Evaluation of Antioxidant, Cytotoxic and Antimicrobial Activity of Phyllanthus Acidus
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Available online on www.ijppr.com International Journal of Pharmacognosy and Phytochemical Research 2016; 8(11); 1751-1758 ISSN: 0975-4873 Research Article Evaluation of Antioxidant, Cytotoxic and Antimicrobial Activity of Phyllanthus acidus Tahira Foyzun*, Koly Aktar, Mohammad Ashraf Uddin Southeast University Lecturer, Southeast University. Available Online: 15th November, 2016 ABSTRACT The aim of present study was to evaluate antioxidant, cytotoxic and antimicrobial activities of methanolic extracts of pulp and seed of Phyllanthus acidus. The antioxidant potential were evaluated in terms of total phenolic content, total flavonoid content and DPPH radical scavenging potential by specific standard procedures. Maximum phenolic (25.672± 0.645 mg gallic acid equivalents/mg of plant extract) and flavonoid (13.893 0.320 mg catechin equivalents/mg of plant extract) contents were found in pulp extract than seed extract. Both the pulp and seed extracts showed the potent antioxidant activity with IC50 value of 5.96 µg and 6.79 µg/mL respectively which are very close to the IC50 value of standard ascorbic acid having 2.16 µg/mL). The cytotoxic activity was evaluated by using brine shrimp lethality bioassay and compared with vincristine sulfate as standard. The cytotoxicity exhibited by both extract was promising with LC50 value of pulp and seed were 6.25 µg/mL and 6.7925 µg/mL respectively comparing with the LC50 value 0.4687525 µg/mL of standard vincristine sulphate as a positive control. Furthermore, the extracts were examined for antimicrobial activity against a panel of microorganisms where both the extracts CMEP and CMES showed mild to moderate antimicrobial activity. Moreover, CMEP have exhibited highest zone of inhibition which was 12mm against Pseudomonous aeruginosa. The results suggest into the plant extracts could be used as a potential therapeutics in many pathological conditions. Keywords: Phyllanthus acidus. CMEP, CMES, Antioxidants, brine shrimp lethality bioassay, antimicrobial activity. INTRODUCTION flavanoids, lignans, phenolics and terpenes15. Phyllanthus The plant kingdom has been the best source of remedies acidus, locally named as Arbaroi in Bangladesh and for curing a variety of diseases. This is why medicinal gooseberry or star gooseberry in India, is an edible small plants have been played a key role in the worldwide yellow berries fruit in the Phyllanthus family. Fruits are maintenance of health. Natural products of higher plants borne in loose clusers, are pale yellow or white, waxy, are an important source of therapeutic agents; therefore, crisp and juicy, and very sour, found in Bangladesh, South many research groups are currently screening the different India, and Southeast Asian countries16-19. Several parts of biological activities of plants1-3. Approximately 20% of the these plants have been used in folk medicine. The roots and plants found in the world have been submitted to seeds are cathartic. The fruit is a liver tonic and a blood pharmacological or biological tests4. The rapid emergence purifier and is used in several vitialed conditions of of multiple drug resistant strains of pathogens to current jaundice, bronchitis, constipation and piles in Ayurvedic antimicrobial agents has generated an urgent intensive system of medicine20. The leaves are useful to treat fever, search for new antibiotics from medicinal plants. Many piles, small pox, blood vomiting, itching and gum medicinal plants have been screened extensively for their infection21. Several therapeutic properties including antimicrobial potential worldwide5-7. Cytotoxic screening antiviral22, antibacterial23, neuroprotective24, antifibrosis25, of plants is the preliminary methods to identify active and anticancer26 activities have also been reported for compounds of plants8,9. In addition a greater interest in the Phyllanthus acidus. Extensive evaluation of traditional antioxidant activity of plant extracts exists because of free medicine for various medicinal activities is an obligatory radicals (e.g. reactive oxygen species) that can be step in the isolation and characterization of the active responsible for several diseases, for example, heart principle and further leading to drug development. In view disease, stroke, arteriosclerosis and cancer, as well as the of these, this study is therefore designed to evaluate the aging process10. Plants (fruits, vegetables, medicinal antibacterial, antioxidant and cytotoxic activities of herbs) contain a wide variety of free radical scavenging Phyllanthus acidus. molecules, such as phenolic compounds, vitamins, terpenoids and some other endogenous metabolites, that MATERIALS AND METHODS are rich in antioxidant activity11-14. Plants belonging to Collection and Identification of the Plant Sample genus Phyllanthus (Euphorbiaceae) produce useful Ripe fruit of Phyllanthus acidus were collected in the secondary metabolites such as alkaloids, tannins, vicinity of Mirpur, Dhaka Bangladesh during the month *Author for Correspondence: [email protected] Tahira et al. / Evaluation of Antioxidant… 3 y = 0.0864x + 0.0105 with occasional sun drying. These were then dried in an R 2 = 0.9997 oven for 24 hours at considerably low temperature (not more than 45⁰C) for better grinding. Dry samples of fruits were ground into a fine powder in a grinding mill. The 2 Gallic acid coarse powder was then stored in an air tight container and kept in cool and dry place for further use. Linear Extraction and Solvent Evaporation 1 (Gallic acid) Absorbance The powdered plant materials were extracted by cold extraction process. Powdered plant materials were taken in an amber colored reagent bottle and soaked in 500mL of 0 methanol. The bottle with its contents were sealed and kept 0 4 8 12 16 20 24 28 32 36 for period of about 7 days with occasional shaking and Concentration (µg/ml) stirring. The whole mixture was then filtered through cotton and Whatman no.1 filter paper and was concentrated with a rotary evaporator under pressure at Figure 1: Standard curve of gallic acid for the 50⁰C temperature to afford crude extract known as crude determination of total phenolic content. methanolic extract (CM). Determination of Total Phenolics Total phenolic content of methanolic extracts of pulp and 30 seed of Phyllanthus acidus were determined employing the method as described by Singleton et al.,196527 involving 25 Folin-Ciocalteu reagent as oxidizing agent and gallic acid CMEP as standard. Firstly, 0.5 ml of plant extract or standard of 20 CMES different concentration solution was taken in a test tube and 2.5 ml of Folin – ciocalteu (Diluted 10 times with 15 water) reagent solution was added into the test tube. Then 2.5 ml of Sodium carbonate (7.5%) solution was added and 0 10 incubated for 20 minutes at 25 C to complete the reaction. GAE/gm GAE/gm Sample of Then the absorbance of the solution was measured at 760 nm using a spectrophotometer against blank. A typical 5 blank solution contained all reagents except plant extract or standard solution. 0 Determination of Total Flavonoids CMEP CMES Total flavonoid content was determined by following the 28 Name of Sample procedure by Dewanto et al., 2002 . Catechin was used as standard and the flavonoid content of the extracts were Figure 2: Total phenolic content (mg/gm plant extract in expressed as mg of catechin equivalent/gm of dried Gallic acid equivalent) of the CMEP and CMES of extract. Firstly, one milliliter of aqueous extract containing Phyllanthus acidus. 0.1 g/ml of dry matter was placed in a 10 ml volumetric flask, then 5ml of distilled water added followed by 0.3ml 3 y = 0.0053x + 0.0606 of 5% NaNO2. After 5 minutes, 0.6 ml of 10% AlCl3 was R2 = 0.9967 added and volume made up with distilled water. The solution was mixed and absorbance was measured at 510 2 Catechin nm. Linear Antioxidant Assay 1 (Catechin) DPPH (1, 1-diphenyl-2-picrylhydrazyl) Radical Absorbance Scavenging Assay The antioxidant activity of different fractions and isolated 0 0 100 200 300 400 500 600 compounds were determined in terms of hydrogen Concentration (µg/ml) donating ability, using the DPPH method with a minor modification28,29. Firstly, 2 ml of methanol solution of plant extract or standard at different concentration was Figure 3: Standard curve of catechin for the taken in a test tube. Then 3 ml of methanol solution of determination of total flavonoids. DPPH was added into the test tube. The test tube was incubated at room temperature for 30 minutes in dark place March 2014. The plant was identified by an expert to complete the reaction. Then the absorbance of the taxonomist. solution was measured at 517 nm using a Preparation of Plant Sample spectrophotometer against blank. A typical blank solution After collecting, the pulps and seeds were washed contained all reagents except plant extract or standard thoroughly in tap water and shade dried for several days solution. IJPPR, Volume 8, Issue 11: November 2016 Page 1752 Tahira et al. / Evaluation of Antioxidant… Table 1: Absorbance of Gallic acid at different Test organisms were collected from the Microbiology concentrations after treatment with Folin-Ciocaltue research laboratory, Department of Pharmacy, Southeast reagent. University. To perform this test the extracts were dissolved Concentration Absorbance Absorbance in the same solvent used for their extraction and sterilized (µg/ml) a b C Mean ± STD by 0.22µm sterile Millipore filter. Then 100 µl inoculums 1 0.078 0.075 0.076 0.076±0.001 (106CFU/ml) was spread on the surfaces of the agar plate 2 0.176 0.171 0.181 0.176±0.005 prepared for the growth of bacteria. The discs (6 mm in 4 0.364 0.368 0.372 0.368±0.004 diameter) contain 10 µl of the extracts as 100mg, 200mg 8 0.722 0.718 0.726 0.722±0.004 and 500mg per disc was placed on the surface of the 16 1.413 1.417 1.423 1.417±0.005 inoculated media. Standard Kanamycin disc (30µg/disc) 32 2.758 2.752 2.764 2.758±0.006 was used as positive control and negative controls were prepared with the same solvents used to dissolve the Determination of Total Antioxidant Capacity sample extracts.