IDENTIFICATION AND ANTIBACTERIAL ACTIVITY OF METHANOL

Open Access Research Journal www.pradec.eu Medical and Health Science Journal, MHSJ ISSN: 1804-1884 (Print) 1805-5014 (Online) Volume 12, 2012, pp.70-77 IDENTIFICATION AND ANTIBACTERIAL ACTIVITY OF METHANOL EXTRACT OF LUFFA ACUTANGULA ROXB. Antibacterial activity of Luffa acutangula Roxb. (angled luffa) has been assayed against some pathogenic bacterial. Fruit powder of angled luffa was macerated with methanol, and the methanol ed sequentially with hexane, chloroform, ethyl acetate and buthanol. Antibacterial activity was evaluated by well diffusion method. Extract with the highest antibacterial activity was identified regarding their class of compounds using phytochemical screening and Thin Layer Chromatography (TLC) method. The antibacterial activity of the was compared with that of the ampicillin used. The methanol inhibited the growth of the P. aeruginosa, E. coli, B. subtilis and S.aureus, but did not inhibit the growth of the E. aerogenes, S. dysentriae and S. thypi. The ethyl acetate showed the highest antibacterial activity against P. aeruginosa, E. coli, B. subtilis and S. aureus, followed by chloroform, buthanol, and hexane, respectively. The ethyl acetate possed phenolic, condensed tannin, flavonoids, saponins and terpenoids. Based on the MIC and the equivalent value of ethyl acetate compared with that ampicillin used, the antibacterial activity of ethyl acetate was lower than with that of the ampicillin used. RESMI MUSTARICHIE 1, LINAR ZALINAR UDIN 2, MUCHTARIDI 1, SUPRIYATNA 1 1 Faculty of Pharmacy, Universitas Padjadjaran, Indonesia 2 Department of Applied Chemistry, Indonesian Institute of Sciences, Indonesia Keywords: Luffa acutangula Roxb., antibacterial activity, well diffusion, methanol, ethyl acetate. UDC: 581.52 Introduction The problems of unwanted side effects of some antibiotics and the urgent need to cure infectious diseases induce scientists to search for new antibacterial drugs derived from plants (Aliero, Aliero, and Buhari, 2008). Cucurbitaceae plants tribes that have been reported among other things provide antibacterial activity of Luffa cylindrica (= Belustru) and Benincosa hispida (= Beligo). Oyetayo et al. (2007) states that the leaves and fruit seeds contain antibacterial compounds of Belustru alkaloids and saponins. Kumar, Swamy, Sanjay et al. (2006) on the other hand reported that fruit Beligo methanol containing triterpenoids and flavonoids could inhibit bacterial growth. In this paper, we report testing the antibacterial activity of methanol of Luffa acutangula Roxb. (angled luffa). This test includes identification of the phytochemical screening and testing of in-vitro antibacterial by hole diffusion method against E. coli, B. subtilis, S. aureus, P. aeruginosa, S. dysentriae and/or S. thypi and Ampicillin was used as the MIC appeal against antibacterial activity. Experimental Materials and instruments All chemicals were used as received without further purification and all solvents were of reagent grade. Test bacteria used were E. coli FNCC 0091, B. subtilis FNCC 0059, S. aureus FNCC 0047, P. aeruginosa FNCC 0063 and S. thypi FNCC 0050. E. aerogenes and E. dysentriae - 70 -

were obtained from Indonesian Institute of Sciences - Bandung. One-way ANOVA with p> 0.05 were applied for data analysis. Flow chart of the experiment is shown in Figure 1. FIGURE 1. FLOW CHART OF THE RESEARCH Angled luffa fruit (1000 g) Dried powder Thinly sliced, dried in an oven for 3 days 55 o C then finally milled Test for antibacterial activity against E.coli, B.subtilis, S. aureus, P. aeruginosa, E. aerugenes, S. dysentriae and S. thypi 1. Macerated with 2.5 L methanol 1x 24 hours, and succesvely by 3 x 30 hours with 850, 990 dan 600 ml methanol. 2. Evaporated by Rotary Evaporator at 40 o C Concentrated methanol (261.6 g) Added 200 ml water:metanol (1:3) Aqueous methanol 1. Multilevel solvent ion using successive hexane, chloroform, ethyl acetate and butanol. 2. Evaporated by Rotary evaporator at 40 oc n-hexane Chloroform Ethyl acetate Butanol Residue/wat er Phytochemical screening: saponin, phenolate, tannin / polyphenol, flavonoid, terpenoid and alkaloid Tests for antibacterial activity against bacteria that can be inhibited by the methanol Antibacterial Determination of antibacterial the highest by comparing the inhibition diameter formed Determination of minimum inhibitory concentration and value appeal to ampicillin The highest antibacterial Confirmation test compound class with the TLC - 71 -

Results and discussion Maceration Medical and Health Science Journal / MHSJ / ISSN: 1804-1884 (Print) 1805-5014 (Online) Maceration method used as ion method provided quite a lot ion and reduced the excessive heat of the samples. Therefore, not many damaged ed compounds conditioned use of methanol as a solvent; all the compounds of plants suspected of being the compound are compound antimicroorganisms dissolved in organic solvents, so that these compounds can be obtained through solvent ion with methanol (Cowan, 1999). It is known that methanol can almost all the compounds including anthocyanin compounds, terpenoids, saponins, lactone, tannins, flavones, and polyphenols. Durmaz, Sagun, Tarakci, and Ozgokce (2006) showed that ion with methanol is a good method for ing plant antibacterial compounds from Allium vineale Linn., Chaerphyllum macropodum Linn. and Prangos ferulacea (L) Lindl. Multiple soaking was useful to optimize the ion of the compounds contained in samples. Tests for antibacterial activity of methanol s Tests for antibacterial activity of methanol s are shown in Table 1. Tests conducted by the hole diffusion method using DMSO as solvent. DMSO is also used as a negative control, since DMSO is a colorless aprotic polar solvent that can dissolve polar and non- that the methanol s inhibited the polar with a very wide range. Table 1 shows growth of E. coli, S. aureus, P. aeruginosa and B. subtilis, but did not inhibit bacterial growth of E. aerogenes and S. dysentriae. Repeated testing with two different concentrations as shown in Figure 2 confirms these results wheree for S. thypi there was not observed growth inhibition. TABLE 1. TESTS FOR ANTIBACTERIAL ACTIVITY OF METHANOL EXTRACT OF LUFFA FRUIT No. Tested Bacteria Test results Sample weight 20 mg/hole Sample weight 15 mg/hole Sample weight 10 mg/hole 1 S. aureus * + + 2 P. aeruginosa + + + 3 E. coli + + + 4 B. subtilis + * + 5 E. aerogenes - - - 6 S. dysentriae - - - 7 S. thypi * * * Note: (+) - positive anti bacterial; (-) - negative antibacterial; (*) - diameter of inhibition is not so clear (dubious) Effect of variation of bacteria in each sample weight and influence of the increasing weight of each sample to bacteria growth inhibition can be detected by data analysis using One-way ANOVA. This study shows that the effect of weight variation test sample generally provides the effect of bacterial variation in inhibiting the growth of bacteria; however, bacteria S.aureus with P. aeruginosa, B. subtilis with S. aureus, P. aeruginosa with B. subtilis gave similar effects in inhibiting the growth of tested bacteria. Effect of increasing the weight of each sample of bacteria showed that increasing sample weight of methanol s from 10 mg/hole to 15 mg/hole had no effect in inhibiting the growth of bacteria S. aureus, E. coli and B. subtilis, but gave effect in inhibiting the growth of bacteria P. aeruginosa marked with significant differences between sample weight 10 mg/hole with a sample weight 15 mg/sample. - 72 -

FIGURE 2. ANTIBACTERIAL ACTIVITY TEST RESULTS OF THE METHANOL EXTRACT OF THE BACTERIUM S. AUREUS, P. AERUGINOSA, E. COLI, B. SUBTILIS AND S. THYPI Diameter of Inhibition (MM) 14 12 10 8 6 4 2 0 10mg/hole 15mg/hole S.aureus P aeruginosa E coli B subtilis S.typhi The above results show that the methanol s inhibited the growth of gram positive bacteria (B. subtilis and S. aureus) and gram negative bacteria (P. aeruginosa and E. coli), and the obtained results are consistent with results from Tomori et al. (2007) showed that the ethanol of the fruit L. breviflora (also Cucurbitaceae) inhibits the growth of the bacterium E. coli, B. subtilis, S. aureus, and P. aeruginosa Tests for antibacterial activity of s from various solvents As shown in the Figure 1, the methanol was ed using a solvent gradually increasing polarity. The was then tested for antibacterial activity (Figure 3). FIGURE. 3 ANTIBACTERIAL ACTIVITY TEST RESULTS OF VARIOUS SOLVENT EXTRACTS OF THE BACTERIUM S. AUREUS, P. AERUGINOSA, E. COLI, AND B. SUBTILIS WITH EXTRACT WEIGHT OF 15 MG/HOLE 14 diameter of inhibition (mm) 12 10 8 6 4 2 0 E coli B subtilis S aureus P aeruginosa hexane chloroform n butanol ethyl acetate water - 73 -

Extracts which had the highest bacterial activity included ethyl acetate followed by chloroform, butanol and hexane. Water s had no antibacterial activity and were marked by the lack of inhibition diameter around the hole. Testing antibacterial active compounds To identify groups of compounds contained in s, chemical screening was carried out with the result shown in Table 2. TABLE 2. THE TEST RESULTS ANTIBACTERIAL COMPOUNDS CONTAINED IN THE VARIOUS EXTRACTS No. Group Test results Compound Methanol Hexane Chloroform Ethyl acetate Butanol tested Extract 1 Alkaloid + - + - + 2 Phenolic + - + + + 3 Saponin + - - + - 4 Tannin* + - + + + 5 Flavonoid + - + + + 6 Terpenoid + + + + - Note: * condensed tannin, (+)= test positive, (-) = test negative As shown in Table 2, hexane which contains only terpenoid compounds has antibacterial activity against all tested bacteria but smallest compared to other s. This indicates that the terpenoid group has contributed in inhibiting the growth of E. coli, B. subtilis, S. aureus, and P. aeruginosa with little strength. The phytochemical screening of the fractions of ethyl acetate was followed by TLC mobile phase chloroform test (Table 3). TABLE 3. TEST RESULTS OF GROUPS OF COMPOUNDS IN THE EXTRACT OF ETHYL ACETATE AND ITS TLC AND PS RESULTS Group PS TLC results Compound results Visible UV 254 nm UV 365 nm Tested Rf Colour Note Rf Colour Note Rf Colour Note Flavonoid + 0.09 Tawny + 0.08 Yellowish + Green 0.39 Greenish + Yellow Tannin & + 0.18 Grayish + 0.18 Grayish + 0.18 Grayish + Phenolic Black Black Black Terpenoid + 0.41 Purple + Saponin + 0.25 Greenish + Blue 0.55 Blue + Note : (+) Test positive, PS = Phytochemical Screening, TLC = Thin Layer Chromatography Cowan (1999) states that alkaloids have antibacterial activity associated with high aromatic compound content which have contributed to form a chelate with bacteria DNA. Flavonoids have pharmacological activity associated with the ability to work as a powerful - 74 -

antioxidant, free radical catchers, chelate with metal forming and interacting with the enzyme (Bylka, Matlawska, and Pilewski, 2004) and flavonoids also. Tannin has antibacterial activity through molecular action, i.e. by forming complexes with proteins through hydrogen bonding and hydrophobic bonding, binding of bacterial peptidoglycan cell wall and inhibiting the activity of the enzyme lactase which is a b- destroying enzyme b-lactam antibiotics (Shimamura, Zhao, and Hu, 2007). Saponins can act as detergents that have a structure binding of hydrophilic molecules and organic molecules to damage the cytoplasmic membrane and kill the bacteria. Terpenoids have antibacterial activity associated with the destruction of the cell membrane by lipophilic compounds. Phenolic compounds can cause denaturation of proteins through adsorption processes involving hydrogen bonds. Comparative antibacterial activity of ethyl acetate with ampicillin Ampicillin is used as a comparator antibacterial activity of the ethyl acetate. The test result of minimum inhibitory concentration (MIC) of ethyl acetate s is shown in Figure 4. FIGURE 4. THE TEST RESULTS OF MINIMUM INHIBITORY CONCENTRATION OF ETHYL ACETATE EXTRACT diameter of inhibition (mm) 16 14 12 10 8 6 4 2 0 E coli B Subtilis S aureus P aeruginosa 0,75mg/micro L 0,30mg/micro L 0,1 mg/micro L 0,28mg/micro L The smallest concentration used was 0.028 mg/µl which can still inhibit the growth of all bacteria. Table 4 is obtained by using concentrations of 0.10 mg/µl or sample weight 2.0 mg/hole up to 0.010 mg/µl or sample weight 0.20 mg/hole. Ampicillin inhibitory concentration test results are shown in Table 5. Results of comparative determination of ethyl acetate and aampicillin are shown in Table 6. It can be concluded that the antibacterial activity of ethyl acetate from the fruit of Luffa is weaker when compared with ampicillin; because the ethyl acetate has a minimum inhibitory concentration greater than the MIC for ampicillin, that is E.coli is 0.003%, B. subtilis - 0.0025%, S. aureus - 0.0079%, and P. aeruginosa - 0.0063%. - 75 -

TABLE 4. THE MIC OF ETHYL ACETATE EXTRACT Concentrations Inhibition diameter (mm) mg/µ L E.coli B. subtilis S. aureus P. aeruginosa 0.10 10.10 ± 0.33 9.78 ± 0.52 10.03± 0.47 12.87 ± 0.34 0.050 8.50 ± 0.51 8.19 ± 0.31 8.43 ± 0.20 9.76 ± 0.31 0.030 7.58 ± 0.53 7.96 ± 0.35 7.88 ± 0.06 8.32 ± 0.35 0.020 6.89 ± 0.29 7.25± 0.16 7.34 ± 0.15 7.60 ± 0.06 0.010 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 Note : Inhibiton diameter of negative control 6 mm TABLE 5. THE MIC OF AMPICILLIN Concentrations Inhibition diameter (mm) mg/µ L E.coli B. subtilis S. aureus P. aeruginosa 1.5.10-5 13.5 ± 0.20 12.24 ± 0.30 13.2 ± 0.14 13.26 ± 0.32 7.6.10-6 10.58 ± 0.47 10.35 ± 0.27 10.12 ± 0.26 10.12 ± 0.14 3.8.10-6 9.90 ± 0.06 9.96 ± 0.10 8.84 ± 0.28 9.30 ± 0.26 1.9.10-6 8.23 ± 0.21 8.82± 0.10 8.05 ± 0.15 7.34 ± 0.25 1.0.10-6 7.76 ± 0.11 7.93± 0.20 7.24 ± 0.09 6.00 ± 0.00 5.0.10-7 7.24 ± 0.09 7.23 ± 0.06 6.00 ± 0.00 6.00 ± 0.00 2.5.10-7 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 1.2.10-7 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 6.00 ± 0.00 Note : Inhibiton diameter of negative control 6 mm Bacteria TABLE 6. THE RESULT OF THE COMPARATIVE DETERMINATION OF ETHYL ACETATE EXTRACT AND AMPICILLIN The actual concentration of ethyl acetate The equivalent concentration of ethyl acetate to Ampicillin The comparative value against Ampicillin E. coli 0.10 mg/µ L 3.2.10-6 mg/µ L 0.0032% B. subtilis 0.10 mg/µ L 2.5.10-6 mg/µ L 0.0025% S. aureus 0.10 mg/µ L 7.9.10-6 mg/µ L 0.0079% P. aeruginosa 0.10 mg/µ L 6.3.10-6 mg/µ L 0.0063% Conclusion Luffa fruit methanol inhibited the growth of B. Subtilis, S. Aureus, P. aeruginosa and E. Coli, but did not inhibit the growth of E. Aerogenes and S. Thypi. Extracts which had the highest antibacterial activity was ethyl acetate followed by chloroform, butanol and hexane s. Chemical screening results obtained from the ethyl acetate did not contain a class of alkaloid compounds, chloroform s did not contain the saponin compound, butanol did not contain saponins and terpenoid compounds and hexane s contained only terpenoid compounds. Antibacterial activity of ethyl acetate luffa fruits is weaker when compared to Ampicillin. - 76 -

References Aliero, A., Aliero, B., and Buhari, U., 2008. Preliminary phytochemical and antibacterial screening of Scadoxus multiflorus," Int. Jor. P. App. Scs., Vol.2(4), pp.13-17 Bylka, M., Matlawska, I., Pilewski, N., 2004. Natural flavonoids as antimicrobial agents, JANA, Vol.7(2), pp.24-31 Cowan, M., 1999. Plant products as antimicrobial agents, Clinical Microbiology Reviews, Vol.12(4), pp.564-68 Durmaz, H; Sagun, E; Tarakci, Z; Ozgokce, F., Antibacterial activities of Allium vineale, Chaerophyllum macropodum and Prangos ferulacea, African Journal of Biotechnology, Vol.5(19), pp.1795-1798 Kumar, G., Swamy, V.B., Sanjay, P. And Kumar, A., 2006. Antimicrobial effect of Benincasa hispida against acne inducing bacteria, AAPSJ, Abstratct retrived in August 22, 2012 at http://www.aapsj.org/ abstracts/am_2006/staged/aaps2006-001774.pdf Oyetayo, F., Oyetayo, V., and Ajewole, V., 2007. Phytochemical profile and antibacterial properties of the seed and leaf of the Luffa plant (Luffa cylindrica), J. Pharmacol. Toxicol., Vol. 2(6), pp.586-89 Shimamura, T., Zhao, W., and Hu, Z., 2007. Mechanism of action and potential for use of tea catechin as an antiinfective agent, Anti-infective Agent In Medicinal Chemistry, Vol.6, pp.57-62 Tomori, O., Saba, A., and Dada-Adegbola, H., 2007. Antibacterial activity of ethanolic of whole fruit of Lagenaria breviflora Robert, J. Anim.Vet.Adv., Vol.6(5), pp.752-57 - 77 -