Screening of Antimicrobials of some Medicinal Plants by TLC Bioautography Middha Himanshu 1* and Parihar Pradeep 2 1Department of Microbiology, DTM College of Biosciences, Bikaner, Rajasthan 2 Lovely Professional University, Phagwara, Punjab Abstract The antimicrobial activity of clove, cinnamon, Datura and Tulsi were evaluated in different solvents by agar well diffusion method. The bioactivity of selected plants material was observed maximum in pet ether against Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Klebsiella pneumonia, except Tulsi. In case of tulsi acetone extract showed maximum activity. The bioautographic results revealed that the max. Zone of inhibition of clove, cinnamon, datura and tulsi were observed at R f 0.639, 0.147, 0.803 & 0.263 respectively which indicates that the compound retained at these R f values having potent antimicrobial activity. Key words Agar well diffusion, antimicrobial activity, bioautography, plant extract. Introduction Natural products from plants are of interest for the discovery of antimicrobial compounds. Because of available antimicrobials failure to treat infectious diseases, many researchers have focused on the investigation of natural products as source of new bioactive molecules. (1, 2) A variety of methods are found for this purpose and since not all of them are based on same principles, results obtained will also be profoundly influenced not only by the method selected, but also by the microorganisms used to carry out the test, and by the degree of solubility of each testcompound. (3, 4) The currently available screening methods for the detection of antimicrobial activity of natural products fall into three groups, including bioautographic, diffusion, and (5) dilution methods. Screening programmes for biologically active natural products require the right bioassays. Detection of compounds with the desired activity in complex plant extracts depends on the reliability and sensitivity of the test systems used. This investigation concerns the antimicrobial screening of Clove, Cinnamon, Datura and Tulsi by bioautographic agar overlay method. The bioautography method aided in the identification & localization of the compounds showed maximum Zone of inhibition. (6) Methodology Collection and Identification of Plant materials All the plant material selected for study is based on their reported traditional use in *Corresponding Author Himanshu Middha 60 P a g e
rural region of India for treating infections. Datura seeds, Clove buds and Cinnamon bark were purchased from the local market of Bikaner while the Tulsi leaves were collected from the Botanical garden of Dungar College, Bikaner. All the plant materials were identified and authenticated by Dr. B. R. Gadi, botanist in the Department of Biological Sciences of Dungar college, Bikaner. Antimicrobial Activity All the plant materials were successively extracted by maceration in petroleum ether, Acetone, Ethanol and then finally with 1000 ml of Hydroalcohol and then dried under vacuum oven. A series of dilutions (25%, 50%, 75% and 100%) were prepared with distilled water. Preparation of Inoculum: Inoculum of Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Klebsiella pneumonia, were prepared in nutrient broth medium and incubated at 37 C for 8 hours. Preparation of Petri dishes: The melted agar was poured into previously sterile Petri dishes immediately after it was taken out from the autoclave to prevent it become hardened and allowed to settle down. Bacterial lawn was spread on nutrient agar plates by using sterile cotton swab. Four wells were made in each Petri dish by digging in agar plate with the help of sterile metallic borer (diameter = 8mm). Procedure: Antibacterial activity of dry extracts of various plant materials in different solvents was determined by well diffusion method. Dilutions (20 μl) of various dry extracts in concentration of 25%, 50%, 75% and 100% were poured in wells of each petridish and marked respectively. All these Petri dishes were incubated at 37±2 C aerobically for 24 hrs. The antimicrobial activity was determined by measuring the zone of inhibition (mm) of the plant extracts against a particular pathogen. Extracts showing zone of inhibition >13 mm were considered that they are possessing antimicrobial activity. Thin Layer Chromatography Selection of plant extract Antimicrobial activity of all the four plant extracts in four different solvents (total sixteen extracts) were determined against four microorganism i.e. Staphylocccus aureus, Proteus vulgaris, Escherichia coli, Klebsiella pneumonia by comparing the zone of inhibition. For further characterization and fractionation one extract of each plant (total 4 extracts) was selected showing highest antimicrobial activity among sixteen extracts. Preparation of mobile phase Different mobile phases were developed by mixing the different solvents in different ratio and filtered through watt man filter paper, using vacuum pump. Solvent Composition Ratio system I Butanol: Acetic acid 4:1 II Toluene: Ethyl Acetate 93:7 III Toluene: Methanol: Acetone: 14:4:1:1 Acetic Acid IV Toluene: Acetic Acid 70:30 V Chloroform: Ethyl Acetate: 7.5 : Formic Acid 6:0.5 VI Ethyl Acetate: Formic Acid: Acetic Acid 100:11: 27 61 P a g e
Procedure: TLC was performed on a precoated silica gel aluminum plate (10 x 20 cm, Silica gel 60 F, Merck) for the chromatographic separation of the extracted plant material. The petroleum ether extract of clove, cinnamon, datura and acetone extract of Tulsi were applied on base line of different TLC plate by using capillary and placed at 45 angle in the development chamber containing mobile phase. The R f values was calculated by using the following formula- Distance travelled by solute R f = Distance travelled by solvent (6, 7) Bioautography assay: Selections of TLC plates TLC chromatogram serves as a chemical fingerprint of the plant extract. For further investigation one mobile phase which showed maximum spots among six solvent systems for each plant extract was selected. The plates were run in duplicate; one set was used as the reference chromatogram and the other was used for bioautography. Procedure A bioautography technique was employed to define the active constituents. Bioautographic method is basically to localize the antibacterial compound from crude extract into chromatogram. The agar-overlay technique was used to conduct the bioautographic assay. In this technique TLC plates were placed in Petri dishes containing nutrient agar (previously treated with selected microorganism) and incubated at 37 C for 24 hrs. The active constituents diffused in to microbial inoculated agar medium and clear zone of inhibition was produced. The width of Inhibition zones and R f values of bioautographic plate was compared with related spots and their R f values of reference plates. Result and Discussion By comparing the different extracts of clove against different bacteria (table 1), it was found that the pet ether extract exhibited good antimicrobial activity against the selected microorganism. The P. vulgaris is more sensitive while K. pneumoniae is less sensitive. Acetone extract possess almost similar activity towards all the selected bacteria. However, ethyl alcohol shows moderate activity against K. pneumoniae and has less activity against remaining three bacteria. S. aureus and P. vulgaris is moderately inhibited by hydroalcoholic extract. S. aureus is more sensitive and K. pneumoniae is less sensitive. This observation clearly indicates that the polarity of antimicrobial compounds makes them more readily extractable by pet ether and using organic solvent does not negatively affect their bioactivity. Since pet ether extract having higher antimicrobial activity against all selected microorganism hence the pet ether extract was selected for further characterization. When we compared the activity of all Cinnamon extracts against selected bacteria (Table 2), the results indicated that all the bacteria were strongly inhibited by the pet ether extract but S. aureus was 62 P a g e
more sensitive and E.coli was less sensitive. Acetone extract exhibited good activity against P. vulgaris and E.coli while S. aureus and K. pneumoniae was moderately inhibited by it. Ethyl alcohol and hydroalcoholic extract does not show any significant effect on the selected bacteria. Hence the pet ether extract was selected for further characterization. Antimicrobial activity of Datura may be due to the presence of tropane alkaloid. The comparative study of different extract of Datura towards selected microorganism (table 3) shows that only pet ether extract has maximum efficiency to inhibit the growth of all selected organisms. Acetone extract has almost similar activity against all microorganisms but less than pet ether extract. Ethyl alcohol extract of datura did not show the activity against K.pnumoniae, E.coli and P. vulgaris while hydro alcohol extract against K.pnumoniae & E.coli. Hence the pet ether extract was selected for further characterization. The data obtained in the present study related to antibacterial activity of Datura stramonium are consistent with previous (9) report, that shows the antibacterial activity against both S.aureus and E.coli. The comparative activity of different extracts of Tulsi on bacteria selected for study is shown in table-4. Results indicated that acetone extract has higher activity against all selected bacteria. The gram positive S. aureus most sensitive and P. vulgaris is least sensitive towards acetone extract. In case of pet ether extract moderate activity was observed towards all the microorganisms. S. aureus is highly sensitive while as P. vulgaris is least sensitive. Ethyl alcohol and hydroalcohol extracts exhibits less but almost same activity towards selected microorganism and showing the zone of inhibition between the ranges of 12.33 to 15.57 mm. Since Pet ether, ethyl alcohol and hydroalcohol extracts shows comparatively lower activity then acetone, hence the acetone extract was selected for further characterization. The R f value of different separated compounds of selected plant materials in various mobile phase composition is shown in table-5. Results indicated that maximum numbers of spots (five) of Clove were observed in Chloroform: Ethyl acetate: Formic Acid (7.5: 6: 0.5) system with R f value 0.969, 0.849, 0.766, 0.714 and 0.639 shown in Figure-5. In case of cinnamon maximum six spots were observed in Toluene: Acetic Acid (70:30) with R f value 0.585, 0.526, 0.372, 0.295, 0.207 and 0.147 (Figure-6). While in datura maximum five spots were observed in Toluene: Methanol: Acetone: Acetic Acid (14:4:1:1) with R f value 0.860, 0.822, 0.803, 0.753 & 0.677 (Figure-7). The result indicates that maximum numbers of spots (nine) of Tulsi were observed in Toluene: Acetic Acid (70:30) system with R f value 0.574, 0.526, 0.371, 0.317, 0.263, 0.179, 0.143, 0.053 and 0.029 shown in Figure-8. For further identification by bioautography a combination of mobile phase was selected in which maximum spots were observed. Thin layer chromatography (TLC) Bioautographic assays 63 P a g e
The results of TLC Bioautography are summarized in Table-6. Clove pet ether extract showed five spots at different R f values in Chloroform: Ethyl acetate: Formic Acid (7.5:6:0.5) solvent system. The compound which retained at R f value 0.639 shows maximum zone of inhibition against S. aureus (Figure-9) while as Cinnamon pet ether extract showed six spots at different R f values in Toluene: Acetic Acid (70:30) solvent system. The compound which retained at R f value 0.147 shows maximum zone of inhibition against S. aureus (Figure-10). In case of datura pet ether extract five spots were showed at different R f values in Toluene: Methanol: Acetone: Acetic Acid (14:4:1:1) solvent system. The compound which retained at R f value 0.803 shows maximum zone of inhibition against S. aureus (Figure-11). Whereas Tulsi Acetone extract showed nine spots at different R f values in Toluene: Acetic Acid (70:30) solvent system. The compound which retained at R f value 0.263 exhibits strong antibacterial activity against S. aureus (Figure-12). References: 1. Recio, M.C.; Rios, J.L. (1989). A review of some antimicrobial compounds isolated from medicinal plants reported in the literature 1978-1988. Phytoter. Res., 3, 117-125. 2. Silver, L.L.; Bostian, K.A. (1993). Discovery and development of new antibiotics: the problem of antibiotic resistance. Antimicrobial Agents Chemother., 37, 377-383. 3. Rios, J.L.; Recio, M.C.; Villar, A. (1988). Screening methods for natural products with antimicrobial activity: a review of the literature. J. Ethnopharmac., 23, 127-149. 4. Vanden Berghe, D.A.; Vlietinck, A.J. (1991). Screening methods for antibacterial and antiviral agents from higher plants. In: Dey, P.M., Harbone, J.D. (eds), Methods in Plant Biochemistry, Academic Press, London, p. 47-69. 5. Cleidson Valgas, Simone Machado de Souza, Elza F A Smânia, (2007). Screening methods to determine antibacterial activity of natural products, Brazilian Journal of Microbiology 38:369-380 6. K. Hostettmann, C. Terreaux, A. Marston and O. Potterat, O. (1997). J. Planar Chromatogr. 10:251-257 7. Hamburger, M.O.; Cordell, G.A. (1987) A direct bioautographic TLC assay for compounds possessing antibacterial activity J. Nat. Prod., 50, 19-22 8. Beghe, W.J.; Kline, R.M. (1972) The use of tetrazolium salts in bioautography procedures J. Chromatogr, 64, 182-184 9. Uzun E., (2004) Traditional medicine in turkey and antimicrobial activities of selected species Journal of Ethan pharmacology, 49:287-296 64 P a g e
Table 1 Comparative antimicrobial activity of different extracts of Clove against different Microorganisms Zone of Inhibition (mm) Microorganism Pet ether Acetone Ethyl alcohol Hydroalcohol S. aureus 23.567 19.467 16.567 20.267 K.pnumoniae 20.767 20.9 20.767 18.833 E.coli 23.833 20.467 17.133 19.9 P. vulgaris 26.567 20.5 15.9 20.067 30 25 Zone of Inhibition 20 15 10 5 0 S. aureus K.pnumoniae E.coli P. vulgaris Clove Extracts Figure 1 - Graphical presentation of Comparative antimicrobial activity of different extracts of Clove against different microorganisms 65 P a g e
Table 2 - Comparative antimicrobial activity of different extracts of Cinnamon against Different microorganisms Zone of Inhibition (mm) Microorganism Pet ether Acetone Ethyl alcohol Hydroalcohol S. aureus 36.1 17.667 12.933 14.467 K.pnumoniae 27.667 17.8 12.967 17.167 E.coli 24.333 19.867 14.6 17.767 P. vulgaris 34.667 22.833 12.633 12.967 Zone of Inhibition 40 35 30 25 20 15 10 5 0 S. aureus K.pnumoniae E.coli P. vulgaris Cinnamon Extracts Figure 2 - Graphical presentation of Comparative antimicrobial activity of different extracts of Cinnamon against different microorganisms 66 P a g e
Table 3 - Comparative antimicrobial activity of different extracts of Datura against Different microorganisms Zone of Inhibition (mm) Microorganism Pet ether Acetone Ethyl alcohol Hydroalcohol S. aureus 14.43 14.73 12.9 14.5 K.pnumoniae 16.57 13.47 0 0 E.coli 17.67 12.53 0 0 P. vulgaris 16.17 14.7 0 12.57 Zone of Inhibition 20 18 16 14 12 10 8 6 4 2 0 S. aureus K.pnumoniae E.coli P. vulgaris Datura Extracts Figure 3 - Graphical presentation of Comparative antimicrobial activity of different extracts of Cinnamon against different microorganisms 67 P a g e
Table 4 - Comparative antimicrobial activity of different extracts of Tulsi against Different microorganisms Zone of Inhibition (mm) Microorganism Pet ether Acetone Ethyl alcohol Hydroalcohol S. aureus 19.1 21.53 14.2 15.57 K.pnumoniae 18.1 20.83 13.3 14.7 E.coli 19.47 20.93 12.8 12.97 P. vulgaris 15.7 16.4 12.5 12.3 25 20 Zone of Inhibition 15 10 5 0 S. aureus K.pnumoniae E.coli P. vulgaris Tulsi Extracts Figure 4 - Graphical presentation of Comparative antimicrobial activity of different extracts of Tulsi against different microorganisms 68 P a g e
Table 5- R f value of Tulsi extracts in different compositions of solvents Solvent system I II III IV V VI Clove Cinnamon Datura Tulsi spots Rf spots Rf spots Rf spots Rf 0.974, 0.923, 2 0.921, 0.278 3 0.871 1 0.921 2 0.82, 0.65 0.985, 0.847, 0.826, 0.735, 0.764, 0.813, 0.758, 0.811, 0.804, 0.789, 1 0.576 3 0.727 4 0.717, 0.703 7 0.775, 0.98, 0.83, 0.860, 0.822, 0.987, 0.949, 0.886, 0.77, 0.76, 0.803, 0.753, 0.822, 0.759, 0.683, 1 0.68 5 0.751, 5 0.677 7 0.664 4 5 0.753, 0.702, 0.681, 0.659, 6 0.585, 0.526, 0.372, 0.295, 0.207, 0.147 3 0.975, 0.766, 0.30 9 0.969, 0.849, 0.766, 0.714, 0.639 1 0.571 1 0.653 1 0.977 1 0.925 3 0.96, 0.608, 0.392 1 0.943 5 0.574, 0.526, 0.371, 0.317, 0.263, 0.179, 0.143, 0.053, 0.029 0.961, 0.633, 0.557, 0.381, 0.366 Visible Light U.V Light Visible Light U.V Light Fig. 5 - Photograph of TLC of clove in Fig. 6 - Photograph of TLC of Cinnamon Chloroform: Ethyl acetate: Formic Acid (7.5:6:0.5) in Toluene: Acetic Acid (70:30) 69 P a g e
Visible Light U.V Light Fig. 7- Photograph of TLC of Datura in Fig. 8 - Photograph of TLC of Tulsi in Toluene: Methanol: Acetone: Acetic Acid (14:4:1:1) Toluene: Acetic Acid (70:30) Table 6 -Bioautographic results of different plant materials Plant Extract Solvent System No. of Spots R f at which max. zone of inhibition occurred Clove (Pet ether) Chloroform: Ethyl acetate: Formic Acid 05 0.639 (7.5:6:0.5) Cinnamon (Pet ether) Toluene: Acetic Acid 06 0.147 (70:30) Datura (Pet ether) Toluene: Methanol: Acetone: Acetic Acid (14:4:1:1) 05 0.803 Tulsi (Acetone) Toluene: Acetic Acid (70:30) 09 0.263 70 P a g e
Fig.- 9 Bioautography of clove against S. aureus Fig.-10 Bioautography of cinnamon against S. aureus Fig.-11 Bioautography of Datura against S. Aureus Fig.-12 Bioautography of Tulsi against S. aureus 71 P a g e