 ppm), 3 (lead 100 ppm), 4 (Pb 50 ppm + O. sanctum 100 ppm) and 5 (Pb 100 ppm + O. sanctum

100 ppm) were made (7 chicks/group). After 12 weeks,a significant (p < 0.01) decrease in total erythrocyte count,

hemoglobin and TLC was noted in the chicks fed on 50 ppm and 100 ppm lead as compared to control but increased

in groups fed with lead and O. sanctum, thus, proving that the herb under study neutralized the toxicity and is

protective in nature37. Genotoxicity in adult male wistar albino rats was induced by lead acetate for 12 weeks. Lead

increased micronuclei in polychromatophilic erythrocytes of bone marrow. Rats were divided into six groups

including control group and the other five groups were fed with low and high doses of lead and O. sanctum. The

group, which was treated with high dosage of O. sanctum in addition to lead, exhibited reduction in lead induced

genotoxicity by reducing number of micronuclei in bone marrow cells38. The ethanolic extracts of B. pinnatum, S.

aromaticum and O. sanctum were used to make polyherbal formulation. Intraperitoneally gentamicin (100 mg/kg/d)

was given for 8 days which resulted in nephrotoxicity in wistar rats. Alcoholic B. pinnatum (200 mg/kg) and

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polyherbal formulation (200 mg/kg) reduced gentamicin induced nephrotoxicity39. Antigenotoxic and anticlastogenic

effect of O. sanctum leaf distillate on human polymorphonuclear leukocytes and human peripheral lymphocytes was

studied. Three doses of distillate (50 μL/mL, 100 μL/mL, and 200 μL/mL) were given. The positive controls used for

inducing genotoxicity and clastogenicity were: MMC (0.29 μmol/L), Potassium dichromate (Cr+6

) 600 μmol/L,

Benzo[a]pyrene (30 μmol/L). The damage to DNA, chromosomal aberration and micronucleus formation was

protected with distillate of O. sanctum leaves (50 μL/mL, 100 μL/mL, and 200 μL/mL). LC-MS showed eugenol,

luteolin and apigenin and they had the protective effect against genotoxicants40.

5.11 Anti microbial Potential Ethanol extracts of O. sanctum, O. majorana, C. zeylanicum and X. armatum were tested for antibacterial activity

against B. subtilis, B. cereus, B. thuringiensis, S. aureus,Pseudomonas spp, Proteus spp, S. typhi, E. coli, S.

dysentriae, K. pneumoniae by agar well diffusion method. The plant extracts showed more activity for gram-positive

bacteria than gram-negative bacteria41. Synergistic effect of acetone extract of O. sanctum and antibiotics (Penicillin,

Gentamicin, Cephalexin, Ciprofloxacin and Tetracycline) was tested against Methicillin Resistant Staphylococcus

aureus (MRSA) by using disc diffusion method. Zone of inhibition increased significantly with all antibiotics42. The

chemical constituents of individual and mixed ethanolic leaf extracts from A. indica and O. sanctum were tested for

antimicrobial activity. The mixed extract showed better activity against fish pathogens indicated by zone of inhibition,

minimum inhibitory and minimum bactericidal concentration43. Liquid inhibition test was used to evaluate the

antibacterial activity of aqueous extract, chloroform extract, alcohol extract and oil of O. sanctum leaves against E.

coli, P. aeruginosa, S. typhimurium and S. aureus. O. sanctum extract was effective against E. coli, P. aeruginosa, S.

aureus and S. typhimurium giving optical density reduction from 0.20 to 0.85, chloroform extract being most effective

against P. aeruginosa giving 0.85 reductions in optical density44. Disc diffusion method was used to test ethanolic

extracts of O. sanctum, A. indica, T. aestivum, P. emblica and S. potatorum for antimicrobial activity. O. sanctum

(82.05% removal of E. coli), A. indica (71.79% removal of E. coli) and T. aestivum (64.1% removal of E. coli) proved

to have the greatest potential. In all herbs, maximum removal of E.coli was found at 30 min contact time onwards45.By

cup diffusion method, aqueous ethanolic extracts of O. sanctum (Tulsi), E. caryophyllata(Clove), A. bidentata

(Datiwan) and A.aindica(Neem) were subjected to in vitro antibacterial assay against human pathogens E. coli, S.

typhi, S. paratyphi, S. aureus, K. pneumoniae, P. aeruginosa. All extracts proved to be potent, the maximum result

being shown by clove46. By disc-diffusion method, antibacterial activity of O. sanctum L. essential oil was evaluated

against E. coli, Klebsiellasp., P. mirabulus, P. aeruginosaand S. aureus. Maximum zone of inhibition was seen against

S. aureus (20.0 mm & 41.5 mm) and minimum against E. coli (10.2 mm & 17.8 mm) for the concentration of 5 μL

and 10 μL oil, respectively. In P. mirabulus, P. aeruginosa and Klebsiellasp, the inhibition zones were 15.1 mm &

26.0 mm, 10.2 mm & 20.0 mm and 11.1 mm & 19.4 mm, respectively, for the same concentration of the essential

oil47. Antimicrobial activity against E. coli and Staphylococcus aureus by O. sanctum, M. koenigiiand A. vulgaris leaf

extracts showed significant results, thus, proving the potential of the herb48.

5.12 Anti cancer Potential Ethanolic extract (mixed in equal proportion) of W. somnifera, O. sanctum and T. cordifolia was given to a patient

under chemotherapy. In-vitro cytogenic analysis showed that these drugs decreased chromosomal aberrations49. The

antitumor mechanism of ethanol extracts of O. sanctum was studied in A549 cells and the Lewis lung carcinoma

animal model. Cytotoxicity against A549 cells was exerted by the extract. Cleavage of poly (ADP-ribose) polymerase

(PARP), releasing cytochrome C into cytosol and activation of caspase-9 and -3 proteins was also observed. Growth

suppression of Lewis lung carcinoma in a dose-dependent manner was observed. Hence, ethanol extract of O. sanctum

has antitumor potential50. A polyherbal formulation NR-ANX-C (composed of the extracts of W. somnifera, C.

sinensis, O. sanctum, shilajith and triphala) was tested for antioxidant and antiulcer potential. The formulation was

tested at 25 and 50 mg/kg dosage. It proved to be more effective than ranitidine in reducing ulcer index and at 50

mg/kg NR-ANX-C showed results similar to omeprazole in preventing ulcer formation. A dose- dependent decrease in

gastric juice volume and total acidity and increase in gastric pH and total adherent gastric mucus was also seen in NR-

ANX-C treated groups. Lipid peroxidation was also reduced. NR-ANX-C can be used as an adjuvant in the treatment

of gastric ulcer51. Methanol induced ulcer in wistar rats was treated with aqueous extract of O. sanctum (100mg/kg and

200 mg/kg) given orally. Antioxidant potential of gastric mucosa increased, thus, preventing mucosal damage and

proving the antiulcer activity of the herb52. Another herbal formulation Prolmmu was tested for anticancer effect on

ethinyloestradiol induced ovarian adenocarcinoma in rats. Increased activities of serum glutamate pyruvate

transaminase (SGPT) and serum alkaline phosphatase (SAP) due to ethinyloestradiol were significantly (p<0.05)

decreased by Prolmmu (500 mg/kg, orally, daily for 4, 8 and 12 weeks after 20, 16 and 12 weeks of EO administration

in groups 3, 4 and 5, respectively). The ovarian tissues of group 2 revealed marked fibrous tissue proliferation of

follicular epithelium. Regeneration, improvement and normalization of ovarian tissues were observed. Prolmmu

proved to have anticancer effect on ethinyloestradiol induced ovarian cancer53.


13

5.13 Anti inflammatory Potential Using carrageenan induced paw edema model, anti-inflammatory activity of O. sanctum leaf paste was studied. Adult

albino rats were divided into 3 groups: control (vehicle), standard (Indomethacin 100 mg/kg) and fresh tulsi paste (500

mg/kg). Drug administration was done orally, 1hr before phlogistic agent. The percent Inhibition was 0%, 76% and

67% for control, standard and test, respectively. O. sanctum gave 88.15% inhibition comparable to 100 mg/kg of

indomethacin54.

5.14 Antioxidant Potential Forty-two broiler chicks (day-old) divided into six groups of seven chicks each were given supplementation of O.

sanctum leaf powder (0.25% and 0.5%), organic selenium (0.3 ppm) and their combinations, to check their effect on

levels of antioxidative enzyme. Levels of Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px) and

Catalase were measured in plasma at the end of 3rd and 6th week of age. Dietary selenium (0.3 ppm) supplementation

increased GSH-Px activity and O. sanctum leaf powder (0.5%) increased SOD and Catalase levels. Combinational diet

of both proved to be more effective, thus, proving to combat oxidative stress in broilers to the best55. DPPH, Nitric

oxide and reducing power assays were used to test the antioxidant activity of O. sanctum leaves. IC50 value of

16.39+0.31 and 16.20+0.33 μg/mL was observed for DPPH and Nitric oxide scavenging assays, respectively. In

reducing power assay significant antioxidant activity was seen56. Ferric reducing antioxidant power (FRAP) assay,

improved ABTS radical cationdecolorization assay and DPPH free radical scavenging assay were used to study

antioxidant activities of O. sanctum and O. basilicum. Folin- Ciocalteau micro method was used to analyze total

phenolic contents. The results were better for O. bascilicum as compared to O. sanctum57. DPPH radical, nitric oxide

radical, superoxide anion radical and hydroxyl radical scavenging assays were used to evaluate the antioxidant and

free radical scavenging activity of aqueous ethanolic (1:1) extract of O. sanctum (AEOS) in various systems in a dose

dependent manner. Aqueous ethanolic (1:1) extract of O. sanctum (50, 100, 200, 300, 400 and 500 μg/mL) showed

38.06, 41.45, 44.83, 49.06, 57.78 and 65.98% inhibition, respectively, on peroxidation of linoleic acid emulsion. IC50

value was found to be 34.21 μg/mL in DPPH radical scavenging assay and 18.69 μg/mL of ascorbic acid. Nitric oxide

radicals were scavenged giving 86.91 μg/mL IC50 value and curcumin had 86.91 μg/mL IC50 value. Superoxide

generated by PMS/NADH-NBT system was scavenged with IC50 value of 73.38 μg/mL and for curcumin IC50 was

24.67 μg/mL. Hydroxyl radical generated by the deoxyribose method was also inhibited with 42.69 μg/mL IC50 value.

Standard Catechin showed IC50 value of 17.71 μg/mL. Aqueous ethanolic (1:1) extract of O. sanctum can be

considered as a natural antioxidant58. O. sanctum plants were treated with paclobutrazol (PBZ) and Abscissic acid

(ABA) to analyze the changes in the enzymatic and non-enzymatic antioxidant responses. Non-enzymatic antioxidants

(ascorbic acid) decreased in the ABA treated plants and increased in the PBZ treated plants along with α-tocopherol

content. Enzymatic antioxidants (ascorbate peroxidase and superoxide dismutase) were also enhanced. When

compared with the control plants, catalase activity increased59.

5.15 Thrombolytic Potential O. sanctum, C. longa, A. indica, A. occidentale along with Streptokinase (positive control) and water (negative

control) were used to investigate thrombolytic activity of herbal preparations. An in vitro thrombolytic model was

used for study. The percentage (%) clot lysis was statistically significant (p<0.0001) when compared with vehicle

control. Moderate clot lysis activity was shown by all plant extracts being 30.01 ± 6.168% for O. sanctum, 32.94 ±

3.663%for C. longa, 27.47 ± 6.943% for A. indica and 33.79 ± 2.926% for A.occidentale. Streptokinase (positive

control) showed 86.2 ± 10.7 % clot lysis effect. Hence, herbal preparations possess in vitro thrombolytic potential60.

5.16 Mast Cell Stabilizing Potential Ethanolic extract of O. sanctum, flavonoid fraction and standard (Prednisolone) were given for 14 days to albino rats

sensitized by horse serum and triple antigen containing B. pertussis. Mast cell of intestinal mesentery was studied.

Mast cell degranulation up to 12.55% and 80.90% was seen in unsensitized and sensitized rats, respectively. Standard

and ethanolic extract inhibited mast cell degranulation to an extent of 72.25% and 62.44% (100 mg/kg body weight)

and 67.24% (200 mg/kg body weight), respectively. By flavonoidal fraction, 54.62% and 60.48% inhibition at 75 and

150 mg/kg body weight, respectively, was observed61.

5.17 Anti cataleptic Potential Anticataleptic potential of a polyherbal formulation NR-ANX-C (containing W. somnifera,O. sanctum, C. sinensis,

triphala and shilajit extracts) in intraperitoneally (1mg/kg) induced haloperidol catalepsy in mice was studied. Five

groups of male albino mice were made. Cataleptic score was measured as the time the animal maintained an imposed

posture. Scopolamine (1 mg/kg) was used to compare the anticataleptic potency of herbal formulation (10, 25 and 50

mg/kg). Oxidative stress and degree of catalepsy was also estimated by superoxide dismutase level in brain tissue.

Minimum cataleptic score was in the NR-ANX-C (25 mg/kg) treated group and minimum SOD activity was in the

same group62. O. sanctum anti-cataleptic activity was studied considering 2.7% ursolic acid in it, which has

antioxidant properties. Haloperidol (1.0 mg/kg i.p.) was used to induce catalepsy. After 15 min, significant reduction

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of cataleptic score was observed on a standard bar test with the standard drug Levodopa (30 mg/kg, i.p), the aqueous

extract (300 mg/kg, i.p) and the alcoholic extract (300 mg/kg, i.p) of the leaves of O. sanctum. Except 0 and 15 min,

the results were significant for alcoholic and aqueous extracts63.

5.18 Anti anxiety Potential O. sanctum extract (100 mg/kg body weight) was evaluated for its effects on restraint stress in rats. Memory

impairment resulted in mice due to 21 days’ stress. Decrease in latency to enter the target quadrant in Morris water

maze test compared to the stressed animals and increase in latency to enter the dark compartment during retention test

in passive avoidance tests both after 24 hours and 48 hours, were the positive results after oral feeding of the extract.

Memory was also improved in stressed rats. O. sanctum, thus, has anti-stress potential64. Antifatigue activity of 70%

alcoholic extract of O. sanctum was investigated in rats. O. sanctum extract (150, 300 and 450 mg/kg b.wt.) was given

every day along with weight-loaded forced swim test on alternate days. Test was conducted for a period of 2 weeks.

O. sanctum lowered malondialdehyde (MDA) and lactic acid levels in liver and muscle tissues. Serum biochemical

parameters also reduced. Best performance against fatigue was observed at 300 mg/kg b.wt. dosage65.Comparative

antidepressant activity of O. sanctum (OS) and imipramine using animal models of depression was carried out. Forced

Swimming Test (FST), Reserpine Reversal Test (RRT), Haloperidol- Induced Catalepsy (HIC) and Pentobarbitone

Sleeping Time (PST) in male wistar rats were used as models of depression study. Imipramine (15 mg/kg/i.p) and

herbal extract of OS (500 mg/kg/p.o) was given. Reduction in immobility time in FST, RRT and protection against

HIC, compared to control, respectively, was observed after single administration. The antidepressant activity of OS

was comparable to imipramine and indicates the potential for its use as an adjuvant in depression treatment66.Thirty-

five volunteers (21 male and 14 female) of average age 38.4 years were given O. sanctum extract (500 mg/capsule,

twice daily, p.o. after meal). Standard questionnaires based on different psychological rating scale at baseline (day 0),

mid-term (day 30) and final (day 60) were used for clinical investigations. Results revealed that O. sanctum

significantly (p<0.001) lessened anxiety disorders, correlated stress and depression and increased the willingness to

adjustment. O. sanctum is an anxiolytic agent67.

5.19 Termite and Mosquito Repellency Potential Antitermitic activity of crude extracts (hexane, butanol, chloroform, methanol, ethyl acetate and water extracts) of

inflorescence, leaf, root and stem of O. sanctum was studied against the termite species, H. indicola. After eleven

days, the result was maximum for ethyl acetate leaves extract giving termite mortality of 84.45 ± 27.21 and minimum

mortality was seen for stem extract of water i.e. 43.89 ± 39.97. Maximum repellency (29.1) was seen for methanol

root extracts while minimum (21.3) for water extracts68. Ether extract of O. sanctum was tested at 150, 200, 230, 250,

300, 350, 400, 450, 500, 550, 650 and 900 mL volume against Anophele, Culex and Ades of adult 3-5 days old

mosquitoes in small net, large net and large room conditions. High concentration of O. sanctum leaf extract showed

greater repellent activity in all net containing mosquitoes, whereas,low concentration of extract showed greater

activity in small net but lesser in large net. Hence, high concentration of O. sanctum leaf extract can be used in

mosquito repellent formulation69. Antiviral activity of methanolic extracts of A. paniculata, C. limon, C. citratus, M.

charantia, O. sanctum and P. citrosum on dengue virus serotype 1 (DENV-1) was evaluated. Maximum non-toxic

dose (MNTD) was in the decreasing order of M. charantia>C. limon>P. citrosum, O. sanctum>A. paniculata>C.

citratus. Antiviral assay showed that A. paniculata had the most viral inhibition followed by M. charantia. Extracts of

O. sanctum and C. citratus showed less inhibition effect70.

5.20 Chemopreventive Potential Study of the chemopreventive activity of ethanolic leaf extract of O. sanctum on cell proliferation, apoptosis and

angiogenesis during N-methyl-N’- nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis was carried out

on rats. Four groups consisting of ten rats each were made. Group 1 rats received MNNG (150 mg/kg body weight) by

intragastric intubation three times (two week interval between treatments). Group 2 rats, in addition to MNNG (150

mg/kg body weight), received ethanolic O. sanctum extract (300 mg/kg body weight) three times/week. Group 3 rats

were given ethanolic O. sanctum extract (300 mg/kg body weight) only. Group 4 was the control. After 26 weeks, all

the rats were killed. O. sanctum extract lessened the symptoms of MNNG-induced gastric carcinomas71.

5.21 Genoprotective Potential The blood of cigarette workers was found to be genotoxic. When they were given essential oil of O. sanctum (12

μg/mL of culture) the toxic effect reduced, thus, showing the genoprotective role of the herb72.

5.22 Plant Disease Resistance Potential Activities of enzymes of rice seeds (Phenylalanine amino lyase (PAL), Catalase, Peroxidase, Polyphenol oxidase

(PPO) and Tyrosinase) were studied. Exposure of fungus R. solani with control was done after the seeds were treated

with leaf extracts of C. citrus and O. sanctum. Then mechanism of different enzymes was studied (0, 24, 48, 72, 96

and 120 hours after fungus exposure). C. citrus resulted in 2-4 fold increase in enzyme activities and O. sanctum in 2-


15

3 fold increase. Ethanolic leaf extracts gave best results73. The pathogenic fungus F. solanif. sp. melongenae was

isolated from infected plant parts. A. indica, A. annua, E. globulus; O. sanctum and R. emodi plant extracts (5, 10, 15

and 20% concentration) were tested to control brinjal wilt pathogen. Considerable reduction in the growth of pathogen

was observed74. Control of powdery mildew of Bhendi (E. cichoracearumDC) was carried out by ten treatments P.

fluorescensI18 (0.2%), P.fluorescens1(0.2%), O. sanctum 10%, Neem Seed Kernel Extract 5%, K2HPO4 50 mM,

Salicylic acid 1mM, O. sanctum 5% + P. fluorescensI18 (0.2%),Neem Seed Kernel Extract 5 % + P. fluorescensI18

(0.2%), Carbendazim 0.1% and control. Two sprays with the time interval of 30 and 60 days after sowing were given.

Neem Seed Kernel Extract 5%+ P. fluorescensI18 0.2% followed by 9.49% with P.fluorescensI18 0.2%, 10.36% with

carbendazim and 11.9% with P. fluorescens-1 gave disease incidence of 8.83% (minimum value). As compared to

control, NSKE 5%+P. fluorescensI18 followed by 42.44% in P. fluorescensI18, 41.84% with 0.1% carbendazim and

39.73% with P. fluorescens-1 also increased the yield to 43.43% over control75.

5.23 Europathogen Resistance Potential Essential oils of C. aromaticus and Rama and Shyama Tulsi were tested for anti-urinary tract infection potential.

Rama Tulsi and C. aromaticus oils proved to be most active against bacteria causing infection, whereas, Shyama Tulsi

was least potent. Concentrations of 0.5 μL/mL-6 μL/mL (MIC) gave best results76.

6. CONCLUSION Plants are used as medicines since the beginning of mankind. In this modern era of research and development, herbal

medicine is not kept unnoticed and plants are still used either as medicine or medicinal substitutes. O. sanctum has

gained extra importance in the field of ayurvedic (herbal) medicine because of its vast pharmacological activities

which are increasing day by day with research. Recently work has been done to compare the phytochemistry and

pharmacology of O. basilicum and O. sanctum77. The present review focuses on the botanical characteristics,

phytochemistry and ethno medicinal and pharmacological applications of the herb. Study on O. sanctum can lead to

improvement of synthetic medicine and can make the availability of low cost drugs to people.

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32. Khogare, D.T.,Lokhande, S.M., Effect of Tulasi (Ocimum sanctum) on Diabetes mellitus, ISRJ, (2011), 1(2),

189-191.

33. Kochhar, A., Sharma, N.,Sachdeva, R., Effect of Supplementation of Tulsi (Ocimum sanctum) and Neem

(Azadirachtaindica) Leaf Powder on Diabetic Symptoms, Anthropometric Parameters and Blood Pressure of

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34. Dineshkumar, B.,Analava, M.,Manjunatha, M., Antidiabetic and Hypolipidaemic Effects of Few Common

Plants Extract in Type 2 Diabetic Patients at Bengal, Int J Diabetes &Metab, (2010), 18, 59-65.

35. Akilavalli, N.,Radhika, J., Hepatoprotective Activity of Ocimum sanctum Linn. Against Lead Induced

Toxicity in Albino Rats, Asian Journal of Pharmaceutical and Clinical Research, (2011), 4(1).

36. Makwana, M.,Rathore, H.S., Prevention of Hepatorenal Toxicity of Acetaminophen with Ocimum sanctum in

Mice, IJPT, (2011), 3(1), 1385-1396.

37. Prakash, A., Singh, S.P.,Varma, R.,Choudhary, G. K., Impact of Lead on Clinicohematological Parameters

After Dietary Oral Administration and Protective Efficacy of Tulsi (Ocimum sanctum) in Cockerels, Indian J.

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38. Sujatha, K.,Srilatha, C.,Rao, T.S.C.,Amaravathi, P., Ameliorated Effect of Ocimum Sanctum on Lead Induced

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39. Jain, S.,Argal, A., Protective Effect of Bryophyllum pinnatum, Syzygium aromaticum and Ocimum sanctum

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(2012), 2(1), 55-59.

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17

41. Joshi, B.,Lekhak, S., Sharma, A., Antibacterial Property of Different Medicinal Plants: Ocimum sanctum,

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Science, Engineering and Technology, (2009), 5(1), 143-150.

42. Imran, M., Lawrence, R.,Alam, M.D.N.,Shariq, M., Kumar, E.J., Synergistic Effects of Ocimum sanctum

Extract and Antibiotics on Methicillin Resistant Staphylococcus aureus (MRSA) Isolated from Clinical

Specimens, Journal of Recent Advances in Applied Sciences, (2012), 27, 99-107.

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Water Purification, International Journal of Engineering Science and Technology, (2011), 3(9), 7170-7174.

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extraction and antimicrobial properties of different medicinal plants: Ocimum sanctum (Tulsi), Eugenia

caryophyllata(Clove), Achyranthes bidentata (Datiwan) and Azadirachta indica (Neem), Journal of

Microbiology and Antimicrobials, (2011), 3(1), 1-7.

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(2008), 6(1&2), 63-65.

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71. Manikandan, P.,Nagini, S., Proliferation, Angiogenesis and Apoptosis-associated Proteins are Molecular

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ISSN (Print): 2220-2625


*Corresponding Author Received 12th August 2012, Accepted 17th January 2013

Piperidine Promoted Regioselective Synthesis of α, β-unsaturated Aldehydes

*A. H. Banday

Department of Chemistry, Islamia College of Science and Commerce, Srinagar-190009, J&K, India.


ABSTRACT An efficient, facile and regioselective synthesis of α,β-unsaturated aldehydes from β-hydroxynitriles is reported. The reaction is

carried out using DIBAL-H and promoted by piperidine under dry conditions at a temperature of -78 oC and can be described as a

concomitant reduction-elimination reaction. The same reaction if carried out in the absence of piperidine gives mainly the

uneliminated reduction product. The products formed are of immense importance as synthons in a large number of chemical

reactions and biological processes.

Keywords: α,β-unsaturated aldehydes, metal hydrides, DIBAL-H, β-hydroxynitriles.

1. INTRODUCTION Aldehydes in general and α,β-unsaturated aldehydes in particular are versatile substrates for the synthesis of a large

number of molecules. Various methods have been used for the synthesis of the synthetically valuable α,β-unsaturated

aldehydes. These methods include the reduction of nitriles, palladium catalysed dehydrosilylation of silyl-enol ethers,

the Wittig reaction etc1-2.

Isobutylaluminium alkyls are the versatile reagents mainly useful for reduction transformations. The varieties

of these alkyls mainly include the diisobutylaluminium alkyl hydride (DIBAL-H) besides the less common

diisobutylaluminium butylated oxy toluene (DIBAL-BOT) and the triisobutylaluminium (TIBAL). In comparison to

metal hydride reducing agents, these catalysts offer various advantages for their use at the commercial scale. These

organometallic catalysts offer conveniences in handling and stoichiometric additions besides offering stereo selectivity

in some applications3-5.

DIBAL-H is a versatile and unique organometallic hydride used for the reduction and preparation of

pharmaceuticals and a large number of other laboratory chemicals. DIBAL-H has replaced most of the metal hydrides

including LiAlH4 and NaBH4 on the commercial scale6-8. This can be attributed to the economic advantage, higher

selectivity, cleaner reductions and high yields of the products. DIBAL-H being a liquid, unlike other metal hydride

reducing agents, is miscible in most of the solvents and can be used for the reduction of a variety of substrates. Being

easily oxidisable in the air, DIBAL-H reactions are easily worked up. It is an electrophilic reducing agent, usually

employed in selective reductions of esters or nitriles to aldehydes; lactones to lactols; α,β-unsaturated carbonyl

compounds to allylic alcohols, at low temperatures (-78oC)6-9. DIBAL-H reduces carbonyl or nitrile groups selectively

in presence of double bonds, halide groups, ethers, nitro groups etc. Keeping all these things into consideration, we, in

continuation of our research program towards use of organometallic reagents for the synthesis of various useful

products12-14, herein report the concomitant synthesis of α,β-unsaturated aldehydes from β-hydroxynitriles using

DIBAL-H as an organometallic reductant under selective conditions.

NC

OAc

H

HOH

NC

OAc

H

HOTHP

CHO

OH

OH

CH3

NC OTHP

CH3

NC

CH3

OHC

a b

a b

1a 2a 3a

1g 2g 3g

Scheme 1: Concomitant conversion of β-hydroxynitriles to α,β-unsaturated aldehydes.a, DHP/p-TSA/DCM (95%); b, DIBAL

neat/Toluene/-78 oC (80%) /Piperidine

mailto:*[email protected]


20

2. RESULTS AND DISCUSSION Keeping in view the importance of α,β-unsaturated aldehydes as versatile precursors for the synthesis of huge number

of useful compounds into consideration, we, in continuation of our interest in the development of novel methodologies

using organometallic catalysts12-14, herein report a facile synthesis of the α,β-unsaturated aldehydes by using DIBAL-

H as the reducing agent in the presence of piperidine as the promoter for concomitant reduction-elimination reaction.

β-hydroxynitriles were commercially obtained and the pregnenolone based β-hydroxynitrile was obtained through

knoevenegal reaction of pregnenolone using well known synthetic strategy15-16. Though there are some methods

already reported for the synthesis of various such steroidal analogs17, our method differs in the strategy employed and

the selectivity of the elimination product. The synthesis of steroid based α,β-unsaturated aldehyde (3g) was reported

by us in a previous communication18, which led to the synthesis of various such analogs as reported in this

communication. It is very pertinent to mention here that when the carbon bearing the nitrile group was primary in

terms of having two hydrogens, the yield of the resulting α,β-unsaturated aldehyde was quite insignificant. The

reaction was carried out by first protecting the hydroxyl group at the β-position with DHP in dichloromethane using

catalytic amounts of p-toluene-sulfonic acid. The DHP protected nitrile was then treated with DIBAL-H in dry toluene

followed by 2 Mol% piperidine at a temperature of -78oC. A very interesting situation was observed in the workup of

the reaction. If the reaction time with DIBAL-H and piperidine was prolonged to 1 hr followed by treatment with

methanol and subsequent workup, the main product was the α,β-unsaturated aldehyde. If however the reaction mixture

was exposed to DIBAL-H for shorter periods (less than 10 min.) and not treated with piperidine after 10 min., the

main product obtained was the corresponding β-hydroxy aldehyde. The reaction mixture was allowed to stand for 2

hrs after workup and then filtered through ceilite to get a crude gummy mass which when chromatographed over silica

gel using Hexane:Ethylacetate, yielded the α,β-unsaturated aldehyde as the main product (>70%).

3. CONCLUSION In conclusion we have successfully demonstrated here, the facile and unprecedented synthesis of industrially and

biologically important α,β-unsaturated aldehydes through concomitant reuction/elimination of β-hydroxynitriles. The

above reduction/elimination reaction was found to be general with regard to various β-hydroxynitriles.

Table-1: DIBAL-H catalyzed concomitant synthesis of α,β-unsaturated aldehydes

a. All compounds were characterized by 1HNMR, IR 13C and Mass spectrometry in CDCI3

OH

CH3

NC

NC

OH

NC

OH

NC

Ph

OH

NC Ph

OH

NC

AcO

H

HOH

CH3

OHC

OHC

OHC

OHCPh

OHC Ph

HO

H

OHC

NC

OH

OHC

Entrya

a. All compounds were charecterized by 1H NMR, IR, 13C and Mass spectrometry in CDCl3.

b

c

d

e

g

a

f

Yield( %)

78

73

70

68

69

70

62

beta-hydroxynitriles alpha-beta-unsaturated aldehydes

Banday

21

4. EXPERIMENTAL 4.1 General Melting points were recorded on Buchi Melting point apparatus D-545; IR spectra (KBr discs) were recorded on

Bruker Vector 22 instrument. NMR spectra were recorded on Bruker DPX200 instrument in CDCl3 with TMS as

internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values were

mentioned in δ (ppm) and coupling constants are given in Hz. Mass spectra were recorded on EIMS (Shimadzu) and

ESI-esquire 3000 Bruker Daltonics instrument. The progress of all reactions was monitored by TLC on 2x5cm pre-

coated silica gel 60 F254 plates of thickness of 0.25mm (Merck). The chromatograms were visualized under UV 254-

366 nm and iodine.

4.2 General procedure for the preparation of 3-(5,6-dihydro-4H-pyran-2-yloxy)-2-methylpropanenitrile (2a) 3-hydroxy-2-methylpropanenitrile (1a) (1.0g, 11.7 mM) was dissolved in CH2Cl4 (10 mL) and treated with

dihydropyran (1.50 mL, 13.2 mM) and p-TSA (10 mg). The resulting mixture was stirred for 2 h at ambient

temperature. Workup and filtration of the crude product through a SiO2 column gave the corresponding

tetrahydropyranyl derivative 1b (1.90 g, 11.3 mM, 97%), which was characterized by 1H NMR, IR, 13 C and mass

spectrometry.

4.3 General procedure for the preparation of methacryldehyde (3a) 3-(5,6-dihydro-4H-pyran-2-yloxy)-2-methylpropanenitrile (1b) (1.0g, 5.9 mM) was taken in dry toluene (20 mL) at -

78 oC under nitrogen. DIBAL (neat, 4 mL) was added dropwise to the above solution and the mixture was stirred for 1

hr. MeOH (5 mL) was added carefully after 1 hr, and the mixture was allowed to warm up to ambient temperature and

then stirring continued overnight. The solution was filtered through Ceilite, the solvent removed, and the residue was

chromatographed on a SiO2 column (15 g, hexane- ethyl acetate ) to give methacryldehyde (3a) (0.28g, 4.6 mM, 78%).

4.4 Spectral data of representative end products 3a and 3g

4.4.1 Methylpropenal (3a)

B.pt: 67-69 C, IR (KBr): 2845 (CH), 1730 (C=O); 1H NMR (CDCl3): 1.70 (s, CH3), 5.93 (d, 1 H), 6.19 (d, 1 H);

9.66 (s, 1H). 13C NMR (500 MHz, CDCl3): 15.1, 132.7, 147.3, 193.3. MS (ESI, m/z 93) (M+Na). Anal. calcd. for

C4H6O: C, 68.55; H, 8.63. Found C, 68.39; H, 8.69.

4.4.2 3α-Hydroxy-5-pregn-20ξ-ene-20-carboxaldehyde (3g)

Mp: 170-172 C, D: (+) 17. IR (KBr): 3600 (OH), 1680(C=O), 1620 (C=C) cm-1; 1H NMR (CDCl3): 0.56 and 0.99

(s, angular CH3), 2.80 (t, 1 H, J = 10 Hz, 17-H), 5.36 (m, 1 H, 3-H); 6.12 (s, 1H), 6.30 (s, 1H), 9.56 (s, 1H). 13C NMR

(500 MHz, CDCl3): 15.5, 20.9, 22.3, 24.0, 28.9, 30.5, 30.7, 39.0, 41.3, 46.4, 54.2, 58.7, 71.7, 75.7, 76.2, 109.9,

125.5, 128.4, 137.9, 145.6, 198.3. MS (ESI, m/z 351) (M+Na). Anal. calcd. for C22H32O2: C, 79.95; H, 10.37. Found

C, 79.89; H, 10.33.

5. ACKNOWLEDGEMENT The authors thank Principal of ICSC for his encouragement.

6. REFERENCES 1. Craig, J. C., Ekwuribe, N. N., Synthesis of α,β-unsaturated aldehydes via 1-aminopropa-1,2-dienes:

mechanistic studies. Tetrahedron Letters, 1980, 21, 27, 2587-2590.

2. Mironov, S. J., Farberov, M. I., Commercial methods of synthesis of α,β-unsaturated aldehydes and ketones.

Russ. Chem. Rev. (1964), 33, 311-15.

3. Ziegler, K., Martin, H., Krupp, F., Metallorganische Verbindungen, XXVII Aluminiumtrialkyle und Dialkyl-

Aluminiumhydride Aus Aluminiumisobutyl-Verbindungen. Justus Liebigs Annalen der Chemie. (1960), 629

(1), 14–19.

4. Galatsis, P., Diisobutylaluminum Hydride in Encyclopedia of Reagents for Organic Synthesis John Wiley &

Sons: New York, (2001).

5. Penny, S. J., Reduction by Alumino- and Borohydrides in organic synthesis. 2nd edition, Viley-VCH, New

York, (1997), 97, P4.

6. Yeh, M. C. P., Lee, Y. C., Young, T. C., A Facile Approach to the Synthesis of Allylic Spiro Ethers and

Lactones. Synthesis, (2006), 3621-3624.

7. Dickson, H. D., Smith, S. C., Hinkle, K. W., A convenient scalable one-pot conversion of esters and Weinreb

amides to terminal alkynes. Tetrahedron Lett. (2004), 45, 5597-5599.

8. Porto, R. S., Vasconcellos, M. L. A., Ventura, E., Coelho, F., Diastereoselective Epoxidation of Allylic Diols

Derived from Baylis-Hillman Adducts. Synthesis, (2005), 2297-2306.

http://iopscience.iop.org/0036-021X/33/6/R01

http://en.wikipedia.org/wiki/Karl_Ziegler

http://en.wikipedia.org/wiki/Justus_Liebigs_Annalen_der_Chemie


22

9. Lee, Y., Akiyama, K., Gillingham, D. G., Brown, M. K., Hoveyda, A. H., Highly Site- and Enantioselective

Cu-Catalyzed Allylic Alkylation Reactions with Easily Accessible Vinylaluminum Reagents. J. Am. Chem.

Soc, (2008), 130, 446-447.

10. Gao, F., Hoveyda, A. H., Selective Ni-Catalyzed Hydroalumination of Aryl- and Alkyl-Substituted Terminal

Alkynes: Practical Syntheses of Internal Vinyl Aluminums, Halides, or Boronates. J. Am. Chem. Soc, (2010),

132, 10961-10963.

11. Busacca, C. A., Raju, R., Grinberg, N., Haddad, N., James, J. P., Lee, H., Lorenz, J. C., Saha, A.,

Senanayake, C. H., Reduction of Tertiary Phosphine Oxides with DIBAL-H. J. Org. Chem., (2008), 73, 1524-

1531.

12. Banday, A. H., Arora, B. S., Alam, M. S., Kumar, H. M. S., A novel ‘Domino-Click approach’ to

unsymmetrical bis-triazoles. Helv. Chem. Acta, (2007), 90, 12, 2368-2374.

13. Shafi, S., Banday, A. H., Ismail, T., Kumar, H. M. S., Domino addition/ N-C heterocyclization of Azides with

allenyl magnesium bromide: Rapid synthesis of 5-butynyl- 1,2,3-triazoles. Synlett, (2007), 7, 1109-1111.

14. Bhat, B. A., Shafi, S., Purnima, B., Banday, A. H., Kumar, H.M.S., Ferrier rearrangement for the synthesis of

PEG-bound 2,3-unsaturated glycopyranosyl amino-acids. Tetrahedron Lett. (2007), 48, 6, 1041-1043.

15. Concepción, P., Medarde, M. M., Feliciano, A. S., A Short Review on Cardiotonic Steroids and Their

Aminoguanidine Analogues. Molecules, (2000), 5, 51-81.

16. Kabat, M. M., Kurek, A., Wicha, J. Cardiotonic steroids. A synthesis of bufadienolides and cardenolides from

3.beta.-acetoxy-5-androsten-17-one via common intermediates. J Org Chem, (1983), 48, 4248-4251.

17. Schonecker, B. et al. Syntheses of precursors of 19-norcardenolides and bufadienolides, Pharmazie, (1986), 41

(5), 320-324.

18. Abid H. Banday A. H., Singh, S., Alam, M. S., Reddy, D. M., Gupta, B. D., Kumar, H. M. S., Synthesis of

novel steroidal D-ring substituted isoxazoline derivatives of 17-oxoandrostanes. Steroids, (2008), 73, 370–

374.


ISSN (Print): 2220-2625


*Corresponding Author Received September 20th 2012, Accepted January 4th 2013

pH Effect on Stoichiometry and Stability of Ferrous Complexes of

(-)-3-(3,4-dihydroxyphenyl)-L-alanine

*N. Fatima, S. Z. A. Zaidi, S. Nisar and M. Qadri

Department Of Chemistry, University Of Karachi, Karachi, Pakistan


ABSTRACT Living organisms contain many bio-molecules that are comprised of oxygen and nitrogen donor atoms and therefore have high

affinity for metal ions, such as iron, present in the body. The stability constants of these chelates are very high in general. One of

such molecules is (-)-3-(3,4-dihydroxyphenyl)-L-alanine which is present in brain to control the normal function of neurons. In

case of neurodisorder disease, Parkinson’s, this molecule is orally administered in the form of tablets. The pH of stomach is acidic

and blood is about neutral, therefore in the present work, the chelation, stoichiometry and stability of iron complexes of (-)-3-(3,4-

dihydroxyphenyl)-L-alanine is studied at acidic to neutral pH. Reaction of this molecule in vitro showed interesting results.

Stoichiometry of the complex is found 1:3 and it remains pH independent (in buffered & non buffered solutions).While stability of

the complex increases with the rise of pH as revealed by molar extinction coefficient values.

Keywords: Iron, Levodopa, stoichiometry, pH effect, molar extinction coefficient.

1. INTRODUCTION Metals by themselves and in proper balance to one another have important biochemical and nutritional functions1-2.

The bioavailability of these minerals is also a very important factor, depending on the food source and also upon the

pH of stomach3-4.

Iron plays significant role for normal brain and nerve function through its involvement in the synthesis of

neurotransmitters5. Severe iron deficiency anemia may result in many diseases6.On the other hand dopamine is

considered basic factor, responsible of behavioral changes in neurodisorder disease7-9. Iron and dopamine both play

significant role in normal function of brain. Also the excess iron is found in brain in Parkinsonian patients but still the

relation of iron and dopamine is ambiguous10-11.

Dopa of dihydroxyphenyl alanine is a precursor of dopamine and norepinephrine. It is levorotatory

stereoisomer of dopa12 while d-enantiomer leads to clinical side effects13. Levodopa is (-)-3-(3,4-dihydroxyphenyl)-L-

alanine, (Fig. 1) is a derivative of Amino acid (alanine) containing catechol moiety. Catechol has high affinity for

metal ions specifically iron, therefore Levodopa is expected to chelate iron strongly14-17.

Fig-1: (-)-3-(3,4-dihydroxyphenyl)-L-alanine Commonly known as Levodopa

In the beginning part of the project, the complex formation of (-)-3-(3,4-dihydroxyphenyl)-L-alanine with iron was

carried. Fe2+ forms intense color complex with (-)-3-(3,4-dihydroxyphenyl)-L-alanine having absorbance maxima in

visible region. Spectral characteristics of the said complex, stoichiometry and effect of pH on complex formation are

explored. Current research is carried to investigate the stability of complex at low pH, which in turn is helpful to

understand the chelation of iron with administered Levodopa in stomach.

2. EXPERIMENTAL A. R. grade reagents were used for all the reactions. CO2 free distilled deionized water was used to prepare the

solution.

2.1 Absorbance maxima: To explore wavelength of the maximum absorbance the complex of Fe2+ (-)-3-(3,4-dihydroxyphenyl)-L-alanine,

0.005mmol of Fe2+ salt solution was mixed with enough excess of levodopa solution (prepared in deionized distilled

water). The stock solution was subjected to scanning in UV-visible region on GENESYS 6 (Thermo Electron

Corporation). Using the resultant spectra, suitable wavelengths were selected for further study. The complex solution

showed absorbance maxima at two wavelengths; 430nm and 730nm. The metal and ligand solutions have no

absorbance at these wavelengths (Fig. 2). All further work was carried out on both wavelengths.

2.2 Molar extinction coefficients: Different dilutions of the stock complex solution were then prepared in deionized distilled water. Absorbance of all

diluted solutions was recorded at selected wavelengths (430 and 730nm). was determined as the slope of straight line


24

on a graph plotted between recorded absorbance against metal concentration. The same work was repeated using

buffer of pH 3.0, 4.0, 5.0 and 6.0.

2.3 Mole ratio Accurate amount of Ferrous ammonium sulfate was taken to prepare metal solution in deionized distilled water, while

the ligand solutions were prepared in buffer solutions of desired pH. At each pH, different aliquots of ligand solution

were added in 0.005mmol metal solution and volume was kept constant for all. The absorbance was recorded at

430nm and 730nm, while temperature was maintained at 25±1°C.

3. RESULTS AND DISCUSSION

3.1 Spectral Characteristics A green colored complex of Fe(II) and levodopa was formed at pH 4.0, 5.0 and 6.0 at 25+1°C except pH 3.0.

Absorbances were recorded at both the selected wavelengths. Spectra in Fig. 3 show drastic change with varying pH

(Fig. 3). No green color was observed in buffer of pH 3.0. Spectra in the above mentioned range has no peak.

Fig-2: Spectra of Fe (II) complexed with Levodopa in non-buffered aqueous solution. Fe(II) = 0.05mmol, 10 fold ligand solution

Fig-3: Spectra of Fe (II) complexed with Levodopa, pH 4.0, 5.0 and 6.0 buffer solutions. Fe (II) = 0.005 mmol, 10 fold ligand

solution

3.2 Molar Absorptivity

Fe(II) and levodopa formed a complex at pH buffer 4.0, 5.0 and 6.0 at 25+1°C and absorbance was recorded at all the

selected wavelengths and molar absorptivities were evaluated (Figure 4-5,Table-1). Two peaks at 430 and 730nm

were found

selected wavelengths increases simultaneously with pH. However in pH 6.0 buffered solutions, peak shift is observed

Fatima et al, 2013

25

-1cm-1.

Table-1: Molar Absorptivity of Fe(II)-Levodopa Complex at different pH at all Selected wavelengths

pH Molar Absobtivity (M

-1cm

-1)

430nm. 730nm. 615nm.

Non buffered 237 302.6 ----

4.0 783.1 633.7 ----

5.0 866.8 796.2 ----

6.0 1834 1735 2528

Fig-4: Molar Absorptivity of Fe (II)-Levodopa Complex, pH 4.0, 5.0 & 6.0, λ = 430nm.

Fig-5: Molar Absorptivity of Fe (II)-Levodopa Complex, pH 4.0, 5.0 & 6.0, λ = 730nm.

3.3 Stoichiometry Applying mole ratio method, In each set, different aliquots of ligand solution were added to 0.005mmol Fe (II)

solution in order to get 0.5-10 times L:M mole ratio. Gradual increase was observed in absorbance with the increase of

ligand to metal mole ratio (Fig 6). At high concentration of ligand the absorbance is independent of Ligand

concentration. Tangent drawn on the curve in Figure 6-9 gives a value of ~3 showing a 1:3 metal to ligand ratio in the

complex.

[Fe(H2O)6]2+

+ H2LD [Fe(H2O)4LD] +2H3O+

[Fe(H2O)4LD] +H2LD [Fe(H2O)2(LD)2]2

+2H3O+

[Fe(H2O)2(LD)2] +H2LD [Fe(LD)3] +2H3O+


26

Stoichiometry of Fe (II) and Levodopa Complex was further studied on pH 3.0, 4.0, 5.0 and 6.0. Consistency was

found in the results as shown in the Figure 7-9 (Table-2). At all the considered pH ML3 complex is formed regardless

of the solution pH.

Table-2: pH effect on the Stoichiometry of Complex

pH Stoichiometry Fe2+

(LD)3

Aqueous (non buffered) 1:3

4.0 1:3

5.0 1:3

6.0 1:3

Fig-6: Stoichiometry of Fe (II)-Levodopa Complex in non-buffered aqueous solution. Fe (II) = 0.005mmol, λ = 730nm.

Fig-7: Stoichiometry of Fe (II)-Levodopa Complex, pH 4.0, 5.0 and 6.0. Fe (II) = 0.005mmol, λ = 430nm.

Fatima et al, 2013

27

Fig-8: Stoichiometry of Fe (II)-Levodopa Complex, pH 4.0, 5.0 and 6.0. Fe (II) = 0.005mmol, λ = 615nm.

Fig-9: Stoichiometry of Fe (II)-Levodopa Complex, pH 4.0, 5.0 and 6.0. Fe (II) = 0.005 mmol. λ = 730nm.

4. CONCLUSIONS Complex formation of Fe (II) L-dopa is studied at pH 3.0, 4.0, 5.0 and 6.0 buffered and also in non buffered media.

Two distinct peaks were found in complex spectra, one at 430nmand another at 730nm. In pH 6.0 buffer, the 730nm

peak sharpens while shifted to 615nm.

At the selected wavelengths, Fe (II):H2L mole ratio plot showed an ML3 complex formation in spite of pH;

evident of stability of the investigated complex. The same result is verified by molar extinction coefficient values that

increase with the increase of pH. In ML3 complex, three levodopa molecules occupied all six positions around the

metal in an octahedral molecule. Thus levodopa acts as bidentate ligand forming a strong chelate.

Levo-dopa forms very strong complex with Fe(II) at stomach pH. The result is consistent with literature. It

showed that the Levodopa in drug molecule is chelated by iron and therefore may not efficiently reach to its required

destination.

The question arises whether this chelation helps the L-dopa to cross blood brain barrier or not. This important

point requires further investigation to be explored.

Since pH of brain is approximately 10. Work at high pH may provide imperative information about chelation

of Fe(II) by this molecule in brain. In-vitro study at high pH is in progress and will soon be published.

5. ACKNOWLEDGEMENT We are indebted to Dean’s Research grant of Science Faculty, University of Karachi that enabled us to carry out this

project.

6. REFERENCES

1. Atkins, P. W., Holker, J. S. E., Holiday, A. K. Oxford Univ. Press; Metals and Metabolism; (1978).

2. DAS, A. K., A Text Book on Medicinal Aspects of Bio-Inorganic Chemistry; (1990), CBS Publishers.

Delhi.

3. Khan, S. Y., Ph.D. Thesis, Department of Chemistry, University of Karachi, Pakistan, (2000).


28

4. Ruth, Pike L., Brown, M. L., Nutrition, An Integrated Approach, 1st ed.,283, John Wiley & Sons. (1984).

5. Beard, J. L., J Nutr., (2001), 131(2S-2), 568S-579S.

6. Lee, G. R., In: Lee, G. R., Foerster, J., Paraskevas, F., Greer, J. P., Rogers, G. M., eds. Wintrobe's Clinical

Hematology, 10th ed., (1999), 979-1070, Baltimore, Williams and Wilkins.

7. Barron, A. B., Maleszka, R., Vander, Meer, R. K., Robinson, G. E., Proc. Natl. Acad. Sci. U.S.A. (2007),

104(5), 1703-7.

8. Vanden Heuval, D. M. A., Pasterkamp R. J. Progress in Neurobiology, (2008), 85, 75-9.

9. Arias-Carrión, O., Pöppel, E., Act Neurobiol Exp., (2007), 67(4), 481-488.

10. Gerlach, M., Ben-Shachar, D., Riederer, P., Youdim, M. B. H., J. Neurochem.,(1994), 63, 793-807

11. Parkinson's Facts. www.parkinson.org. Miami: National Parkinson's Foundation, Inc. 1996-2000

12. Renth, E.O. Agewandte Chemie, (1975), 14(7), 361.

13. Ross, B. M., Moszcynska, A., Ehrlich, J., Kish, S., J. Neuroscience, (1998), 83, 791-798.

14. Iffat, A. T., Maqsood, Z. T., Fatima, N., J. Chem. Soc. Pak., (2005), 27(2), 174-177.

15. Fatima, N., Pak. J. Chem. (2012), 2(2), 91-98.

16. Fatima, N., Maqsood, Z. T., J. Saudi Chem. Soc. (2005), 9(3), 519-528.

17. Gulzar, S., Fatima, N., Maqsood, Z. T., J. Saudi Chem. Soc., (2005), 9(1), 113-118.


ISSN (Print): 2220-2625


*Corresponding Author Received September 14th 2012, Accepted January 3rd 2013

Estimation of Chromium in Effluents from Tanneries of Korangi Industrial Area

*R. Parveen, M. Ashfaq,

1J. Qureshi, S. M. M. Ali and M. Qadri

Department of Chemistry, University of Karachi, Pakistan

1King Abdul Aziz University, Saudi Arabia


ABSTRACT The samples were collected from the tanneries located in Korangi industrial area, have high chromium concentration exceeding

the tolerable limit.The effluents from tanneries are directly disposed off into streams without any treatment or ineffective method

are used for treatment. The concentration of chromium was found to be 18.57-.170.12 ppm in residue and 15.20-185.50 ppm in

filtrate of korangi industrial effluent in 2011. The conductance varies from 6.7 to 175 S/m which shows the high concentration of

ionic species. The pH of samples was found to be mostly alkaline (7.0-8.9) except in T1 of January.

Key words: tanneries effluents, Chromium (VI) and (III) potential, conductance, pH

1. INTRODUCTION Chromium (Cr) is an environmentally important metal used in a range of industrial processes1.Chromium as an

environmental toxin increases in the system by electroplating, metal finishing, chromate preparation, leather tanning

etc. The Cr (III) and Cr (VI) are predominant oxidation states of chromium present in the environment. These two

main oxidation states significantly differ in biological, geochemical and//toxicological properties1. The Cr (VI) is

soluble, toxic and carcinogenic1, while Cr (III) over a narrow concentration range, is considered vital for mammals.

The Latter maintain many metabolic processes of glucose, lipid and protein, whereas Cr (VI) is reported to have a

toxic effect on humans. It is mutagenic, carcinogen, cause of allergic contact dermatitis6. Chromium (ΙΙΙ) is

characteristically treated by increasing the pH of the industrial effluent through adding chemicals and coagulants such

as lime and Fe compounds in order to recover precipitated chromium hydroxide. Numerous studies have reported

99.5% recovery using this method7-8.

Chromium (III) sulphate salts mostly are used as tanning agent, consequential in severe groundwater contamination

around tanneries, which is oxidized into Cr (VI)9. The Brazilian environmental legislation states that total Cr in final

effluents10 should not exceed 0.5 mgl-1. The limits of 0.5 and 50 mg l-1 for Cr (III) and Cr (VI) were only specified for

the domestic supplied water, respectively11. Therefore, the determination of total Cr is still very useful, even when

speciation is necessary, one formis determinate and the other form is calculated by difference. Iwata et al12 used photo-

reduction method on the surface of titanium oxide (TiO2) films of dilute Cr(VI) solutions at an adjusted pH 2 for a

period of 7 days. It was found that concentration of Cr (VI) ion decreased from 10ppm to 0.03ppm. Three different

analytical methods comprising colorimetric method with 1, 5-diphenyl-carbazide, electro-thermal, atomic absorption

spectrometry (ET AAS) and flame atomic absorption spectrometry were utilized dy to determine traces of chromium

(Cr) in synthetic tannery effluent from laboratory scale treatment process variation13. Rajmond14 use a mixed bed ion

exchange column for the simultaneous determination of Cr (III) and Cr (VI) using UV detector from environmental

samples such as rainwater and galvanic sediments. The method was conventional from the linearity, limit of detection,

limit of quantification, and the influence of sample pH. Similarly, Jen-Fon et al15 used a reversed-phase ion – pair

high-performance liquid chromatographic method for simultaneous determination of Cr (III) and Cr (VI) in an

aqueous solution. The Cr (III) was first chelated with ethylenedi-aminetetraacetic acid (EDTA) and separated with C8–

column using an effluent containing acetonitrile and tetrabutylammonium ion. The separated species were then

monitored with a UV – detector at 242nm.

In another work Noroozifar and Khorasani16 established a method that is very specific, selective, simple and

inexpensive for the speciation of Cr (VI) and Cr (III) from spiked natural water samples and effluent samples from

leather treatment plant. The method is based on the quantitative extraction of chromate and Cr (III) as

tetrabutylammonium – chromate ion-pair in methyl isobutyl ketone (MIBK), and then back extraction and pre-

concentration with an acidic diphenylcarbazide (DPC) solution. Whereby Cr (VI) – DPC complex was determined by

a spectrophotometer at 548nm.

The present work is to estimate the concentration of chromium in effluents of tanneries in Korangi Industrial

Area Karachi. We focused on chromium as it is the major require constituent of tanneries.

2. MATERIAL AND METHOD The digestion of samples was carried out in Nitric acid (Merck AR Grade) and Perchloric acid (70% Merck AR

Grade). Standard solutions of chromium for calibration curve were prepared by Chromium solution 1000µg/ml in

1wt% HCl (Sigma Aldrich, Atomic Absorption Grade).


30

Samples were collected from various sampling units' sites from outlets of different Tanneries at distance of 5-10 feet

in Korangi Industrial Area. The locations of tanneries are reported as GPRS locations [Table-1].These samples were

collected and stored in plastic bottle of 50 cm3, coded and brought to laboratory for chromium analysis.

Portion of sample of about 40 ml filtered through whatman 40. Physical measurements have been done by the

parameters such as pH, conductivity and potential of the filtered effluent samples. The pH of the samples was

measured with Jenway 3320 pH meter. Conductivity values were determined by the use of Jenway 4010 conductivity

meter. Table-1: Locations of samples sites

Sites Degree Feet Inch Direction

T1 24° 5.7' 19.18" North

67° 07' 07.07" East

T2 24° 51' 19.69" North

67° 07' 9.79" East

T3 24° 51' 13.44" North

67° 7' 15.37 East

T4 24° 51' 9.82" North

67° 7' 11.13" East

T5 24° 51' 18.02" North

67° 7' 11.66" East

T6 24° 7' 14.80" North

67° 7' 14.80" East

For chromium analysis atomic absorption spectrophotometer was used (Model: Perkin - Elmer AA3100).

Nitric acid and Percholric acid were used as oxidizing agents to destroy matrix. 40 ml of sample solution was filtered

through Whatman’s 40 filter paper.10 ml nitric acid (60%) was added to the filtrate and heated until got a clear

solution. Finally the volume of the filtrate was made up to the mark in 50.0 ml volumetric flask with double distill de-

ionized water.

Residue collected on the Whatman´s 40 filter paper, was dried in oven and digested in 10 ml nitric acid (60%) and

heated to frothing. Subsequently, 3 ml Percholric acid (70%) was added to dissolve residue completely. Then 1:1 ratio

of HCl and water was used to make up the volume up to 50 ml of solution.

3. RESULTS AND DISCUSSION The major sources of chromium in sea water are effluents from industries. The different sources of chromium are

reported by Ismail et al17.

The concentration of chromium was found to be 18.75-170.12 ppm in residue and 15.2- 185.5 ppm in filtrate

of Korangi Industrial effluent in 2011 [Table-2]. This variation of concentration is found according to the availability

of raw material. The lowest concentration in filtrate and residue found in tannery coded as T1 15.02±0.76 ppm in

January and T1 (18.57±0.21ppm) in March respectively [Table-2]. The highest value of chromium concentration

estimated in filtrate of T4 (185.75±1.03 ppm) and residue of T6 (170 1.30ppm) in November [Table-2].The highest

concentration of chromium was observed in month after Eid Qurban, when the highest amount of raw material is

available. The mean concentration of chromium in the water is higher than the limit (0.05mg/l) recommended by the

World Health Organization (WHO) (1989) in drinking water. The levels of chromium in effluents below than 1 mg/l

as recommended by the Federal Ministry of Environment (FMENV, 1991)18.The level of chromium in the collected

effluents is much higher than recommended value indicated by Federal Ministry of Environment. In tanneries, oxide

of Cr(VI) is used for softening and oxidation process. The minimum pH for removing chromium from the tannery

wastewater by sodium hydroxide, calcium hydroxide and magnesium oxide as precipitating agent is pH 8-919.The pH

variation is found to be 7 to 8.7.

In the pH range of 7, hexavalent chromium exists as CrO42-

, HCrO41-and Cr2O7

2- ions. These ions are

relatively soluble and therefore mobile in the environment. In basic pH tetrahedral, yellow CrO42- chromate ion is the

prevailing specie formed by CrO3, while the orange-red Cr2O72- ions are in equilibrium with HCrO4

1- in the pH range of

2 - 620. Mostly the pH range of samples is basic; therefore the most likely form of the hexavalent chromium in the

effluent is CrO42-as the pH of samples are mostly basic.

The reason of a T1 in January having acidic pH might be due to accidentally matching the time of sample

collection with the time when the acid were drain off during the tanning process.

More obvious reason for this high level of industrial pollution shows effluent are dispose off directly to the

stream without any treatment. The other reason of high concentration of chromium is the use of already polluted

ground water in different industries. This is actually due to leaching from dump sites, poor sanitation and industrial

activities21. As its concentration is diluted by distance from source to sea but still its level is harmful to aquatic biota

and Mangroves along the coastal areas22. Conductance of the samples varied between 6.7 to 179 S/m. The

Parveen et al, 2013

31

conductance may be attributed to the presence of Na+, Cl- and other metal ions. As NaCl salt is extensively used in

tanneries for leather preservation.

The values of the potential varied between -90 to +709 mV of different industrial effluent. All tanneries in

different months have high positive potential, except T1 in July and T3 in March and May samples have negative

potential. Potential can also give us idea about dominating oxidation state of chromium.

Table-2: The concentration of chromium in korangi Industrial are in different months form different tanneries

Sample Month pH Conductance

(S/m)

Potential

(mV)

Cr in filtrate

(ppm)

Cr in residue

(ppm)

T1

1 January 3.57 132 158 15.20 0.76 36.39 0.56

2 March 7.96 134 10 53.12 0.85 18.57 0.21

3 May 7.92 122 155.9 59.52 0.68 36.15 0.13

4 July 8.50 102 -50.6 89.25 0.85 78.72 1.00

5 Sept 7.95 95 92 72.89 0.978 59.12 0.85

6 Nov 7.85 94 33.7 181.11 0.94 147.83 0.23

T2

1 January 8.16 64.6 125 49.35 0.75 33.65 0.85

2 March 8.05 54.4 135 40.12 0.73 42.45 0.35

3 May 8.01 40.5 110 58.50 0.56 36.13 0.85

4 July 8.72 37.2 157 75.75 0.95 42.83 0.86

5 Sep 8.41 49.7 159 78.50 1.1 40.89 0.92

6 Nov 8.70 44.6 110 96.25 1.6 122.13 1.20

T3

1 January 7.94 89.5 152.5 33.51 0.66 24.15 0.52

2 March 8.01 75 -90 35.25 0.74 36.14 0.23

3 May 8.09 9.6 -89 41.75 0.56 22.35 0.34

4 July 7.58 17.6 138 90.75 0.75 82.89 0.93

5 Sep 7.52 20.1 130 77.50 0.95 62.13 0.76

6 Nov 7.96 20.9 140 162.25 0.85 136.12 1.00

T4

1 January 8.63 6.9 234 52.59 0.87 43.41 0.65

2 March 8.68 6.7 709 63.75 0.95 34.59 0.55

3 May 8.71 6.8 222 58.25 0.72 22.48 0.75

4 July 7.44 39.8 268 102.75 0.97 89.15 0.86

5 Sep 7.01 39.6 254 109.82 0.75 71.25 0.83

6 Nov 7.96 40.8 266 185.50 1.03 150.95 0.75

T5

1 January 8.43 31.42 150 37.25 0.83 47.25 0.95

2 March 7.99 11.51 178 51.25 0.45 48.92 0.81

3 May 7.9 26.8 169 70.51 0.65 49.78 0.35

4 July 7.44 39.8 268 103.23 1.1 95.75 0.76

5 Sep 7.01 39.6 254 99.52 0.87 83.78 0.95

6 Nov 7.96 40.8 266 158.61 0.87 142.10 0.89

T6

1 January 7.07 27.90 177 70.20 0.53 52.14 0.56

2 March 7.09 17.5 180 85.35 0.89 49.13 074

3 May 7.21 115 169 73.31 0.45 50.12 0.98

4 July 7.07 179 235 102.03 0.76 92.45 0.86

5 Sep 7.09 75.5 290 93.45 0.95 99.89 0.92

6 Nov 7.21 125 211 162.15 0.98 170.12 1.30

The reduction reaction of chromium +6 to chromium +3 occurs at -170 mV. No clear picture can be obtained from

these redox potential values, as different redox couple may also exist. Mayer et al22 used redox potential data to

control effluent the concentration of ammonia23.The Two way ANOVA by Minitab software was applied to the data.

There is a significant difference found with respect to month both in residue and filtrate, Fcal=77.55, 36.19

respectively. Whereas insignificant difference between sites was observed for filtrate Fcal =2.693 while it is

significant for residue Fcal 5.12 shown in Table 3 and 4.


32

Table-3:Two-way Anova for Filtrate Sources of Variances Sum of Square Degree of Freedom Mean Square Fcrit

Sites 2738.251279 5 547.6502558 2.639641278

Months 37551.39877 5 7510.279754 36.19909648

Residuals 5186.786746 25 207.4714698

Total 45476.4368 35

Chromium toxic effects are associated with long term low level exposure to pollutants common in our environment:

air, water, food and numerous consumer products23-24. Exposure to chromium is associated with many chronic diseases

such as dermatitis, ulcers and perforation of the nasal septum and respiratory illness as well as increased lung and

nasal cancer25.The adsorption methods is developed in the optimized conditions like pH, shaking time and amount of

adsorbent. The concentration of Cr after removal was determined by atomic absorption spectrophotometer26.

Table-4: Two-wayAnova for residue

Sources of Variances Sumof Square Degree of Freedom Mean Square Fcrit

Sites 2084.685946 5 416.9371892 5.126618057

Months 31538.21992 5 6307.643984 77.55816076

Residuals 2033.198029 25 81.32792117

Total 35656.1039 35

4.CONCLUSION Keeping in view the toxic effect as chromium as pollutant from tanning industries, it was found that all the samples

from the tanneries located in Korangi industrial had high chromium values greater than the tolerable limit.They were

introduced into streams directly, due to lake of disposal regulation and non-availability of treatment. The public are

unaware about the water quality in Pakistan. This problem is increasing day by day. Efforts have been made and new

methods have been suggested for controlling these problems, but yet not are successful as due to expensive or not

completely effective to remove all the pollutants including chromium.

Government authorities must be strict against the law violating industries and people’s awareness program

must have to be started otherwise we have to pay the consequences.

5. Acknowledgement:

We are thankful to Dean Faculty of Science, University of Karachi, for Providing us grant to support the project.

6. REFERENCES 1. Fendorf, S. E., Lamble, G. M.,Stapleton, M. J., Kelley, M. J. and Sparks, /D.L.Environ. Sci. Technol., (1994),

28, 284.

2. Ackerly, D. F., Gonzalez, C. F., Park, C. H., Balke, I. R., Keyhan, M. and Martin, A. Appl. Environ.

Microbiol., (2004), 70, 873.

3. Stein, K., Schwedt, G. Fresenius, J. Anal. Chem., (1994), 350, /38.

4. Cespo´n-Romero, R. M., Yebra-Biurrun, M. C. and Ber-/mejo-Barrera, M. P. Anal. Chim.Acta, (1996), 327,

37.

5. Sule, P. A. and Ingle, J. D. Anal. Chim. Acta, (1996), 326, 85.

6. Sperling, M., Welz, S. and Xu, B. Anal. Chem., (1992), 64, 3101.

7. Nancy, J. S., Industrial pollution control issues and techniques. 2nd Ed., Van Nastrand Reihold New york,

(1992), 324.

8. Nemerow, N. Industrial and hazardous waste treatment. VNR, New York, (1991), 409.

9. Thakur, I. S., Verna, P. and Upadhyaya, K. C. Biochem. Biophys. Res. Comm., (2001), 286, 109.

10. FEEMA, Standards for liquid effluent discharge, Environmental Agency of Rio de Janeiro state, NT-202.R-

10, Rio de Janeiro, Brazil, 1986.

11. CONAMA. Standards for domestic water supply, Environmental National Council of Brazil, Brazil,

ResolutionNo20, 1986.

12. Iwata, T., Ishikawa, M., Ichino, R. and Okido, M. Surface and Coating Technology, (2003), Volumes 169–

170.

13. Monteiro, M. I. C.,Fraga, I. C. S.,Yallouz, A.V.,de Oliveira, N. M. M.and Ribeiro, S. H. Talanta, (2002), 58,

629.

14. Rajmond, M. J. Liq. Chromat. & Related Tech., (2005), 28, 2849.

15. Jen-Fon Jen., Guang-Lien, Chi-Shi Ou Yang and Shih-Ming Yang. Analyst, (1993), 118, 1281.

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16. Noroozifar, M. and Khorasani-Motlagh, M. Anal. Sci., (2003), 19, 705.

17. Ismail, S., Saifullah, S. M. andKahn, S. H. J. Chem. Soc. Pak., (2006), 28, 426.

18. World Health Organization (WHO). Guidelines for Drinking-waterQuality. Health Criteria and Supporting

information. World Health reviewing Organization, Geneva, (1989), 2.

19. Esmaeili, A., Mesdaaghi Nia, A. and Vazirinejad, R. Am. J. Appl. Sci., (2005), 2, 1471.

20. Ibrahim, M. Biosciences Research communications, (2008), 20,293.

21. Raza, R. and Maqsood, Z. T. J. Chem. Soc. Pak., (2005), 27, 258.

22. Myers M., Myers, L. and Okey, R. The use of Oxidation –Reduction Potential as a mean of Controlling

Effluent of Ammonia Concentration in an Extended aAeration Activated Sludge. University of Utah,Water

Environment Foundation. (2006), 5901-5925.

23. ATSDR, Toxicological profile for chromium. US Department of Healthand Human Service, Public Health

Service. 2008, 61.

24. Atiq-ur-Rahman, S. and Iqbal, M. Z. Pak. J. Chem., (2011), 1,43.

25. Pandey, V.C., Singh, J.S., Kumar, A., Tewari, D. D. Clean – Soil, Air, Water, (2010), 38, 1116.

26. Tahir, H.,Yasmeen, G., Akhtar, N., Sultan M. and Qadri M. Pak. J. Chem., (2012), 2,1.


ISSN (Print): 2220-2625


*Corresponding Author Received 8th October 2012, Accepted 5th February 2013

Determination of 106

Ru,134/137

Cs, and 241

Am concentrations and action level in the

foodstuffs consumed by inhabitants of Iraq

*H. N. Majeed and A. K. Hasan

Education College for girls, Kufa University, Al-Najaf Al-Ashraf, Kufa, Iraq.


ABSTRACT The specific activity concentrations of (106Ru, 134/137Cs, and 241Am) nuclides in 40 imported foodstuffs which collected randomly in

January 2012 from all Iraqi cities markets were studied.

The rang of specific activity concentrations of 106Ru varies from (37.930±6.16) Bq kg-1 (S No. :17: Turkey Kidney bean) to

99.735±9.99 Bq kg-1 (S No.:32: Egypt Broad bean), with average value 71.667±8.47 Bq kg-1. For 134Cs varies from 0.200±0.45 Bq kg-

1 (S No. :19 : Ukraine Chick-pea) to 2.365±1.54 Bq kg-1 (S No. :33 : Peru Broad bean) with average value (0.988±0.99) Bq kg-1.The

activity concentrations of 137Cs varies from 0.164±0.40 Bq kg-1 (S No.:19 : Ukraine Chick-pea) to 5.291±2.30 Bq kg-1 ( S No.: 39:

Uzbekistan Mung bean) with average value 1.460±1.21, then for 241Am the activity concentrations varies from 0.029±0.17 Bq kg-1 (S

No.:23 : Iran Chick-pea) to 1.248±1.12 Bq kg-1 (S No.:40: Canada Green peas) with average value 0.399±0.63. All the values were

less than the World average concentrations [15,17]. The high contributor for 106Ru, 134/137Cs, and 241Am radionuclides were in Broad

bean and other foodstuffs (which contained Brown grit, White grit, Mung bean and Green peas) as a 12%, Broad bean as 14%, corn as

a 19% and other foodstuffs with 15% respectively

The lowest contributor of 106Ru, 134/137Cs, and 241Am radionuclides in the studied foodstuffs were 6% in cowpea, 7% in semolina, 5%

in lentil and 4% in lentil respectively.

The action level of the 106Ru, 134/137Cs, and 241Am radionuclide’s for three age groups have been calculated and the foodstuffs were

within the range permitted and free of any radiation and thus there was no seriousness in dealing with.

Keywords: The specific activity concentrations, the action level, foodstuffs, Iraqi markets and 106Ru, 134/137Cs, and 241Am

radionuclides.

1. INTRODUCTION Radiation from natural sources gives more than 80 % of the total exposure received by the average member of a

population and a portion of this exposure comes from dietary intake. The natural radioactivity elements are distributed

everywhere in the environmental with different concentrations, their concentrations have been found to depend on the

local geological condition and as such they vary from one place to another. It is necessary to monitor release of

radioactivity into the environment in order to be able to provide an appropriate protection of humans1-3.

There are four main components of general background radiation, Natural radioactivity in food and water and

inhaled air, Natural terrestrial radiation from our immediate environment, including buildings, Natural cosmic radiation

from Sun, stars and from galactic and intergalactic plasma and Medical and industrial applications. The biological effect

of ionizing radiation, such as gamma rays, X rays, and fast electrons is often nearly proportional to the absorbed radiation

energy; that is, it is proportional to the radiation dose4. Knowledge of natural radioactivity in man and his environment is

important since naturally occurring radionuclides are the major source of radiation exposure to man5. Radioactive nuclides

present in the natural environment enter the human body mainly through food and water and these measurements serve as

the basic standards against which occupational exposures are assessed6.

Although many different kinds of radionuclides can be discharged following a major nuclear emergency some are

very short lived and other do not readily transfer into food. Radionuclide s generated in nuclear installations and that

could be significant for the food chine include:

radioactive hydrogen (3H), carbon (14C), technetium (99Tc), sulphur (35S), cobalt (60Co) strontium (89Sr and 90Sr),

ruthenium (103Ru and 106Ru), iodine (131I and 129I), uranium (235U) plutonium (238Pu, 239Pu and 240Pu), caesium (134Cs and 137Cs), cerium (103Ce), iridium (192Ir), and americium (241Am).The radionuclides of most concern for possible transfer to

foods have been considered when setting the Codex Guideline levels described below. Of immediate concern is iodine-

131, it is distributed over a wide area, found in water and on crops and is rapidly transferred from contaminated feed into

milk. However, iodine-131 has a relatively short half-live and will decay within a few weeks. In contrast, radioactive

caesium which can also be detected early on, is longer-lived (Cs-134 has a half life of about 2 years and Cs-137 has a half

life of about 30 years) and can remain in the environment for a long-time. Radioactive caesium is also relatively rapidly

transferred from feed to milk. Uptake of caesium into food is also of long term concern7.

Intervention in emergency exposure situations is carried out on the basis of intervention and action levels.

Intervention levels (IL) are expressed in terms of the dose that is expected to be avoided or averted over time by a specific

mailto:*[email protected]


35

protective action associated with the intervention. Action levels (AL) are defined in terms of the dose rate or activity

concentration above which protective or remedial actions are generally recommended. Action levels for food and water

correspond to the radionuclide concentrations that could lead to an individual receiving a dose equal to a specified

intervention level, assuming that the contaminated portion of the diet remain sat the action level for the duration of the

assessment period4.

In this paper the specific activity and action level of 106Ru, 134/137Cs, and 241Am radionuclides in 40 samples of

foodstuffs from Iraqi markets for three age groups were studied, the reason of this study is to investigation the foodstuffs

of Iraqi market as well as to calculate the action level and the validity of such material for human ingestion as a preventive

measure, all of the collected samples have been product in 2012 and still in use to 2014. The studying foodstuffs

considered essential items in breakfasts and lunch meals for most simple Iraqi families which mixture two or three type of

these foodstuffs to do the meals.

2. EXPERIMENTAL PROCEDURES In order to measure the natural radioactivity in foodstuffs a total of 40 samples were collected randomly in January 2012,

from all Iraqi cities, the sample were crushed to fine grain size and sieved in order to homogenize it and remove big size.

The powdered samples were packed in a marinelli beaker, one kilogram from each sample and sealed tightly cap, each

sample was counted for 5 hour on the Gamma spectrometer with scintillation detector 2" 2" inch NaI (Tl) from

SPECTRUM TECHNIQUES, INC.US, the background spectra was also collected for the same period of time and

subtracted from the sample spectra.

The detector was calibrated using six radionuclides with eight γ-ray lines emitted ranged from 80keV for Ba-133

to 1332.5 keV for Co-608-9 which has been done for calculation the efficiency calibration see fig-1.

The detector was surrounded by a lead shield to reduce the background of the system. The activity of a specific

radionuclide with a gamma energy transition could be expressed using the following equation10-11.

].[......

1kgBqtmI

N

tmI

NA

netnet (1)

Where netN is the net counts (area under the specified energy peak after back ground subtraction) in (C/s), netN is the

random error in (C/s), is the efficiency of the detector, Iγ is the transition probability of the emitted gamma ray, t is the

time (in sec) for spectrum collected and m is the sample weight in (kg).

The specific activity concentrations of the 106Ru, 134/137Cs, and 241Am, were

determined by the γ-ray transitions8-9 are as follows:

1. 106Ru (1050.41) keV.

2. 134Cs (604.720) keV.

3. 137Cs (661.657) keV.

4. 241Am (59.5409) keV.

The action level for a given radionuclide within a particular food and age group is calculated as12:

jkikj

kjifDCM

ILAL

,,

,, (2)

Fig. (1a):Energy calibration curve of 2"×2" NaI(Tl) detector.

E = 1.4311Ch- 9.8696

R2 = 1

0

200

400

600

800

1000

1200

1400

0 200 400 600 800 1000

Channel Number

Energ

y (

keV

)

Fig. (1b):Full energy peak eff iciency as a function of gamma ray energy for

2"×2"NaI(Tl) detector.

ε = 7.3448(E)-0.8561

R2 = 0.8678

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0 200 400 600 800 1000 1200 1400

Energy (keV)

Effic

iency %

Majeed et al, 2013

36

Where kjiAL ,,is the action level for radionuclide i in food group j and age group k in (Bq kg-1), IL is the intervention level

(Sv), Mj,k is the mass of food group j consumed by age group k over the assessment period (kg/year), DCi,k is the ingestion

dose coefficient for radionuclide i and age group k (Sv Bq-1) and fi = contamination factor, equivalent to intake of food

group j assumed to be uniformly contaminated to the full value of kjiAL ,, .

Both the mass of food consumed and the radionuclide ingestion dose coefficients are age-specific, and their

values reflect the classification of the population into a limited set of representative age groups. The above definition of

the action level results in a distinct value for each radionuclide, age and food group combination12-13.

If several radionuclides are present in a sample the following summation criterion must be satisfied:

1i

i

AL

A (3)

Where Ai is the measured activity of radionuclide i, and ALi is its corresponding action level11.

The radiological impact of a particular radionuclide released into the environment during a nuclear emergency is a

function of its abundance and its environmental, biological and radiological properties. The radionuclides which are most

likely to be predominant contributors to dose through ingestion following a nuclear reactor accident are dependent on the

type of facility and the severity of the event, typically, those having the most significance to dose from the ingestion of

contaminated food and water are 89/90Sr, 103/106Ru, 131I, 134/137Cs, 238/239Pu and 241Am12-7. This group of radionuclides is most

likely to be of concern in terrestrially produced foods. Biological concentration processes in fresh water and marine

systems can result in very rapid transfer and enrichment of specific radionuclides12-15. The gamma radionuclide’s which

are measured by NaI detector was (106Ru, 134/137Cs, and 241Am).

3. RESULTS AND DISCUSSION Iraqi families dependent in Primary shape in preparing meals on mixing three types or more of foodstuffs per meal

therefore which may be a major cause of the overflow allowed the concentration.

The specific activity concentrations of ( 106Ru,, 134/137Cs, and 241Am) nuclides in 40 imported foodstuffs which

collected randomly from the Iraqi markets were in table 1.& fig. (2) the rang of specific activity concentrations of 106Ru

varies from (37.930±6.16) Bq kg-1 (S No. :17: Turkey Kidney bean) to 99.735±9.99 Bq kg-1 (S No.:32: Egypt Broad

bean), with average value 71.667±8.47 Bq kg-1. For 134Cs varies from 0.200±0.45 Bq kg-1 (S No. :19 : Ukraine Chick-pea)

to 2.365±1.54 Bq kg-1 (S No. :33 : Peru Broad bean) with average value (0.988±0.99) Bq kg-1.

The specific activity concentrations of 137Cs varies from 0.164±0.40 Bq kg-1 (S No.:19: Ukraine Chick-pea) to 5.291±2.30

Bq kg-1 ( S No.: 39: Uzbekistan Mung bean) with average value 1.460±1.21, for 241Am the specific activity concentrations

varies from 0.029±0.17 Bq kg-1 (S No.:23 : Iran Chick-pea) to 1.248±1.12 Bq kg-1 (S No.:40: Canada Green peas) with

average value 0.399±0.63. All the values were less than the world average concentrations16-18.

Fig. (2 a): The specif ic activity of 106Ru in Bq.kg-1.

0.000

55.000

110.000

1 4 7 10 13 16 19 22 25 28 31 34 37 40

Sample No.

Count/S

ec.

106Ru

Fig. (2 b): The specif ic activity of 134Cs in Bq.kg-1.

0.000

1.250

2.500

1 4 7 10 13 16 19 22 25 28 31 34 37 40

Sample No.

Count/sec.

134Cs

Fig. (2 c): The specif ic activity of 137Cs in Bq.kg-1.

0.000

3.000

6.000

1 4 7 10 13 16 19 22 25 28 31 34 37 40

Sample No.

Count/S

ec.

137Cs

Fig. (2 d): The specif ic activity of 241Am in Bq.kg-1.

0.000

0.500

1.000

1.500

1 4 7 10 13 16 19 22 25 28 31 34 37 40

Sample No.

Count/S

ec.

241Am


37

Table-1: The specific activity concentrations of (106Ru, 134/137Cs, and 241Am). radionuclide s for each sample in (Bq. kg–1).

Sample No. Type Origin country 106Ru 134Cs 137Cs 241Am

1 Rice, (272) India 76.574±8.75 1.123±1.06 1.033±1.02 0.273±0.52

2 Rice,(Alwalima) India 61.629±7.85 1.058±1.03 2.881±1.70 0.131±0.36

3 Rice, (1121Sella) India 83.882±9.16 0.608±0.78 0.625±0.79 0.049±0.22

4 Rice, (Alaella) India 87.097±9.33 1.044±1.02 1.745±1.32 0.317±0.56

5 Rice Thailand 93.533±9.67 0.649±0.81 0.522±0.72 0.600±0.77

6 Semolina Turkey 63.991±8.00 1.465±1.21 0.860±0.93 0.345±0.59

7 Semolina Italy 57.489±7.58 0.533±0.73 1.764±1.33 0.513±0.72

8 Semolina Syria 77.339±8.79 0.559±0.75 0.928±0.96 0.659±0.81

9 Semolina Lebanon 78.627±8.87 0.379±0.62 0.181±0.43 0.498±0.71

10 Semolina Saudi Arabia 70.428±8.39 0.759±0.87 1.017±1.01 0.518±0.72

11 Semolina Kuwait 71.140±8.43 0.702±0.84 1.284±1.13 0.697±0.83

12 Wheat (type 1) Turkey 74.821±8.65 1.427±1.19 1.530±1.24 0.529±0.73

13 Wheat (type 2) Turkey 67.333±8.21 1.252±1.12 0.738±0.86 0.682±0.83

14 Kidney bean China 87.188±9.34 1.017±1.01 1.034±1.02 0.546±0.74

15 Kidney bean Argentina 83.600±9.14 1.084±1.04 1.392±1.18 0.271±0.52

16 Kidney bean Kyrgyzstan 78.271±8.85 1.574±1.25 1.497±1.22 0.527±0.73

17 Kidney bean Turkey 37.930±6.16 0.723±0.85 0.898±0.95 0.807±0.90

18 Kidney bean Lebanon 45.937±6.78 0.672±0.82 0.738±0.86 0.266±0.52

19 Chick-pea Ukraine 41.138±6.41 0.200±0.45 0.164±0.40 0.260±0.51

20 Chick-pea India 49.604±7.04 0.631±0.79 1.558±1.25 0.077±0.28

21 Chick-pea Italy 82.172±9.06 0.980±0.99 0.225±0.47 0.380±0.62

22 Chick-pea Russia 79.154±8.90 0.954±0.98 2.118±1.46 0.260±0.51

23 Chick-pea Iran 82.857±9.10 0.284±0.53 1.307±1.14 0.029±0.17

24 Red lentil Turkey 85.081±9.22 0.725±0.85 0.658±0.81 0.046±0.22

25 Grain lentil Canada 78.693±8.87 0.800±0.89 0.690±0.83 0.191±0.44

26 Grain lentil Lebanon 72.941±8.54 1.800±1.34 0.790±0.89 0.139±0.37

27 Grain lentil Iran 67.114±8.19 0.341±0.58 1.144±1.07 0.207±0.46

28 Cowpea Peru 47.991±6.93 1.972±1.40 0.615±0.78 0.111±0.33

29 Cowpea Madagascar 40.608±6.37 0.704±0.84 0.841±0.92 0.376±0.61

30 Cowpea Iran 46.559±6.82 0.918±0.96 1.360±1.17 0.242±0.49

31 Broad bean Syria 69.934±8.36 1.207±1.10 0.710±0.84 0.153±0.39

32 Broad bean Egypt 99.735±9.99 1.171±1.08 2.018±1.42 0.657±0.81

33 Broad bean Peru 84.527±9.19 2.365±1.54 4.199±2.05 0.934±0.97

34 Corn Turkey 75.515±8.69 1.512±1.23 1.241±1.11 0.066±0.26

35 Corn Argentina 96.310±9.81 0.735±0.86 3.618±1.90 0.191±0.44

36 Corn Saudi Arabia 91.836±9.58 1.121±1.06 3.220±1.79 1.020±1.01

37 Brown grit Lebanon 84.193±9.18 0.826±0.91 3.479±1.87 0.501±0.71

38 White grit Turkey 46.414±6.81 0.850±0.92 0.759±0.87 0.100±0.32

39 Mung bean Uzbekistan 68.146±8.26 1.634±1.28 5.291±2.30 0.563±0.75

40 Green peas Canada 79.331±8.91 1.150±1.07 1.738±1.32 1.248±1.12

Average 71.667±8.47 0.988±0.99 1.460±1.21 0.399±0.63

min. 37.930±6.16 0.200±0.45 0.164±0.40 0.029±0.17

max. 99.735±9.99 2.365±1.54 5.291±2.30 1.248±1.12

The relative contributions to concentrations of 106Ru, 134/137Cs, and 241Am radionuclides in 40 samples of foodstuffs shown

in fig.(3), we can notice that the high contributor for 106Ru were in Broad bean and other foodstuffs (which contained

Brown grit, White grit, Mung bean and Green peas) as a 12%, for 134Cs Broad bean the high contributor was in

Broad bean as 14%, for 137Cs the high contributor was in corn as a 19% while for 241Am, also the other foodstuffs were

high contributor with 15% ratio. The lowest contributor of 106Ru, 134/137Cs, and 241Am radionuclides in the studied

foodstuffs were 6% in cowpea, 7% in semolina, 5% in lentil and 4% in lentil respectively.

The action level result of the determination of 106Ru, 134/137Cs, and 241Am radionuclides in 40 samples of foodstuffs

from Iraqi markets for three age groups were presented in table-2. The mass of food group j consumed by age group k

Majeed et al, 2013

38

over the assessment period (kg/year) were (M=499, 519 and 450 kg/year) for adult, (12-19) year and (5-11) groups

respectively. The ingestion dose coefficient for radionuclide i and age group k (Sv Bq-1) were as in table-319.

Table-2: The action level for 106Ru, 134/137Cs, and 241Am radionuclide within afood stuffs and three age groups.

Sample No. Foodstuff type The action level AL (Bq kg

-1)

For adult For (12- 19) year For (5-11) year

1 Rice, (272) 0.0656 0.0826 0.194

2 Rice,(Alwalima) 0.0551 0.0695 0.160

3 Rice, (1121Sella) 0.0661 0.0852 0.210

4 Rice, (Alaella) 0.0732 0.0914 0.213

5 Rice 0.0830 0.1038 0.241

6 Semolina 0.0563 0.0696 0.158

7 Semolina 0.0559 0.0688 0.153

8 Semolina 0.0719 0.0889 0.201

9 Semolina 0.0669 0.0832 0.194

10 Semolina 0.0638 0.0789 0.179

11 Semolina 0.0685 0.0840 0.186

12 Wheat (type 1) 0.0695 0.0857 0.192

13 Wheat (type 2) 0.0660 0.0808 0.178

14 Kidney bean 0.0761 0.0940 0.215

15 Kidney bean 0.0726 0.0921 0.217

16 Kidney bean 0.0766 0.0961 0.218

17 Kidney bean 0.0475 0.0570 0.116

18 Kidney bean 0.0415 0.0518 0.119

Chick-pea 0.0374 0.0472 0.111

Chick-pea 0.0406 0.0511 0.120

Chick-pea 0.0683 0.0852 0.200

Chick-pea 0.0656 0.0816 0.189

Chick-pea 0.0635 0.0812 0.200

24 Red lentil 0.0632 0.0802 0.196

25 Grain lentil 0.0632 0.0798 0.191

26 Grain lentil 0.0591 0.0740 0.174

27 Grain lentil 0.0540 0.0677 0.161

28 Cowpea 0.0408 0.0506 0.115

29 Cowpea 0.0402 0.0496 0.110

30 Cowpea 0.0441 0.0554 0.126

31 Broad bean 0.0567 0.0714 0.169

32 Broad bean 0.0907 0.1125 0.256

33 Broad bean 0.0896 0.1083 0.227

34 Corn 0.0642 0.0825 0.197

35 Corn 0.0792 0.0992 0.233

36 Corn 0.1012 0.1261 0.277

37 Brown grit 0.0782 0.0970 0.219

38 White grit 0.0385 0.0485 0.114

39 Mung bean 0.0768 0.0955 0.205

40 Green peas 0.0872 0.1052 0.221

Average 0.0645 0.0803 0.184

Min. 0.0374 0.0472 0.110

Max. 0.1012 0.1261 0.277

The intervention level (Sv) and contamination factor were (1mSv and 0.2) for other commercial Foods and Beverages for

all age group12.

The AL for adult group one was (0.0645) Bq kg-1 average with minimum value (0.0374) Bq kg-1 in sample no. 19

and maximum (0.1012) Bq kg-1

in sample no. 36 fig. (4a), AL for (12-19) year, group two was (0.0803) Bq kg-1

average

with minimum value (0.0472) Bq kg-1 in sample no.19 and maximum value (0.1261) Bq kg-1 in sample no. 36 fig. (4b)

while AL for (5-11) year group three was (0.184) Bq kg-1 average with minimum value (0.110) in sample no.19 and

maximum value (0.277) Bq kg-1 in sample no 36. fig. (4c).


39

As shown the foodstuffs were within the range permitted and free of any radiation and thus there was no

seriousness in dealing with20-22

Fig-3: The relative contributions to concentrations of 106Ru, 134/137Cs, and 241Am radionuclide s in 40 samples of foodstuffs.

Table-3: Age–specific committed effective dose coefficients for ingestion18

Radionuclide Half -life Ingestion Dose Coefficient to Age (Sv/Bq)

Adult (12-19) year (5-11) year 106Ru 1.01y 7.0e-09 8.6e-09 1.5e-08 134Cs 2.06 y 1.9e-08 1.9e-08 1.4e-08 137Cs 30 y 1.3e-08 1.3e-08 1.0e-08

241Am 432 y 2.0e-07 2.0e-07 2.2e-07

Fig-4: The action level (AL) for 106Ru,134/137Cs, and 241Am radionuclide within a Food stuffs and three age groups, (4a) for adult, (4b)

for (12- 19) year and (4c) for (5-11)year.

106Ru

Rice,

11%Semolina

10%

Wheat

10%

Kidney bean

9%Chick-pea

9%

lentil

11%

Cow pea

6%

Broad bean

12%

Corn

12%

Other

10%

134Cs

Rice,

9% Semolina

7%

Wheat

12%

Kidney bean

10%Chick-pea

6%lentil

9%

Cow pea

11%

Broad bean

14%

Corn

11%

Other

11%

137Cs

Rice,

9% Semolina

7%Wheat

7%

Kidney bean

7%Chick-pea

7%lentil

5%Cow pea

6%

Broad bean

15%

Corn

19%

Other

18%

241Am

Rice,

7% Semolina

13%

Wheat

14%

Kidney bean

12%Chick-pea

5%

lentil

4%

Cow pea

6%

Broad bean

14%

Corn

10%

Other

15%

Fig.(4a)

0.0000

0.0600

0.1200

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

Sample No.

∑(Ai/ALi)≤1

Fig.(4b)

0.0000

0.0700

0.1400

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

Sample No.

∑(Ai/ALi)≤1

Fig.(4c)

0.000

0.150

0.300

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

Sample No.

∑(Ai/ALi)≤1

Majeed et al, 2013

40

5. CONCLUSIONS The specific activity concentrations of (106Ru,, 134/137Cs, and 241Am) nuclides in 40 imported foodstuffs were coincident

with world average concentrations. The high contributor for 106Ru, 134/137Cs, and 241Am radionuclides were in Broad bean

and other foodstuffs (which contained Brown grit, White grit, Mung bean and Green peas) as a 12%, Broad bean as 14%,

corn as a 19% and other foodstuffs with 15% respectively

The lowest contributor of 106Ru, 134/137Cs, and 241Am radionuclides in the studied foodstuffs were 6% in cowpea,

7% in semolina, 5% in lentil and 4% in lentil respectively.

The action level of the 106

Ru, 134/137

Cs, and 241

Am radionuclide’s for three age groups were within the range

permitted and free of any radiation and thus there was no seriousness in dealing with.

6. REFERENCES 1. Carter, M. “Radionuclides in the Food Chain”, New York: Springer-Verlag, 58-71. (1988).

2. Hasan, A. K., Majeed, H. N. and Hassan, S. A. ” Measurements of Natural Radiation in soil of the College of

Education for girls, University of Kufa, Al-najaf Al-ashraf, Iraq “,Pak. J. Chem., 1(4):1-6, (2011).

3. Harb, S., Salahel Din, K., Abbady, A. and Saad, N.” The annual dose for qena governorate population due to

consume the animal products” Proceedings of the4thEnvironmental Physics Conference, 10-14 March, Hurghada,

Egypt, (2010).

4. IAEA, “Natural and induced radioactivity in food”, (2002).

5. UNSCEAR, “Sources and effects of ionizing radiation”. New York: United Nations, (2000).

6. Asefi, M., Fathivand, A., Amidi, A., Najafi, A. “Determination of 226Ra and 228Ra concentrations in foodstuffs

consumed by inhabitants of Tehran city of Iran” Iran. J. Radiat. Res. 3, 3, (2005).

7. International Food Safety Authorities Network (INFOSAN),”Information on nuclear accidents and radioactive

contamination of foods”, (2011).

8. IAEA, “Update of x ray and gamma ray decay data standards for detector calibration and other applications”,

Volume 1, Vienna, (2007).

9. IAEA, "Guidelines for radioelement mapping using gamma ray spectrometry data", Vienna, (2003).

10. Ebaid,Y., Rom. Journ. Phys. 55, 69, 74, (2010).

11. Al-Sulaiti, H., Regan, P., Bradley, D., Matthews, M., Santawamaitre, T. and Malain, D., "Preliminary

Determination of Natural Radioactivity Levels of the State of Qatar using High Resolution Gamma ray

spectrometry", IX Radiation. Physics & Protection Conference, 15-19 November Nasr City - Cairo, Egypt (2008).

12. “Canadian Guidelines for the Restriction of Radioactively Contaminated Food and Water Following a Nuclear

Emergency, Guidelines and Rationale “, Canada, (2000).

13. IAEA, “Radioactive fallout in food and agriculture”, IAEA-TECDOC-494., Vienna (1989).

14. IAEA, “Intervention criteria in a nuclear or radiation emergency”, Safety Series No. 109, Vienna, (1994).

15. IAEA, “Measurement of radionuclides in food and the environment”, Vienna, (1989).

16. Nuclear Safety Commission” Evaluation of Environment Radiation Monitoring Results”, (2011).

17. Notification of provisional regulation values in foods MHLW, Mar. 17, 2011.

18. Agency for Toxic Substances and Disease Registry (ATSDR). (2004). Toxicological profile for americium.

Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service.

19. ICRP, “Age-dependent doses to members of the public from intake of radionuclides”: Part 5 Compilation of

ingestion and inhalation dose coefficients. ICRP Publication 72. Ann. ICRP 26, No. 1, Pergamon Press, Oxford,

(1996).

20. World Health Organization. Derived intervention levels for radionuclides in food (1988).

21. Food and Agriculture Organization, FAO Food Balance Sheets, 1979-1981. FAO, Rome, (1984).

22. United Nations Sources and Effects of Ionizing Radiation. United Nations Scientific Committee on the Effects of

Atomic Radiation, 1993 Report to the General Assembly, with scientific annexes. United Nations sales

publication E.94.IX.2. United Nations, New York, (1993).


ISSN (Print): 2220-2625


*Corresponding Author Received 27th August 2012, Accepted 2nd January, 2013

The Effects of Squeezed Grapes Residue on the Preservation of Stored Fried Potato

Chips

*N. M. Salih AL-Janabi, A. M. Mahmed AL-Samraee and

1W. S. Ulaiwi

*Department of food science, College of Agriculture, Baghdad University, Iraq. 1Basic Sciences Section, College of Agriculture, Baghdad University, Iraq.


ABSTRACT The squeezed grapes residue (SGR) was used as antioxidant. The oxidative activity SGR estimated in vitro by measuring the

reducing power and scavenging of hydrogen peroxide. The concentration of SGR was 200ppm which was added to frying oil to

prepare potato chips. The antioxidant activity was measured at 45oc for a storage periods 0, 3, 6, 9, 12 days. It was found that the

peroxide (pov) and thiobarbuytric acid (TBA) values by using the SGR were lower than of the control and similar to BHT

treatment (the (pov) values of control, BHT and SGR were 8.00, 6.00 and 6.00 meq /kg respectively). At the end of storage, TBA

values of control, BHT and SGR were 1.17, 1.01 and 1.01mg/kg. The antioxidant activity of SGR was higher as compared with

control, but similar to synthetic antioxidant (BHT.) Therefore, we are recommended SGR as natural antioxidant to be used in food

systems.

Keywords: Natural preservatives, Waste of Grapes Juice, Potato Chips, Antioxidant

1. INTRODUCTION Residues such as peels and seeds that result from fruit juice production may contain substantial amounts of valuable

natural antioxidants and antimicrobials. Grape skin and seeds are rich source of healthy promoting polyphenols,

including flavan-3- ols of different degree of polymerization that are potent free radical scavengers which is useful to

be used as preventative agents against cancer, cardiovascular diseases and premature aging1. Grape (Vitis vinifera) is

one of the world’s largest fruit crops, with an approximate annual production of 58 million metric tons2. Grape seeds

contain lipid, protein, carbohydrates and 5-8% polyphenols depending on the variety. The polyphenols are mainly,

flavonoids, including: gallic acid, monomeric flavan-3-ols catechin, epicatechin, gallocatechin, epigallocatechin,

epicatechin-3-o-gallate, procynidin (dimmers, trimmers and more highly polymerized procynidins). The most

abundant phenolic compounds which were isolated from grape seeds are catechins, epicatechin, procynidin and some

dimers and trimers3. Various herbs, vegetables and fruits contain numerous phytochemicals in addition to phenolic

compounds4-5. Many of these phytochemicals possess significant antioxidants capacities that are associated with lower

incidense and lower mortality rates of cancer in several human cohort4. Natural antioxidants capacities substances

(NAS) usually have aphenolic moiety in their molecular structure, its NAS can act as antioxidants, or have synergistic

effect when used together with phenolic antioxidants, which can be obtained from plant material, food waste,

microorganisms and animal cells6-7. The word “antioxidant” means the implicitly restricted to chain breaking

inhibition of lipid peroxidation8. Microbial activity is a primary mode of deterioration of many foods and is often

responsible for the loss of quality and safety, concern over pathogenic and spoilage microorganisms in food is

increasing due to the increase in outbreaks of food borne disease9. Currently there is a growing interest to use natural

preservatives, like plant extracts for increasing the shelf life of foods, as these possess a characteristic flavor,

antioxidant activity as well as antimicrobial activity10. Some potato chips companies have responded to the criticism,

both informal and legal, by investing in research and development to modify existing recipes and create health

conscious products also marketed baked potato chips as an alternative with lower fat content11-12. The present study

aimed to use the squeezed grapes residue as a cheep (byproduct) natural preservative to increase the shelf-life of

potato chips.

2. MATERIALS AND METHODS Waste of grape juice (SGR): was obtained from a selling shop of grape juice in Baghdad. It was washing (3 times) and

naturally dried. Sun flower oil without any synthetic antioxidants was obtained from plant oils Iraqi general company.

Potato: was purchased at a local market in Baghdad (Iraqi potato variety).

Dried waste grape juice were crushed and extracted in a soxhlet extractor with petroleum ether (60-80oc for 6

hr) to extract the fatty material. The defatted SGR powder extracted in a soxhlet apparatus for 8 hr separately with

methanol: water: acetic acid (90:9.5:0.5). The extracts were filtered and concentrated under vacuum to get crud

extract13.

Total phenolic, free phenolic and tannin in squeezed of grape juice extract (SGR) were determined quantitively

by using titration method14-15. Assessment antioxidant activity of (SGR) in vitro were Measured by two methods,

scavenging hydrogen peroxide16 and reducing power17.



42

Preparation of potato chips peeled potatoes were cut in to slices using a slicer machine (Turkish-origin/kenod). The

slices were macerated in dis- water with (2.5gm/lL) salting for 15 min at room temp (22oc), and it were rinsed under

running tap water. Then the excess water was drained before being fried, after that the slices were divided into three

allots, the first: counted as a control. The second: potato was fried in an oil (sun flower) with 200ppm of SGR, and the

third with BHT (2%) as synthetic antioxidant at 195oc min in an electric container fryer. Fried potato chips were

drained while cooling to remove excess oil. Three samples of potato chips were placed in an incubator at 45oc for 12

days, stored and periodically used for analysis18.

Extraction of oil from potato chips:- the stored potato chips were crushed, n-hexane added and the mixture was

shaken twice for 30 min in a stomacher at room temperature in a dark place. The extract were filtered (what man no.4)

to obtain particles free extract and then concentrated with a rotary evaporator 35c. The extracted oil analysis was

based on: Peroxide value (pov)19 and thiobarbutiric acid (TBA)20.

3. RESULTS AND DISCUSSION The total phenols were 13.312% (Tannin 8.32 %and free phenols 4.992%) in (SGR) extract. Most of the phenolic or

polyphenolic compounds in nature have antioxidative activities, like tocopherol, flavonoid and other organic acids21.

[Fig -1] shows the comparison of the scavenging of peroxide abilities of (SGR) extract with ascorbic acid were (42.91,

101.4, 107.2 and 115.7)% for (SGR) at concentration (2,4,6 and 8) mg/ml respectively, while were in the following

order (101.20, 120.64, 183.80 and 199.19)% for vit. C at the same concentrations mentioned above. The ability of

scavenging was increasing with the increase of concentration of SGR extract and vit. C. scavenging of hydrogen

peroxide by phenolic compounds due to donor of electrons22.

Fig-1: Antioxidant activity of SGR by Scavenging of peroxide

The results showed that (SGR) extract was effective nearly as BHT at con. 40 mg/ml the reducing power for it was

88.60 % for BHT and 88.91% for SGR. while reducing power for PG less than 66.66 %. Reducing power activity of

(SGR) extract was enhanced by increasing its concentration [Fig -2]. Reducing power indicates compounds that are

electron donors, which can act as primer and secondary antioxidant23. These results agreed with result of24 Vit. C had

Scavenging peroxide activity at 1.0 mlmole concentration reached 70.4% and gallic acid 93.3% at the same

concentration24.

Fig-2: Antioxidant activity of SGR (6%) by Reducing power for 12 days

0

50

100

150

200

250

2 4 6 8

Scav

en

gin

g ac

tivi

ty

Con.mg/ml

Vit. C

SGR

0

20

40

60

80

100

10 20 30 40

Re

du

cin

g p

ow

er

Con.mg/ml

BHT

SGR

PG

Salih Al-Janabi et al, 2013

43

Determination of (pov) can be used as an oxidation index during the early stages of lipid oxidation as the primary

products (hydroperoxids). [Fig- 3] shows increasing in pov during storage and the control was the highest, followed by

(SGR) and BHT treatment which were 10.00, 8.00, 8.00 meq/kg respectively. After 6 days of storage pov of (SGR)

treatment was the slowest than of control and BHT, but after 9 days the pov decreased for all treatment. Control, SGR

and BHT [Fig-3] but pov of the (SGR) was lowest than the BHT and control, while after 12 days of storage pov of

(SGR) decreased progressively and the peroxide value was 8.00 meq/kgm for control which was highest than BHT

and (SGR) treatments [Fig-3].

Fig-3: Peroxide value (pov) on potato chips containing SGR (200ppm) stored at 45OC

In the present study we have applied waste of grape juice to characterize the direct preservation food matrix. We have

also compared it with a synthetic antioxidant (BHT). Plant extract with antioxidant effect have been tested in several

food systems like ground beef25, ground beef patties26, fish meat system27. SGR used in this study to know the effect

of it on the oxidative stability of fried potato chips.

TBA values of potato chips storage are shown in [Fig-4], after 6 days of storage potato chips at 45OC:TBA

value of control treatment was highest than that of BHT and SGR treatments, the TBA value in SGR was 6.52 mg/kg

where TBA value of SGR 5.74 mg/kg approach to TBA value of BHT 5.64 mg/kg after 6 days of storage.

Fig-4: Thiobarbitric acid value (TBA) on potato chips containing squeezed grapes residue (200ppm) stored at 45 OC

After the storage period finished (12days), SGR and BHT treatment exhibited similar results. However, control

treatment result in a higher increase. Some studies provided that the oligomeric procyanidine from grape seed and pine

bark, bilberry and ginko exhibited total antioxidant activities in the range of 5.12 -2.57 mHTrolox. An indication of

valuable antioxidant capacity28, in other studies about the natural antioxidants (NA) used of oleoresin rosemary, Sage

extract and citric acid improved the sensory acceptability of potato chips during 5 days repeated deep fat frying29.

control

SGR

BHT0

1

2

3

4

5

6

7

03

69

12

pe

roxi

de

val

ue

Days

control

SGR

BHT

control

SGR

BHT0

1

2

3

4

5

6

7

0 36

912

Thio

bar

bu

tiri

c ac

id v

alu

e

Days

control

SGR

BHT


44

Also presence of rosemary extract or ascorbyl palmitate in the frying oil caused a marked reduction in the rate of loss

of the tocopherols30. These results were contemporary to result in precedent study, provided antioxidant activity of

SGR extract (in vitro) like antioxidant activity of BHT and PG by estimate (pov) through β-carotene bleaching test31.

The antioxidant activity of SGR due to phenolic compounds moiety. Phenolic compounds have antioxidant activity

manly due to their redox properties, which can play an important role in adsorbing and neutralizing free radicals32.

4. CONCLUSION We thought waste of grape juice can be used as abiological coats, coating material and preservative of food as

antioxidant and antimicrobial, these results showed that adding 200 ppm of SGR to potato chips were good level as a

natural antioxidant due to decreasing peroxide values and TBA value, even though SGR may be a source of phenolic

compounds.

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6. Dugan, L. R., MA, USA, (1980), pp263-282.

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9. Tauxe, R. V., Dariy. Food Environmental Sanitation, (1997), 17:788-795.

10. Smid, E. J. and Gorris, L. G. M. "Natural antimicrobials for food preservation. In M. Shafiurr Rahman (Ed),

Handbook of food preservation (pp.285-308)". New York: Marcel Dekker, (1999).

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wsj.com/article", (2010).

12. Niddk, Win Notes, "http//win-niddk.nih.gov/notes/article 19", (1998).

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Pak. J. Chem. 3(1):45-47, 2013 Short Communication

ISSN (Print): 2220-2625


*Corresponding Author Received 21st January 2013, Accepted March 5th 2013

A Visual Demonstration of Solvent Effect in Chemical Kinetics through Blue Bottle

Experiment

*R. Azmat

Department of Chemistry, University of Karachi, Pakistan


ABSTRACT In the study of chemical kinetics, usually solvent effect was explained to show the consequences on rate of reaction theoretically

which is difficult to understand for under graduate students. The blue bottle experiment as a “one day activity” can be used to

explain well visually the solvent effect through demonstration of color change. Kinetics of reduction of methylene green by

sucrose and mannose in pure and aqueous methanol medium in presence of NaOH has been investigated for demonstration of

solvent effect. The two sugars sucrose and mannose were selected for the experiment those acts as a reducing agents in a basic

solution and reduces the methylene green into colorless form. The progress of this reduction reaction was followed by the color

changes that the methylene green goes through in variable percentage of alcohol. When the bottle is shaken the oxygen in the air

mixes with the solution and oxidizes the methylene green back to its intermediate state (purple). The color of the solution will

gradually change and become purple (intermediate) and then colorless in 5-10% methanol but in pure methanol color transition

were Blue-> purple-> pink indicate the color due to the alcoholic medium. It was observed that increase in percentage in the

solvent composition decrease the rate of reduction. The pink color continues due to alcoholic medium which may be attributed

with the solvent effect. The observed variation in reading with solvent compositions has been interpreted in terms of interactions

of media with the reacting species and the transitions state involved in this reaction.

Keywords: Kinetics, color change, transitions state

1. INTRODUCTION Solvents can in fact influence rates of reaction and order of a chemical reaction. A solvent effect is the group of effects

that a solvent has on chemical reactivity. Solvents can have an effect on solubility, stability and reaction rates and

choosing the appropriate solvent allows for thermodynamic and kinetic control over a chemical reaction.

Solvent interaction with reacting species related to the nature of product, if products more polar then the

reactants, the reaction rate will accelerate in polar solvent but if reactants are polar then the products, rate of reaction

will decreases in presence of polar solvent. If both reactant and products are non polar, polarity of solvents will have

no influence on the rate of the reaction and the rate is independent of the nature of the solvent. When reactants interact

with the solvent and are solvated, leading to lowering the potential energy of the reactants, then the activation energy

increases which lowers the reaction rate. The dielectric constant of the medium plays an important role in case of ionic

reactions

This experiment was designed in such a way that students of under graduate classes can learn and observed

effect of medium visually with color loss of the dye.

2. EXPERIMENTAL 1. Before starting the visual demonstration of blue bottle experiment a “theoretical concept of redox reaction” of

about 20 - 30 minutes was delivered.

2. All required solution of dye, reducing sugar and alkaline medium (NaOH) were prepared by usual methods as

reported earlier1.

3. Shaking of solution allow to dissolve the oxygen in reaction mixture which give blue color.

4. Upon standing color permanently change in aqueous media.

5. The solvent effect were monitored via different percentages of alcohol and pure alcohol

6. Upon standing in pure alcohol pink color permanently exists.

2.1 The demonstration of methodology 1. Flask which contains dye, mannose and sodium hydroxide solution was shaking vigorously to show that blue

color appear due to the dissolve oxygen from atmosphere.

2. The blue color of the dye showed the oxidized state of the dye or oxidation of dye occurs.

3. Now allow to stand the flask, blue color will change into the color less which showed that oxygen is

consumed by the sugar which converted into respective acid and now hydrogen from sugar acid will

abstracted by the dye or “H” will added into the dye molecule for its reduction and reduction is indicated by

the color less state after addition of hydrogen1 (Figure). 4. Variable percentages of alcohol and pure alcohol were used for the preparation of solutions of all reagents for

the demonstration of solvent effects.



46

3. RESULTS AND DISCUSSION Dyes are the best indicators of rate of chemical reaction, they provide help in determining order of reaction through

their color change ability by clock method1-3. The change of color of the dye is related with the presence and absence

of oxygen, also related with the medium which is in chemical kinetics refers as a solvent effect. The study of solvent effect in chemical kinetics usually explained to show the consequences on rate of reaction

theoretically which is difficult to understand for under graduate students. The famous blue bottle experiment can now

be used to explain well visually the solvent effect through demonstration of color change4. This will make the

chemistry easy and interesting for the students. Kinetics of reduction of methylene green by sucrose and mannose in

pure and aqueous methanol medium in presence of NaOH has been investigated for demonstration of solvent effect.

The two sugars sucrose and mannose were selected for the experiment which acts as a reducing agent in a basic

solution and reduces the methylene green into colorless form. The progress of this reduction reaction was followed by

the color changes that the methylene green goes.

When the bottle is shaken the oxygen in the air mixes with the solution and oxidizes the methylene green back

to its intermediate state (purple). Note the effect of reduction of methylene green by varying the volume of each

parameter. The color of the solution will gradually change and become purple (intermediate) and then colorless in 5-

10% methanol but in pure methanol color transition were Blue-> purple -> pink color due to the alcoholic medium.

When the solution is shaken gently it will turn back to purple (intermediate) in 5-10% methanol but in pure methanol

pink color reappears5-8. The pink color continues due to alcoholic medium which may be attributed with the medium

effect. This cycle can be repeated many times. After a while the indicator seems to "wear out". When this happens add

more methylene green to enhance the color changes. It was observed that increase in % in the solvent composition

decrease the rate. The observed variation in reading with solvent compositions has been interpreted in terms of

interactions of media with the reacting species and the transitions state involved in this reaction. This is may be due to

a polar solvent decreases the reaction rate if the reactants are more polar than the products. Either the reactant or the

product or the activated complex interacts with the solvent, there may be considerable influence on the rate of the

reaction8. When the reactants interact with the solvent and are solvated leading to lowering the potential energy of the

reactants then the activation energy increases lowering the reaction rate.

4. CONCLUSION It was concluded that the persistence of pink color in pure alcohol indicate the solvent effect on rate of reduction

reaction. This experiment can be used as a one day open activity for learning and teaching Chemistry in an easiest

way.

Fig-1: A visual solvent effect

5. REFERENCES

1. Azmat, R., Irshad, M. and Farooqi, I., Pak. J. Chem. (2011), 1(1):19-21. 2. Azmat, R., “Reduction of Methylene Blue with Reducing Sugars” Publisher VDM Verlag Dr. Müller e. K.

(2009).

3. Azmat, R. and Uddin, F., Chinese Journal of Chemistry. (2009), 27(7):1237-1243. 4. Azmat, R. and Uddin, F., Canadian J. Pure and Appl. Sci. (2009), 2(1) 275-279. 5. Azmat, R. Uddin, F. and Mohammed, F. V. Appl., J. of Chemical Research. (2008), 6: 7-21.

Azmat

47

6. Azmat, R., Uddin, F. Appl., J. of Chemical Research. (2010), 13(5):72-54. 7. Ahmed, K., Uddin, F. and Azmat, R. Chinese Journal of Chemistry (2009), 27(7): -1229-1242. 8. Azmat, R., Naz, R. and Qamar, N. (2011), J. Chem. 1(2):90-91, 2011.

Pak. J. Chem Guide for Authors


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