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Evaluation of inhibitory effect of Vernonia cinerea L. leaf extracts on different

fungal species

Article in International Journal of Pharmacy and Pharmaceutical Sciences · March 2013

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 Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 5, Issue 2, 2013

Research Article

EVALUATION OF INHIBITORY EFFECT OF VERNONIA CINEREA L. LEAF EXTRACTS ON

DIFFERENT FUNGAL SPECIES

P. DHANALAKSHMI1, A. JAYA PRAKASH PRIYA1, E. SAGADEVAN2, YAMINI SUDHA LAKSHMI3, A. MANIMARAN4,

S. SINDHU2 AND P. ARUMUGAM2
1Biocontrol and Microbial Metabolites Lab, CAS in Botany, University of Madras, Maraimalai Campus, Chennai 600025, 2Bioproducts
Division, Armats Biotek Private Limited, Guindy, Chennai 600032, 3Dept. of Biochemistry, Professor Dhanapalan College of Arts and
Science, Kelambakkam, Chennai 603103, 4Aarupadai Veedu Institute of Technology, Paiyanoor, Chennai 603104.
Email: [email protected]
Received: 30 Jan 2013, Revised and Accepted: 25 Mar 2013
ABSTRACT
Objective: Fungal infection is a very serious cosmetic problem which is of utmost concern globally, one such infection being Dandruff. Finding anti-
dandruff molecules from natural sources is the current research focus among cosmetic researchers. Medicinal plants may offer a valuable source for
antidandruff molecules. Hence, in the present study, Vernonia cinerea L. was studied for its antidandruff activities.
Methods: Initially, V. cinerea plant leaves were extracted with different solvents and studied for their antifungal activity against Candida albicans,
Candida parapsilosis and Candida tropicalis. Furthermore, the antidandruff activity of ethyl acetate extract was studied and compared with a
commercial antidandruff shampoo. In addition, the ethyl acetate extract was subjected to preliminary phytochemical screening and thin layer
chromatography to elucidate the bioactive principles present in the extract.
Results: From the data obtained, it was found that the ethyl acetate extract of V. cinerea leaves has shown very good antifungal activity at all
concentrations tested. Also, the ethyl acetate extract exhibited excellent antidandruff activity against Pityrosporum ovale and Pityrosporum
folliculitis. Ethyl acetate extract of V. cinerea has exhibited increased synergistic antidandruff activity with commercial product. Comparatively, the
V. cinerea extracts alone or in combination with commercial product exhibited more antidandruff activity against P. folliculitis. Qualitative
phytochemical tests, thin layer chromatography of ethyl acetate extracts demonstrated the presence of common alkaloids, carbohydrates and
glycosides, glycosides, saponins, proteins, amino acids. Moreover, phenols were found to be the major active constituent in V. cinerea which
constitutes as a good source of antidandruff agents.
Conclusion: The results of the study suggest that the leaves of Vernonia cinerea can be further exploited for its therapeutic use against several fungal
infections.
Keywords: Vernonia cinerea, Antifungal activity, Antidandruff activity, Phytochemicals.

INTRODUCTION bioactive compounds are alkaloids, flavonoids, tannins and phenolic

compounds [10]. In the present study, an attempt was made to
Dandruff is a common scalp problem that occur when dead skin is investigate the potential antidandruff activity of Vernonia cinerea
shed, producing irritating white flakes and possibly an itchy scalp. (L.) as an alternative strategy.
Hair fall is very common in dandruff sufferers [1, 2]. Generally,
dandruff is thought to represent the mildest form of seborrheic Vernonia cinerea (Family: Asteraceae) is a terrestrial annual erect
dermatitis of the scalp. Its pathogenesis remains to be entirely herb, widely distributed in India, Bangladesh, Sri Lanka and Malay
elucidated, although the yeast organism Malassezia has been island [11]. The plant is extensively used in indigenous medicine as
proposed as an etiological factor [3]. The genus Malassezia belongs stomachic and for cold, asthma and bronchitis [12]. The leaf juice
to the basidiomycetous yeasts and is classified in the Malasseziales extract is used to treat skin diseases and treating dysentery in
(Ustilaginomyces, Basidiomycota) [4]. Among the different children [13], where as the roots of the plant are used for the
Malassezia species, M. globosa and M. restricta have been most treatment of eruptive boils, quicker healing of accidental wounds,
closely associated with dandruff in humans [5]. Malassezia may filariasis and toxic viral fevers. However the antidandruff activity of
stimulate cytokine production by keratinocytes (epidermal cells that the same plant has not been explored yet. Hence, to promote the use
synthesize keratin), further contributing to the inflammatory of medicinal plants as potential sources of antimicrobial compounds
component of seborrheic dermatitis and dandruff [6]. It is also it is necessary to thoroughly investigate their composition and
stated that, Malassezia sp. may produce tryptophan metabolites activity [14]. Therefore, the present study was taken up to assess the
which are active on the aryl hydrocarbon receptor, thus produce crude leaf extracts of V. cinerea for its antidandruff action against
inflammation [7]. Malassezia sp.

At present, many chemical substances are used for treating dandruff MATERIALS AND METHODS
by controlling the abundance of yeasts on the scalp. The main active Plant material
agents used currently for controlling dandruff include imidazole
derivatives such as ketoconazole and other compounds. They act by The fresh plant of Vernonia cinerea (Linn.) was collected from fields
removing the scales, reduce Malassezia species adherence to located in Bharathiar University Campus at Coimbatore, India. The
corneocytes and restrain its growth. Though a wide variety of leaves were separated from the steam, washed with tap water,
antifungal agents are available for the treatment of dandruff, a rinsed with distilled water, and air-dried for 1h. Then the leaves
complete cure is far from reach. Further, most of the available drugs were shade dried in room temperature for one week. They were
are either fungistatic or expensive in nature and phytochemicals are ground into powder and subjected to extraction with different
less potent [8]. As fungal resistance to synthetic antibiotics is in rise solvents.
[9] an alternative in the way of herbal medicine in controlling the Plant extracts preparation
same is the need of the hour. The medicinal value of plants can be
attributed to some chemical substances which produce a definite The ground leaves were extracted with different solvents such as,
physiological action on the human body. The most important of such chloroform, ethyl acetate and methanol following the method of
 Dhanalakshmi et al.
Int J Pharm Pharm Sci, Vol 5, Issue 2, 414-416

Eloff [15]. The extraction of the leaf powder was done with solvents Thin Layer Chromatography
in the ratio of 1:10 under shaking condition. The extracts were
collected in different conical flasks and the same was repeated thrice The ethyl acetate extract of V. cinerea was loaded on pre-coated
to attain maximum extraction. Then the solvents were condensed to silica plates which were developed using the solvents ethyl acetate
concentrate the extracts obtained. The concentrated extracts were and hexane (1:9). The spots were identified both in the UV light, far
weighed and re-dissolved in respective solvents to yield 10mg/mL light and in the iodine chamber. Then Rf value was calculated as the
solutions for further analysis. ratio of distance traveled by the solute to the distance traveled by
the solvent.
Antifungal activity
Phytochemical screening of V. cinerea crude extract
Well diffusion assay [16]
Qualitative phytochemical tests for the identification of alkaloids,
Potato Dextrose agar was prepared, sterilized and poured in the Petri phenols, flavonoids, terpenoids, steroids and saponins were carried
dishes (9 cm). 24 h growing culture of Candida albicans (MTCC 183), out for all the extracts (freshly prepared) by the methods described
Candida parapsilosis (MTCC 2509) and Candida tropicalis (MTCC 184) previously [11, 18].
were swabbed on the solidified plates and wells (10mm dia.) were made
onto them using a sterilizes cork borer. The different concentrations RESULTS AND DISCUSSION
(250µg, 500µg, 750µg and 1000µg) of the crude extracts were placed in Dandruff is a universal disorder affecting the scalp and can be a
the wells in triplicates. Dimethyl sulphoxide was used as control in the discomforting situation. Presently existing treatment options have
present study. The plates were then incubated at 37 ºC for 24 h, and the certain confines, either due to reduced efficacies or due to
zone of inhibition was measured, if any. acquiescence issues. Furthermore, these drugs are incapable to
Anti-Dandruff Activity avoid recurrence, which is familiar bothersome clinical problem. The
antifungal activity of V. cinerea leaf extracts showed positive results
The Antidandruff activity of the plant extract was carried-out by the against all the tested fungal pathogens, C. albicans, C. parapsilosis
method of Kumar et al., [17] with small modifications. Broth cultures and C. tropicalis (Table 1). Methanol extracts failed to inhibit few
of the organisms (Pityrosporum ovale, Pityrosporum folliculitis) were fungal pathogens at the lower concentrations tested. Chloroform
swabbed over the surface of Potato Dextrose Agar. Wells of 8 mm extracts at all concentrations have exhibited moderate antifungal
diameter were cut at the surface of the agar and 100 μl of the above activity against all the tested pathogens. Among different solvents
prepared samples (10mg/ml) were loaded on the wells respectively. used, the ethyl acetate extract had shown maximum inhibition as
Plates were incubated at 30 ⁰C for 2-3 days. The zone of inhibition compared to methanol and hexane extract and chosen for further
was measured and recorded. analysis.

Table 1: Antifungal activity of different solvent extracts of V. cinerea leaves

S. No. Concentration (µg/ml) Zone of inhibition (mm)
Methanol extract chloroform extract Ethyl acetate extract
C. paropsilosis

C. paropsilosis

C. paropsilosis
C. tropicalis

C. tropicalis

C. tropicalis
C. albicans
C. albicans

C. albicans

1 250 - - - - - 10 11 12 -
2 500 - 11 11 11 11 10 12 13 13
3 750 13 15 11 12 12 12 15 16 15
4 1000 14 18 12 12 12 12 17 20 18
The ‘-’ sign denotes no visible zone of inhibition was obtained

The ethyl acetate extract of V. cinerea recorded MIC value of potential as shown in Table 2. This part of study seems to have
250µg/ml (with a zone of inhibition of 11mm) exhibited very good importance in finding effective antidandruff activity from a
antifungal activity against the tested fungal cultures (Table 2). In commonly available plant V. cinerea.
general, increasing concentrations of all the extracts including ethyl
acetate extracts exhibited increased trend of antifungal activity. Table 2: Antidandruff activity of ethyl acetate extract of V.
Previous reports also suggested the effectiveness of ethyl acetate cinerea leaves
extracts against a wide range of bacteria and fungi [19, 20, 21].
S. Sample Zone of Inhibition (mm)
Medicinal plant extracts are the promising sources of antifungal No. P. ovale P. foliculitis
drugs, even though they have relatively mild effect against human 1 Plant extract 19 20
pathogenic fungi when compared with commercial synthetic 2 Commercial product 21 24
antifungal drugs [22, 23]. An array of synthetic antifungal agents is 3 Plant extract + Commercial 22 25
available for the treatment of dandruff, though a complete cure is product
still far from reach. The fungi responsible for dandruff often
develops resistance to chemical agents and synthesis of such Plants are found to be rich sources of valuable of bioactive
chemicals remains expensive and posing threat to environment due metabolites such as tannins, saponins and phenolics which make
to effluents and byproducts. Herbal extracts with antifungal activity them to have marvelous medicinal properties. Phytochemical
against many fungal pathogens may offer a very good source for screening is an important approach in profiling the active
antidandruff compounds. Hence, an attempt was made to find out ingredients of either whole plant or their solvent extracts of
the synergistic antidandruff activities of the ethyl acetate extract of different parts. Phytochemical screening is an indispensable element
V. cinerea with commercial products. For this, Pityrosporum ovale in herbal technology. In the present study, various qualitative tests
and Pityrosporum folliculitis were chosen as tested organisms. The were performed with ethyl acetate extracts of V. cinerea. The
synergistic study was performed by making the mixtures of V. chromatogram developed with 15 % ethyl acetate in hexane
cinerea extract and commercial product (shampoo) in equal revealed the presence of ten major compounds at R f value of 0.22;
proportions. Results indicated that their effects (commercial 0.34; 0.48; 0.57; 0.68; 0.74; 0.80; 0.88 and 0.97 as visualized under
product and V. cinerea extract) were similar in its inhibitory iodine vapor and UV illumination. The broad-spectrum antimicrobial

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 Dhanalakshmi et al.
Int J Pharm Pharm Sci, Vol 5, Issue 2, 414-416

activities of the plant extract may be, possibly, due to the presence of indolo [3,2-b] carbazole are selectively produced by Malassezia
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shown in table 3. Review of the literature on the phytochemical Dermatol, 2008, 128,1620-1625.
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