Venkata Smitha P et al. / Journal of Pharmacy Research 2012,5(3),1734-1738 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Screening of Antimicrobial and Antioxidant Potentials of Acacia caesia, Dillenia pentagyna and Buchanania lanzan from Maredumilli Forest of India Venkata Smitha P 1, Murali Mohan Ch 2*, Prameela Kandra 2, Sravani R 2 and Raju B. Akondi 3 1Department of Biochemistry, GITAM Institute of Science, GITAM University,Visakhapatnam – 530 045, A.P., India 2Department of Biotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam – 530 045, A.P., India 3Department of Pharmacology, St.Peters institute of Pharmaceutical Sciences, Warangal -506001, A.P., India Received on:10-12-2011; Revised on: 15-01-2012; Accepted on:17-02-2012

ABSTRACT Despite the numerous advances in medicine, the prevalence of infectious diseases continues to rise due to emergence of antibiotic resistant pathogens, which are attributed to the widespread use of antibiotics. Antioxidants help to deal with oxidative stress which is caused by free radical damage. So search for new antimicrobial and antioxidant agents from plants has now- a- days gained an importance. Methanol extracts of Acacia caesia (bark), Dillenia pentagyna (bark) and Buchanania lanzan (bark) were screened for antimicrobial potential against seven bacterial species (Bacillus cereus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella sp, Staphylococcus aureus) three fungal species (Aspergillus niger, penicillium sp. and Trichoderma viride) by disc diffusion method along with minimum inhibitory concentration (MIC) against bacterial sp. The extracts were also evaluated for their in vitro antioxidant activity using FRAP method. B. lanzan showed highest zone of inhibition (7-11 mm) followed by D. pentagyna (7-10 mm) and A. caesia (7-10 mm). The phytochemical analysis had showed the presence of saponins, flavanoids, steroids, cardiac glycosides, alkaloids, tannins and phenolics. This is the first report on the antioxidant property of A. caesia and D. pentagyna. These plants are having excellent antioxidant potential of 84.74±0.32 mg GAE/g and 109.91±0.18 mg GAE/g respectively. Results obtained in this work justify that these plants have broad spectrum antimicrobial activity and a potential source of new classes of antibiotics and antioxidants.

Keywords: Antibacterial activity, antioxidant activity, minimum inhibitory concentration, phytochemicals, maredumilli

INTRODUCTION Infectious diseases are the leading cause of death world-wide. Antibiotic cular, neurological diseases, cancer and oxidative stress dysfunctions [8, 9]. resistance has become a global concern. The clinical efficacy of many existing Therefore, the search for exploitation of natural antimicrobial agents and antibiotics is being threatened by the emergence of multidrug-resistant patho- antioxidants, especially of plant origin, has greatly increased in recent years. gens. Many infectious diseases have been known to be treated with herbal remedies throughout the history of mankind. Natural products, either as Buchanania lanzan Spreng. (Family: Anacardiaceae) commonly known as pure compounds or as standardized plant extracts, provide unlimited oppor- Char in hindi is a Sub deciduous tree, 13-17m high and up to 1.3m in girth, tunities for new drug leads because of the unmatched availability of chemical dark grey bark, leaves alternate, petiolate, very coriaceous or hard sessile diversity [1]. There is a continuous and urgent need to discover new antimi- greenish white flowers and stone hard bi- valved kernel has a pleasant sweet- crobial compounds with diverse chemical structures and novel mechanisms ish acidic flavor; found throughout the hot dried parts of India [10]. All parts of of action for new and re-emerging infectious diseases [2] .The fact that the plant are used for the treatment of various disorders. The oil from the microorganisms nowadays tend to develop resistance towards drugs, coupled seeds is used to reduce granular swelling of the neck [11, 12]. Ointment is made to the undesirable side effects of certain antibiotics offer considerable poten- from the kernel which is used to relieve itch and prickly heat. The gum from tials for the development of new effective antimicrobial and antioxidant the bark used for treating diarrhea and intercostals pains and leaves are used agents; medicinal plants are a prolific source [3]. In recent years, there has for promoting wound healing [13, 14]. The rhizome of B. lanzan finds an impor- been a gradual revival of interest concerning the use of medicinal and aromatic tant place in indigenous medicine as an expectorant, diuretic and carminative plants in developed as well as in developing countries, because plant-derived [15]. It is also found to have anticancer [16] antihypertensive [17] and larvicidal drugs are reported to be safe and without any side-effects especially when activity [18]. It is used for the treatment of various skin disorders, rheumatism compared with synthetic drugs [4,5]. It has been proved that various plants and diabetes mellitus [19, 20]. extracts posses bacteriostatic and bactericidal effects [6] and most of these plants contain many active compounds. The most important of these bioactive Dillenia pentagyna Roxb. of Dilleniaceae family is an endangered tree species constituents of plants are alkaloids, tannins, flavanoids, and phenolic com- accounts for many ethnical uses. The tribal’s and folk communities use vari- pounds [7]. Most of these secondary metabolites other than possessing anti- ous parts of it for treatment of their different ailments and diseases, viz. microbial potential, can also act as potent antioxidants. Antioxidants are delivery (bark), bone fracture (leaf), body pain (root), piles (leaf), diabetes closely related with the prevention of degenerative illness such as cardiovas- (bark), diarrhea and dysentery (bark) [21]. A wide range of pharmacological and biological activities was exhibited by the secondary metabolites isolated from plants belonging to Dilleniaceae [22]. *Corresponding author. Dr. Ch. Murali Mohan, Acacia caesia Linnaeus is a leguminous perennial climbing shrub belonging to Associate Professor, the family Mimosaceae, and is native to south-east Asia. In different parts of Department of Biotechnology, India, the tender leaves are widely used in culinary preparation, and the GITAM Institute of Technology, prickly stem for fencing agricultural fields. The stem bark forms copious GITAM University, froth when rubbed with water and thus used as soap, while its decoction as [23] Visakhapatnam – 530 045, lice killer . The juicy extract from the stem bark is well-known among the Andhra Pradesh, India

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1734-1738 Venkata Smitha P et al. / Journal of Pharmacy Research 2012,5(3),1734-1738 Mizo tribes of north-east India as remedy to gastrointestinal infections. The Screening of antimicrobial activity: crude extract of the stem bark indeed showed profound anthelmintic effects Antimicrobial activity of organic extracts of the plant samples were evalu- on Raillietina echinobothrida, causing extensive damage in the worm’s tissue ated by the paper disc diffusion method [25]. For determination of antibacte- such as in the tegumental layer, muscle tissue and parenchyma [24]. rial activity, overnight grown bacterial cultures were adjusted to 0.5 McFarland turbidity standards [26]. For the determination of antifungal activity, all the The present study aimed at evaluating the antibacterial potentiality of fungal isolates were first adjusted to the concentration of 106 cfu/ml. The methanolic extracts of barks of three medicinal plants against bacteria and bacterial and the fungal broth cultures of 100 µl each were inoculated onto fungi. The minimum inhibitory concentration (MIC) for antibacterial activ- nutrient agar and potato dextrose agar plates respectively through spread ity was determined. Other objectives includes search of active phytochemicals plate method. Firstly stock solutions (100 mg/ml) of each individual plant present in the methanolic extracts of these plants and to obtain a preliminary extract were prepared separately. Sterile filter paper (What man filter paper idea about the chemical nature of the active ingredient/ingredients involved in No.1) discs of diameter 9 mm were prepared and 10 µl of each extract the antimicrobial potentiality. The antioxidant activity of these extracts is dilutions were impregnated onto the discs and carefully placed at the centre also evaluated. of the previously seeded plates with 0.5 McFarland and 106 cfu/ml cultures of bacteria and fungi respectively with sterile forceps. Disc with solvent MATERIALS AND METHODS alone was served as control. Streptomycin (10 µg/ml) for bacteria and Albendazole (10 µg/ml) for fungi was used as standard antimicrobials for Collection and identification of plant material: comparison. Bacterial culture plates were then incubated at 37°C for 24 h Three plant species were selected in the present investigation. The plants while the fungal cultures are incubated at (25°C –27°C) for 48 h. Antimicro- and their parts selected were Acacia caesia (Mimosaceae) (bark), Dillenia bial activity was determined by measurement of zone of inhibition of growth pentagyna (Dilleniaceae) (bark), Buchanania lanzan (Anacardiaceae) (bark) around each paper disc (mm). For each extract (test) three replicate trials were collected from Maredumilli forest located near Rampachodavaram were conducted against each organism. Each zone of inhibition was measured (Mandal), Rajahmandry (district), Andhra Pradesh (State), South India with with a ruler and compared with standard [27]. the help of a local Ayurvedic Doctor. The plants were identified at Kovel Foundation, Visakhapatnam with reference to passport data book of NBPGR Minimum inhibitory concentration (MIC): (National Bureau of plant genetic resources) under the Division of plant The minimum inhibitory concentration (MIC) of the extracts was estimated exploration and collection, New Delhi. A. caesia Collection No. KF V 7209 against bacterial organisms only which showed better zone of inhibition. It and Accession No. 29, D. pentagyna collection No. KF GSBVK 7175 and was carried out by the method as described as earlier [28]. The determination Accession No. 95 and Buchanania lanzan Collection No. KF VS 7274 and of the MIC was done with different dose levels of (10, 25, 50, 100 mg/ml) of Accession No. 94. extracts. To 0.5 ml of varying concentration of the extracts 10 ml of nutrient broth was added and then a loopful of the test organism previously diluted to Extraction of plant material: 0.5 McFarland turbidity standard bacterial isolates was inoculated into the The crude extracts of the three plant material were prepared separately by test tubes. The procedure was repeated on the test organisms using the methanol as solvent as described below. standard antibiotic streptomycin. Tubes containing nutrient broth only seeded with the test organism serve as control. Tubes containing bacterial Solvent extraction: cultures were then incubated at 37°C for 24 h. The minimum inhibitory The plant material was brought to laboratory and washed under running tap concentration was taken as the tube with the least concentration of the water and blotted with filter paper then shade dried on laboratory benches extract with no visible growth. by putting news papers. After complete drying the plant material was then ground into powder by using hand mill. The powdered plant material (10 g) Preliminary phytochemical screening: was placed in a Soxhlet extractor and was exhaustively extracted using 100 ml Preliminary phytochemical tests for identification of Tannins, Phenols, Sa- of methanol (60 – 80°C). The crude extracts were respectively concentrated ponins, Alkaloids, Steroids, Cardiac glycosides and Flavonoids were carried in vacuo at 40°C using a rota vapor. The crude extracts thus obtained were out for all the extracts. The extracts were analyzed for the presence of preserved in freezer at -20°C until use. The successive extractive values were Alkaloids, Saponins, Cardiac glycosides [29]; Tannins & phenols, Flavanoids 2.95 g, 3.73 g and 3.60 g respectively for the barks of A. caesia, D. pentagyna [30, 31]; Steroids [32]. and B. lanzan. The crude plant extracts were labelled as A. caesia (bark) (A), D. pentagyna (bark) (B), B. lanzan (bark) (C). In-Vitro Antioxidant study:

Test microorganisms and microbial culture: Ferric reducing antioxidant power (FRAP) method [33] Bacterial cultures of Bacillus cereus, Escherichia coli, Proteus vulgaris, The ferric reducing property of the extract was determined by taking 1 ml of Pseudomonas aeruginosa, Bacillus subtilis, Salmonella sp., Staphylococcus different dilutions of standard solutions of Gallic acid (10 -100 µg/ml) or aureus and fungal cultures of Aspergillus niger, penicillium sp. and Tricho- methanolic extract that has adjusted to come under the linearity range (500 derma viride were obtained from laboratory isolates. µg/ml) was taken in 10 ml volumetric flasks and mixed with 2.5 ml of potas- sium buffer (0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The Bacterial strains were cultivated at 37°C and maintained on nutrient agar mixture was incubated at 50ºC for 20 min. Then 2.5 ml of 10% trichloroacetic slant at 4°C.Fungal strains were cultivated at 25°C and maintained on potato acid was added to the mixture to stop the reaction. To the 2.5 ml of above dextrose agar slants at 4°C. All the cultures were tested for their purity. Pure solution 2.5 ml of distill water is added and then 0.5 ml of 0.1% of FeCl3 was cultures thus obtained were kept on respective agar slants at 4°C until needed. added and allowed to stand for 30 min before measuring the absorbance at They were sub-cultured once in every month. Each inoculum was prepared 593 nm. The absorbance obtained was converted to Gallic acid equivalent in by inoculating the stock culture into freshly prepared media. All the bacterial mg per gm of dry material (GAE/g) using Gallic acid standard curve [34] (Fig strains were incubated at 37°C for 24 h and fungi at 27°C for 48 h. The test 3). organisms were grown overnight in respective broth media.

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1734-1738 Venkata Smitha P et al. / Journal of Pharmacy Research 2012,5(3),1734-1738

R2 values represented mean data set of n=3 Fig. 3 Standard curve of Ferric reducing antioxidant power (FRAP) method. Fig. 1 Bar chart showing results of antibacterial susceptibility of test RESULTS organisms to methanol extracts of Acacia caesia (A) ; Dillenia pentagyna The present study was conducted to investigate antibacterial properties of (B) ; Buchanania lanzan (C). three selected plants from Maredumilli forest, which is less studied and used in Indian folk medicine. Herbal remedies play a fundamental role in tradi- tional medicine in rural areas of India where the therapeutic treatment of choice as antiseptic, anti-inflammatory and in treatment of infectious dis- eases including diarrhea. In present study, attempt was made to correlate traditional herbal medicinal knowledge held by the Indian native people with modern scientific laboratory-based assay. Antimicrobial screening of metha- nol extracts of barks of A. caesia, D. pentagyna and B. lanzan are presented in Table 1 and Fig.1 & 2. The present study revealed that the extracts of three plants showed potent antibacterial and antifungal activity against all refer- ence microbial strains. B. lanzan exhibited highest antibacterial activity (7-12 mm) against the reference strains except for E. coli and S. aureus. Antifungal activity of 6 mm, 8 mm and 10 mm was exhibited against A. niger, Penicillium sp. and T. viride respectively. D. pentagyna showed maximum antibacterial activity of (7-10 mm) against maximum strains except for P. vulgaris and antifungal activity of 7 mm, 8 mm and 8 mm was exhibited against A. niger, Penicillium sp. and T. viride respectively. Fig. 2 Bar chart showing results of antifungal susceptibility of test Table: 1 Results of antimicrobial activity of methanol crude plant ex- organisms to methanol extracts of Acacia caesia (A) ; Dillenia pentagyna tracts of three different plants against pathogenic bacteria and fungi. (B) ; Buchanania lanzan (C).

Zones of inhibition (Mean ± SD) (mm)a A. caesia showed antibacterial activity of (7-10 mm) against five strains Microorganisms* Crude plant extracts tested b tested and no activity against E. coli and Salmonella sp. It showed no anti- A B C Antibiotics† positive standards fungal activity against the three tested fungal strains. The results of minimum inhibitory concentration (MIC) of the extracts were showed in Table 2. Bacterial Strains Among three plant extracts the MIC values are lower for B. lanzan (25 mg/ Escherichia coli 0 10 ±1.2 0 15 ±0.9 Bacillus subtilis 7 ±0.7 8 ±1.4 8 ±0.4 15 ±0.4 ml) against B. cereus and Salmonella sp. (50 mg/ml) against P. aeruginosa Bacillus cereus 10 ±0.6 9 ±0.9 12±0.8 15 ±0.6 and (100 mg/ml) against P. vulgaris and B. subtilis followed by D. pentagyna Staphylococcus aureus 7 ±0.9 7 ±0.4 0 20 ±0.8 which showed (50 mg/ml) against E. coli, B. cereus and Salmonella sp. & Pseudomonas aeruginosa 10 ±0.6 7 ±0.9 9±0.5 19 ±0.6 (100 mg/ml) against B. subtilis, S. aureus and P. aeruginosa. A. caesia showed Proteus vulgaris 7±0.4 0 7 ±1.2 20 ±0.3 Salmonella sp. 0 9 ±0.6 12 ±1.5 18 ±1.0 (50 mg/ml) against B. cereus & P. aeruginosa, (100 mg/ml) against B. subtilis, Fungal Strains S. aureus & P. vulgaris. The preliminary phytochemical analysis of the Trichoderma viride 0 8 ±0.4 10 ±0.9 0 methanol extracts of these plants showed the presence of saponins, flavanoids, Penicillium Sp 0 8 ±0.8 8 ±0.3 20 ±0.6 steroids, cardiac glycosides, alkaloids, tannins and phenolics showed in Table Aspergillus niger 0 7 ±0.7 6 ±0.9 10 ±1.6 3. aValues are mean of three replicates ± SD Capacity of FRAP method bPlant names: A- Acacia caesia; B- Dillenia pentagyna; C- Buchanania lanzan. At low pH, measuring the change in absorption at 593 nm can monitor † Antibiotics used as positive Standards: Streptomycin (for bacterial strains); reduction of a ferric complex to the ferrous form, which has an intense bluish Albendazole (for fungal strains) green color. The change in absorbance is directly related to the combined or

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1734-1738 Venkata Smitha P et al. / Journal of Pharmacy Research 2012,5(3),1734-1738 Table: 2 Minimum inhibitory concentrations (MIC) Leaf extracts of the plant species, A. caesia L. by using different alcoholic Plant Extract Test Organism I II III IV solvents were tested against the ten human pathogenic bacteria and results support the therapeutic importance of A. caesia in curing infectious diseases A Bacillus cereus + + * - and encourage the extensive use of it in health care practices [39].The present Pseudomonas aeruginosa ++ + * - study provides the first evidence on its antioxidant activity. Bacillus subtilis + + + * Staphylococcus aureus + + + * Proteus vulgaris + + + * D. pentagyna commonly found tree was taken to explore the antimicrobial B Escherichia coli ++ + * - and antioxidant properties as there was very limited work done on the plant. Bacillus subtilis ++ + + * Bacillus cereus + + * - Terpenoids and flavonoids were the most well known among the secondary Staphylococcus aureus + + * metabolites isolated from plants belonging to Dilleniaceae, though a large Pseudomonas aeruginosa + + + * amount of alkaloid was also searched [22, 40]. Rhammentin 3-glucoside also Salmonella sp. + + * - isolated from the D. pentagyna [22]. Two new flavonoid glycosides, naringenin C Bacillus cereus + * - - Salmonella sp. + * - - 7-galactosyl and dihedral quercetin 5-glactoside were isolated from D. Bacillus subtilis + + + * pentagyna have been found to exhibit cytotoxic and lymphocytic activity [41]. Pseudomonas aeruginosa + + * - A new diterpene, dipoloic acid isolated from the stem of D. pentagyna exhib- Proteus vulgaris + + + * ited cytotoxic activity [41]. This plant is shown to possess highest antioxidant Plant names: A- Acacia caesia; B- Dillenia pentagyna; C- Buchanania lanzan. activity due the presence of these flavonoids. I = 10 mg/ml, II = 25 mg/ml, III =50 mg/ml, IV=100 mg/ml *=MIC, - = No growth, + = little growth, ++ =Dense growth B. lanzan showed highest zone of inhibition among the three plants. Prelimi- Table: 3 Results of preliminary phytochemical screening of plant extracts. nary phytochemical testing which is carried using chemicals for the identifi- cation of various phytoconstituents suggest the presence of glycosides, Tests A B C tannins, phenolic compound and steroids in the bark extract of B. lanzan, Tannins and Phenols - + + which may be responsible for their antibacterial effects. Still, further analy- Saponins + + - ses of the antioxidant capacity of the beneficial compounds of these plants alkaloids + + - are needed for a better understanding of how to implement them in functional Steroids - - + Cardiac glycosides - - + medicine. The broad spectrum of antimicrobial activity may help to discover Flavonoids + + - new chemical classes of antibiotic substances that could serve as selective agents for infectious diseases, chemotherapy and control. A- Acacia caesia; B- Dillenia pentagyna; C- Buchanania lanzan; + = Pres- ence; - = Absence ACKNOWLEDGEMENTS: “total” reducing power of the electron-donating antioxidants present in the The authors are grateful to Mr. Subba Rao, Tribal Doctor, Tallapalem (Vil- reaction mixture. lage), Maredumilli, East Godavari, A.P., India for helping in plant collection and also Kovel Foundation for providing assistance in plant collection and Here the FRAP showed the results of methanol extracts of A. caesia (bark), authentication. Authors are also thankful to Dept. of Microbiology, Andhra D. pentagyna (bark) and B. lanzan (bark) that of 84.74±0.32mg equivalent to Medical College, Visakhapatnam for providing microbial cultures. Authors Gallic acid (GAE)/g of sample, 109.91±0.18mg GAE/g and 49.05±1.02mg are also grateful to Management and Head, Dept.of Biotechnology, GIT, GAE/g of sample respectively shown in Table 4.The present study has GITAM University for providing necessary facilities. shown that these three plant extracts exhibited maximum antioxidant capac- ity. Considering all the three plants, bark of D. pentagyna registered maxi- REFERENCES: 1. Ujjwal N, Ruchi S, Rashmi KM, Raju KC, Lipid Content and in vitro mum activity followed by bark of A. caesia and B. lanzan. antimicrobial activity of oil seeds of some Indian Medicinal Plants, Table: 4 Results of Ferric reducing antioxidant power (FRAP) method Current Research in Bacteriology, 1, 2008, 1-6. 2. Parekh J, Chanda SV, In vitro antimicrobial activity and phytochemi- Plant extracts Conc. of extracts mg GAE/g of extracts cal analysis of some Indian medicinal plants, Turkish Journal of Biology, 31, 2007, 53-58. A 500µg/ml 84.74±0.32 3. 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Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1734-1738