|Year : 2010 | Volume
| Issue : 1 | Page : 46-50
Antibacterial, antifungal and free radical scavenging activity of Croton gibsonianus Nimm. Grah. (Euphorbiaceae)
KS Vinayaka1, D Swathi2, TR Prashith Kekuda2, K Bhagath3, N Mallikarjun2
1 Department of Studies and Research in Applied Botany, Jnanasahyadri, Shankaraghatta - 577 451, Karnataka, India
2 Department of Studies and Research in Microbiology, Sahyadri Science College (Autonomous), Shivamogga - 577 203, Karnataka, India
3 Department of Chemistry, National Pre-University College, Barkur, Udupi, Karnataka, India
|Date of Web Publication||8-Dec-2010|
T R Prashith Kekuda
Department of Studies and Research in Microbiology, Sahyadri Science College (Autonomous), Shivamogga - 577 203, Karnataka
Background: Croton gibsonianus Nimm. Grah is a shrub belonging to the family Euphorbiaceae and grows in the under-story of the evergreen forests of the Western Ghats. The present study was performed to investigate the antibacterial, antifungal and free radical scavenging potentials of the methanol extract of C. gibsonianus leaves. Materials and Methods: The powdered leaf material was subjected to soxhlet extraction using methanol. The antibacterial and antifungal activities of the methanolic extract were determined by the agar well diffusion method. The free radical scavenging activity was performed using the DPPH free radical scavenging assay. Results: The extract was found to cause marked inhibition of Pseudomonas aeruginosa followed by Escherichia coli and Staphylococcus aureus. Among fungi, Aspergillus niger was inhibited to a greater extent, followed by Chrysosporium indicum, Candida albicans and Trichophyton rubrum. The inhibition of test fungi was dose-dependent. The radical scavenging activity was found to be concentration dependent and the IC 50 value for the extract was found to be 43.78 μg/ml. A phytochemical analysis of the extract showed the presence of saponins, tannins, glycosides and terpenoids. Conclusion: The methanolic extract of C. gibsonianus leaves could be used in the treatment of bacterial and fungal infections and damage caused by free radicals. The presence of various phytochemicals might be responsible for these activities of the extract. Further studies on isolation of constituents from the extract and their biological activities are under investigation.
Keywords: Agar well diffusion method, Croton gibsonianus Nimm. Grah, dermatophytes, DPPH assay, soxhlet extraction
|How to cite this article:|
Vinayaka K S, Swathi D, Prashith Kekuda T R, Bhagath K, Mallikarjun N. Antibacterial, antifungal and free radical scavenging activity of Croton gibsonianus Nimm. Grah. (Euphorbiaceae). J Nat Pharm 2010;1:46-50
|How to cite this URL:|
Vinayaka K S, Swathi D, Prashith Kekuda T R, Bhagath K, Mallikarjun N. Antibacterial, antifungal and free radical scavenging activity of Croton gibsonianus Nimm. Grah. (Euphorbiaceae). J Nat Pharm [serial online] 2010 [cited 2013 Dec 10];1:46-50. Available from: http://www.jnatpharm.org/text.asp?2010/1/1/46/73589
| Introduction|| |
Antibiotics provide the main basis for the therapy of microbial infections. However, the high genetic variability of these pathogenic microbes enables them to rapidly develop antibiotic resistance. In recent years, development of multi-drug resistance in the pathogenic microbes has created major clinical problems in the treatment of infectious diseases and other problems such as toxicity of certain antimicrobial drugs on the host tissue, which triggered interest in the search of new antimicrobial substances/drugs of plant origin. Phytomedicines have shown great promise in the treatment of various diseases including viral infections. Single and polyherbal preparations have been used throughout human history for the treatment of various types of illness. ,,, Considering the rich diversity of plants, it is expected that screening and scientific evaluation of plant extracts for their antimicrobial activity may provide new antimicrobial substances. Free radicals are chemical species containing one or more unpaired electrons that makes them highly unstable and causes damage to other molecules by extracting electrons from them in order to attain stability. Free radicals contribute to more than 100 disorders in humans, including atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, cancer and acquired immunodeficiency syndrome. In recent years, much attention has been devoted to natural antioxidants and their association with health benefits. ,, Croton gibsonianus Nimm. Grah is a shrub belonging to the family Euphorbiaceae that grows up to 1.5-2 m in length. It is prevalent in the under-story of the evergreen forests of the Western Ghats. Branchlets are stellately pubescent; leaves opposite, elliptic oblong, glandular-serrate, apex shortly acuminate, base rounded with a pair of glands. Inflorescence is terminal racemes. The flowers are unisexual, with male flowers above and female flowers at the base. The ovary is globose, densely hairy and the fruit is tricarpellary and is a berry. Flowering and fruiting occur between October and December [Figure 1].  The present investigation was undertaken to determine the antibacterial, antifungal and free radical scavenging potentials of the methanol extract of C. gibsonianus leaves.
| Materials and Methods|| |
lection and Identification of the Plant Material
The plant material was collected in the Hulikal Ghat region (alt-2115, lat-13Ί 44' N to 75Ί 01' E), Karnataka (Western Ghats region of Southern India) during May 2009 and authenticated by Prof. K. G. Bhat, Udupi, Karnataka, India. The voucher specimen (KU/AB/KSV/2032) was deposited in the University herbaria at the P.G. Department of Studies and Research in Botany, Shankaraghatta, Karnataka, for future reference.
Extraction and Phytochemical Analysis
The leaf material was washed thoroughly with running tap water, shade dried, powdered and used for extraction. A known amount of the powdered leaf material (500 g) was subjected to soxhlet extraction and exhaustively extracted with methanol for about 48 h. The extract was filtered and concentrated in vacuum under reduced pressure using a rotary-flash evaporator and dried in a desiccator. The methanolic extract was subjected to preliminary phytochemical screening to screen for the presence of various secondary metabolites in the solvent extracts. ,
Preparation of the Extract for Antibacterial and Antifungal Activity
For screening the antibacterial and antifungal activities, the condensed methanol extract was dissolved in 10% dimethyl sulfoxide (DMSO) to get concentrations of 2.5, 5.0 and 10.0 mg/ml of DMSO.
Antibacterial Activity of the Methanol Extract
The efficacy of the methanol extract was tested against bacteria, namely Staphylococcus aureus MTCC-902, Escherichia More Details coli MTCC-405 and Pseudomonas aeruginosa MTCC-1934 by the agar-well diffusion method.  The bacterial cultures were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh. In this method, 24 h-old nutrient broth cultures of the test bacteria were swabbed uniformly on solidified sterile nutrient agar plates using a sterile cotton swab. Then, aseptically, wells of 6 mm diameter were bored in the inoculated plates with the help of a gel puncher and the extracts (2.5, 5.0 and 10.0 mg/ml of 10% DMSO), standard (chloramphenicol, 1 mg/ml) and control (10% DMSO) were added separately into the respectively labeled wells. The plates were incubated at 37ºC for 24 h in an upright position and the zone of inhibition formed around the well was recorded. The experiment was carried out in triplicates to get the average reading.
Antifungal Activity of the Methanol Extract
Fungi, namely Trichophytum rubrum (MTCC3272), Chrysosporium indicum (MTCC4965), Candida albicans (MTCC4748) and Aspergillus niger (MTCC1884), were tested for their susceptibility to the solvent extracts by the agar-well diffusion method.  The fungal cultures were obtained from IMTECH, Chandigarh, India. The fungal inocula were aseptically swabbed on sterile and solidified Sabouraud dextrose agar plates. Then, aseptically, wells of 6 mm diameter were bored in the inoculated plates with the help of a gel puncher and the extracts (2.5, 5.0 and 10.0 mg/ml of 10% DMSO), standard (Flucanozole, 1 mg/ml) and control (10% DMSO) were added separately into the respective labeled wells. The plates were incubated at 28ºC for 72 h in an upright position and the zone of inhibition formed around the well was recorded. The experiment was carried out in triplicates to get the average reading.
Free Radical Scavenging Activity of the Methanol Extract
The DPPH assay was employed to determine the free radical scavenging activity of the methanol extract.  Different concentrations of the methanol extract and ascorbic acid (standard), namely 10, 25, 50, 100 and 250 μg/ml were prepared in methanol. DPPH (0.002% in methanol) was used as a free radical. Equal volume of different concentrations of the solvent extracts and DPPH were mixed in clean and labeled test tubes separately and the tubes were incubated at room temperature in the dark for 30 min. The optical density was measured at 517 nm using a UV-vis spectrophotometer. The degree of stable DPPH* decolorization to DPPHH (reduced form of DPPH) yellow indicated the scavenging efficiency of the extract. The scavenging activity of the extract against the stable DPPH* was calculated using the following equation:
Scavenging activity (%) = A - B / A x 100
where, A is the absorbance of DPPH and B is the absorbance of the DPPH and extract combination.
| Results|| |
Preliminary phytochemical analysis of the methanol extract of C. gibsonianus showed the presence of saponins, tannins, glycosides and terpenoids. The result of the antibacterial activity of the methanol extract is shown in [Table 1]. Results were recorded as presence or absence of zones of inhibition around the well. The inhibitory zone around the well indicated the absence of bacterial growth, which was reported as positive, and absence of a similar zone was reported as negative. In this study, the extract has shown inhibition of the test bacteria in a dose-dependent manner. The extract was found to cause a greater inhibition of P. aeruginosa, followed by E. coli and S. aureus. Inhibition caused by the standard antibiotic was marked on S. aureus. The standard antibiotic caused a greater inhibitory activity than the methanol extract. No inhibition of the test bacteria was observed in case of the control. It appears that, overall, the bacteria were found to be sensitive to the extract. The reasons for this could be that the components of the plant material were active against bacteria and are most often were obtained through solvent extraction.
|Table 1 :Antibacterial activity of the methanol extract of C. gibsonianus|
Click here to view
The inhibitory activity of the methanol extract against test fungi was revealed by the presence of growth inhibition zones around the well, and is depicted in [Table 2]. Among fungi, marked inhibition was observed in case of A. niger, followed by C. indicum, C. albicans and T. rubrum. The inhibition of test fungi was dose-dependent. T. rubrum was found to be the least affected one.
The antioxidant activity of different concentrations of the methanolic extract and standard (ascorbic acid) is shown in [Figure 2]. The extract exhibited a marked antioxidant activity by scavenging DPPH* (free radical) and converting it into DPPHH. A dose-dependent radical scavenging activity of the extract was observed. The scavenging activity of ascorbic acid was greater than that of the methanol extract. The IC 50 value for the extract was found to be 43.78 μg/ml.
|Figure 2 :Free radical scavenging activity of the methanol extract of C. gibsonianus|
Click here to view
| Discussion|| |
Infectious diseases caused by bacteria, fungi, viruses and parasites remain a major threat to public health despite tremendous progress in human medicine. Their impact is particularly great in developing countries because of the relative unavailability of medicines and the emergence of widespread drug resistance.  Interest in natural products with antimicrobial properties has revived as a result of the current problems associated with the use of antibiotics.  Over 50% of all modern clinical drugs are of natural plant origin, and natural products play an important role in drug development programs in the pharmaceutical industry.  The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids and phenolic compounds.  Plant-derived products have received attention in recent years due to their diverse pharmacological activities.  The antimicrobial activities of tannins,  flavonoids,  saponins,  terpenoids  and alkaloids  have been documented. In the present study, phytoconstituents, namely glycosides, tannins, terpenoids and saponins, were detected in the extracts, which may account for the antibacterial and antifungal activities. Dermatomycoses are the superficial fungal infections in humans and are caused by dermatophytes, a group of filamentous fungi. These fungi invade and draw nutrients from the keratinized tissues such as skin, hair and nails. Among the dermatophyte genera, Trichophyton, Microsporum and Epidermophyton are most important. As the dermatophytes have developed resistance to antimycotic drugs, there is an urgent need for non-toxic, safe and cost-effective antifungal agents. , In view of this, the present study highlights the possible use of plants in the treatment of fungal infections like Aspergillosis, Candidiasis and Dermatomycoses, as the test fungi were found to exhibit susceptibility to the extract. In this study, the extract has shown inhibition of the test bacteria that are known to cause infections in humans. Most of these bacteria have already developed resistance against several antibiotics. Thus, the extract could be used against antibiotic-resistant bacteria.
Free radicals are found to be a product of normal metabolism. Although oxygen is essential for the aerobic forms of life, oxygen metabolites are highly toxic. As a consequence, reactive oxygen species (ROS) are known to be implicated in many cell disorders and in the development of many diseases, including cardiovascular diseases, atherosclerosis, chronic inflammation, etc. , Although organisms have endogenous antioxidant defences produced during normal cell aerobic respiration against ROS, other antioxidants are taken both from natural and synthetic origin.  Antioxidants that can inhibit or delay the oxidation of an oxidizable substrate in a chain reaction, therefore, appear to be very important.  Synthetic antioxidants are widely used, but their use is being restricted nowadays because of their toxic and carcinogenic effects. Thus, interest in finding natural antioxidants, without any undesirable effect, has increased greatly.  There are several methods available to assess the antioxidant activity of the compounds. An easy, rapid and sensitive method for the antioxidant screening of plant extracts is the free radical scavenging assay using 1, 1, diphenyl-2-picryl hydrazyl (DPPH) stable radical spectrophotometrically. In the presence of an antioxidant, the DPPH radical obtains one more electron and the absorbance decreases.  DPPH is a relatively stable, nitrogen-centered free radical that easily accepts an electron or hydrogen radical to become a stable diamagnetic molecule.  DPPH radicals react with suitable reducing agents, as a result of which the electrons become paired-off forming the corresponding hydrazine. The solution therefore loses color stoichometrically depending on the number of electrons taken up.  In this study, the scavenging activity of the methanol extract was found to be dose dependent, i.e. higher the concentration, higher was the scavenging activity. Although the DPPH radical scavenging abilities of the extracts were lower than that of ascorbic acid, the study showed that the extract has proton-donating abilities and could serve as a free radical inhibitor or scavenger, acting possibly as a primary antioxidant.
| Conclusion|| |
A marked antibacterial, antifungal and free radical scavenging activity of the methanol extract of C. gibsonianus was observed in this study. The extract, in a suitable form, may be used in the treatment of microbial diseases and radical damage. The presence of various phytochemicals might be responsible for these activities of the extract. This is the first report on these promising potentials of C. gibsonianus. Further studies on the isolation of constituents from the extract and their biological activities are under investigation.
| References|| |
|1.||Davies J. Inactivation of antibiotics and the dissemination of resistance genes. Science 1994;264:375-82. |
|2.||Idose O, Guthe T, Willeox R, Deweck AL. Nature and extent of penicillin side reaction with particular reference to fatalities from anaphylactic shock. Bull World Health Organ 1968;38:159-88. |
|3.||Maddux MS, Barrere SL. A review of complications of amphotericin-B therapy: Recommendations for prevention and management. Drug Intell Clin Pharm 1980;14:177-81. |
|4.||Adwan G, Abu-Shanab B, Adwan K, Abu-Shanab F. Antibacterial effects of nutraceutical plants growing in Palestine on Pseudomonas aeruginosa. Turk J Biol 2006;30:239-42. |
|5.||Ali SS, Kasoju N, Luthra A, Singh A, Sharanabasava H, Sahu A, et al. Indian medicinal herbs as sources of Antioxidants. Food Res Int 2008;41:1-15. |
|6.||Kumpulainen JT, Salonen JT. Natural Antioxidants and Anticarcinogens in Nutrition, Health and Disease. UK: The Royal Society of Chemistry; 1999. p. 178-87. |
|7.||Cook N, Samman S. Flavonoids- Chemistry, metabolism, cardioprotective effects, and dietary sources. J Nutr Biochem 1996;7:66-76. |
|8.||Ramaswamy SN, Rao RM, Govindappa DA. Flora of Shimoga district, Karnataka. Prasaranga, Manasagangothri. Mysore, Karnataka: Mysore University; 2001. p. 531. |
|9.||Manjunatha BK, Patil HS, Vidya SM, Kekuda TR, Mukunda S, Divakar R. Studies on the antibacterial activity of Mucuna monosperma DC. Indian Drugs 2006;43:150-2. |
|10.||Parekh J, Chanda SV. In vitro antimicrobial activity and phytochemical analysis of some Indian medicinal plants. Turk J Biol 2007;31:53-8. |
|11.||Adeniyi BA, Ayepola OO. The phytochemical screening and antimicrobial activity of leaf extracts of Eucalyptus camaldulensis and Eucalyptus torrelliana (Myrtaceae). Res J Med Plant 2008;2:34-8. |
|12.||Ravikumar YS, Mahadevan KM, Kumaraswamy MN, Vaidya VP, Manjunatha H, Kumar V, et al. Antioxidant, cytotoxic and genotoxic evaluation of alcoholic extract of polyalthia cerasoides (roxb) Bedd. Environ Toxicol Pharmacol 2008;26:142-6. |
|13.||Okeke IN, Laxminarayan R, Bhutta ZA. Antimicrobial resistance in developing countries: Part 1: recent trends and current status. Lancet Infect Dis 2005;5:481-93. |
|14.||Abu-Shanab B, Adwan G, Abu-Safiya D. Antibacterial activities of some plant extracts used in Palestine in popular medicine. Turk J Biol 2004;28:99-102. |
|15.||Nair R, Kalariya T, Chanda S. Antibacterial activity of some selected Indian medicinal flora. Turk J Biol 2005;29:41-7. |
|16.||Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian medicinal plants. Afr J Biotechnol. 2005;4:685-8. |
|17.||Gupta M, Mazumder UK, Kumar RS, Sivakumar T, Vamsi ML. Antitumor and Antioxidant status of Caesalpinia bonducella against Ehrlich ascites Carcinoma in swiss albino miceJ Pharmacol Sci 2004;94:177-84. |
|18.||Doss A, Mubarack HM, Dhanabalan R. Pharmacological importance of solanum trilobatum. Indian J Sci Tech 2009;2:41-3. |
|19.||Mandalari G, Bennett RN, Bisignano G, Trombetta D, Saija A, Faulds CB, et al. Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry. J Appl Microb 2007;103:2056-64. |
|20.||Avato P, Bucci R, Tava A, Vitali C, Rosato A, Bialy Z, et al. Antimicrobial activity of saponins from Medicago sp.: Structure-activity relationship. Phytotherapy Res 2006;20:454-7. |
|21.||Funatogawa K, Hayashi S, Shimomura H, Yoshida T, Hatano T, Ito H, et al. Antibacterial activity of hydrolyzable tannins derived from medicinal plants against Helicobacter pylori. Microbiol Immunol 2004;48:251-61. |
|22.||Navarro V, Delgado G. Two antimicrobial alkaloids from Bocconia arborea. J Ethnopharmacol 1999;66:223-6. |
|23.||Macura AB. In vitro susceptibility of dermatophytes to antifungal drugs: A comparison of two methods. Int J Dermatol 1993;32:533-6. |
|24.||Emmons CW, Binford CH. Medical Mycology. 3 rd ed. Philadelphia: Lea and Febiges; 1974. p. 117-67. |
|25.||Gutteridge JM. Free radicals in disease processes: A compilation of cause and consequence. Free Radic Res 1993;19:141-58. |
|26.||Knight JA. Diseases related to oxygen-derived free radicals. Ann Clin Lab Sci 1995;25:111-21. |
|27.||Rechner AR, Kuhnle G, Bremmer P, Hubbard GP, Moore KP, Rice-Evans CA. The metabolic fate of dietary polyphenols in humans. Free Radic Biol Med 2002;33:220-35. |
|28.||Halliwell B, Gutteridge JM, Cross CE. Free radicals, antioxidants, and human disease: Where are we now? J Lab Clin Med 1992;119:598-620. |
|29.||Koleva II, Vanbreek TA, Linssen JP, Groot AD, Evstatieva LN. Screening of plant extracts for antioxidant activity: A comparative study on the three testing methods. Phytochem Anal 2002;13:8-17. |
|30.||Jayaprakasha GK, Singh RP, Sakariah KK. Antioxidant activity of grape seed extracts on peroxidation models in vitro. J Agric Food Chem 2001;55:1018. |
|31.||Blois MS. Antioxidant determinations by the use of stable free radical. Nature 1958;29:1199. |
[Figure 1], [Figure 2]
[Table 1], [Table 2]