Department of Pharmacology, Faculty of Pharmacy, University of Karachi.
*Faculty of Pharmacy, Hamdard University, Karachi.
The aim of present investigation was to evaluate antifungal activity of
ethanolic extract of Raphanus sativus L. var. caudatus. The antifungal
activity using agar disk diffusion method was determined against six
fungal strains; Aspergillus niger, Trichphyton rubrum, Microsporum
different concentrations (50, 100, 200 and 400μg/ml) of Raphanus
fungi and zone of growth inhibition were measured. Raphanus
fungi at all tested concentrations except Fusarium lini which was inhibited only at higher
concentrations (400 and 800μg/ml). It is noted that with increase in concentration, the zones
of growth inhibition were also increased. Thus Raphanus caudatus could be a lead for
development of antifungal agents in future.
KEYWORDS: Raphanus caudatus, ethanolic extract, antifungal activity, Miconazole.
Medicinal plants are used for various diseases around the globe. In fact, almost all the
cultures of world depend upon herbs for primary health care because they are economical,
easily available with fewer side effects (Mukhergi et al. 2007). It was evaluated that about
25% of various type of herbal constituents have been extracted from plants (Mukhtar et al.
Raphanus sativus L. Var. caudatus belongs to the family Brassicaceae, is a plant of
the radish genus Raphanus. These are basically Radish pods purple or green in color, are
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 5.210
Volume 4, Issue 11, 429-437 Research Article ISSN 2278 – 4357
Article Received on
16 Sep 2015,
Revised on 5 Oct 2015,
Faculty of Pharmacy,
consumed for properties attributed to Raphanus. These are known as Mungraa or Sungraa in
Pakistan & India (Khare, 2007).
Radish pods are rich in ascorbic acid, folic acid and potassium. They are a good source of
suggested to be the functional food since it was reported to have several kinds of minerals,
vitamins and some active pharmaceutical metabolites such as sulforaphene and sulforaphane
(Songsak and Lockwood, 2002, Pokasap, et al. 2013). Both isothiocyanate compounds have
proven role against prostate, breast, colon and ovarian cancers by virtue of its cancer-cell
growth inhibition and cytotoxic effects on cancer cells (Holst and Williamson, 2004). RC
contained phenolic compounds which showed antioxidant activity (Charoonratana, et al.
2014). Radish is found to be antimicrobial (Abdou, et al. 1972), anti-fungal (Terres et al.
1992), antiurolithiatic (Vargas, et al. 1999), anti-inflammatory (Moon and Kim, 2012) and
antioxidant (Takaya, et al. 2003). The leaf, seed and root of Raphanus sativus are claimed to
have various medicinal uses (Gutiérrezand and Perez, 2004). Fatty acids are the major
nutritional composition of interests in seed. Other nutritional components include minerals,
vitamin, proteins and polysaccharides (Sham, et al. 2013).
Among plants, Brassicaceae have been reported having significant antifungal
properties, its seeds are also reported for antifungal activities with different antifungal
components (Duke and Ayensu, 1985, Bown 1995). In contrast RC, one of the varieties of
radish, has not been reported against fungal infections so far. The present study is designed to
explore antifungal potential of RC.
MATERIAL AND METHODS
Fresh and healthy pods of Raphanus sativus L. were collected from District Karachi
(Pakistan) and were identified from herbarium, University of Karachi, Pakistan. The plant
pods were washed thoroughly with double distilled water to avoid contamination.
The plant pods were dried in air under shade at room temperature and stored in properly
The dried pods were grinded with the help of mechanical grinder. Extraction of plant pods
were carried out with ethanol using soxhlet apparatus (Davey et al. 2010). 100 grams of
powdered pods of plant were placed in thimble of soxhlet apparatus and extracted by 500 ml
of ethanol. All extracts were filtered using autoclaved Whatmann’s filter paper and
centrifuged at 2400 × g for 15 minutes (Wittschier et al. 2009). All the extracts were dried in
rotary evaporator at 45° C until semi- solid extract obtained. Percentage yield of extract was
calculated. The extract was stored at 4
C in the refrigerator until further use. Four different
IN-VITRO ANTIFUNGAL ACTIVITY
Following six different fungi were selected for antifungal evaluation:
Aspergillus niger (ATCC 1015), Trichphyton rubrum (ATCC MYA 4438), Microsporum
canis (ATCC 10214), Fusarium lini (NRRL 2204), Candida glabrata (ATCC 90030) and
Candida albicans (ATCC 36082). The yeasts and molds were grown in Sabouraud dextrose
agar and PDA media, respectively, at 28°C. The stock cultures were maintained at 4°C.
In vitro antifungal activity of ethanolic extract of Raphanus caudatus against six pathogenic
fungi were evaluated by the agar disk diffusion test (Bauer et al 1966; Rios et al 1988). The
extract was dissolved in DMSO (dimethyl sulfoxide), sterilized by filtration using sintered
glass filter and stored at 4°C. For the determination of zone of inhibition, fungal strains were
taken as a standard antibiotic for comparison of the results. Four concentrations (50, 100, 200
and 400μg/ml) of Raphanus caudatus extract and standard drug (Miconazole) were prepared
in double-distilled water using nutrient agar tubes. The zones of inhibition of fungal growth
were determined by measuring sizes of inhibitory zones (including the diameter of disk) after
7 days at 28°C. All experiments were carried out in triplicate, the solvent ethanol was also
evaluated alone for its inhibitory effect and it has shown no inhibitory effect at tested
In the present study, four different concentrations of RC ethanolic extract against six fungal
strains were evaluated. The results were measured as zone of growth inhibition. The extract
has shown significant antifungal activity against all fungi at all tested concentrations except
extract. It is noted that with increase in concentration, the zones of growth inhibition were
also increased. Antifungal effects of standard drug Miconazole and RC extract against
different fungal strains are presented in table 1 and 2 respectively. The zones of inhibition for
different fungal strains were found in the range of 17 -28 mm and 14 – 20mm for Miconazole
and RC ethanolic extract respectively (Figure 1-2).
Table 1: Antifungal activity of Miconazole (Standard drug) against different fungal
Zone of inhibitions (mm)
Values are Mean + SD of three experiments.
Values are Mean + SD of three experiments; - indicates no zone of inhibition.
Statistical differences of results for antifungal assay were carried out by ANOVA (Analysis
of variance) test. Results with p value < 0.05 were taken as significant. All data manipulation
and statistical analysis were carried out by using Statistical Package for Social Sciences
(SPSS for Windows version 20, SPSS inc., Chicago, IL, USA).
Fungal infections are very common in individuals with compromised immune systems and
especially in the diabetic patients.
Additionally very old and very young individuals are also
Plants have enriched profile of antimicrobial constituents for example flavonoids, alkaloids,
polysaccharides, coumarins, glycosides, lignans, saponins, polyines, thiophenes, proteins and
polyphenolics (Jassim and Naji, 2003). It has been estimated that approximately 250000 to
500000 species of plants exist but few of them have been evaluated for their antifungal
potential. The plants could be a lead for the development of antifungal compounds as they
have already been inhibiting phytopathogenic fungi to which they are exposed in their
environment (De Lucca et al 2005).
Due to its high yield and excellent nutritional value Radish has been grown all over the world
potential. Earlier, the ethanolic extract of root juice of radish exhibited antifungal activity
against Candida albicans (Caceres, 1987). In another study, Takasugi et al 1987 isolated
spirobrassinin, one of the phytoalexin from Raphanus sativus L. var. hortensin which showed
substantial antifungal potential. Two peptides rich in cystein designated as RsAFP1 and
RsAFP2 were isolated from radish showed notable antifungal activity against different fungi
(Terras et al 1992). Moreover antifungal activity of these peptides have been suggested via
receptor mediated mechanism (Thevissen et al 1996).
Figure1: Antifungal activity of Miconazole against different fungal strains.
Figure2: Antifungal activity of RC ethanolic extract against different fungal strains.
Shin and Hwang, 2001 isolated two antifungal substances named as RAP1 (Raphanus
Their structure and molecular masses were determined using different chromatographic and
non-chromatographic techniques. Both substances were effective fungicidal agents especially
against Candida albicans and Saccharomyces cerevisiae. The antifungal activity of RC in the
present investigation could be related to the presence of peptides.
Lee et al 2013 showed that ethyl acetate extract of Korean radish leaves contain significant
activities against different gram positive bacteria’s.
It has been reported that isothiocyanate compounds in Raphanus and other members of
presence of sulphoraphane, sulphoraphene and different other isothiocyanates have been also
reported in the pods of Raphanus (Songsak and Lockwood 2002). In addition Ferulic acid and
caffeic acid in Raphanus also exhibited antifungal activities. Thus the occurence of these
compounds in RC could be linked to its antifungal activity in the present study.
Radish and its seeds are reported to have 2S albumin showed antifungal activity by enhancing
In the present investigation,
has shown remarkable invitro
antifungal potential. In vitro study gives role model for screening of drugs and so helps in
further evaluations of activities of drugs. Further research including in vivo studies is
required to elucidate the antifungal phytochemicals of the plant with their target of action.
Fitoterapia, 2002; 73: 209-216.
P. Pocasap, N. Weerapreeyakul, and S. Barusrux. Cancer preventive effect of Thai rat-
B. Holstand and G. Williamson. A critical review of the bioavailability of glucosinolates
and total phenolic contents in raphanus sativus pod International Journal of Pharmacy and
Pharmaceutical Sciences, 2014; 6.
of Allium sativum, Allium cepa, Raphanus sativus, Capsicum frutescens, Eruca sativa,
Allium kurrat on bacteria. Qualitas Plantarum et Materiae Vegetabiles, 1972; 22: 29-35.
Broekaert. In vitro antifungal activity of a radish (Raphanus sativus L.) seed protein
homologous to nonspecific lipid transfer proteins. Plant physiology, 1992; 100:
S. R. Vargas, G. S. Perez, G. Perez, S.M. Zavala, and G.C. Perez. Antiurolithiatic activity
P. D. Moon and H. M. Kim. Anti-inflammatory effect of phenethyl isothiocyanate, an
Y. Takaya, Y. Kondo, T. Furukawa, and M. Niwa. Antioxidant constituents of radish
sprout (Kaiware-daikon), Raphanus sativus L. Journal of agricultural and food chemistry,
2003; 51: 8061-8066.
R.M.P. Gutiérrezand R.L. Perez. Raphanus sativus (Radish): their chemistry and biology.
The Scientific World Journal, 2004; 4: 811-837.
T.T. Sham, A.C.Y. Yuen, Y.F. Ng, C.O. Chan, D.K.W. Mok and S.W. Chan. A review of
the phytochemistry and pharmacological activities of Raphani semen. Evidence-Based
Complementary and Alternative Medicine, 2013.
Mukherjee PK, Kumar
V, Houghton PJ. Phytotherapy Research. Screening of Indian
medicinal plants for acetylcholinesterase inhibitory activity, 2007; 21(12): 1142–1145.
Mukhtar M, Arshad M, Ahmad M, Pomerantz RJ, Wigdahl B, Parveen Z. Antiviral
potentials of medicinal plants. Virus Res, 2008; 131(2): 111-120.
Davey MR, Anthony P. 2010. Plant Cell Culture: Essential Methods. 1
Wittschier N, Faller G, Hansel A. Aqueous extracts and polysachharides from liquorice
roots (Glycchriza glabra L.) inhibit adhesion of helicobacter pylori to human gastric
mucosa. J. Ethnopharmacol, 2009; 125: 218–228.
Jassim SAA, Naji MA. Novel antiviral agents: a medicinal plant perspective. Journal of
Takasugi, M., Monde, K., Katsui, N., & Shirata, A. Spirobrassinin, a novel sulfur-
containing phytoalexin from the daikon Rhaphanus sativus L. var. hortensis
(Cruciferae). Chemistry letters, 1987; 8: 1631-1632.
Cáceres, A., Girón, L. M., Alvarado, S. R., & Torres, M. F. Screening of antimicrobial
diseases. Journal of Ethnopharmacology, 1987; 20(3): 223-237.
Thevissen, K., Ghazi, A., De Samblanx, G. W., Brownlee, C., Osborn, R. W., &
thionins. Journal of Biological Chemistry, 1996; 271(25): 15018-15025.
SHIN, H. K., & HWANG, C. W. New antimicrobial activity from Korean radish seeds
De Lucca, A. D., Cleveland, T. E., & Wedge, D. E. Plant-derived antifungal proteins and
peptides. Canadian journal of microbiology, 2005; 51(12): 1001-1014.
Bown, D. 1995. Encuclopaedia of herbs and their uses. Dorling Kindersley, London.
Duke, J. A., & Ayensu, E. S. (1985). Medicinal plants of China (Vol. 2). Reference
Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by
standardized single disc method. Am J Clin Pathol, 1966; 36: 493–6.
Rios JL Recio MC, Villar A. Screening methods for natural products with antimicrobial