M. Kannan et al.

/ Journal of Pharmacy Research 2012,5(5),2418-2421

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

Phytochemical, antibacterial and antioxident studies on medicinal plant Solanum torvum.

M. Kannan*1 B. Dheeba 2 , S. Gurudevi 1 and A. J. A. Ranjit Singh3 Department of Microbiology, V.H.N.S.N, College, Virudhunagar 626001. Tamil Nadu, India. 2 Department of Chemistry and Biosciences, Srinivasa Ramanujan Centre, SASTRA University, Kumbakonam. Tamil Nadu, India. 3 Department of Advanced Zoology and Bio-technology, Sri.Paramakalyani College, Alwarkurichi. Tamil Nadu, India.
1

Received on:15-01-2012; Revised on: 22-02-2012; Accepted on:12-04-2012 ABSTRACT

Objectives: The present study planned to investigate the phytochemical, antimicrobial and antioxident properties of Solanum torvum. Methods In this study the plant Solanum torvum was powdered and the powder was subjected to soxhlet, extraction with petroleum ether and methanol. The various phytochemical constituents like alkaloids, flavonoids, glycosides, phenols, saponins and sterols were identified in selected plant extract by TLC and HPLC. The antibacterial activity and antioxidant activity of the plant was analysed using Fentons reagent free radicle scavenging studies measured colorimetrically at 620 nm. Results Preliminary phytochemical investigation revealed the presence of Saponins, glycosides, tannins, alkalaids, volatile oils and flavonoids. The antibacterial and antioxidant activity was expressed at varying concentration and dose dependent. KEY WORDS: Solaum torvum, HPLC, Fentons method and free radicles. 1. INTRODUCTION Plants have been used in traditional medicine for several thousand years1 . The knowledge of medicinal plants had been accumulated in the course of many centuries based on different medicinal systems such as Ayurveda, Unani and Siddha. In India, it is reported that traditional healers use 2500 plant species and 100 species of plants serve as regular sources of medicine. During the last few decades there had been an increasing interest in the study of medicinal plants and their traditional use in different parts of the world. Documenting the indigenous knowledge through ethanobotanical studies is important for the conservation and utilization of biological resources 2 . Plants have an almost limitless ability to synthesize aromatic substances mainly secondary metabolities of which atleast 12,000 have been isolated a number estimated to be less than 10% of the total. In many cases these substances serve as the molecules of plant defense against predation by microorganisms, insects and herbivores. Further, some of the phytochemical which may involve in plant odour (terpenoids), pigmentation (tannins and quinines) and flavour (capsacin)3 . However, several of these molecules possess medicinal properties. Some important medicinal plants used in Ayurveda, Unani, Siddha and in folk medicine for treating several ailments including microbial infections, diarrhoea and diabetes 4 . The genus Solanum was large and complex, made up about 1,700 species worldwide5 . The genus Solanum was probably derived from the Latin name of a plant that was used medicinally for treatment of epilepsy. The genus Solanum was considered to be one of the largest and most complex among the Angiosperms. The genus is well represented in Brazil and is widely distributed from North to South in diverse phytogeographic regions and is widely used in folk medicine, commonly know as jurubeba the name derived from the Tupi-guarni word yubeba, which refers to the presence of prickles in

some of them. Spreading or sprawling shrubs 2-3 m tall, prickles 3-7 mm long, slightly hooked, laterally flattened, scattered on stems, both leaf surfaces and main veins, sprase on agen and mature growth all parts pubescent with stellate hairs, spares on upper leaf surface. Leaves simple, alternate, broadly ovate elliptic, variable in size, 10-15cm long 8-10cm wide, staminate flowers. Berries few to 10 in clusters, drab yellow, brownish at maturity, mucilaginous, drying with age, globose, 1-1.5cm in diameter, pedicels 1-1.5 cm long, thickened below calyx, calyx not much enlarged. Seeds numerous, drab brownish, flattened, discoid, 1.5-2mm long, slightly reticulate, Self compatible6 . Solanum torvum was used in many Ayurvedic treatments; it has seeing diuretic and digestive properties. It used in the treatment for coughs, liver diseases. Solanum torvum used to reduce body heat and strengthening the body 7 . The plants are considered to be promising source of medicine in the traditional healthcare system. The efficacy and safety of herbal medicine have turned the major pharmaceutical population towards medicinal plants research. The present work was carried out to investigate the phytochemical, antimicrobial and antioxidant activities of selected Solanum torvum plant extracts. 2. MATERIALS AND METHODS 2.1 Extract Preparation The health plant leaves of Solanum torvum was collected from Western Ghats of Madurai range, Tamil Nadu, India. They were collected in early morning and were washed in tap water and shade-dried for 10 days. The shade dried plant material was powdered using mixer grinder and that powder was subjected to Soxhelet extraction with petroleum ether and methanol (60o C) for 24 hrs. Each solvent extract was distilled and condensed at 40o C. The condensed extract was stored at room temperature in air tight bottles and used for further studies. 2.2 Phytochemical Studies The presence of bioactive phytocompounds was secondary metabolites from the leaves of Solanum torvum and Solanum nigrum were qualitatively analysed by thin layer chromatography. Solid phase of silica gel were kept in hot air oven in 100o C for 20 minutes. Silica powder was mixed with petroleum ether

*Corresponding author.
Kannan Marikani, P.G. Department of Microbiology, V.H.N.S.N.College, Virudhunagar-626 001, TamilNadu, India.

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and makes slurry. 20x 20 cm TLC glass plates covered with that slurry and allowed to air dried. After drying the plates were kept in hot air oven in 72o C for 1 hr. After developing the plates the condensed filtrate was spotted using capillary tube. The different spots were separated using a different solvent mixture act as mobile phase. 2.2.1 TLC study of alkaloids The powdered leaves of Solanum torvum was wetted with a half diluted NH4 OH and lixiviated with Et OAc for 24hrs at RT. The organic phase is separated from the acidified filtrate and basified with NH4 OH (pH 11-12). It is extracted with chloroform (3x), condensed by evaporation and used for chromatography. The alkaloid spots were separated using the solvent mixture chloroform and methanol (15:1). The colour and hRf value of the separated alkaloids were recorded both under Ultra Violet (UV 254nm) and visible light after spraying with Dragendorffs reagent. 2.2.2 TLC study of flavonoids One gram powdered leaves of Solanum torvum was extracted with 10ml methanol on water bath (60o C/5min). The filtrate was condensed by evaporation, added a mixture of water and EtOAc (10:1mL), and mixed thoroughly. The EtOAc phase thus retained is used for chromatography. The flavonoid spots were separated using chloroform and methanol (19:1) solvent mixture. The colour and hRf value of these spots were recorded under ultraviolet (UV254nm) light. 2.2.3 TLC study of glycosides The powdered leaves of Solanum torvum was extracted with 70% EtOH on rotary shaker (180 thaws/min) for 10hr. 70% lead acetate is added to the filtrate and centrifuged at 5000rpm/10min. the Supernatant was further centrifuged by adding 6.3% Na2 Co3 at 10000 rpm/10min. the retained supernatant was dried, redissolved in chloroform and used for chromatography. The glycosides were separated using EtOAc-MeOH-H2 O (80:10:10) solvent mixture. The colour and hRf values of these spots were recorded by observing under ultraviolet (UV254nm). 2.2.4 TLC study of phenols The powdered leaves of Solanum turvum was lixiviated in methanol on rotary shaker (180 thaws/min) for 24h. The condensed filtrate was used for chromatography. The phenols were separated using chloroform and methanol (27:0.3) solvent mixture. The colour and hRf values of these phenols were recorded under visible light after spraying the plates with FolinCiocalteus reagents heating at 80o C/10min. 2.2.5 TLC study of saponins Two grams of powdered leaves of Solanum torvum was extracted with 10ml 70% EtOH by refluxing for 10min. The filtrate was condensed, enriched with saturated n-BuOH, and thoroughly mixed. The butanol was retained, condensed and used for chromatography. The saponins were separated using chloroform, glacial acetic acid, methanol and water (64:34:12:8) solvent mixture. The colour and hRf values of these spots were recorded by exposing chromatogram to the iodine vapours. 2.2.6 TLC study of sterols Two grams of powdered leaves of Solanum torvum was extracted with 10ml methanol in water bath (80o C/15min). The condensed filtrate is used for chromatography. The sterols were separated using chloroform, glacial acetic acid, methanol and water (64:34:12:8) solvent mixture. The colour and hRf values of these spots were recorded under visible light after spraying the plates with anaisaldeyde-sulphuric acid reagent and heating (100o C/6min). 2.2.7 High Performance Liquid Chromatography (HPLC) HPLC analysis was carried out for the component separated in thin layer chromatography. It was performed on Schimedzu, Spintrom HPLC-530 avaliable in Science Instrumentation Centre, Cecri, Karikudi (TN-India). The results were recorded. 2.3 ANTIMICROBIAL STUDIES 2.3.1 Preparation of inoculum The test microorganisms, gram positive Staphylococcus aureus (MTCC Acc.No.7443) and Bacillus subtilis (MTCC Acc.No.441) and gram negative Escherichia coli (MTCC Acc.No.476), Salmonella species (MTCC Acc.No.53) and Pseudomonas aeruginosa (MTCC Acc.No.424) bacteria were obtained from MTCC culture. The organisms were inoculated into Nutrient broth and incubated at 37o C for overnight. The bacterial cells were harvested by centrifuging at 5000rpm for 15mints. The pellet formed was washed twice with PBS and the cells were counted by haemocytometer. The bacterial cells were diluted to approximately 105 CFU/ml before use8 . 2.3.2 Agar well diffusion method The antibacterial activity of the leaf extracts was determined using agar well diffusion method by following the published procedure with slight modification9 . Nutrient agar was inoculated with the given microorganisms by spreading the bacterial inoculums on the media. Wells (8mm diameter) were punched in the agar and filled with plant extracts. Control wells containing neat solvents (negative control) and standard antibiotic tetracycline (Positive control). The plates were incubated at 37o C for 18 hours and the antibacterial activity was assessed by measuring the diameter of the zone of inhibition. The relative antibacterial potency of the given preparation was calculated by comparing its zone of inhibition with that of the standard drug tetracycline. 2.4 Determination of Antioxidant property The antioxidant activity of the methanolic extract of Solanum torvam was studied. Standardized solution of Fe- EDTA complex (Fentons reagent) reacts with hydrogen peroxide by Fenton type of reaction leading of the formation of hydroxyl radicals (OH). These reactive oxygen species degrades O-Toluidine to produce purple colour complex. Antioxidants in the sample suppress the degradation of O-Toluidie. This reaction is measured colorimetrically at 620nm. The inhibition of colour development is measured as antioxidant activity. Prepare 1 in 100 dilutions of plant extracts and Label the tubes for different extracts (E). Take another set of tubes for extract control (Eo). Among them three tubes are labeled as blank (B), Control (C) and Standard (S). Add 1.5 ml of H 2 O and 2.5ml of Buffer and O-Toludine for Blank (B). In standard (S) 0.5ml of each H 2 O2 , Fenton Reagent, H 2 O, Buffer, O-Toludine were taken. In test sample 0.5ml of each Ascorbate, H 2 O2 , Fenton Reagent, H2 O, Buffer, O-Toludine were added. In Extract control (Eo ) tubes 0.5 ml extract, 1.5ml H2 O, 0.5ml Buffer were taken. In plant Extract (E1 ) tubes 0.5ml of Solanum torvum and Solanum nigrum extract, 0.5ml of H 2 O2, 0.5ml of Fenton Reagent, 0.5ml Buffer and 0.5ml O-Toludine were taken. During estimation all samples were incubated for 30 min at 37o C before adding buffer of 0.5ml and O-Toludine of 0.5ml. After addition of buffer and O-Toludine all the samples were kept in Incubator for 30 min at 100o C. To estimate the antioxident activity the OD value was taken at 620nm. 3. RESULTS AND DISCUSSION 3.1 Phytochemical studies Preliminary phytochemical investigation revealed the presence of Saponins, glycosides, tannins, alkalaids, volatile oils and flavonoids, as indicated in

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Table-1. The results showed that Solanum torvum demonstrated for the presence of all phytocompounds tested, except the absence of tannins in petroleum ether extract of Solanum torvum. The presence of some of these compounds had been demonstrated previously by other researchers10,11 . For example, the presence of alkaloids in the leaf of Solanum torvum and the absence of tnnins in the leaf of Solanum torvum has been demonstrated. However some of the results obtained are not in agreement with the previous findings. This might be due to climatic and environmental factors. The curative properties of medicinal plants are perhaps due to the presence of various secondary metbolites such as alkaloids, flavonoids, glycosides, phenols, saponins, sterols etc. Thus the preliminary screening test may be useful in the detection of the bioactive principles and subsequently may lead to the drug discovery and development. Further, these tests facilitate their quantitative estimation and qualitative separation of pharmacologically active chemical compounds.
Table 1: Preliminary screening of secondary metabolites from Solanum torvum
S. No. 1 2 3 4 5 6 7 Secondary metabolite Alkaloids Flavonoids Glycosides Phenols Saponins Sterols Tannins Name of the test Wagners test NaOH test Molisch test Ferric chloride test Froth test Salkowski test Gelatin test Solanum torvum + + + + + + + + + + + + + -

3.2 Antibacterial activity The antimicrobial activities of the methanol extracts of Solanum torvum and are determined against five bacterial strains. The results were compared with those produced by the standard antibiotic Tetracycline 1mg/ml. The results of the sensitivity are summarized in table 3. All strains showed sensitivity toward methanolic extract. Among the gram negative bacteria E. coli, Salmonella and Pseudomonas aeruginosa showed promising sensitivity 2.6 to 30mm to methanolic extract. The Gram positive bacteria Bacillus subtilis and Staphylococcus aureus were showed promising sensitivity toward methanolic extract. The high concentration (100%) of Solanum torvum effective against Pseudomonas aeroginosa (30.0 mm), E.Coli (28.2mm), Staphylococcus aureus (25.0mm), Bacillus subtilis (18.3mm) and Salmonella species (15.8mm). Antibacterial activity that may be due to the presence of alkaloids, flavonoids, phenols, saponins and sterols. The biologically active compounds are screened by dissolving the crude powder on various compound specific solvents confirmed by the TLC. The antibacterial activity was expressed at varying concentration and dose dependent. The various concentration of (100%, 80%, 60%, 40%, 20%) methanol extracts of showed significant activity against all the bacteria tested. The methanol extract of Solanum leaves which showed activity against all bacterial species tested at all the dosages.
Table 3: Antimicrobial activity of methanol extracts from leaves of Solanum torvum
Sl. No. Pathogens Zone of inhibition (mm) 100% 80% 60% 40% 28.2 18.3 25 15.8 30 23.1 16 17.6 13 18.4 18.5 14.7 15.2 10.3 15 8 10.5 9.6 4.1 10.7 C 20% 5.7 4.2 3.6 2.6 4.8 Solvent control Tetracycline 1mg/ml 34 22 28 10 17

(+) Positive; (-) Negative, (I) Methanol, (II) Petroleum ether

The TLC profiles of secondary metabolites (Alkaloids, Flavonoids, Glycosides, Phenols, Saponins and Sterols) are tabulated in the Table-2. Among the six groups of phytochemicals determined from the leaves of Solanum torvum. The flavonoids are found to be the most abundant one followed by Alkaloids and Glycosides. But the Phenals, Saponins and Sterols were low in concentration. The data of secondary metabolites of Solanum torvum revealed the presence of six, pink to intense black coloured (Plate 1) secondary metabolites with hRf values 74.49, 83.33, 69.39, 33.33, 46.09, 46.09 and 21.05. The qualitative HPLC flavonoids profile were detected at a wavelength of 254 nm due to sharpness of the peaks and proper baseline and recorded its (Rt min), percent area and heights were recorded. The plant Solanum torvum showed the single major peak at 3.973 Retention time. The HPLC studies proved the presents of the bioactive compound in the selected sample. Therefore, the data generated from these experiments have provided the chemical basis for the wide use of this plant as therapeutic agent for treating various ailments. However, there is need to further carryout advanced hyphenated spectroscopic studies in order to elucidate the structure of these compounds. Further more, this data maybe handy in probing of active biocompounds of this plant in the future.
Table 2: TLC Profile of phytochemicals presenting in Solanum torvum
S.No.Plant Name Colour of the spot Name of the Secondary metabolites Compound Alkaloids Flavonoids Glycosides Phenols Saponins Sterols R f value

1 2 3 4 5

Escherichia coli Bacillus subtilis Staphylococcus aureus Salmonella species Pseudomonas aeruginosa

3.3 Antioxidant Studies The level of antioxidant activity in the Solanum torvum was determined by using the Fenton reagent by free radicle scavenging studies (Table 4). The free radicle scavenging activity of Solamun torvum was compared with Ascorpic acid test control. It showed the does deperdest variation was observed in anti oxidant studies; the selected plants have the moderate antioxidant activity. The bioactive compounds of the Solanum torvum have antioxidant proparties and were relatively difficult to measure each antioxidant compound separately. Therefore, several different methods have been delayed to evaluate the antioxidant activity of biological samples 12,13,14 . Solanum torvum was exhibiting strong antioxidant activity in Fentons reagent assay, also showed strong activity in the free radical scavenging assay. From these results could suggest that the consumption of this plant extract could possibly after some dietary benefits since they contain constituents, which are able to protect against lipid peroxidation and to scavenge free radicals. The moderate antioxidant activity of Solanum torvum indicated by the Fentons reagent assay, is possibly due to their essential oil constituents15 . One of the previous studies showed the antimicrobial compounds in ethylacetate extract from Solanum torvum especially towards Pseudomonas aeruginosa. Nosocomial
Table 4: Analaysis of antioxidant activity from the various concentration of the plant extract of Solanum torvum
S. No. 1 2 3 4 Samples Standard Test control Plant Extract control Plant Extract 0.5 ml 0.036 (- 93.93%) 0.593 0.471 (- 20.57%) 0.635 -7.08% 1.0 ml 0.175 (- 70.14%) 0.586 0.56 (- 4.44%) 0.688 -17.40% 1.5 ml 0.237 (- 62.55%) 0.633 0.627 (- 0.95%) 0.708 -11.85%

Solanum torvum

Pink Yellow Pink Blue Light yellow Intense black

74.49 83.33 69.39 33.33 46.09 21.05

Percentage of results given in paranthesis was compared with the test control.

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infections caused by these gram negative bacteria are amongst the most difficult to treat with conventional antibiotics 16,17,18 and its thus possible that these plants may yield drugs that could improve the treatment of infections caused by the microorganisms. The presence of the steroidal alkalid solasodine was potentially an important starting material for the synthesis of steroid hormones, is characteristic of the genus Solanum19,20 . The wide distribution, anticancer properties 21,22,23 of the glyco alkaloids and molluscicidal activity24 of the crude extracts, led us to the selection of these Solanum species. The present work conform the presence of bioactive compound in Solanum torvum . It can possible to act as a potential immunomodulator. However, the existence of antimicrobial compounds in methanol extracts from Solanum torvum was effective against Pseudomonas aeruginosa and Salmonella species. However further study in view of elucidating the composition of its active metabolites are worth looking into, in order to confirm its bacteriostatic mechanism of antimicrobial action and antioxidant activity. 4. REFERANCES 1. Abu-Rabia, A., 2005. Urinary diseases and ethnobotany among pastoral nomads in the middle East. Journal of Ethnobiology and Ethnomedicine 1:4. 2. Lev. E., 2006 Ethno diversity within current ethno pharmacology as part of Isradi traditional mediline. Journal of Ethnobiology and ethnomedicine. 2:4 3. Kannan, M., Ranjit singh. A.J.A, Ajith Kumar. T.T., Jegatheswari. P., and Subburalu. S.: 2008. Studies on immuno- bioactivities of Nyctanthes arbortristis (Orleaceae). African Journal of microbiology research. 088-091. 4. Soforowo, A., 1983. Medicinal plants and Traditional Medicine in Africa. Wiley, London. 5. Wagner, W.L., Herbast D.R and Sohmer S.H., 1999. Manual of the flowering plants of Hawai, 2 vols. 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Gilliver.M. and Wilson. L., 1987. Antitumor effects of glycoalkaloids isolated from Solanum Sodomacum. Planta. Med. 53: 34-36. 23. Cham, B.E., 1994. Solasodine glycosides as anti-cancer agents: preclinical and clinical studies. Asian Pac J. Pharmacol. 9: 113118. 24. Silva, TMS., Batista. M.M., camara C.A. and Agra M.F., 2005C. Molluscidal activity of some Brazilian Solanum spp. (Solanaceae) against Biomphalaria glabrata. Ann Trop Med Parasitol. 99 : 419425.

Source of support: Nil, Conflict of interest: None Declared

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