Exploration of cytotoxic and antioxidant potential of Antigonon leptopus (Family: Polygonaceae)

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1 World Journal of Pharmaceutical Sciences ISSN (Print): ; ISSN (Online): Published by Atom and Cell Publishers All Rights Reserved Available online at: Original Article Exploration of cytotoxic and antioxidant potential of Antigonon leptopus (Family: Polygonaceae) Lopamudra Pradhan and Sunita Bhatnagar* Medicinal and aromatic division, Regional Plant Resource Centre, Ekamra kanan, Nayapalli, Bhubaneswar, India ABSTRACT Received: / Revised: / Accepted: / Published: Antigonon leptopus leaf extracts were explored for their phytochemical, antioxidant and cytotoxic activity. Four solvent extracts namely hexane, chloroform, ethyl acetate and methanol extracts were prepared. All extracts showed the presence of one or the other important class of compounds, like hexane was positive for flavonoids, whereas sapponins and tannins were present in methanol extract. Ethyl acetate extract showed very good antioxidant activity in TLC based assay and mild to good antioxidant activity in DPPH radical scavenging, FRAP and Nitric oxide radical scavenging assays. Ethyl acetate extract also exhibited mild cytotoxic activity (55%) in brine shrimp assay at a higher dose of 200microgram/ml. Key Words: Antigonon leptopus, Cytotoxic, Antioxidant, Alkaloids INTRODUCTION Antigonon leptopus is commonly known as Mexican Creeper and coral vine. The coral vine is found in coastal areas of Andhra Pradesh, India. The tubers are edible have a nut like flavor, and are used in Mexico for food [1]. Traditionally the leaves of A. leptopus reduce swelling, and a tea from the leaves can be made to treat diabetes and the blossoms are used for treating high blood pressure [2]. Plant also possesses anticoagulant, analgesic, antithrombin, antiinflammatory, antidiabetic and lipid peroxidation, antihelmintic and anticonvulsant activity [3]. The vine is used in treating cough and throat constrictions. In Chinese traditional medicine, it is used for the treatment of nephritis, hepatitis and colitis [4]. In view of the folklore use in the treatment of a number of diseases and the presence of flavonoidal glycosides in the leaves of A.leptopus, the present study was undertaken to assess the biological importance of the same by exploring the phytochemical, cytotoxic and antioxidant profile of the plant. MATERIALS AND METHODS Collection and processing of plant material: Fresh leaves of medicinal plant (Antigonon leptopus) were collected from the medicinal germplasm garden of Regional Plant Resource Centre (RPRC), Bhubaneswar. The leaves were dried in shade. After drying, the leaves material were grinded using mechanical blender (Lexus make) in to fine powder. Same was used for preparing extracts. Solvent extracts were prepared by Soxhlet extraction method. About 20 gm of powder was uniformly packed into a thimble and extracted with 250 ml of solvents starting from nonpolar to polar solvents. Solvents used were hexane, chloroform, ethyl acetate and methanol. After extraction, extracts were concentrated under vacuum at lower temperature of 40 ºC using Rotary Evaporator (Bucci make). Concentrated extracts were kept in tight screw cap vials and were stored in a refrigerator at 4ºC till further use. Phytochemical analysis: Phytochemical tests were conducted as per the methods of Trease et al [5] and Harborn [6]. Phytochemical test: Test for alkaloid: Mayer s Test: 100 µl extract was taken in a test tube to which 2 ml of dilute HCl was added and 1ml of Mayer s reagent was added in drop wise manner. Yellow buff or cream colour precipitate indicates the presence of alkaloid. Wagner s Reagent: To small amount of extract solution 2 ml dilute HCl and 1 ml of Wagner s *Corresponding Author Address: Dr. Sunita Bhatnagar, Medicinal and aromatic division, Regional Plant Resource Centre, Ekamra kanan, Nayapalli, Bhubaneswar, India; bhasunita@gmail.com

2 reagent was added drop wise. The reddish brown precipitate shows the presence of alkaloid. Dragendroff s Reagent: To 200 µl of extract, 2 ml of dilute HCl and 1ml of reagent was added in a test tube, the orange brown precipitate shows the presence of alkaloid. Test for flavonoids: To 5 ml of extract, 1 ml of 10% NaOH solution was added, yellow colour turning to colourless is an indication of presence of flavonoids. Test for anthraquinones: To 1ml of extract, 2 ml of 5% KOH was added. Pink colour shows the presence of anthraquinone. Test for saponins: About 2 ml of 1%NaHCO 3 was added to 1 ml of extract and was shaken vigourously. Lather like formation persistent for some time is indication of presence of saponins. BEA Benzene: Ethanol: Ammonium hydroxide (90:10:1) [Non polar/basic] EMW Ethyl acetate: Methanol: Water (40:5.4:4) [Polar/neutral] CEF Chloroform: Ethyl acetate: Formic acid (5:4;1) [Acidic] The precoated TLC sheets 60 F 254 (Merck Company) were activated at C for 10 minutes. The samples were then spotted with the help of micro tips leaving 2 cm from the bottom of the sheet. All the above mentioned solvents were used for TLC chromatographic separation of solvent extracts. After drying of sheets DPPH solution was sprayed. Yellow bands in purple background represent the antioxidant bands of the extract [7]. Rf values of all the antioxidants was calculated using the following formula. Retardation factor (R f) = Distance travelled by the compounds / Total distance travelled by the solvents. Test for tannin: 1gm of sample was added with 100ml of distilled water boiled and cooled. Then 1% of FeCl 3 was added drop wise to the aqueous solution. Green black precipitate shows the presence of tannins. Test for terpenoids: 400 µl of chloroform was added to 1ml of extract. Then 23 drops of H 2SO 4 was added. Reddish brown color shows the presence of terpenoids. Test for starch: 1gm of dried powder was taken and grinded thoroughly using mortal pestle with 10 ml of distilled water and filtrate alcoholic iodine solution was added to the filtrate. Blue colour indicates the presence of starch. Test for phlobatannin: Fresh leaves powder of plant was grinded with distilled water to make aqueous solution. Then the mixture was filtered and filtrate was taken as sample. 1ml of aqueous 1% HCl was added to the 1ml of sample followed by boiling. A red precipitate is indication of phlobatannin. Test for cardiac glycoside (Keller Killani test): About 5 ml of the extract was mixed with 2 ml of glacial acetic acid containing 1 drop of FeCl 3 solution. To this 1ml of concentrated H 2SO 4 was slowly from the sides of the test tube. Formation of brown ring at the interphase is the indication of presence of cardiac glycosides. Antioxidant activity TLC based antioxidant assay: Three types of solvents were prepared for TLC chromatography technique. Quantitative antioxidant assay: Ferric reducing antioxidant power assay (FRAP ASSAY): Total antioxidant activity was measured by ferric reducing antioxidant power (FRAP) assay of Benzie & Strain [8] and William et al [9]. DPPH radical scavenging assay: A reaction mixture containing 50µl of 1mM DPPH solution and 100 µl of various concentrations of plant extracts (31.25, 62.5, 125, 250, 500 and 1000μg/ml) were prepared in methanol. Control consisted of only 100µl of methanol and 50 µl of DPPH. Samples were incubated in dark for 30 min at room temperature. The yellow colour chromophore formed was measured at 517nm using Multiplate Reader. Ascorbic acid was used as standard. Nitric oxide radical scavenging Assay: Sodium nitroprusside 5 mm was prepared in phosphate buffer (ph 7.4). To 1 ml of various concentrations of test compound, sodium nitroprusside 0.3 ml was added. The test tubes were incubated at 25 ºC for 5 hr after which, 0.5 ml of Griess reagent was added. The absorbance of the chromophore was read at 546 nm. The experiment was performed in triplicates. Cytotoxic activity of solvent extracts: Cytotoxic activity was conducted using brine shrimp assay [10]. Brine shrimp (Artemia salina) eggs were incubated for 48hrs (1.8gm of black salt in 100ml of distilled water) to get the desired growth of the larvae for biological evaluation. Stock solution of different extracts was prepared at a concentration of 10μg/ml. Extract was evaluated at the doses 25, 358

3 50 & 100μg/ml. For each dose level three replicates were used. Motility, readings were taken every hour up to 4hours. After 24hrs, number of live parasites was counted in all the samples, percentage inhibition was calculated by comparing the treated samples with the controls. Standard deviation was also calculated. RESULTS AND DISSCUSION Moisture content of leaf samples were found to be 75 percent and yield of methanol extract was highest followed by chloroform, ethyl acetate and hexane respectively. As can be observed from the Table 2, Fresh leaves showed the presence of alkaloids, tannins, terpenoids, sapponins and phlobotanins. Hexane extract consisted of only flavonoids. Study is in confirmation with the earlier reports in which Flavonoidal glycosides have been reported from the leaf of Antigonan leptopus[11]. Chloroform extracts showed the presence of cardiac glycosides and terpenoids whereas ethyl acetate was the richest extract consisting of five class of phytochemicals. Methanol showed the presence of tannins and saponins. Presence of terpenoids, alkaloids, tannins and sapponins is significant as all of these have known medicinal potential [12]. Antioxidant activity of Antigonan leptopus Qualitative Antioxidant Screening: Qualitative antioxidant activity was conducted using TLC based DPPH assay. DPPH is a stable free radical and accepts an electron, or hydrogen radical to become a stable diamagnetic molecule. The intensity of the yellow colour depends on the amount and nature of radical scavenger present in the plant extract. All the extracts were subjected to this qualitative screening and ascorbic acid was used as standard. Three solvents were selected on the basis of polarity. All the extracts showed antioxidant bands with the highest number of 9 bands in ethyl acetate extract. Thus, ethyl acetate extract showed highest number of probable antioxidant molecules. (Table 2). Quantitative antioxidant activity DPPH free radical scavenging assay: Ethyl acetate extract exerted an inhibition of 84.32%, methanol extract exerted an inhibition 88.17% and that of ascorbic acid was 92.72% at 500 μg/ml. The IC 50 of ethyl acetate extract was calculated i.e µg/ml and methanol extract µg/ml, while that of ascorbic acid was μg/ml. Although methanol and ethylacetate extracts showed good antioxidant activity but were not as effective as ascorbic acid. Reason could be that crude extract is a mixture of a number of molecules, some of which act synergistically where 359 as others act antagonistically with one another and hence pure compounds have more activity as compared with the crude (Fig 1). Ferric reducing antioxidant power assay: The antioxidant can donate an electron to free radicals, which leads to the neutralization of the radical. Reducing power was measured by direct electron donation in the reduction of Fe 3 to Fe 2. The product was visualized by forming the intense Prussian blue colour complex and then measured at 593 nm. A. leptopus extract showed concentrationdependent reducing power. However, its reducing power was weaker than that of ascorbic acid, which exhibited the strongest reducing power (Fig 2). FRAP value of ethyl acetate and methanol was more than 2, which is however considered good activity. Nitric oxide radical scavenging assay: Nitric oxide (NO. ) is an important chemical mediator generated by endothelial cells, macrophages, neurons, etc. and involved in the regulation of various physiological processes. Excess concentration of nitric oxide is associated with several diseases such as vascular collapse, various carcinoma and ulcerative colitis. Oxygen reacts with the excess nitric oxide to generate nitrite and peroxy nitrite anions (ONOO ), which act as free radicals [13]. In the present study, the plant extracts compete with oxygen to react with nitric oxide and thus inhibits generation of the anions. Standard used was quercetin and none of the extracts showed better activity as compared to quercetin. Thus, overall it can be concluded that Antiogonan leptopus does possess mild antioxidant activity and a number of antioxidant molecules as observed in quantitative tests. Brine shrimp mortality assay (cytotoxicity): Artemia salina is one of the convenient organisms for toxicity tests in vitro due to its robust nature and easy maintenance in the laboratory condition. The bioassay study with Artemia larvae was carried out as short term toxicity test and only on freshly hatched nauplii. As can be observed from Fig 4, chloroform and ethyl extracts showed dose dependent activity and all the extracts showed mild activity. CONCLUSION Moisture content of leaves of Antigonon leptopus was found to be % and methanol extract showed the highest yield (7.82%) as compared to other extracts. Cytotoxic activity of all the extracts was mild, Qualitative as well as quantitative antioxidant results of the ethyl acetate extract were promising. As ethyl acetate extract has shown promising antioxidant potential and in qualitative

4 analysis it showed a number of antioxidant bands, hence same extract could be explored further for the isolation of antioxidant molecules. Thus, study has provided lead for the isolation of antioxidant principles. Acknowledgements: We would like to thank Forest and Environment Department, Govt of Odisha for providing Laboratory facilities for the work. TABLE 1: Phytochemical analysis of leaf extract Phytoconstituent Fresh leaves Hexane Chloroform Ethyl acetate Methanol Alkaloid Flavonoid Anthraquinone Tannin Cardiac glycoside Terpenoids Saponins Phlobatannin Starch () presence of phytoconstituennt, () absence of phytoconstituent TABLE 2: Qualitative tlc analysis of Antigonon leptopus in leaves Extract Rf value/no of bands EMW CEF BEA Ascorbic acid Hexane Chloroform Ethyl acetate Rf value no separation 0.21 no separation no of bands Rf value 0.43, , 0.78, 0.57, , 0.25, 0.37, 0.74, 0.68,0.59 no of bands Rf value 0.31, 0.38, 0.70, 0.07, 0.55, 0.81, 0.84, 0.08, 0.25, 0.37, no of bands , 0.53, 0.45, 0.40, Rf value 0.65, 0.84, ,0.19, 022, 0.63, no of bands Methanol Rf value Streak 0.54, 0.25, 0.15 no separation no of bands

5 Fig 1: DPPH radical scavenging assay of solvent extracts of Antigonan leptopus Fig 2. FRAP assay of Antigonon leptopus Fig 3: Nitric oxide radical scavenging assay of Antigonon leptopus 361

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