Objective: Increasing usage of medicinal plants for trapeutic purposes, made a unique place for this branch of complementary medicine. While Echinophora Platyloba has a folkloric usage, unfortunately has not yet reached an eligible palce. This study conducted to evaluate the effect of extract of Echinophora Platyloba on some common dermatophytes. Methods: 4 tubes containing diluted solutions of hydroalcoholic extract (35, 50, 150 and 250 mg/ml) have been prepared and one tube of 0 mg/mL as control hydroalcoholic. Using agar dilution method, fugi were coincubated with diluted solutions of the extract and the growth results were recorded after 21 days. Results: Trichophyton (T) schenlaini and T. verucosum were totally sensitive. T. rubrum and Microsporum (M) gypsum were totally resistant. T. mentagrophyte, M. canis and Epidermophyton flucosum were resistant to 35, 50 and 150 mg/ml dilutions, but they were sensitive to 250 mg/ml. T. violaseum was resistant in 35 and 50 mg/ml but sensitive to 150 and 250 mg/ml. Conclusion: 3 concentrations of hydroalcohalic extract (35, 50 and 150 mg/ml) may have clinical application. On the base of this study, the plant in the mentioned dilutions showed anti fungal effect against T. schoenlieinii, T. verucosum. The plant extract showed weaker antifuongal against T. mentagrophytes, Microsporum canis and Epidermophyton flocusm but this may have not good anti fungal effect aginst T. rubrum and T. gypseum. The next step of study may be the production of drug product and clinical assay

Avijgan, Majid.

Hafizi, Massoud.

Nilfrooshzadeh, Mohammad Ali.

Saadat, Mehdi.

shahrekord university of medical scinces

Original ArticleAntifungal Effect of Echinophora Platyloba’ s Extract against Candida albicansMajid Avijgan a*, Massoud Hafizi b, Mehdi Saadat b and Mohammad Ali Nilforoushzadehc aDepartment of Infectious and Tropical Diseases. Shahr-e-Kord University of Medical Sciences, Shahr-e-Kord, Iran. bShahr-e-kord University of Medical Sciences, Shahr-e-Kord, Iran. cIsfahan Skin and Leishmaniasis Diseases Research Center, Isfahan, Iran.AbstractThe present study was undertaken to investigate the effectiveness of the ethanolic extract of Echinophora platyloba DC. on Candida albicans.Using the agar dilution method, the growth condition of standard Candida albicans ATCC 10231, cultured on the media containing plant extracts at different concentrations (1, 2, 4, 8, 16, 32, 64, 128 and 256 mg/ml) was studied. The results were recorded twenty one days after the incubation period, maximum time for the growth of fungi.Results showed that the extract of Echinophora platyloba, equal or above the concentration of 2mg/ml, effectively inhibits the growth of Candida albicans. In other words it shows growth on media containing 1mg/ml of the extract. Results of the present study revealed a great promise in the application of Echinophora platyloba extract against Candida albicans. It is concluded that the plant studied could be a good antifungal source.Keywords: Candida albicans; Echinophora platyloba; Ethanolic extract; Yeast; Traditional Medicine.IntroductionTraditional Medicine, in some countries Like Iran has a major therapeutic role, and traditional healers have been using different plants to treat people for thousand of years. In Iran, in the last century, there has been a decline in the application of traditional medicine. However, fortunately the use of alternative medicines has increased the interest of pharmacologists and herbalists over the past decade. Historically, plants have provided a source of inspiration for novel drug compounds, as plant derived medicines have made large contributions to human health and well being (1). Natural products have served as a major source of drugs for centuries, and about half the pharmaceuticals in use today are derived from natural products (2). One study has reported that 25 to 50% of current pharmaceuticals are derived from plants (3). Microbiologists and natural-product chemists are trying to discover more about phytochemicals, which could be developed for treatment of infectious diseases (3). There are some folkloric herbs still in use which are being used without any scientific evidence. one of them is Echinophora platyloba DC. It has ten species (4) and in Chahar mahal va bakhtiary province, its’ main use is as a food seasoning (5) and also to prevent the fungal growth on some food like tomato paste and pickled cucumber. Among ten species, four of them are endemic in * Corresponding author:   E-mail: avijgan@yahoo.com Copyright © 2006 by School of PharmacyShaheed Beheshti University of Medical Sciences and Health ServicesIranian Journal of Pharmaceutical Research (2006) 4: 285-289Received: October 2005Accepted: April 2006Avijgan M, Hafizi  M, Saadat M and Nilforoushzadeh MA/ IJPR 2006, 4: 285-289Iran, including E. orientalis, E. sibthorpiana, E. cinerea and E. platyloba (4). A previous study, has shown the presence of components such as flavonoid, alkaloid and saponin (6). Another study showed a growth inhibitory effect with this extract, against some common dermatophytes (7). The lack of pharmacological and clinical data on the majority of herbal medicinal products is a major impediment to the integration of herbal medicines into conventional medical practices. For valid integration, pharmacological and clinical studies should be conducted on those plants lacking such data (8). A mong them Echinophora platyloba could be considered.Considering the folkloric application of this herb as a food seasoning (4) and also as an additive material to prevent the growth of fungal on some food like tomato paste and pickled cucumber, there is a lack of information on the antifungal action of Echinophora platyloba. This study is an attempt to determine the antifungal activity of Echinophora platyloba, on standard Candida albicans ATCC 10231.ExperimentalMaterialThe plants were collected from the southwestern parts of Iran (Shahr-e-Kord). A voucher specimen of plant was deposited at the Herbarium of the Faculty of Sciences, Isfahan University, Isfahan, Iran. The aerial parts were separated, shade-dried and crushed into powder using a pestle and mortar and kept in airtight light-protected containers. Preparation of extractThe percolation method was used to obtain the crude extract (9). A quantity (100 gr) of powdered plant was soaked in 500 ml of 96% ethanol for 3 days with frequent agitation. Ethanolic extract was prepared by maceration of the dried plant materials in 96% ethanol (the plant material to solvent ratio was 1:5). The mixture was filtered and the crude extract collected. The crude extract was then distilled at 48ºC using a water bath. The semi-dried extract was collected for further drying in an oven at 37ºC. The Final crude dry extract was equal to 10 gr per 100 gr of the plant. Preparation of media18×150 ml media tubes were used in this study. The method used for study was the agar dilution method (Macro-dilution). On the basis of company instructions, the base medium was made of Sabouraud dextrose agar (Merck, Germany). In contrast to the instructions, we added half the amount of distilled water, but later the remaining volume of water was added along with the antibiotics. After autoclaving the medium, antibiotics were added to make Sabouraud, Chloramphenicole and Cyclohexamide (SCC) medium. [Chloramphenicol (Lorestan Company, Iran) was used to prevent bacterial growth and Cyclohexamide (Oxoid, USA) to inhibit saprophytic fungal growth].Preparation of dilutionAt temperature of 37ºC, 10 g of crude extract was mixed with 10 ml of distilled water to obtain concentration of 1000 mg/ml. On the basis of serial dilution method (Table 1), different concentrations of extract were achieved. At this step, 5 ml of extract in various concentrations were added to 5 ml of the SCC medium to reach the final extract concentration (Table 2).This mixture was placed into screwed tubes, was shaken and stored until a semi solid form obtained. MicroorganismThe microorganism used was Candida Tubes 1 2 3 4 5 6 7 8 9 10 11Distilled water  (ml) 8 4 4 4 4 4 4 4 4 4 8Extract (ml) 8 4 4 4 4 4 4 4 4 4 0Concentration (mg/ml) ≈ 512 256 128 64 32 16 8 4 2 1 0Table 1. Difference concentrations of the extract.*• In order to obtain a serial dilution, half the total  volume of each extract column (tube) was placed in the next extract column (tube),        except for the 11th tube .The half volume of the 10th tube was discarded.Antifungal Effect of Echinophora Platyloba’ s Extract...albicans ATCC 10231, purchased from the Biotechnology Research Center of Iran.Anti- fungal activity of plant extractSome fresh cultured colonies (cultured colonies less than one week after passage on the new medium) of Candida albicans were added to sterile normal saline to obtain 0.5 McFarland turbidity standard. Finally 200 microlitres of this fungal suspension were added to the media and cultured (with streak method) using a sterile loop and then incubated at room temperature in a closed condition (at the hood) and observed weekly for 21 days.The 10th tube was made without any extract (control for media) and the 11th tube without a fungal suspension (control for contamination).The concentration of extract in the first tube, which inhibits the growth of Candida albicans, was recorded as the minimal inhibitory concentration.ResultsOn the basis of this study, Candida albicans is sensitive to a minimum dilution of 2 mg/ml of ethanolic extract of Echinophora platyloba. There is an overt growth of yeast in the control tube (10th tube) and the tube containing 1 mg/ml of extract, but no growth of any fungi in the other tubes (dilutions of 2, 4, 8, 16, 32, 64, 128 and 256 mg/ml) and the control tube for contamination (11th tube) (Table 3).All the concentrations extract (except 1mg/ml) at 2, 4, 8, 16, 32, 64, 128 and 256 mg/ml demonstrated growth inhibitory activity against Candida albicans. The concentration of 2 mg/ml is minimum inhibitory dilution of this study. DiscussionThere is no previous report on evaluation of this plant concerning its’ activity against Candida albicans. Although, the antibacterial effect of another species of this family (E. sibthrobia) has been reported (5). This study was designed to evaluate the activity of Echinophora platyloba extract against Candida albicans. The potential of this extract to inhibit Candida albicans ATCC 10231 growth in at least an MIC of 2 mg/ml concentration revealed a reasonable effect against Candida albicans.The studied plant is one of its’ four species, being endemic of Iran, which grows in many parts of the country (10). One study has reported that this plant contains saponin, alkaloid and flavonoid (6). Fungal agents are susceptible to extract’s Tubes 1 2 3 4 5 6 7 8 9 10 11SCC media (ml) medium 5 5 5 5 5 5 5 5 5 5 5Milliliter of each column from table 15 5 5 5 5 5 5 5 5 5 ml of distilled water5Final concentration of extract in the medium256 128 64 32 16 8 4 2 1 0 256Addition of the fungal suspension+ + + + + + + + + + -Table 2.  Final concentrations of the extract within the mediaTubes 1 2 3 4 5 6 7 8 9 10 11SCC media (ml) medium 5 5 5 5 5 5 5 5 5 5 5Milliliter of each column from table 15 5 5 5 5 5 5 5 5 5 ml of distilled water5Final concentration of extract in the medium (mg/ml )256 128 64 32 16 8 4 2 1 0 256Addition of the fungal suspension+ + + + + + + + + + ---Growth of Candida albicans --- --- --- --- --- --- --- --- +++ +++ ---Table 3.  Final result of fungal growth in each extract concentration within the mediaAvijgan M, Hafizi  M, Saadat M and Nilforoushzadeh MA / IJPR 2006, 4: 285-289components like saponins. Some steroidal saponin, like CAY-1, have shown to be a potent fungicide against Candida albicans by disrupting the membrane integrity of the fungal cells, at concentrations below the threshold of mammalian cell toxicity (11, 12). Also, in one study on saponins obtained from Hedera taurica, the antibacterial and antifungal action of saponins was investigated (13). This study revealed that saponins H1 and H2 had no antimicrobial effect, but saponin Sx1 possessed in vitro antifungal activity with respect to Candida albicans, C. krusei and C. tropicalis (13). Further more, the isolated alkaloid from Schizozygia coffaeoides has a potent antifungal effect in comparison to other components (14). Considering that this plant has saponins, flavonoids and alkaloids, these findings could be related to the effect of these components.Thymus vulgarlis and Populus candicans essential oils, which are not usually considered to be particularly antifungal, have been found in a study to be highly inhibitory at normal therapeutic concentrations (15). A similar finding was noted in our study, showing the effective antifungal effect of Echinophora platyloba against Candida albicans.Herbal medicines, as independent or complementary drugs, are helpful in the treatment of some diseases. For example the therapeutic effect in patients treated with a combination of Agastache lotion and western medicine was significantly better in curing skin tinea and genital candidiasis than when they were used alone (16). The essential oil of Agastache rugosa, combined with ketoconazole, may be particularly useful against Blastoschizomyces capitatus, a rare pathogenic fungus documented to cause severe and fatal mycoses in immunocompromised patients (17). There are some active component extracts, such as the Ginseng radix extract and the extract obtained from the Japanese herb, Juzen-taiho-to, which had been demonstrated to enhance the anti-Candida activity of macrophages in vitro and prolong the survival time of C. albicans-infected ones (18).On the basis of folklore application of this herb, which is used as a food seasoning and also to prevent fungal growth in tomato paste, as well as the results of this study, it is confirmed that (1) (2)(3)(4)(5)(6)(7)(8)(9) (10)(11)(12)(13)El Astal ZY, Ashour AERA and Kerrit AAM. Antimicrobial activity of some medicinal plant extracts in Palestine. Pak. J. Med. Sci. (2005) 21: 187-193Clark AM. Natural Products as a resource from new drugs. Pharm. Res. (1996) 13: 1133-44Cowan MM. Plant products as antimicrobial agent. Clin. Microbiol. Rev.  (1999) 12: 564-82Rechinger KH. Flora Iranica. 162 Akademische Druck- und Verlagsanstalt, Austria (1987) 72Sadrai H, Asghari G and Yaghobi K. Evaluation of ethanolic extract and essential oil of Echinophora platyloba on isolated ileum of rat. Pajouhesh dar Pezesheki (2002) 7: 150-155Nourozi M. Evaluation of Phytochemical and Antimicrobial Effect of Echinophora platyloba. Pharm. D. Thesis. Tehran University of Medical Sciences, Tehran (1989) 35-40Avijgan M, Saadat M and Nilforoushzadeh MA. Effect of extract of Echinophora Platyloba on some common dermatophyts. Proceeding of the Second Symposium of Medicinal Plants; 2005 Jan 26-27; Tehran, IranFong HH. Integration of herbal medicine in to modern medical practices issues and prospects. Braz. J. Med. Biol. Res. (2000) 33: 179-89Samsam Shariat H. Extraction of Components of Herbal Medicines. Mani Press, Isfahan (1992) 25Mozafarian V. Family of Umbilifera in Iran. Jungle and pastur, Tehran (1983) 259-260Stary F and Jirasek V. A Concise Guide in Color Herbs. Hamlry Press, Prague (1977) 15-25De Lucca AJ, Bland JM, Vigo CB, Cushion M, Selitrennikoff CP, Peter J, Walsh TJ. CAY-I, a fungicidal saponin from Capsicum sp. Fruit. Med. Mycol. (2002) 40: 131-7Mel’nichenko EG, Kirsanova MA, Grishkovets VI, this plant has an effective role in the inhabitation of fungal growth specially against Candida albicans.In conclusion, the studied plant could provide some activity against Candida albicans, however, it is not known that which component of the extract is responsible for this effect. Further studies using isolated constituents instead of the whole extract should be carried out. AcknowledgmentsWe thank the Research Council of Shahr-e-kord, Isfahan University of Medical Sciences, Skin Diseases and Leishmaniasis Research Center for their financial support. The authors would also like to thank Maria Gillies for her kind language revision of the final manuscript.ReferencesAntifungal Effect of Echinophora Platyloba’ s Extract ...Tyshkevich LV and Krivorutchenko Iul. Antimicrobial activity of saponins from Hedera taurica Carr. Mikrobiol. Z. (2003) 65: 8-12Kariba RM, Houghton PJ and Yenesew A. Antimicrobial activities of a new schizozygane indoline alkaloid from Schizozygia coffaeoides and the revised structure of isoschizogaline. J. Nat. Prod. (2002) 65: 566-9McFadden R. Comparison of the inhibitory effects of various plants extracts on the growth of Candida albicans in vitro. Eur. J. Herbal Med. (1995) 1: 26-31Na M, Li Ly and Yang YD. Anti-fungal test of composite agastache lotion on seven pathogenic fungi and its clinical application. Zhongguo Zhong Xi Yi Jie He Za Zhi (2003) 23: 414-6(14)(15) (16) This article is available online at http://www.ijpr-online.comShin S and Kang CA. Antifungal activity of the essential oil of Agastache rugosa Kuntze and its synergism with ketoconazole. Lett. Appl. Microbiol. (2003) 36: 111-5Akagawa G, Abe S, Tansho S, Uchida K and Yamaguchi H. Protection of C3H/HE J mice from development of Candida albicans infection by oral administration of Juzen-taiho-to and its component, Ginseng radix: possible roles of macrophages in the host defense mechanisms. Immunopharmacol. Immunotoxicol. (1996) 18: 73-89(17) (18)

Anti fungal effect of Echinophora platyloba extract on some common dermathophytes

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