TLC BASED PHYTOCHEMICAL ANALYSIS AND ANTIOXIDANT ACTIVITY OF SENNA ALATA

treatment of skin diseases. In the present study chloroform, methanol, petroleum ether and aqueous extracts were used for phytochemical analysis, TLC profiling and antioxidant activity of Senna alata. The phytochemical analysis of Senna alata indicated presence of tannins, saponins, flavonoids, alkaloids, terpenoids, steroids, cardiac glycosides and quinones in Senna alata. In TLC analysis maximum 6 bands were found in leaf extract, 5 bands in stem extract and 4 bands were found in root methanol extract. The plant showed good antioxidant activity in methanol and aqueous based leaf, stem and root extract which may be due to the presence of flavonoids and phenolic compounds. The plant has potent antioxidant and antimicrobial properties so characterization of bioactive molecules of the plant should be beneficial for formulation of drugs. Flavonoids and phenol important role in antioxidant activity of plants neutralizing free radicals Rutin and quercetin is a natural flavones derivative having strong free radical scavenging property used in treatment of several diseases. The pharmacological properties of rutin antibacterial, antiviral, antitumour, anti-protozoal, anti-allergic, anti-inflammatory, hypolipidaemic and vasoactive Gallic acid is obtained from tannic acid by hydrolysis with H 2 SO 4 and the biological properties of gallic acid includes radical scavenging, anti-fungal, anti-cancer, anti-inflammatory, and chemoprotective Abo et al. reported that Senna alata was used in treatment of skin disorders such as ringworm, hernia, blennorrhagia, diabetes, constipation, hemorrhoids, intestinal parasitosis and syphilis. The different biological properties exhibited by the compound give motivation for isolation of bioactive molecules from natural sources. to formation of reactive oxygen species and antioxidant may proteins, and lipids methanol, aqueous Presence of anthroquinones, phenols, Senna DPPH radical scavenging


ISSN: 2320-5407
Int. J. Adv. Res. 8 (11), 1099-1107 1100 n-dotriacontanol, kaempferol, n-triacontanol (Liu et al., 2009), aloe-emodin-O-β-glucoside, kaempferol, aloeemodin, rhein methyl ester (Duong et al., 2013), 2, 5, 7, 4' tetrahydroxy Isoflavone and 5, 7-dihydroxy-3', 4'dimethoxy flavones (Rahman et al., 2015), kaempferol (Rhazri et al., 2015), and volatile oil (Igwe and Onwu ,2015) Presence of secondary metabolites in the plant exhibits various pharmacological properties such as antifungal, antitumor, antioxidant, antibacterial, anti-inflammatory, analgesic, and immune stimulating activities have been reported from its leaf, stem, flower, root-bark and seed extracts. Isah et al. reported that Senna alata is used for curing of skin infections, diabetes mellitus, sickle cell anemia and malaria. Abubakar et al. reported ellagitannin, n-hexadecanoic acid, naphthalene, phenolic acids, purine and xanthone compounds in Senna alata. Traditionally leaf part of the plant is used in treatment of skin diseases such as ringworm, itching, scabies, eczema, ulcers and other related disease (Igoli et al.2005). Whole parts of the plant have medicinal values such as leaves were used for skin infection, seeds as anti-helminthic, roots for uterus disorder (Herman et al. 1978). Esimone reported that extracts of plant are used for cosmetic formulation in several skin care products and dehydrated leaves were used in herbal formulation such as herbal soaps, shampoos, tincture and herbal tea. Bejoy et al. reported that the extracts of plants are used as antiinflammatory, diuretic and CNS depressants. The plant is known for antifungal, antidiabetic, anticancer, antiinfammatory, anti-genotoxic, hepatoprotective, hypolipidemic, antioxidant, antimicrobial and anti-estrogenic effects 14 . Flavonoids and phenol play important role in antioxidant activity of plants by absorbing and neutralizing free radicals (Sarkar et al. 2014). Rutin and quercetin is a natural flavones derivative having strong free radical scavenging property used in treatment of several diseases. The pharmacological properties of rutin include antibacterial, antiviral, antitumour, anti-protozoal, anti-allergic, anti-inflammatory, hypolipidaemic and vasoactive (Calabro, 2005). Gallic acid is obtained from tannic acid by hydrolysis with H 2 SO 4 and the biological properties of gallic acid includes radical scavenging, anti-fungal, anti-cancer, anti-inflammatory, and chemoprotective (Soong and Barlow,2006). Abo et al. reported that Senna alata was used in treatment of skin disorders such as ringworm, hernia, blennorrhagia, diabetes, constipation, hemorrhoids, intestinal parasitosis and syphilis. The different biological properties exhibited by the compound give motivation for isolation of bioactive molecules from natural sources. Oxidative stress arises due to imbalance between formation of reactive oxygen species and antioxidant defense system which may damage DNA, carbohydrate, proteins, and lipids (Halliwell and Gutteridge, 1992). Antioxidant is a defense mechanism that prevents the oxidation of other substances in low concentration the reactive oxygen species include hydroxyl radical, hydrogen peroxide, nitric oxide superoxide anion and per oxy-nitrite anion (Nagendrappa, 2005). The aim of the present study was to evaluate the phytochemical, TLC analysis and antioxidant potential of the plant in chloroform, methanol, petroleum ether and aqueous extracts.

Collection and identification of plants:
The leaf, stem and root parts of the plant were collected from Durg district, Chhattisgarh, India and authenticated by Botanical Survey of India, Allahabad. The parts of the plant were washed; shade dried and grinned to make powder Powdered samples were used for preparation of extracts in soxhlet apparatus.

Preparations of Extracts:
10gms of each powder was taken in conical flask and 100ml of each solvent (methanol, chloroform, petroleum ether, aqueous) were added separately. Soxhlet apparatus is used for preparation of extracts with different solvents of their increasing polarity. Extraction were done until the solvent in soxhlet became colorless then each extract was collected in separate bottles. The extracts were stored at -20 o C and used for phytochemical analysis.

Qualitative phytochemical analysis:
Phytochemical screening was carried out by following the standard protocol of Brindha et al. and Edeoga et al. for the presence of alkaloids, glycoside, steroids, tannins, flavonoids, saponin quinones, terpenoids and gum.

TLC analyses:
Thin layer chromatography is used for separation of bioactive molecules of the plant Silica gel "G" is used for preparation of TLC plates, homogeneous suspension of silica gel was spayed over the plates and dried in air. TLC plates were activated in hot air oven at 110 o C for 30 min and after cooling the plates were used for chromatography. Methanol based leaf extracts were used as sample and the mobile phase contain ethyl acetate: methanol: water in ratio (36:36:28). The plates were visualized under UV-Transilluminator for detection of spots and the Rf values were calculated by using following formula Rf value = Distance travelled by solute/ Distance travelled by solvent DPPH-Method: DPPH-free radical scavenging activity of methanol, chloroform, petroleum ether and aqueous extract of Senna alata were examined by following the standard protocol (Koleva et al., 2002). DPPH in its radical form absorbs at 517 nm but it absorption decreases due to reduction by an antioxidant or a free radical species. DPPH solution (03 mM) was prepared in methanol and kept in dark bottle to protect it from light. Control solution was prepared by dissolving 1ml of DPPH in 3ml of methanol solution. Test sample contain varying concentration (02, 04, 06, 08, 1 mg/ml) of different extracts and 1ml of DPPH was added and final volume of 4ml is makeup with methanol. The tubes were incubated in dark for 30 minutes and reading was taken at absorbance 517nm in UV-Spectrophotometer. Ascorbic acid was taken as standard using methanol as diluents. The percentage inhibition by sample treatment is determined by the formula:  (Table 1, 2 and 3) showed presence of tannin, saponin, alkaloid, flavonoid, cardiac glycoside, steroids, terpenoids, quinones whereas gum was found absent in all parts and extracts of the plant. In leaf extract of Senna alata glycoside and alkaloid was found in petroleum ether, tannin, flavonoid, glycoside, quinones and steroid was found in chloroform extract. In methanol extract all the phytocompounds was present except gum whereas in aqueous extract saponin, tannin, flavonoids and quinones was found. In stem extracts cardiac glycoside, alkaloids and quinones was found present in petroleum ether and chloroform extract where as tannin, terpenoid, flavonoid, glycoside and quinones was found in methanol extract whereas saponin, tannins and quinones were found in aqueous extract. In root extract saponin and alkaloid was found in petroleum ether extract, tannin glycoside, alkaloid and quinones was found in chloroform extract, saponin, tannin, steroid, glycoside and quinones was found in methanol extract. Tannin (Table 3) Fig 1:-Showing TLC profiling of leaf, stem and root methanol extract of Senna alata.

Antioxidant activity:
The antioxidant activity of leaf, stem and root extracts of plant was estimated by DPPH free radical scavenging activity. DPPH method is fast, easy and cheap method to measure the antioxidant activity in food samples (Kirtikar and Basu, 1975).Ascorbic acid is used as standard and absorbance was taken at 570nm. The optical density of the sample decreases due to decolorization of purple colour of DPPH to yellow colour which may be due to pairing of DPPH free radical with free radical species of sample (Ghosh, 1998). In leaf based extract of Senna alata the antioxidant activity was found in methanol, aqueous and petroleum ether extract and absent in chloroform extract. As, on increase the concentration of sample the optical density decreases due to increased percentage of scavenging of free radicals. The antioxidant activity of ascorbic acid was found highest. In stem extract antioxidant activity was found in methanol, aqueous and chloroform extract where as in root part methanol and aqueous based extracts. Methanol and aqueous extract showed potent antioxidant activity in leaf, stem and root part which may be due to presence of flavonoids and phenolic compounds. Panichayupakaranant et al. reported antioxidant activity in methanolic extracts of Senna alata by DPPH method. Pharmacological and clinical studies suggested that flavonoids exhibit radical scavenging activity against free radicals and acts effectively in inhibiting oxidation of lipoproteins and prevents various human diseases (Ames, 1993;Heinonen et al., 1998;Rice Evens, 1996). Senna alata showed significant scavenging effect on the DPPH free radical and the optical density of the sample decreases with increasing concentration of sample extract which may be due to presence of flavonoids and phenolic compound. The percentage of free radical scavenging activity was calculated by using formula and the radical scavenging activity increases with increasing sample extracts. Antioxidant activity is evaluated mostly by DPPH method due to difference in free radical scavenging activity of different compounds the rate of reaction of DPPH differs in different compounds (Janaszewska and Bartosz, 2002

Conclusion:-
Senna alata is a traditional medicinal plant known for its ethno pharmacological features and valued for laxative effect and used in treatment of several skin diseases such as ringworm and scabies. Many authors have contributed for detection of bioactive molecules of this plant. In the present study we have detected phytocompounds in four extracts from leaf, stem and root part by phytochemical and TLC analysis. The secondary metabolites detected in plant include tannin, saponin, alkaloid, glycoside, quinones, flavonoid and steroids in different extracts. The plant showed potential antioxidant activity in methanol and aqueous based leaf, stem and root extracts. Our finding suggests the importance of plant used as a source of natural antioxidant to prevent oxidative stress and also used to inhibit antimicrobial growth. Thus this study should beneficial for detection and structure elucidation for pharmaceutically lead compound.