In Vitro Antioxidant Activity and Antibacterial Activity of Ethyl Acetate Extract of Medicinal Tree Species, Anacardium occidentale L. and Mangifera indica L

This study was undertaken to evaluate the antioxidant property and antibacterial activity of ethyl acetate extract of mature leaves and flower of the medicinal tree species, Anacardium occidentale and Mangifera indica. 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH) free radical scavenging method and agar well diffusion method was used to detect antioxidant activity and antibacterial activity respectively. Various plant extracts of both the tree species studied were found to have potent antioxidant activity against DPPH and antibacterial activity against five human pathogens such as Pseudomonas aeruginosa; Salmonella typhi; Bacillus subtilis; Escherichia coli and Staphylococcus aureus. Therefore ethyl acetate extracts of mature leaf and flower extract of both A.occidentale and M.indica can be considered as a new potential source of natural antioxidants and antibacterial agent for pharmaceutical industries.

During the last few decades there has been an increasing demand in the study of medicinally important traditional plants in different parts of the world. A.occidentale is a tropical tree indigenous to Brazil, a member of the family Anacardiaceae, which is now widely grown in other tropical countries like India and is a multi-purpose plant (Togun 1977). Many parts of this tree are used in traditional medicine. Commercially important two parts are cashew nut for diet and the liquid from nut shell (CNSL) for various industrial and medical applications (Joseph 1975;Pillai et al. 1990). Anacardic acid is having very much demand in the international market (Rodrigues et al. 2004). Apart from this, a large number of other phenolic compounds are founds in very small quantities in the shell (Miraliakbari and Shahidi 2008). Phenol is seen throughout the plant system though it is mainly concentrated in the nut shell. The mango (Mangifera indica L.) is one of the choicest fruit crops of tropical and sub-tropical regions of the world, especially in Asia. Its popularity and importance can easily be realized by the fact that it is often referred as 'King of fruits' in the tropical world (Singh et al. 1991). Mangiferin is the most important phenolic compound in mango leaves, bark, peels and kernels and is present in particularly high quantities in young leaves . Mangiferin has many biological activities, including anticancer, antimicrobial, anti-allergenic, anti-inflammatory, Print ISSN : 1974ISSN : -1712 Online ISSN : 2230-732X analgesic, immunomodulatory and hypolipidemia, as well as antioxidant activity (Masibo and He 2008).
Antioxidants act as a defense mechanism that protect against oxidative damage and include compounds to remove repair damaged molecules. It can prevent the oxidation caused by free radicals and sufficient intake of antioxidants is supposed to protect against diseases. Plants are the source of medication for preventive, curative, protective purposes. Many of these herbal medicines are finding this way into the world market as alternatives to prescribed allopathic drugs currently available to treat various disorders and aliments.
The development of new microbial agents against resistant pathogens is increasing interest. Therefore, the ethyl acetate extract from different parts of medicinal plants used locally in folk medicine was evaluated for antimicrobial activity. Antimicrobial agents are used to inhibit the activity of microorganisms. The use of plant extracts and phytochemicals, with established antimicrobial properties, could be of great significance in preventive and/or therapeutic approaches. In view of the above, the present study has been made to investigate the antioxidant potential as well as the antimicrobial activities of a traditionally used medicinal tree plants such as A. occidentale L. and M. indica L.

Plant material
The plant materials (mature leaves and flower) of A. occidentale and M. indica were collected from mother stock trees. The shade dried mature leaves and flower of the study species were made into fine powder.

Preparation of plant extracts
The shade dried plant materials were ground and used for preparing extracts. Powdered samples were extracted with ethyl acetate by maceration and kept it for a period of 24 hrs at room temperature at a ratio of 1:100 (g:ml). Homogenized samples were centrifuged at 10,000 rpm for 15 minutes and supernatants were pooled. Then the extract was filtered to remove all undissolved matter, including cellular material and other constituents that are insoluble in the extraction solvent and each extract was concentrated in a rotary evaporator to remove ethyl acetate. The residue thus obtained was dissolved in ethyl acetate and stored at 4-8°C in a refrigerator for further analysis (Muthusamy et al. 2013;Harborne 1998)

DPPH radical scavenging activity
The 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH) scavenging activity was measured (Kumarasamy et al. 2007). Ethyl acetate extract of the samples at various concentrations (20, 40, 60, 80 and 100 µg/ml) was added separately to each 1ml of 0.1mM ethyl acetate solution of DPPH and allowed to stand for 20min. Absorbance at 517nm using spectrophotometer was measured. Ascorbic acid was used as standard. The corresponding blank reading was also taken and DPPH radical scavenging activity was calculated by using the following formula: Where A 0 and A 1 stand for absorption of the control sample and absorption of tested extract solution respectively. The control samples contained all the reagents except the extract. IC 50 value is the concentration of sample required to scavenge 50% of DPPH free radical and was calculated from the % inhibition versus concentration sigmoidal curve, using a non-linear regression analysis.

Statistical analysis
All analyses were carried out in triplicate and the data were reported as means ± SD. The data were subjected to one way analysis of variance (ANOVA) and the significance of the difference between means was determined by Duncan's Multiple Range Test (P<0.05) using the statistical software package (SPSS for Windows, ver.17, 2008).

Antioxidant activity
The  (Silva et al. 2016). The free radical scavenging activity of young leaves, barks, roots and kernels of M. indica were also studied (Samba et al. 2018). Mangiferin is the major phenolic compound present in mango leaves ).
The antioxidant activity of different plant part extracts of A. occidentale and M. indica were also expressed in terms of IC 50 (µg/ml) values (Table 2) and it ranged from 97.34±1.33µg/ml to   the  Table 3. The control (ethyl acetate) showed no zone of inhibition against five typical bacterial strains (Fig. 1). Among the two extracts of A. occidentale tested, flower showed maximum zone of inhibition (24mm) against S. typhi. Flower showed same range of inhibitory action against B. subtilis and E. coli with a zone of inhibition of 19mm (Table 3 & Fig. 1).

C-control
It was observed that ethanol and ethyl acetate leaf extract also showed zone of inhibition against S. aureus and E. coli (Chbisika et al. 2014). Thus the present study is in conformity with early reports. The antibacterial activities of different plant parts of A. occidentale are mainly due to the presence of a phenolic lipid known as anacardic acid (Sujatha et al. 2011).
Result of the present study also revealed that ethyl acetate extract of mature leaves of M. indica showed zone of inhibition against P. aeruginosa, B. subtilis, E. coli, S. typhi and S. aureus. It was observed that among the two extract of M. indica, ethyl acetate extract of mature leaves showed comparatively high antimicrobial activity against P. aeruginosa with a zone of inhibition 24mm. It also showed 23mm zone of inhibition against S. aureus, 17mm zone of inhibition against B. subtilis (Table 3 & Fig. 1).
It was also observed that mature leaf extract of M. indica showed maximum zone of inhibition (24mm) against P. aeruginosa compared to the mature leaf extract of A. occidentale (19mm) ( Table 3 & Fig.  1). This study was also supported by early study, there the antibacterial activity of flower of M.indica against E.coli was investigated (Poongothai and Rajan 2013). It was also confirmed that ethanolic and methanolic extract of mango leaf showed relatively high zones of inhibition against S. typhi. (Zakaria et al. 2006). Plant extracts with antibacterial activities have therapeutic potential to heal several infectious diseases and are related with lesser side effects compared to the synthetic drugs.

CONCLUSION
This research provides information about the antioxidant property and antibacterial activity of various plant parts of A. occidentale and M. indica. Hence, it is identified that these two species can be used as a source for the manufacturing of drugs of scavenging property and antibacterial activity. However, large scale in vivo studies are required to confirm the scavenging property and antimicrobial activity before going for commercialization.