IJPSR (2014), Vol. 5, Issue 11 (Research Article) Received on 23 April, 2014; received in revised form, 16 June, 2014; accepted, 31 July, 2014; published 01 November, 2014 BIOLOGICAL STUDIES OF THE BARK OF ALBIZIA LEBBECK (L.) BENTH Jerin Tasnim 1 *, Anamika Saha 1, Shamim Ahmed 2, Nasim Sultana 2, Tanvir Muslim 1 and Md. Azizur Rahman 1 Department of Chemistry, University of Dhaka 1, Dhaka-1000, Bangladesh. Analytical Research Division, BCSIR Laboratories 2, Dhaka, Dhaka-1205 Bangladesh. Keywords: Antimicrobial activity, Albizia lebbeck, cytotoxicity, DMSO, free radical-scavenging activity, DPPH. Correspondence to Author: Jerin Tasnim Department of Chemistry University of Dhaka Dhaka-1000, Bangladesh E-mail: tasnimjerin@gmail.com ABSTRACT: The phytochemical screening of the bark of Albizia lebbeck (L) showed positive test for tannin, alkaloid, flavonoid, terpenoid, phlabotannin, saponin, steroid and cardiac glycoside. In this article an attempt was made for biological studies (the antimicrobial activity, cytotoxicityand free radical-scavenging activity) of different extracts (hexane, methanol, ethyl acetate, water and butanol extract) of the bark of Albizia lebbeck. The antimicrobial sensitivity of the extracts was tested using the disc diffusion method. The cytotoxic effects of five extracts (hexane, methanol, ethyl acetate, water and butanol extract) of the bark of Albizia lebbbeck were carried out. Free radical scavenging activity of the bark of Albizia lebbeck was performed. All the extracts showed moderate anti microbial activity and gram positive bacteria were more sensitive than gram negative bacteria. Significant cytotoxic activity was noticed and hexane extract was found the most lethal to brine shrimp nauplii. This result revealed that the highest cytotoxic constituent may be non polar in nature. Good free radical scavenging activity was observed in all the extracts, and hexane extract was found be the most potent. INTRODUCTION: Albizia lebbeck (L.) Benth belongs to Fabaceae (alt. Leguminosae) family. The Fabaceae or Leguminosae, commonly known as the legume, pea, or bean family, with 730 genera and over 19,400 species 1. It is a deciduous tree about 18-30m long with a trunk 50cm to 1m in diameter, gray colored bark 2, leaves are evenly 2- pinnate and the leaflets are in 5-9 pairs, flowers are stalked, white fragarant in globose umbellate heads 2-3.8cm diameter, stamens are longer than the corolla 3. QUICK RESPONSE CODE DOI: 10.13040/IJPSR.0975-8232.5(11).4969-74 Article can be accessed online on: www.ijpsr.com DOI link: http://dx.doi.org/10.13040/ijpsr.0975-8232.5(11).4969-74 Albizia lebbeck is used as general and universal antidote. Preliminary phytochemical screening of Albizia lebbeck was done earlier 4. The plant is considered as the most potent alexipharmic and every part of it is prescribed for the treatment of bites and stings from venomous animals. It possesses anti-inflammatory activity 5, favorable response in all kinds of allergic conjunctivitis 6, anti bacterial activity against infectious diarrhea 7, 8. The alcoholic extract of the bark revealed moderate anthelmintic activity against in vitro human ascaris lumbricoides 9. Bark is antihelminthic, antispermatogenic 10, antiasthmatic 11, antidiabetic 12, relives toothache, and strengthens the gums and the teeth, used in leprosy, skin diseases. The root is astringent and used in ophthalmia, hemicranias, antihelminthic, rat bite and shows antifungal activity 13. The seeds are anti-tumor, aphrodisiac, International Journal of Pharmaceutical Sciences and Research 4969
brain tonic, used for gonorrhea and tuberculous glands; the oil is applied topically in leucoderma (Yunani) 14, 15. The pods show antispermatogenic effect 16, 17. The leaves are good for syphilis, ophthalmic diseases, night blindness anxiolytic activity and nootropic activity 18, 19. They are reported to be good fodder with 17-26% crude protein; 100 kg of leaves yield 11-12 kg of digestible protein and 37 kg of digestible carbohydrate 20. In the area of apiculture its flowers are fragrant and attract bees. It is highly regarded by beekeeper for the light colored honey its nectar provides 21. Albizia lebbeck has nitrogen fixing woody legume. The nitrogen fixation increases with the age of the plant 22. The present study deals with the biological investigation of different extracts of the bark of the plant Albizia lebbeck. The antimicrobial activity, cytotoxicity and free radical-scavenging activity of the bark of this plant is being reported here. MATERIALS AND METHODS: Chemicals and solvents: The reagents used in this work were of analytical grade (Merck and BDH). All the solvents were distilled before use. Sample collection: The bark of Albizia lebbeck (locally known as Shirish) was collected from Dhaka city, Bangladesh. The taxonomy of the plant was confirmed by consulting with Prof. Dr. Md. Abul Hassan, Department of Botany, University of Dhaka. A voucher specimen of this plant was deposited in the Bangladesh National Herbarium (BNH), having ACCESSION NO DACB 36551. The bark was first cleaned and dried below 40 0 C. The dried bark was grinded to powder by a cyclotec grinder (200 meshes) and the powder was stored in an airtight bottle, and this was used throughout the investigations. Extraction: The powdered bark (800 gm) was extracted with methanol (3 2.5 Liter) at room temperature for 48 hours. Then it was filtered and the filtrate was concentrated below 40 C with a rotary evaporator to obtain a dry mass. Dry mass of methanol extract (~40 gm) was triturated with distilled water (200 ml) and then the solution was partitioned with n-hexane followed by ethyl acetate and lastly with n-butanol. Antimicrobial screening: Five extracts (hexane, methanol, ethyl acetate, water and butanol extract) of the bark of Albizia lebbeck were used for the test. The antimicrobial sensitivity of the extracts 23, 24 was tested using the disc diffusion methods and the sample concentration was 400 g per disc. Both gram-positive (Staphylococcus aureus, B. cereus) and gram-negative (Escherichia coli, p. Aervgin and Salmonella typhi) organisms were taken for the test. The test samples were weighed accurately and calculated amounts of the solvents were added accordingly using micropipette to the dried samples to get desired concentrations. The test samples were applied to previously sterilized discs using adjustable micropipette under septic conditions. The sample discs, the standard antibiotic discs and the control discs were placed gently on the previously marked zones in the agar plates preinoculated with test bacteria and fungi. The plates were then kept in a refrigerator at 4 0 C for about 24 hours upside down to allow sufficient diffusion of the materials from the discs to the surrounding agar medium. The plates were then inverted and kept in an incubator at 37 0 C for 24 hours. The antimicrobial potency of the test agents are measured by their activity to prevent the growth of the micro-organisms surrounding the discs which gives clear zone of inhibition. After incubation, the antimicrobial activities of the test materials were determined by measuring the diameter of the zones of inhibition in millimeter with a transparent scale. In this investigation, chloramphenicol (30 g/disc) standard disc was used as the reference. Free radical-scavenging activity of Albizia lebbeck: Free radical scavenging activity of the bark of Albizia lebbeck was carried out using 1, 1- diphenyl-2-picrylhydrazyl (DPPH) 25. Methanol solution (1.0 ml) of the extract at different concentration was mixed with 1.0 ml of a DPPH in methanol solution (20µg/ml). The antioxidant potential was assayed from the bleaching of purple colored methanol solution of DPPH radical by the plant extract as compared to that of ascorbic acid by UV spectrophotometer. DPPH radical scavenging activity is described as IC 50, which is the concentration of samples to International Journal of Pharmaceutical Sciences and Research 4970
produce 50% reduction of the DPPH. 200μl of various concentrations of the extracts in methanol was added to 2 ml of a 0.004% methanol solution of DPPH. After 30 min incubation period at room temperature the absorbance was read against a blank at 517 nm. Inhibition free radical DPPH in percent (I%) was calculated as follows: (I%) = (1 A sample /A blank ) X 100 Where A blank is the absorbance of the control reaction (containing all reagents except the test material). Extract concentration providing 50% inhibition (IC 50 )was calculated from the graph plotted inhibition percentage against extract concentration. Ascorbic acid was used as positive control. Tests carried out in triplicate and average value was taken. Brine shrimp lethality bioassay: 4 mg of each of the test samples (methanol, butanol, ethyl acetate, n-hexane and aqueous extract) were taken and dissolved in 200 µl of pure dimethylsulfoxide (DMSO) in vials to get stock solutions. Then 100 µl of solution was taken in test tube each containing 5ml of simulated seawater and 10 shrimp nauplii 26. Thus, final concentration of the prepared solution in the first test tube was 400µg/mL. Then a series of solutions of varying concentration was prepared from the stock solution by serial dilution method. In each case 100µl sample was added to test tube and fresh 100µl DMSO was added to vial. Thus the concentrations of the obtained solution in each test tube were as- 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781 and 0.391 g/ml. In the present study vincristine sulphate was used as the positive control. After 24 hours, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. From this data, the percent of lethality of the brine shrimp nauplii was calculated for each concentration. RESULTS AND DISCUSSION: Antimicrobial screening: The antimicrobial sensitivity of five different extracts (hexane, ethyl acetate, butanol, methanol and water extract) of the bark of Albizia lebbeck was tested using sample concentration 400mg per disc. Standard disc of Chloramphenicol (30 mg per disc) was used as reference. After incubation the antimicrobial activity of test material were determined by measuring the diameter of zones of inhibitions in milimeter (mm) and following results (Table 1) were found. TABLE 1: ANTIMICROBIAL ACTIVITY OF DIFFERENT EXTRACTS (WATER, HEXANE, ETHYL ACETATE, METHANOL AND BUTANOL EXTRACT) OF THE BARK OF ALBIZIA LEBBECK SHOWING ZONE OF INHIBITION IN DIAMETER (mm) Extract Staphylococcus aureus Salmonella typhi B. cereus p. aervgin Escherichia coli Hexane 8 0 8 9 11 Methanol 10 0 9 10 8 Ethylacetate 13 0 10 9 5 Water 6 0 0 0 0 Butanol 11 0 12 15 10 Chloramphenicol 22 28 20 18 25 From these results (Figure 1), it appears that all the extracts of Albizia lebbeck have moderate antimicrobial activity 27. Butanol extract was found to be most effective for both gram-positive and gram-negative bacteria and water extract showed least activity. Also some other factors for example, the extraction methods into column volume and culture medium composition ph and incubation temperature can influence the results 28. International Journal of Pharmaceutical Sciences and Research 4971
FIGURE 1: ANTIMICROBIAL ACTIVITY OF DIFFERENT EXTRATS OF ALBIZIA LEBBECK TABLE 2: IC 50 VALUES OF STANDARD AND DIFFERENT EXTRACTS OF BARK OF ALBIZIALEBBECK Sample Methanol Extract 1.05 Hexane Extract 2.54 Ethyl acetate Extract 0.83 Butanol Extract 0.32 Water Extract 1.41 Ascorbic acid 0.58 IC 50 (μg/ml) organism can be used as a convenient informant for screening and fractionation in the discovery of new bioactive natural products. Free radical-scavenging activity: The antioxidant potency of five extracts (water, hexane, ethyl acetate, methanol and butanol) of the bark of Albizia lebbeck was studied using 1, 1-diphenyl-2- picrylhydrazyl. IC 50 value was calculated from the graph plotted inhibition percentage against extract concentration and results are given below (Table 2). The results (Figure 2) show that all the tested plant extracts have moderate to potent antioxidant activity 29. The hexane extract exhibited highest radical scavenging potentiality with an IC 50 value of 2.54µg/mL compared to ascorbic acid having IC 50 value of 0.58µg/mL, which is a well-known antioxidant compound. Water and methanol extract also showed good antioxidant activity (IC 50 value 1.41µg/mL and 1.05µg/mL respectively). Other extracts (ethyl acetate and butanol) also showed moderate antioxidant activity. Brine shrimp lethality bioassay: Bioactive compounds are almost always toxic at higher dose. Thus, in vivo lethality in a simple zoological FIGURE 2: IC 50 VALUES OF DIFFERENT EXTRATS OF ALBIZIA LEBBECK TABLE 3: LC 50 VALUES OF STANDARD AND DIFFERENT EXTRACTS OF BARK OF ALBIZIA LEBBECK Sample LC 50 ( g/ml) Methanol Extract 5.88 Hexane Extract 2.95 Ethyl acetate Extract 4.78 Butanol Extract 4.16 Water Extract 5.62 Vincristine sulphate 0.35 Crude extract of hexane, ethyl acetate, butanol, methanol and water were screened by brine shrimp lethality bioassay for probable cytotoxic activity of Albizia lebbeck. Plotting of log of concentration International Journal of Pharmaceutical Sciences and Research 4972
versus percent mortality for all test samples showed an approximate linear co-relation. From the graphs, the median lethal concentration (LC 50, the concentration at which 50% mortality of brine shrimp nauplii occurred) was determined for the samples (Table 3). The degree of lethality was found to be proportional to the concentration ranging from lower to higher. The LC 50 value of hexane extract was found to be 2.95 g/ml, at concentration 0.49 g/ml, which showed the highest toxicity. From this, it can be said that the highest toxic constituent may be non polar in nature (Figure 3). brine shrimp lethality bioassay it reveals that the crude extracts have moderate to potent cytotoxic activity. To justify this cytotoxic potency, cellline assay also recommended. From these results it can be concluded that the traditional use of the bark of Albizia lebbeck are not totally scientifically useless. Further investigation on this plant may contribute to the field of medicine. ACKNOWLEDGEMENT : Authors are thankful to Analytical Research Division, BCSIR Laboratories, Dhaka-1205, Bangladesh; for providing necessary laboratory facilities, and express their gratitude to Department of Chemistry, University of Dhaka, Dhaka-1000, Bangladesh; for giving support and guidance in the work. FIGURE 3: LC 50 VALUES OF DIFFERENT EXTRATS OF ALBIZIA LEBBECK It appears from the result that all the test samples were lethal to brine shrimp nauplii. Comparison with positive control vincristine sulphate signifies that cytotoxicity exhibited by the crude extract of hexane, ethyl acetate, butanol, methanol and water are promising and they might have antitumour or pesticidal compounds 30. However, further study with specific case may establish its potentiality and discovery of new cytotoxic compounds CONCLUSION: The antimicrobial screening of five different extracts of the bark of Albizia lebbeck was done by disc diffusion methods using both gram positive and negative bacteria. From this result it can be reported that all the extracts of Albizia lebbeck have considerable antimicrobial activity. The antioxidant potency of five extracts of the bark of Albizia lebbeck was studied using 1, 1- diphenyl-2-picrylhydrazyl, which showed good antioxident potency specially hexane extract showed the highest potency. From the results of the REFERENCES: 1. Stevens, P. F. (2001 onwards). Angiosperm Phylogeny Website Version 9, June, 2008 Mobot.org. 2. Kumar N and Toky OP. Variation in chemical contents of seed, and foliage in Albizialebbek (L.) Benth. of different provenances. AgroforestrySystems1994; 25(3): 217-225.. 3. Mishra SS, Gothecha VK and Sharma A. Albizia lebbeck: A short review. Journal of Herbal Medicine and Toxicology 2010; 4(2): 9-15. 4. Bobby MDN, Wesely EG, Jhonson M. High performance thin layer chromatography profile studies on the alkaloids of Albizialebbeck. Asian Pacific journal of Tropical Biomedicine 2012; 2(1): S1-S6. 5. Babu NP, Pandi kumar P and Ignacimuthu. Antiinflammatory activity of Albizia lebbeck Benth. an ethnomedicinal plant, in acute and chronoic animal models of inflammation. Journal of Ethnopharmacology 2009; 125(2): 356-360. 6. Nurul IM, Mizuguchi H, Shahriar M et. al. Albizia lebbeck suppresses histamin signaling by the inhibition of histamine H 1 receptor and histidine decarboxylase gene transcriptions. International Journal Immunopharmacology 2011; 11(11): 1766-1772. 7. Acharyya S, Patra A, Bag PK. Evaluation of the Antimicrobial Activity of Some Medicinal Plants against Enteric Bacteria with Particular Reference to Multi-Drug Resistant Vibriocholerae. Tropical Journal of Pharmaceutical Research 2009; 8(3): 231-237. 8. Bobby MN, Wesely EG and Jhonson A. In vitro antibacterial activity of leaves extracts of Albizia lebbeck Benth against some selected pathogens. Asian Pacific Journal of Tropical Biomedicine 2012; 2(2): 859-862. 9. Raj RK. Screening of indigenous plants for anthelminticaction against human Ascarislumbricoides, part II. Indian Journal of Physiology and Pharmacology1975; 19(1): 47-49. 10. Gupta RS, Kachhawa JBS and Chaudhary R. Antispermatogenic, antiandrogenic activities of Albizia lebbeck (L.) Benth bark extract in male albino rats. Journal of Phytomedicine 2006; 13(4): 277-283. 11. Yadav SS, Galib, Patgiri B, Prajapati PK. Clinical efficacy of two different samples of Shirishavaleha in Tamaka International Journal of Pharmaceutical Sciences and Research 4973
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