{"id":1371,"date":"2015-03-25T07:40:41","date_gmt":"2015-03-25T07:40:41","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=1371"},"modified":"2020-04-25T10:59:49","modified_gmt":"2020-04-25T10:59:49","slug":"antifungal-activity-of-vitex-agnus-castus-in-vitro-study","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol3no1\/antifungal-activity-of-vitex-agnus-castus-in-vitro-study\/","title":{"rendered":"Antifungal Activity of Vitex Agnus Castus-In Vitro Study"},"content":{"rendered":"

Introduction <\/strong><\/p>\n

Pathogenic fungi, dermatophytes have the ability to invade keratinized tissues of animals, humans and cause a disease, dermatophytosis, which is the commonest human contagious fungal disease (Esquenazi et al 2004; Sidat et al 2006). The antimicrobial properties of certain Indian medicinal plants were reported based on traditional use (PerumalSamy et al 1998, 1999), and a few attempts were made on inhibitory activity against certain pathogenic fungi.\u00a0 Due to the increasing development of drug resistance in human pathogens as well as negative effects of certain antimicrobial agents, there is a need to search for new antifungal agent without toxicity and side effect.\u00a0 In this study we focused on the in vitro<\/em> screening of antifungal activity of Vitexagnuscastus<\/em> (agnuside) against dermatophytes and opportunistic pathogens.<\/p>\n

Material and Methods<\/strong><\/p>\n

Plant Material<\/strong><\/p>\n

Leaves of\u00a0Vitexagnuscastus<\/em> were collected from botanical garden, University of Madras, Maduravoil (Chennai, Tamil Nadu, India) during June-July 2007.\u00a0 A specimen was deposited at department herbarium, Sathyabama University.\u00a0 Collected plant material was air-dried under shade at room temperature, ground with an electric grinder into fine powder and stored in airtight containers.<\/p>\n

Table 1: Antifungal activity of isolated compounds (MIC, \u00b5g\/ml)<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Tested Fungi\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Agnuside\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Flu<\/strong><\/td>\n<\/tr>\n
Trichophytonmentagrophytes<\/em> 66\/01\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 –\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <12.5<\/td>\n<\/tr>\n
Epidermophytonfloccosum<\/em> 73\/01\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 31.25\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <12.5<\/td>\n<\/tr>\n
T. simii<\/em> 110\/02\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 62.5\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <12.5<\/td>\n<\/tr>\n
Curvularialunata<\/em> 46\/01\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 250\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <12.5<\/td>\n<\/tr>\n
Aspergillusniger<\/em> MTCC 1344\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 –\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <12.5<\/td>\n<\/tr>\n
Botrytis cinerea\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em>–\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 NT<\/td>\n<\/tr>\n
T. rubrum<\/em> MTCC 296\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 62.5\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <12.5<\/td>\n<\/tr>\n
Magnaporthegrisea<\/em>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 >500\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 NT<\/td>\n<\/tr>\n
C. alicans\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em>125\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <12.5<\/td>\n<\/tr>\n
Scopulariopsis<\/em> sp. 101\/01\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 500\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <12.5<\/td>\n<\/tr>\n
NT- Not test; Flu- Fluconazole (standard)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Extraction and isolation<\/strong><\/p>\n

Air-dried powdered of V. agnus<\/em> (1.5 kg) was extracted with ethyl acetate (2 x 2l) for 48 h at room temperature (\u00b1 25oC).\u00a0 The ethyl acetate crude extract was filtered and evaporated under reduced pressure. The total concentrated (160 gm) was chromatographed on a silica gel column (Merck 70\u2013230 mesh, 800 gm, 3.5 i.d.x60 cm) and successively eluted with stepwise gradient of hexane-ethyl acetate system (0%, 5%, 10%, 20%, 30%, 50%, 70% and 100%). Eleven fractions were collected and each fraction was spotted on a precoated Silica gel 60 F254, 0.25mm thick TLC plate (Merck) and eluted in hexane: ethyl acetate (4:6) and fractions with similar Rf values in TLC pattern were pooled together. Fraction-7 (7.3 g) showed signi\u00fbcant antibacterial (MIC) and DPPH free radical inhibition. For further separation bioactive substance was chromatographed on a silica gel column and eluted with a stepwise gradient of Isoproponal alcohol\u2013methanol (8:2) solvent system, and an active isolate of 3.5 g was obtained. This active isolate was subjected to spectral analysis. 1\u00a0H and 13\u00a0C NMR spectra were recorded with a JEOL 300MHz FT NMR spectrometer (H1) 75, MHz (13C) and chemical shifts were given in ppm. IR spectra were taken on a Perkin Elmer FT-IR spectrophotometer and mass spectra on a JEOL GC-MASS spectrometer.<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 1:\u00a0 TLC of Agnuside (Single spot)<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Phytochemical analysis<\/strong><\/p>\n

The presence of phytochemicals alkaloids (Draggendorff\u2019s), flavonoids (Shibat\u2019as reaction), saponins (Frothing test), tannins (5% ferric chloride), terpenoids (2,4-dinitro-phenyl hydrazine),\u00a0 glycosides (Fehling\u2019s solution), steroids (Liebermann\u2019s Burchard test) were evaluated according to the methods described by Edeoga et al. (2005).<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\n\n

Figure 2: Agnuside<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Fungal strains<\/strong><\/p>\n

The following fungi were used for experiments:Trichophytonrubrum<\/em> MTCC 296, T. mentagrophytes<\/em> 66\/01, T. simii<\/em> 110\/02, Epidermophytonfloccosum<\/em> 73\/01, Scopulariopsis<\/em> sp. 101\/01 Aspergillusniger<\/em> MTCC 1344, Botyritiscinerea,Curvularialunata<\/em>46\/01, Magnaporthegrisea<\/em> and Candida albicans<\/em> MTCC 227.<\/p>\n

Preparation of fungal spore<\/strong><\/p>\n

The filamentous fungi were grown on Sabouraud Dextrose Agar (SDA) slants at 28oC for 10 days and the spores were collected using sterile doubled distilled water and homogenized. Yeast wasgrown on Sabouraud Dextrose Broth (SDB) at 28oC for 48 h.<\/p>\n

Antifungal assays<\/strong><\/p>\n

The antifungal activity was performed according to the standard reference method (NCCLS, 2002). The extracts were dissolved in 2% dimethyl sulfoxide (DMSO). The initial concentration of extract was 1mg\/ml. The initial test concentration was serially diluted two-fold. Each well was inoculated with 5\u00b5l of suspension containing 104\u00a0spore\/ml of fungi. The antifungal agent Fluconazole was included in the assays as positive controls; the plates were incubated for 24h up to 9 days at 27\u00b0C for dermatophytes strains. MIC was defined as the lowest extract concentration, showing no visible fungal growth after incubation time.<\/p>\n

Results <\/strong><\/p>\n

Isolation of active compound<\/strong><\/p>\n

One active compound was obtained by using bio-assay guided fractionation (Fig. 1). Structural determination of this compound was done using different spectral technique and confirmed as agnuside (Fig. 2). It had IR absorptions at 3429 (br, hydroxyl), 1700 (Carbonyl), 1637 (olefinic region) and 1274 (C-O) Cm-1.\u00a0 1H NMR (CDCL3, 300 MHZ) spectrum showed the peaks at 6.88-7.88 ppm, which corresponds to the aromatic proton.\u00a0 The peak appears in the range in between 3.40 to 5.20 ppm assigned to carbon connected to oxygen atom. 13 C NMR spectrums showed the peaks at 115.85 to 162.52 ppm correspond to the aromatic carbon.\u00a0 The carbon connected oxygen atom appears in the range between 62.52 to 96.25 ppm.\u00a0 The carbonyl peaks are assigned to 165.23 ppm.\u00a0 The EI-MS (70 eV) of the compound showed molecular ion peaks at 464 (M) +.<\/p>\n

Phytochemical analysis<\/strong><\/p>\n

Phytochemical analysis revealed the presence of triterpenoids, flavonoids and glycosides<\/p>\n

Antifungal assay<\/strong><\/p>\n

Agnuside showed potent activity against tested fungi (Table 1) namely E. floccosum<\/em>(31.25 \u00b5g\/ml), T. simii<\/em>(62.5 \u00b5g\/ml), C. lunata<\/em>(250 \u00b5g\/ml), T. rubrum<\/em>(62.5\u00b5g\/ml), C. albicans<\/em>(125 \u00b5g\/ml) and Scopulariopsissp<\/em>(500 \u00b5g\/ml).<\/p>\n

Discussion<\/strong><\/p>\n

Screening of antimicrobial activity provided the required preliminary observation to select crude plant extracts with potentially useful properties for further chemical and pharmaceutical investigation.<\/p>\n

Conclusion<\/strong><\/p>\n

Results of the present work indicate that the plant species assayed possess antifungal properties.\u00a0 This explains the use of these plants in folk medicine for the treatment of various diseases whose symptoms might involve fungal infections, and underline the importance of the ethno botanical approach for the selection of plants in the discovery of new bioactive compounds.\u00a0 We found significant antifungal activity of agnuside of Vitexagnus-castus<\/em> (leaves).<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Edeoga, H.O., Okwu, D.E., and Baebie, M., Phytochemical constituents of some Nigerian medicinal plants. Afri.J. Biotechnol. <\/em>4:\u00a0685-688 (2005).<\/li>\n
  2. Esquenazi, D., Alviano, C.S., DeSouza, W., Rozental, S., The influence of surface carbohydrates during in vitro infection of mammalian cells by the dermatophyteTrichophytonrubrum. Research in Microbiology, <\/em>155: 144-153 (2004).<\/li>\n
  3. National Committee for Clinical Laboratory Standards., Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A, Wayne, Pa (2002).<\/li>\n
  4. PerumalSamy, R., Ignacimuthu, S., and Sen, A., Screening of 34 Indian medicinal plants for antibacterial properties, J. Ethnopharmacol., <\/em>62: 173-181 (1998).<\/li>\n
  5. Perumalsamy, R., Ignacimuthu, S., Patric Raja, D., Preliminary screening of ethnomedicinal plants from India. Journal of Ethnopharmacology<\/em>, 66: 235-240 (1999).<\/li>\n
  6. Sidat, M.M., Correia, D., Buene, T.P.,
    \nTineacapitis among rural school children of the district of Magude in Maputo province, Mozambique. Mycoses,<\/em>49: 480-483 (2006).<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

    Introduction Pathogenic fungi, dermatophytes have the ability to invade keratinized […]<\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[8],"tags":[],"class_list":["post-1371","post","type-post","status-publish","format-standard","hentry","category-vol3no1"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/1371","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=1371"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/1371\/revisions"}],"predecessor-version":[{"id":33091,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/1371\/revisions\/33091"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=1371"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=1371"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=1371"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}