Pharmacognostic and Physicochemical Evaluation of the Different Varieties of Sesamum indicum L. –...

Research Journal of Pharmacognosy and Phytochemistry. 6(2): April-June, 2014, 99-106

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ISSN 0975-2331 (Print) www.anvpublication.org 0975-4385 (Online) RESEARCH ARTICLE

Pharmacognostic and Physicochemical Evaluation of the Different Varieties of Sesamum indicum L. – A Comparative Study

Shantha T.R.*, Shubhashree M.N., Prathapa Reddy M., Venkateshwarlu G. National Ayurveda Dietetics Research Institute, Ashoka Pillar, Jayanagar, Bangalore-560011

*Corresponding Author E-mail: shanthatr57@gmail.com

ABSTRACT: Sesame is a condiment which is used in every household for culinary, religious and medicinal purposes. The present study deals with pharmacognostical and physicochemical evaluation of different varieties (black, white and brown) of Sesame (Sesamum indicum Linn.). The study revealed that the different varieties of Sesame seeds, microscopically shows all most similar characters, however, distribution of oil globules varies only in the case of black variety seeds. Oil globules and reddish contents are more in case of white variety when compared to other brown and black. Comparative study has taken up to identify the different varieties of Sesame with respect to its macroscopy, microscopy and other parameters like physico-chemical, preliminary phytochemical, fluorescence and TLC studies.

KEYWORDS: Sesame, Varieties of sesame, Pharmacognosy, Thin layer chromatography.

INTRODUCTION: Sesame (Sesamum indicum Linn.) belonging to the family Pedaliaceae is an erect glandular pubescent, annual herb branching from the base, growing up to a height of 95cms. Fruits (capsules) are 2-5 cm in long and 0.5-2 cm in diameter, quadrangular, oblong, compressed, capsules deeply 4 grooved, dehiscent to half way down. Seeds are many, flat, ovate, obovoid, compressed, black or white1. Sesamum is believed as one of the ancient cultivated crop, condiment and is used for extraction of edible oil in the world2. In India it is known as Sesame/Gingely in English, til in hindi, ellu in kannada, eellu-cceti in Tamil, car-elu, chitrallu in Malayalam and Nuvvulu in Telugu. Tila/Sesame in Sanskrit, means a small particle. The word Taila, the sanskrit term for oil is derived from Tila. Sesamum seeds have been an essential article in Hindu religious ceremonies and have been referred to as homadhanya and pitrutarpana in ancient scripts3. The Sesame seeds are antibacterial, antifungal and also used as poultice. These seeds are having the phytosterols 400-413 mg/100g, which are believed to reduce cholesterol levels in blood, enhance the immune response and decrease the risk of certain cancers, and also contains three times more calcium than a comparable measure of milk.

Received on 18.01.2014 Modified on 28.02.2014 Accepted on 06.03.2014 ©A&V Publications All right reserved Res. J. Pharmacognosy & Phytochem. 6(2): April-June 2014; Page 99-106

The seed oil is mildly laxative, emollient and demulcent and the refined oil is rich in antioxidant components like lignans, which are responsible for greater shelf-life, flavor and taste4. The seeds have desirable physiological effects including anti oxidant activity, blood pressure and serum lipid lowering potential as proven in experimental animals and humans5. Sesamum oil is also useful in the industrial preparations of perfumery, cosmetics and in pharmaceuticals as a vehicle for drug delivery, insecticides, paints and varnishes6. Sesame seed is a rich source of edible oil. There seems to be a correlation between seed coat colour and oil content. Lighter coloured variety yields more oil than dark coloured ones. Besides, the oil obtained from lighter coloured seed is cleaner and therefore considered superior7. Ayurvedic Description and uses of Sesamum seed and oil: Ayurveda considers Sesame (Tila) as madhura rasa pradhana and Kashaya (Astringent), katu (Pungent), Tikta (Bitter) Madhura (Sweet) as anurasa. It has guru (Heavy) and snigdha (Unctous), sukshma (Minuteness) guna (Qualities) vyavayi (spreads quickly) vikasi (dilatation of smaller vessels) attains katu vipaka (Post digestive transformation). It is ushna (hot) in potency and himasparsha (cold to touch). It is sthairyabalavarnakara (Bestows stability, strength and colour), keshya (promotes hairgrowth), tvachya (good for the skin) stanya (aids lactation), vranaropana (wound healing), dantya (Strengthens the teeth), alpamutrakruth (reduces the output of urine) grahi (Constipating), vatagna (Alleviates vata), agnideeptikara (Improves appetite), matiprada (Promotes intelligence).

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It is utilized in the treatment of vranaropana (wound healing) bhagna (fracture), arshas (piles), atisara (diarrhoea) dantaroga (dental diseases), Anartava (amenorrhea), vatashonitha (gout), udara (ascities) palita (premature greying of hair), for keshasamvardana (Growth of hair) dantadaurbalya (dental disorders), grahani, etc.8.

However, no comparative pharmacognostic work has not been found on the different varieties of Sesame, hence study has been taken up for evaluating changes among the three varieties of sesame with respect to macroscopy, microscopy, physicochemical, fluorescence and TLC studies.

MATERIALS AND METHODS: Different varieties of sesame were procured from the local market of Bangalore (India) and identified as Sesamum indicum L. following the local floras. Dried the samples of all the 3 varieties were powdered separately and used for carrying out Microscopical, preliminary phytochemical and UV studies9. Dried seeds were soaked in 70% alcohol for 24 hours, and were used to take free hand sections, cleared with chloral hydrate solution and water, stained with safranin according to the standard prescribed methods10,11. Photomicrographs were captured with Nikon Digital camera. Powder of the all 3 varieties of sesame was used for chemical analysis. Physico chemical standards were determined according to the standard procedures For Thin Layer Chromatography (TLC) the powdered seeds were extracted with petroleum ether (40-600C), petroleum ether (60-800c), Chloroform and Ethanol. All the extracts were subjected to TLC in various solvents systems using precoated silica gel GF 254 on aluminium sheet. The solvents used for TLC were of analytical grade13.

Preliminary Phytochemical screening of the seeds were carried out according to the standard methods14.

RESULTS: Macroscopic characters: The seeds are flattened/ compressed, ovoid, pear shaped pointed at one end, 3-4mm long, 1.5 -2 mm broad and 1 mm thick, black, buff coloured, whitish, finely punctuate with four delicate, longitudinal ridges, hilum is located at pointed end. Colour varies from light straw to dark brownish black. Taste bitter, oily without any characteristic smell. Microscopical Characters: Transeverse section of the seed is oval in outline, shows outer epidermis characterized by thin walled palisade cells, where anticlinal walls being more or less wavy, cells contain spherical mass of crystals of calcium oxalate crystals. The testa consists of collapsed cells with yellowish membrane inside. The endosperm and cotyledons consists of cellulosic, polygonal parenchyma containing fixed oil and small aleurone grains. Starch grains are absent. Powder: Powder of all the three varieties of Sesamum shows different colours, like black, light brown and white depending on the colour of seeds. Oily touch with bitter taste and slightly fibrous, when treated with chloral hydrate and water observed under the microscope, different fragments of tissues were observed, with abundant oilgloubles, epidermal cells with crystals, cotyledon cells with oilgloubles and aleurone grains, where these findings helps in the identification of all the 3 varieties of seeds.

Microscopical characters Different varieties of Til Seeds:

Black variety Til Seeds Brown Variety Til Seeds White Variety Til Seeds

Macroscopy Macroscopy Macroscopy

T.s of the seed 10xX40x T.s of the seed 10xX10x T.s of the seed 10xX40x

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T.s of the seed showing palisade region

with crystals. 10xX40x T.s of the seed showing palisade region with crystals 10xX40x

T.s of the seed showing palisade region With crystals 10xX40x

Epidermal cells surface view with calcium oxalate crystals. 10xX40x

Epidermal cells surface view with calcium oxalate crystals10xX40x

Cotyledon portion with abundant aleurone grains. 10xX40x

Cotyledon region with oilgloubles 10xX10x Cotyledon region with oilgloubles10xX40x

Cotyledon region with oilgloubles10xX40x

Palisade and cotyledon portion. 10xX10x Palisade and cotyledon portion.

10xX10x Palisade and cotyledon portion. 10xX10x

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Reddish content,oilgloubles and Aleurone

grains in cotyledon region 10xX40x Aleurone grains oilgloubles in cotyledon

region 10xX40x Aleurone grains oilgloubles in cotyledon

region 10xX40x

Powder study

Abundant oilgloubles 10xX40x Abundant oil globules10xX40x Abundant oil globules

10x X 40x

Cotyledon cells filled with oilgloubles and

aleurone grains. 10x X 40x Cotyledon cells filled with oilgloubles

and aleurone grains. 10x X 40x Cotyledon cells filled with oilgloubles and

aleurone grains. 10x X 40x

Epidermal cells in surface view with crystals. 10x X 40x

Epidermal cells in surface view with crystals. 10x X 40x

Epidermal cells with crystals. 10x X 40x

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Different varieties of til seeds, microscopically shows all most similar characters, only in case of black variety til seeds, it varies in the distribution of oil globules. Oil globules and reddish contents are more in case of black variety when compared to other brown and white varieties. Other microscopic characters are all most same, without any much notifying characters. Macroscopically colour of the seed coat varies from all the three varieties like black seed coat in black variety, light brown in brown variety, White in white variety seed. Diagnostic characters: Presence of abundant oil globules in cotyledon cells. Presence of thin walled parenchymatous cells with

abundant aleurone grains. Presence of polygonal cells with reddish content and

small prism shaped crystals. Presence of spherical mass of crystals of calcium

oxalate in palisade cells of epidermis. Absence of starch grains in the cotyledon portion. Table-1: Reaction of different varieties of seed powder with different chemical reagents under long wave length (UV Studies).

Chemical reagents

Black variety

White variety Brown variety

Con Hcl Light green Light green Light green Con H2SO4

Yellow colour

Light yellow Light yellow

5/Fecl3 Light brown

Light brown Light brown

Iodine solution

Dark brown Dark brown Dark brown

Potassium Hydroxide

Green Light blue Bright Fluroscence with white colour

Sodium hydroxide

Bluish green

Light blue Bright Flouroscence. with white colour

Physico chemical analysis: Ash values were used to determine the quality and purity of the crude drugs. The different ash values such as total ash and acid-insoluble ash, pH, Loss on drying, Alcohol soluble and water soluble extractive values were determined as per the procedure given in the Ayurvedic pharmacopoeia of India (Anonymous, 2010).The results are shown table-2. Preliminary Phytochemical Analysis: The dried powder of different varieties of til seeds was extracted successively with petroleum ether (40-600C), petroleum ether (60-800c), Chloroform and Ethanol by Soxhlet apparatus. The extracts were filtered while hot and concentrated under reduced pressure with the help of Rotary evaporator and weighed. The concentrated extracts of the seeds were subjected to various qualitative chemical tests for the presence or absence of different phytoconstituents viz, alkaloids, carbohydrates, flavonoides, proteins, resins, saponins, tannins and terpenoids etc. by usual prescribed methods15. The results are shown in table-3.

Thin Layer Chromatography: The Petroleum ether (40-600C), Petroleum ether (60-800C), CHCl3 and EtOH extracts were subjected to Thin Layer Chromatography (TLC). All the three varieties Petroleum ether (40-600C) extracts with the solvent system Hexane: Ethyl acetate (1:9) showed the similar ~Rf values like 0.13, 0.19, 0.25, 0.48 and 0.71 in Iodine vapour medium. White variety Petroleum ether (60-800C) extract with the solvent system Hexane: Ethyl acetate (1:9) showed the ~Rf values like 0.01, 0.08, 0.125, 0.57 in Iodine vapour medium. Black variety Petroleum ether (60-800C) extract with the solvent system Hexane: Ethyl acetate (1:9) showed the ~Rf values like 0.01, 0.08, 0.125, 0.3, 0.36 and 0.57 in Iodine vapour medium. Brown variety Petroleum ether (60-800C) extract with the solvent system Hexane: Ethyl acetate (1:9) showed the ~Rf values like 0.01, 0.08, 0.16, 0.3, 0.36, 0.425, and 0.57 in Iodine vapour medium. All the three varieties Chloroform extracts with the solvent system Hexane: Ethyl acetate (2:8) showed the same ~Rf values like 0.03, 0.07, 0.13, 0.30, 0.38 and 0.85 in Iodine vapour medium. All the three varieties Ethanol extracts with the solvent system Hexane: Ethyl acetate (2:8) showed the similar ~Rf values like 0.23, 0.30, 0.37 and 0.80 in Iodine vapour medium (Except one spot Rf 0.46 of brown variety). Thin layer chromatography studies revealed that under different solvent systems all the 3 varieties of til seeds exhibited almost same Rf values with different intensity, which indicates that 3 varieties are macroscopically different, but as per the TLC studies they are almost similar however in petroleum ether 60-800C extract black variety showed two more spots (Rf 0.30, 0.36) and brown variety showed three more spots (Rf 0.30, 0.36 and 0.425) than the white variety likewise in Ethanol extract brown variety showed one more spot (Rf 0.46) than the white and black variety. All the details are shown in table-7. Fluorescence studies: The fluorescence behaviour of the powder of all the 3 samples in different solutions and different extract obtained by successive Solvent extraction towards day light and short ultra violet light were observed, which showed distinct characteristic features distinguishing all the three samples. The details are shown in Table 4, 5 and 6. Table- 2: Physiochemical parameters of seeds of Sesamum indicum L.

S.No. Name of the parameter

White Black Brown

1 Description white black Light brown 2 Foreign matter ≤ 1.0% ≤ 1.0% ≤ 1.0% 3 pH (10% w/v aq.

solution) 4.10 4.20 4.15

4 Loss on drying at 1050C

3.34% 7.12% 5.6%

5 Total ash 2.89% 5.23% 4.8% 6 Acid-insoluble

ash 0.52% 0.19% 0.40%

7 Water-soluble extractive

8.94% 9.57% 9.35%

8 Alcohol-soluble extractive

13.87% 44.85% 16.85%

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Table -3: Preliminary phytochemical tests for different solvent extracts of Sesame indicum L. seeds S.No. Natural product test White Black Brown 1 Alkalods - - - 2 Carbohydrates + + + 3 Flavonoids - - - 4 Triterpenoids + + + 5 Proteins + + + 6 Resins + + + 7 Saponins + + + 8 Steroids + + + 9 Tannins + + + 10 Phenols + + +

Table- 4: Fluorescence analysis of seeds powder with Different reagents: White variety seeds:

S.No. Reagents Day light Short wave Long wave 1 Powder as such cream Dull brown Light brown 2 Powder + water Light cream Light brown Greenish brown 3 Powder + 1N NaOH Brown Light green Brown 4 Powder + 1N HCl Light brown Pale green Light brown 5 Powder + 1N NaOH in methanol Brown Yellowish brown Brown 6 Powder + 50% KOH Yellowish brown Green Brown 7 Powder + 50% H2SO4 Dark brown Greenish brown Brown 8 Powder + Conc.H2SO4 Black Dark black Black 9 Powder + 80% HNO3 Yellowish brown Yellowish green Yellowish brown 10 Powder + Conc.HNO3 Yellowish red Yellowish brown Yellowish brown 11 Powder + CH3COOH Cream Dull brown Dull brown 12 Powder + Iodine water Yellowish black Greenish brown Dark brown

Table- 5: Fluorescence analysis of seeds powder with different reagents: Brown variety seeds:

S.No. Reagents Day light Short wave Long wave 1 Powder as such Light brown Brown Brown 2 Powder + water Light brown Light brown Light brown 3 Powder + 1N NaOH Brown Green Yellowish brown 4 Powder + 1N HCl Light brown Pale green Light green 5 Powder + 1N NaOH in methanol Brown Pale green Greenish brown 6 Powder + 50% KOH Brown Yellowish green Yellowish green 7 Powder + 50% H2SO4 Dark brown Dark green Black 8 Powder + Conc.H2SO4 Black Black Black 9 Powder + 80% HNO3 Yellowish brown Greenish brown Black 10 Powder + Conc.HNO3 Reddish brown Light green Blue 11 Powder + CH3COOH Light brown Light brown Brown 12 Powder + Iodine water Brownish red Greenish brown Blue

Table- 6: Fluorescence analysis of seeds powder with different reagents: Black variety seeds:

S.No. Reagents Day light Short wave Long wave 1 Powder as such Brown Brown Greenish brown 2 Powder + water Brown Brown Dull green 3 Powder + 1N NaOH Black Black Black 4 Powder + 1N HCl Brown Brown Dark green 5 Powder + 1N NaOH in methanol Blakish brown Blackish brown Green 6 Powder + 50% KOH Black Black Black 7 Powder + 50% H2SO4 Black Black Black 8 Powder + Conc.H2SO4 Black Black Black 9 Powder + 80% HNO3 Red Greenish black Black 10 Powder + Conc.HNO3 Reddish brown Greenish black Reddish black 11 Powder + CH3COOH Brown Brown Brown 12 Powder + Iodine water Blackish brown Greenish brown Black

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Table- 7: TLC analysis of different solvent extracts of Sesamum indicum L. seeds TLC photographs

Extract Petroleum ether

40-600C extract Petroleum ether 60-800 C extract

CHCl3 extract EtOH extract

Solvent system used for TLC

Ethyl acetate: Hexane (1:9) Ethyl acetate: Hexane (1:9)

Ethyl acetate: Hexane (2:8) Ethyl acetate: Hexane (2:8)

Major Rf values White, Black and Brown: 0.13, 0.19, 0.25, 0.48 and 0.71;

White: 0.01, 0.08, 0.125, 0.57; Black: 0.01, 0.08, 0.125, 0.3, 0.36 and 0.57 Brown: 0.01, 0.08, 0.16, 0.3, 0.36, 0.425, and 0.57

White, Black and Brown: 0.03, 0.07, 0.13, 0.30, 0.38 and 0.85

White, Black: 0.23, 0.30, 0.37 and 0.80; Brown: 0.23, 0.30, 0.37, 0.46, 0.81;

Viewing medium Iodine chamber (vapour) Iodine chamber (vapour)

Iodine chamber (vapour Iodine chamber (vapour

DISCUSSION: The seeds of Sesamum indicum Linn. were traditionally valued for their oil and oilcakes. In the recent years it has gained additional importance as a source of protein with antioxidant properties. The protein content of sesame seed is approximately 25%, which is low in lysine (3.1% of protein), adequate amount of tryptophan, which is limiting in many oil seeds, but rich in sulfur-containing amino acids like methionine and cystine. Because of its characteristic amino acid composition sesame seed protein is regarded as an excellent protein source for supplementing many vegetable proteins such as soybean and peanut to increase their nutritional value, the flour also impart desirable characteristics when incorporated into products such as ice cream, frozen dessert, sausage, baked food and confectionary14. Sesame seed has about 17% seed wight as hull, which is high in oxalic acid (2-3%), calcium and crude

fibre. Oxalic acid could comple with calcium and reduces its bioavailability, therefore, seed hull is to be removed if sesame meal is used for human food15 . The sesame oil may contain as high as 1.9% of total sterols as a rich source of phytosterols in which β-sitosterol is the most abundant one16, which inhibits the growth of colon cancer cells17, prostrate cancer cells18, and breast cancer cells19. The chemical composition of sesame seed varies with the variety, origin, color and size of the seed. The fat content of sesame seed is around 50% where as the protein content is around 25%. Sesame seed contains high levels of fat and protein20. Because of close structures of phytosterols and cholesterol, phytosterols are therefore acts as competitors for cholesterol absorption, therefore consumption of phytosterols may lower blood cholesterol and thus protect from cardiovascular diseases21.

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Earlier different varieties of sesame seeds have been reported small differences in the percentage of different chemicals present in all the varieties. The black sesame seeds oil contains less tocopherols than oils from brown or white Sesame seeds, which are responsible for the extra oxidative stability of sesame oil22,23. The white variety sesamum seeds contain more quantity of lignin compounds than the black and brown varieties, in which sesamin and sesamolin are the two major lignans, sesamin has been found in other plants also but, sesamolin is characteristic of sesame and has not been found in plants other than Sesamum, sesamin content was always higher than sesamolin24. The total contents of lignan glycosides in white sesame seeds were around 100-170 mg/100-g seeds and in black it varied greatly i.e. 6.4 to 361.3 mg/100-g seeds, but both varieties contains sesaminol triglucoside as the major lignan glycoside25. A significant difference in lignan glycoside contents among different varieties was noticed as white sesame seeds contained an average of 84.5 mg sesaminol in 100 g seeds, and the black variety seeds conatains 113.2mg of sesaminol per 100 g seeds26. The present study revealed that the different varieties of Sesame seeds, microscopically shows all most similar characters, however, distribution of oil globules varies only in the case of black variety seeds. Oil globules and reddish contents are more in case of white variety when compared to other brown and black. These Pharmacognostical studies and physico-chemical data, TLC fingerprint profile and fluorescence analysis data evolved from the present investigation may be utilized for the standardization of the sesamum seeds in order to check and ensure the quality of the drug in quality control laboratories and also for laying down Pharmacopoeial standards for the seeds of sesamum (Sesamum indicum Linn.).

ACKNOWLEDGMENT: The authors are thankful to the Director General, CCRAS, New Delhi for providing necessary facilities to carry out the work.

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