pentagyna and Buchanania lanzan from Maredumilli Forest of India. Venkata Smitha P ..... Botany, 2nd edition=, New York, Mc Graw-Hill Book company lnc,. 1952. .... Seo Y, Hoch J, Abdel-Kader M, Malone S, Derveld I, Adams H,. Werkhoven ...
Venkata Smitha P et al. / Journal of Pharmacy Research 2012,5(3),1734-1738
Research Article ISSN: 0974-6943
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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 Department of Biochemistry, GITAM Institute of Science, GITAM University,Visakhapatnam – 530 045, A.P., India 2 Department of Biotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam – 530 045, A.P., India 3 Department of Pharmacology, St.Peters institute of Pharmaceutical Sciences, Warangal -506001, A.P., India 1
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 resistance has become a global concern. The clinical efficacy of many existing antibiotics is being threatened by the emergence of multidrug-resistant pathogens. Many infectious diseases have been known to be treated with herbal remedies throughout the history of mankind. Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity [1]. There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases [2] .The fact that microorganisms nowadays tend to develop resistance towards drugs, coupled to the undesirable side effects of certain antibiotics offer considerable potentials for the development of new effective antimicrobial and antioxidant agents; medicinal plants are a prolific source [3]. In recent years, there has been a gradual revival of interest concerning the use of medicinal and aromatic plants in developed as well as in developing countries, because plant-derived drugs are reported to be safe and without any side-effects especially when compared with synthetic drugs [4,5]. It has been proved that various plants extracts posses bacteriostatic and bactericidal effects [6] and most of these plants contain many active compounds. The most important of these bioactive constituents of plants are alkaloids, tannins, flavanoids, and phenolic compounds [7]. Most of these secondary metabolites other than possessing antimicrobial potential, can also act as potent antioxidants. Antioxidants are closely related with the prevention of degenerative illness such as cardiovas-
*Corresponding author. Dr. Ch. Murali Mohan, Associate Professor, Department of Biotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam – 530 045, Andhra Pradesh, India
cular, neurological diseases, cancer and oxidative stress dysfunctions [8, 9]. Therefore, the search for exploitation of natural antimicrobial agents and antioxidants, especially of plant origin, has greatly increased in recent years. Buchanania lanzan Spreng. (Family: Anacardiaceae) commonly known as Char in hindi is a Sub deciduous tree, 13-17m high and up to 1.3m in girth, dark grey bark, leaves alternate, petiolate, very coriaceous or hard sessile greenish white flowers and stone hard bi- valved kernel has a pleasant sweetish acidic flavor; found throughout the hot dried parts of India [10]. All parts of the plant are used for the treatment of various disorders. The oil from the seeds is used to reduce granular swelling of the neck [11, 12]. Ointment is made from the kernel which is used to relieve itch and prickly heat. The gum from the bark used for treating diarrhea and intercostals pains and leaves are used for promoting wound healing [13, 14]. The rhizome of B. lanzan finds an important place in indigenous medicine as an expectorant, diuretic and carminative [15] . It is also found to have anticancer [16] antihypertensive [17] and larvicidal activity [18]. It is used for the treatment of various skin disorders, rheumatism and diabetes mellitus [19, 20]. Dillenia pentagyna Roxb. of Dilleniaceae family is an endangered tree species accounts for many ethnical uses. The tribal’s and folk communities use various parts of it for treatment of their different ailments and diseases, viz. delivery (bark), bone fracture (leaf), body pain (root), piles (leaf), diabetes (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]. Acacia caesia Linnaeus is a leguminous perennial climbing shrub belonging to the family Mimosaceae, and is native to south-east Asia. In different parts of India, the tender leaves are widely used in culinary preparation, and the prickly stem for fencing agricultural fields. The stem bark forms copious froth when rubbed with water and thus used as soap, while its decoction as lice killer [23]. The juicy extract from the stem bark is well-known among the
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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 crude extract of the stem bark indeed showed profound anthelmintic effects on Raillietina echinobothrida, causing extensive damage in the worm’s tissue such as in the tegumental layer, muscle tissue and parenchyma [24]. The present study aimed at evaluating the antibacterial potentiality of methanolic extracts of barks of three medicinal plants against bacteria and fungi. The minimum inhibitory concentration (MIC) for antibacterial activity was determined. Other objectives includes search of active phytochemicals present in the methanolic extracts of these plants and to obtain a preliminary idea about the chemical nature of the active ingredient/ingredients involved in the antimicrobial potentiality. The antioxidant activity of these extracts is also evaluated. MATERIALS AND METHODS Collection and identification of plant material: Three plant species were selected in the present investigation. The plants and their parts selected were Acacia caesia (Mimosaceae) (bark), Dillenia pentagyna (Dilleniaceae) (bark), Buchanania lanzan (Anacardiaceae) (bark) were collected from Maredumilli forest located near Rampachodavaram (Mandal), Rajahmandry (district), Andhra Pradesh (State), South India with the help of a local Ayurvedic Doctor. The plants were identified at Kovel Foundation, Visakhapatnam with reference to passport data book of NBPGR (National Bureau of plant genetic resources) under the Division of plant exploration and collection, New Delhi. A. caesia Collection No. KF V 7209 and Accession No. 29, D. pentagyna collection No. KF GSBVK 7175 and Accession No. 95 and Buchanania lanzan Collection No. KF VS 7274 and Accession No. 94. Extraction of plant material: The crude extracts of the three plant material were prepared separately by methanol as solvent as described below. Solvent extraction: The plant material was brought to laboratory and washed under running tap water and blotted with filter paper then shade dried on laboratory benches 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) was placed in a Soxhlet extractor and was exhaustively extracted using 100 ml of methanol (60 – 80°C). The crude extracts were respectively concentrated in vacuo at 40°C using a rota vapor. The crude extracts thus obtained were preserved in freezer at -20°C until use. The successive extractive values were 2.95 g, 3.73 g and 3.60 g respectively for the barks of A. caesia, D. pentagyna and B. lanzan. The crude plant extracts were labelled as A. caesia (bark) (A), D. pentagyna (bark) (B), B. lanzan (bark) (C). Test microorganisms and microbial culture: Bacterial cultures of Bacillus cereus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella sp., Staphylococcus aureus and fungal cultures of Aspergillus niger, penicillium sp. and Trichoderma viride were obtained from laboratory isolates. Bacterial strains were cultivated at 37°C and maintained on nutrient agar slant at 4°C.Fungal strains were cultivated at 25°C and maintained on potato dextrose agar slants at 4°C. All the cultures were tested for their purity. Pure cultures thus obtained were kept on respective agar slants at 4°C until needed. They were sub-cultured once in every month. Each inoculum was prepared by inoculating the stock culture into freshly prepared media. All the bacterial strains were incubated at 37°C for 24 h and fungi at 27°C for 48 h. The test organisms were grown overnight in respective broth media.
Screening of antimicrobial activity: Antimicrobial activity of organic extracts of the plant samples were evaluated by the paper disc diffusion method [25]. For determination of antibacterial activity, overnight grown bacterial cultures were adjusted to 0.5 McFarland turbidity standards [26]. For the determination of antifungal activity, all the fungal isolates were first adjusted to the concentration of 106 cfu/ml. The bacterial and the fungal broth cultures of 100 µl each were inoculated onto nutrient agar and potato dextrose agar plates respectively through spread plate method. Firstly stock solutions (100 mg/ml) of each individual plant extract were prepared separately. Sterile filter paper (What man filter paper No.1) discs of diameter 9 mm were prepared and 10 µl of each extract dilutions were impregnated onto the discs and carefully placed at the centre 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 alone was served as control. Streptomycin (10 µg/ml) for bacteria and Albendazole (10 µg/ml) for fungi was used as standard antimicrobials for comparison. Bacterial culture plates were then incubated at 37°C for 24 h while the fungal cultures are incubated at (25°C –27°C) for 48 h. Antimicrobial activity was determined by measurement of zone of inhibition of growth around each paper disc (mm). For each extract (test) three replicate trials were conducted against each organism. Each zone of inhibition was measured with a ruler and compared with standard [27]. Minimum inhibitory concentration (MIC): The minimum inhibitory concentration (MIC) of the extracts was estimated against bacterial organisms only which showed better zone of inhibition. It was carried out by the method as described as earlier [28]. The determination of the MIC was done with different dose levels of (10, 25, 50, 100 mg/ml) of 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 0.5 McFarland turbidity standard bacterial isolates was inoculated into the test tubes. The procedure was repeated on the test organisms using the standard antibiotic streptomycin. Tubes containing nutrient broth only seeded with the test organism serve as control. Tubes containing bacterial cultures were then incubated at 37°C for 24 h. The minimum inhibitory concentration was taken as the tube with the least concentration of the extract with no visible growth. Preliminary phytochemical screening: Preliminary phytochemical tests for identification of Tannins, Phenols, Saponins, Alkaloids, Steroids, Cardiac glycosides and Flavonoids were carried out for all the extracts. The extracts were analyzed for the presence of Alkaloids, Saponins, Cardiac glycosides [29]; Tannins & phenols, Flavanoids [30, 31] ; Steroids [32]. In-Vitro Antioxidant study: Ferric reducing antioxidant power (FRAP) method [33] The ferric reducing property of the extract was determined by taking 1 ml of different dilutions of standard solutions of Gallic acid (10 -100 µg/ml) or methanolic extract that has adjusted to come under the linearity range (500 µg/ml) was taken in 10 ml volumetric flasks and mixed with 2.5 ml of potassium buffer (0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50ºC for 20 min. Then 2.5 ml of 10% trichloroacetic acid was added to the mixture to stop the reaction. To the 2.5 ml of above solution 2.5 ml of distill water is added and then 0.5 ml of 0.1% of FeCl3 was added and allowed to stand for 30 min before measuring the absorbance at 593 nm. The absorbance obtained was converted to Gallic acid equivalent in mg per gm of dry material (GAE/g) using Gallic acid standard curve [34] (Fig 3).
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R2 values represented mean data set of n=3
Fig. 3 Standard curve of Ferric reducing antioxidant power (FRAP) method.
RESULTS The present study was conducted to investigate antibacterial properties of three selected plants from Maredumilli forest, which is less studied and used in Indian folk medicine. Herbal remedies play a fundamental role in traditional medicine in rural areas of India where the therapeutic treatment of choice as antiseptic, anti-inflammatory and in treatment of infectious diseases 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 methanol 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 reference 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. Table: 1 Results of antimicrobial activity of methanol crude plant extracts of three different plants against pathogenic bacteria and fungi. Microorganisms*
Zones of inhibition (Mean ± SD) (mm)a Crude plant extracts tested b A B C Antibiotics† positive standards
Bacterial Strains Escherichia coli Bacillus subtilis Bacillus cereus Staphylococcus aureus Pseudomonas aeruginosa Proteus vulgaris Salmonella sp. Fungal Strains Trichoderma viride Penicillium Sp Aspergillus niger
a
0 7 ±0.7 10 ±0.6 7 ±0.9 10 ±0.6 7±0.4 0
10 ±1.2 8 ±1.4 9 ±0.9 7 ±0.4 7 ±0.9 0 9 ±0.6
0 8 ±0.4 12±0.8 0 9±0.5 7 ±1.2 12 ±1.5
15 ±0.9 15 ±0.4 15 ±0.6 20 ±0.8 19 ±0.6 20 ±0.3 18 ±1.0
0 0
8 ±0.4 8 ±0.8
10 ±0.9 8 ±0.3
0 20 ±0.6
0
7 ±0.7
6 ±0.9
10 ±1.6
Values are mean of three replicates ± SD Plant names: A- Acacia caesia; B- Dillenia pentagyna; C- Buchanania lanzan. † Antibiotics used as positive Standards: Streptomycin (for bacterial strains); Albendazole (for fungal strains) b
Fig. 1 Bar chart showing results of antibacterial susceptibility of test organisms to methanol extracts of Acacia caesia (A) ; Dillenia pentagyna (B) ; Buchanania lanzan (C).
Fig. 2 Bar chart showing results of antifungal susceptibility of test organisms to methanol extracts of Acacia caesia (A) ; Dillenia pentagyna (B) ; Buchanania lanzan (C).
A. caesia showed antibacterial activity of (7-10 mm) against five strains tested and no activity against E. coli and Salmonella sp. It showed no antifungal activity against the three tested fungal strains. The results of minimum inhibitory concentration (MIC) of the extracts were showed in Table 2. Among three plant extracts the MIC values are lower for B. lanzan (25 mg/ ml) against B. cereus and Salmonella sp. (50 mg/ml) against P. aeruginosa and (100 mg/ml) against P. vulgaris and B. subtilis followed by D. pentagyna which showed (50 mg/ml) against E. coli, B. cereus and Salmonella sp. & (100 mg/ml) against B. subtilis, S. aureus and P. aeruginosa. A. caesia showed (50 mg/ml) against B. cereus & P. aeruginosa, (100 mg/ml) againstB. subtilis, S. aureus & P. vulgaris. The preliminary phytochemical analysis of the methanol extracts of these plants showed the presence of saponins, flavanoids, steroids, cardiac glycosides, alkaloids, tannins and phenolics showed in Table 3. Capacity of FRAP method At low pH, measuring the change in absorption at 593 nm can monitor reduction of a ferric complex to the ferrous form, which has an intense bluish green color. The change in absorbance is directly related to the combined or
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Venkata Smitha P et al. / Journal of Pharmacy Research 2012,5(3),1734-1738 Table: 2 Minimum inhibitory concentrations (MIC) Plant Extract
Test Organism
I
II
III
IV
A
Bacillus cereus Pseudomonas aeruginosa Bacillus subtilis Staphylococcus aureus Proteus vulgaris Escherichia coli Bacillus subtilis Bacillus cereus Staphylococcus aureus Pseudomonas aeruginosa Salmonella sp. Bacillus cereus Salmonella sp. Bacillus subtilis Pseudomonas aeruginosa Proteus vulgaris
+ ++ + + + ++ ++ + + + + + + + + +
+ + + + + + + + + + + * * + + +
* * + + + * + *
* * * * * * * *
B
C
+ * + * +
Plant names: A- Acacia caesia; B- Dillenia pentagyna; C- Buchanania lanzan. I = 10 mg/ml, II = 25 mg/ml, III =50 mg/ml, IV=100 mg/ml *=MIC, - = No growth, + = little growth, ++ =Dense growth Table: 3 Results of preliminary phytochemical screening of plant extracts. Tests
A
B
C
Tannins and Phenols Saponins alkaloids Steroids Cardiac glycosides Flavonoids
+ + +
+ + + +
+ + + -
A- Acacia caesia; B- Dillenia pentagyna; C- Buchanania lanzan; + = Presence; - = Absence “total” reducing power of the electron-donating antioxidants present in the reaction mixture. Here the FRAP showed the results of methanol extracts of A. caesia (bark), D. pentagyna (bark) and B. lanzan (bark) that of 84.74±0.32mg equivalent to Gallic acid (GAE)/g of sample, 109.91±0.18mg GAE/g and 49.05±1.02mg GAE/g of sample respectively shown in Table 4.The present study has shown that these three plant extracts exhibited maximum antioxidant capacity. Considering all the three plants, bark of D. pentagyna registered maximum activity followed by bark of A. caesia and B. lanzan. Table: 4 Results of Ferric reducing antioxidant power (FRAP) method Plant extracts A B C
Conc. of extracts 500µg/ml 500µg/ml 500µg/ml
Leaf extracts of the plant species, A. caesia L. by using different alcoholic solvents were tested against the ten human pathogenic bacteria and results support the therapeutic importance of A. caesia in curing infectious diseases and encourage the extensive use of it in health care practices [39].The present study provides the first evidence on its antioxidant activity. D. pentagyna commonly found tree was taken to explore the antimicrobial and antioxidant properties as there was very limited work done on the plant. Terpenoids and flavonoids were the most well known among the secondary metabolites isolated from plants belonging to Dilleniaceae, though a large amount of alkaloid was also searched [22, 40]. Rhammentin 3-glucoside also isolated from the D. pentagyna [22]. Two new flavonoid glycosides, naringenin 7-galactosyl and dihedral quercetin 5-glactoside were isolated from D. pentagyna have been found to exhibit cytotoxic and lymphocytic activity [41]. A new diterpene, dipoloic acid isolated from the stem of D. pentagyna exhibited cytotoxic activity [41]. This plant is shown to possess highest antioxidant activity due the presence of these flavonoids. B. lanzan showed highest zone of inhibition among the three plants. Preliminary phytochemical testing which is carried using chemicals for the identification of various phytoconstituents suggest the presence of glycosides, tannins, phenolic compound and steroids in the bark extract of B. lanzan, which may be responsible for their antibacterial effects. Still, further analyses of the antioxidant capacity of the beneficial compounds of these plants are needed for a better understanding of how to implement them in functional medicine. The broad spectrum of antimicrobial activity may help to discover new chemical classes of antibiotic substances that could serve as selective agents for infectious diseases, chemotherapy and control. ACKNOWLEDGEMENTS: The authors are grateful to Mr. Subba Rao, Tribal Doctor, Tallapalem (Village), Maredumilli, East Godavari, A.P., India for helping in plant collection and also Kovel Foundation for providing assistance in plant collection and authentication. Authors are also thankful to Dept. of Microbiology, Andhra Medical College, Visakhapatnam for providing microbial cultures. Authors are also grateful to Management and Head, Dept.of Biotechnology, GIT, GITAM University for providing necessary facilities. REFERENCES: 1.
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mg GAE/g of extracts 84.74±0.32 109.91±0.18 49.05±1.02
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A- Acacia caesia; B- Dillenia pentagyna; C- Buchanania lanzan.
DISCUSSION The present study provides evidence for the antimicrobial and antioxidant activities of three plants that have significant application in traditional medicine. The barks, which are mostly used as a source of medication in traditional medicine was considered to examine the properties of the plants. The results confirmed that the extracts obtained with polar solvent methanol showed antimicrobial activity to the test organisms. This is explained by the fact that the secondary metabolites identified in the extracts which are known to have antimicrobial properties are also polar compounds and may have been easily extracted by the polar solvent since it is known that “like dissolves like”. Of the many medicinal plants, many species of Acacia are found to have diverse photochemical compounds of medicinal properties [35-38].
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Source of support: Nil, Conflict of interest: None Declared
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