Pharmacognostical studies and evaluation of total phenolic and flavonoid contents of traditionally utilized fruits of Solanum torvum Sw.

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1 Indian Journal of Natural Products and Resources Vol. 2(2), June 2011, pp Pharmacognostical studies and evaluation of total phenolic and flavonoid contents of traditionally utilized fruits of Solanum torvum Sw. Muhammad Arif* and Sheeba Fareed Faculty of Pharmacy, Integral University, Kursi Road, Lucknow , Uttar Pradesh, India Received 18 June 2010; Accepted 22 March 2011 The fruits of Solanum torvum Sw., commonly known as Shoo-shoo bush, Devil s fig, Wild egg, are pungent in taste, edible, used as a vegetable and are regarded as an essential ingredient in Thai cuisine. In Tripura and West Bengal fruits are crushed or cooked with dry fish (sidhol) and taken with warm rice to cure rheumatic pain. The fruit has been used ethnomedicinally as a tonic and haematopoietic, anti-microbial agent and for the treatment of pain. A decoction of fruits is given for cough ailments and is considered as useful in liver and spleen enlargement. The fruit has sedative, diuretic and digestive in action. In view of the traditional and medicinal importance, the present study deals with pharmacognostical evaluation of fruit including morphological and histological studies. Determination of physicochemical constants, fluorescence analysis and phytochemical profile including TLC fingerprint and estimation of total phenolic and flavonoid contents has also been done. Keywords: Solanum torvum, Shoo-shoo bush, Devil s fig, Wild egg, Histochemical, Phenolics, Flavonoid. IPC code; Int. cl. ( ) A61K 36/00 Introduction Solanum torvum Sw. (Family-Solanaceae) is a small shrub native of Mexico, Peru and Venezuela. It is widely distributed in India, West Indies, Bermuda and Indonesia other than western desert area, Malaya, China, Philippines and tropical America 1. The plant is an erect spiny shrub also known as Shoo-shoo bush, Devil s fig, Wild egg plant usually 2-4 m tall having edible fruits is commonly available in the markets. Fruits are used as a vegetable and they are regarded as an essential ingredient in Thai cuisine 2. The plant is cultivated in the tropics for its very tasty immature fruits. In Tripura and West Bengal fruits are crushed or cooked with dry fish (sidhol) and consumed with warm rice to cure rheumatic pain 3. The fruit has been used ethnomedicinally as a tonic and haematopoietic, anti-microbial agent and for the treatment of pain 4-5. A decoction of fruits is given for cough ailments and is considered useful in liver and spleen enlargement, and possesses sedative, diuretic and digestive properties 6-7. Phenolic and flavonoid compounds are the secondary metabolites which synthesize in plants and located mainly in pericarp of the fruits 8. They posses different biological properties such as: anti-oxidant, *Correspondent author arif_sweet@rediffmail.com Phone: Mobile no: anti-inflammatory, immuno-modulatory, mast cells stabilizing, blood pressure and cholesterol lowering activity 9. Steroidal alkaloids, chlorogenone, neochlorogenone, isoflavonoid sulfate and steroidal glycosides, 22-O-spirostannol (Torvonin-A) are the previously isolated classes of constituents In view of the traditional and medicinal importance, the pharmacognostical investigation of the plant fruit was carried out and results are presented. Materials and Methods The fruits of the plant were collected in the month of June 2009 from the campus of Dibrugarh University, Assam and identified by Prof. Muhibul Islam from the Department of Life Sciences, Dibrugarh University, Assam. A voucher specimen no DU/PS/HRB-03/2009 was deposited in this laboratory herbarium for further reference. The fresh fruit was used for the examination of macroscopic and microscopic characters whereas the dried fruit powder was used for determination of physico-chemical parameters. A thin transverse sections (T.S.) of different part of the fruit were cut by free hand sectioning. Histochemical colour reactions tests of powdered fruit were carried out with Ruthenium red for mucilage, weak iodine solution for starch, Millon s reagent for protein, Dragendorff s reagent for the alkaloidal

2 ARIF & FAREED: PHARMACOGNOSTICAL STUDIES OF SOLANUM TORVUM SW. FRUITS 219 substance, NaOH (Aqs) for flavonoids, Aqs Ferric Chloride for the phenolic compounds 14. Physicochemical parameters like loss on drying, total ash, acid insoluble ash, water soluble ash and crude fibre contents were performed as per Indian Pharmacopoeia 15. Successive soxhlet extractives of the drug were carried out with various solvents like petroleum ether, chloroform, ethyl acetate, methanol and water. The colour/consistency of the extractives was observed. The different extractives were subjected to preliminary phytochemical analysis for the presence of various phytoconstituents Fluorescence analysis of the powder and extracts were carried out 18. TLC fingerprint of different extractives were analysed. The mobile phase solvent system Chloroform:Methanol:Formic acid was used in ratio of (60:40:2). Anisaldehyde-suphuric acid reagent was used as detecting agent. The R f values were compared with standard Digoxin and Rutin 19. Determination of total phenolic contents Total soluble phenolic components in the different extractives of S. torvum fruit were determined with Folin Ciocalteu reagent using gallic acid as a standard 20. Suitable aliquots of the different extractives were taken in a test tube and made up to the volume of 1 ml with distilled water. Then, 0.5 ml of Folin-Ciocalteu reagent (1:1 with water) and 2.5 ml of sodium carbonate solution (20%) were added sequentially in each tube. The tubes were vortexed, allowed to stand in dark for 1 h with intermittent shaking and absorbance was measured at 760 nm. The phenolic content from calibration curve prepared by repeating the operation using 1 ml of gallic acid solution at concentrations (25, 50, 100, 200, 300, 500 µg/ml) in distilled water. Determination of total flavonoid contents Total flavonoid content was estimated by aluminum chloride colorimetric method 21. Suitable aliquot of each extractive were added in 10 ml volumetric flask containing 4 ml distilled water and mixed with 0.3 ml of 5% sodium nitrite and after 5 min add 0.3 ml of 10% aluminum chloride. At 6 th minute add 2 ml 1M-NaOH and made up the volume 10 ml with distilled water. Then the mixtures were allowed to stand at room temperature for 30 min with intermittent shaking and absorbance was measured at 514 nm. The calibration curve was prepared by preparing rutin solutions at concentrations 25, 50, 100, 200, 300 and 500 µg/ml in distilled water. Results and Discussion Macro and microscopic characters The plant is an erect spiny perennial shrub usually 2 or 3 m in height and 2 cm in basal diameter, but may reach 5 m in height and 8 cm in basal diameter. The shrub usually has a single stem at ground level, but it may branch on the lower stem (Plate 1). The fruit is simple, fleshy, shriveled when dry berry. Fruiting calyx lobes less than half length of mature fruit, spines absent (Plate 2). Mature fruits 4-16 per inflorescence; globular; mm in diam., yellowish-green, 2-4-locular, placenta stalked, mesocarp moist but not juicy; exocarp yellowishgreen, mm thick. Peduncle mm long, mm thick at mid-point. Seeds are white or pale yellow when mature, mm diam, embedded in pithy pulp (Plate 2). TS of fruit peduncle The detailed TS of peduncle passing through the centre are almost circular in outline with (star shape) five ridges and show following characters (Plate 3a). Plate 1 Flowering twig of Solanum torvum plant Plate 2 Fruits and seeds of Solanum torvum

3 220 INDIAN J NAT PROD RESOUR, JUNE 2011 Outline of the peduncle shows the presence of ridges and furrows. Epidermis is single layered with thick cuticle followed by 5-6 layers of collenchymas bellow the ridges and furrows. There are two types of (unicellular covering and glandular) trichomes, present on epidermal layer. Middle cortex consists of thick walled 5-6 layered collenchymatous cells. The secondary vascular tissues of peduncle are fundamentally similar to stem. They form continuous cylinder and the primary xylem gets lignified and completely embedded in it. Pith is large and made up of round parenchymatous cells 22. TS of fruit Epicarp is the outermost layer of the fruit composed of radially elongated cells with thin cuticle. The outer mesocarp tissue of the pulp lying underneath the epicarp is composed of 6-8 rows of parenchymatous cells occasionally followed by sclerenchymatous patches. The outer 2-3 layers have brownish colouring matter and the inner cells being larger have thin walled. The endocarp getting merged into parenchymatous tissue of pithy pulp in which the seeds are embedded. Endodermis is not very distinct (Plate 3b-c). Testa of the seed is composed of groups of thin walled tangentially elongated collapsed cells. Endosperm is thin walled parenchymatous cells containing masses of aleurone grains and fat globules (Plate 3d). Powder characteristics and histochemical colour reactions : The fruit powder is brownish green in colour having aromatic odour, pungent taste and rough and fragmented texture. After pressing a little amount of powder between filter paper, a little greasy stain was found, indicates the presence of fatty oils, after shaking powder with water in a test tube persistent forth was formed, indicate the presence of saponins. Behaviour of the powdered fruits with different chemical reagents is shown in Table 1. Majority of the tissues, cells and cell contents were studied and drawn directly from the microscope with the help of camera lucida (Plate 4) and observations are as follows: a) The numerous dark brown colour fragments of epicarp in surface view composed of a single layered elongated cells and fragments show the presence of striated cuticle. b) The fragments of the mesocarp, underlying epicarp are thin walled, unlignified, parenchymatous, frequently broken usually consisting orange oily globules with a diam. of 10.8 µm. c) The fragments of the mesocarp are (in sectional view) filled with microsphenoidal crystals of Ca-oxalate with 11 µm diameter. Plate 3 T.S. of fruit peduncle (3a), T.S. of fruit (diagrammatic) (3b), T.S. of fruit (cellular) (3c), T.S. of seed (3d)

4 ARIF & FAREED: PHARMACOGNOSTICAL STUDIES OF SOLANUM TORVUM SW. FRUITS 221 Table 1 Histochemical colour reactions of powdered fruit of S. torvum with different chemical reagents Reagents powdered fruit Ruthenium red solution Phloroglucinol Conc. HCl Iodine solution Millon s reagent Dragendorff s reagent Conc. NaOH (Aq) Frothing test Aq. Ferric Chloride Salvoski s test Constituents Colour Degree of intensity Mucilage Lignified cells Starch Crystalloids inclusions of protein Alkaloids Flavonoids Saponins Phenolics / Tannins Steroids High, Moderate, Negative Pink Pink Blue Brick red Reddish brown colour alkaloids Wine colour Persistent froth Smoke colour Orange color at the junction of two layer d) The sclereids of the endocarp which occur in groups, beaded thick walled, elongated (in surface view) and have orange oily globules. e) The epidermis composed of thin-walled elongated cells with numerous unicellular and bicellular covering and glandular trichomes. Ash values including total ash, acid insoluble ash and water soluble ash of fruit powder were done as per Indian Pharmacopoeia. The results are shown in Table 2. Successive extractive values with different Table 2 Quantitative standards of powdered fruit of S. torvum Loss on Drying Parameters Values of 3 Replicates (%) w/w Ash Value a) Total ash b) Acid insoluble ash c) Water soluble ash Crude fibre contents Mean ± SD 12.76± ± ± ± ±0.467 Plate 4 Identifying characters of powdered fruit

5 222 INDIAN J NAT PROD RESOUR, JUNE 2011 solvents of powdered fruit were done. The colour, consistency and percentage of extractive values in triplicate and their mean values ± SD was calculated with reference to the air dried drug (Table 3). Table 3 Extractive values for powdered fruits of S. torvum with different solvents Type of solvent Petroleum ether Chloroform Ethyl-acetate Methanol Water Colour & consistency Resinous yellow Semisolid Green Semisolid yellowish green Sticky yellowish brown Sticky dark brown Values of 3 replicates (%) w/w Mean ± SD 0.77± ± ± ± ±0.030 Table 4 Fluorescence analysis of the powdered fruit of S. torvum S. No. Treatment Colour in day light Colour in Short UV (254 nm) The fluorescence analysis of powdered drug as well as its extractives in day light, short UV and long UV were examined. The observations are given in Table 4. The presence or absence of different phytoconstituents, viz. alkaloids, reducing sugar, glycosides, flavonoids, terpenoids, steroids, tannins and phenolic compounds were detected by usual prescribed methods and results are given in Table 5. A brown coloured spot (R f 0.94) corresponding to digoxin was visualized in chloroform and ethylacetate fractions. The methanolic fraction shown greenish brown spot (R f 0.86) and it was compared with standard drug rutin. The number of spots and colour of TLC fingerprints is shown in (Plate 5). The phenolic contents of different extractives of S. torvum fruit are given in Table 6 and their absorbance are shown in Fig. 1. The highest concentration of phenolic content was present in the methanolic and water fractions. The total content of phenolic compounds in methanolic fraction was µg and water fraction was µg gallic acid equivalent/10 mg extract. Fig. 2 indicates the absorbance of flavonoids contents of different Colour in Long UV (365 nm) 1. Dry powder Brownish green Slatey (Grayish) colour Dark green 2. Powder 1M-NaOH alcoholic Light brown Coffee brown Fine particles gives dark brown colour 3. Powder 1M-NaOH aqueous Brown Wine colour Dark brown 4. Powder 1 M-HCl aqueous Grey colour Light green Dark green 5. Powder 50% H 2 SO 4 Yellowish green Green Ash colour Fluorescence analysis of the powdered fruit extracts of S. torvum i. Pet. ether (40-60 o C) Light yellow Golden yellow Milky white ii. Chloroform Greenish brown Coffee colour Yellowish green iii. Ethyl acetate Wine colour Golden yellow Orange colour iv. Methanol Brown Dark brown Green v. Water Dark brown Black Dark green Table 5 Qualitative phytochemical analysis of various extracts of fruit of S. torvum Type of constituents Pet. Ether Chloroform Ethyl-acetate Methanol Water 1. Alkaloids 2. Reducing sugar 3. Glycoside a) Cardiac glycoside b) Saponin glycoside c) Anthraquinone d) Flavonoids glycosides 4. Tannins/Phenolic 5. Phytosterol 6.Triterpenoids 7. Protein 8. Oils/Resins () present and () absent.

6 ARIF & FAREED: PHARMACOGNOSTICAL STUDIES OF SOLANUM TORVUM SW. FRUITS 223 Table 6 Determination of total phenolic and flavonoids contents of different extractives of S. torvum Extracts/ Fractions Phenolic contents a Flavonoids contents b Pet. Ether -- Chloroform Ethyl-acetate Methanol Water All values are expressed as mean ± SEM. (n = 3). a µg of gallic acid equivalent per 10 mg of extract. b µg of rutin equivalent per 10 mg of extract. Plate 5 TLC fingerprints of different extractives of fruit extractives of S. torvum fruit. The content of flavonoids in methanolic fraction was µg and water fraction was µg rutin equivalent/10 mg extract (Table 6). The methanolic and water fraction was found to have highest phenolic and flavonoid content whereas in pet-ether and chloroform fractions no phenolic and flavonoids contents were found. Fig 1 Total phenolic content of different extracts of S. torvum fruit (Results are expressed in mean ± SEM of three parallel measurements). Conclusion S. torvum is a plant used as a traditional medicine and can be well exploited for various pharmacological activities. Amongst the chemical classes present in this plant species, alkaloids, saponins glycosides, steroids, flavonoids and phenolic compounds stand as a class of major importance in the development of new drugs. The present investigation has stated important standardisation parameters of S. torvum fruits. Qualitative and quantitative microscopical characters, ash and extractive values, phytochemical screening and TLC fingerprint profile of different extractives of the fruit would be of useful in authenticating S. torvum fruit. Acknowledgements The authors wish to thank Prof. H.H. Siddiqui, Advisor, Faculty of Pharmacy, Integral University, Lucknow for his valuable suggestions and facilities provided for the study. We also express our thanks to Dr. Mohd. Zeeshan, Dept.of Biotechnology, Integral University, Lucknow for assistance in microscopical parameters. Fig. 2 The total flavonoids content of different extracts of S. torvum fruit (Results are expressed in mean ± SEM of three parallel measurements). References 1 Chopra RN, Nayar SL and Chopra IC, Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research, New Delhi, 1956, 230.

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