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In vitro and in vivo evaluation of anti-dandruff activity of formulated polyherbal

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 P.Suresh Kumar et al. / Journal of Pharmacy Research 2010, 3(12),2956-2958
Research Article
ISSN: 0974-6943 Available online through
www.jpronline.info
In vitro and in vivo evaluation of anti-dandruff activity of formulated polyherbal hair oil
P.Suresh Kumar 1*, S.Sucheta 1, A.Umamaheswari 2, V.Sudarshana Deepa 1
1
Department of Biotechnology, Anna University of Technology, Tiruchirappalli -620024, Tamilnadu, India
2
Department of Pharmaceutical Technology, Anna University of Technology, Tiruchirappalli -620024, Tamilnadu, India
Received on: 15-08-2010; Revised on: 18-09-2010; Accepted on:13-11-2010
ABSTRACT
Dandruff is a major cosmetic problem, caused by lipophilic, dimorphic and yeast like fungus Pityrosporum ovale (P. ovale) and Candida albicans (C. albicans). In
this present study, polyherbal hair oil (PHO) was prepared by using six methanolic crude extracts of herbs namely - Albizia amara , Achyranthes aspera, Cassia
fistula, Cassia auriculata, Datura stramoniuum and Azadirachta indica to evaluated for the antidandruff activity. The Minimum Inhibitory Concentration (MIC)
of PHO against Pityrosporum ovale and Candida albicans was found to be 1.0 mg/ml and 5.0 mg/ml respectively. The zone of inhibition of the PHO was observed
as 15 mm diameter for Pityrosporum ovale and 13 mm for Candida albicans. Preclinical trials were performed with human volunteers. There was a clear
symptomatic relief from dandruff, observed after 10 days usage of PHO in the volunteers. Methylene blue reductase test also confirmed the significant antidandruff
efficacy of the PHO. From the above studies, it was concluded that the formulated PHO is effective and safe in the management of dandruff.

Key words: Dandruff, Pityrosporum ovale, Candida albicans, Methylene blue reductase test, Polyherbal hair oil.

INTRODUCTION
Dandruff is a problem that possesses serious concern in both developed and
developing countries. Dandruff manifests as profuse white to silvery powdery
scales in the scalp region often with moderate to severe itching. Hair falling is
also very common in most of the dandruff sufferers[1]. Pityrosporum ovale is a
yeast-like lipophilic basiodiomyceteous fungus considered to be the causative
agent for dandruff. The present work was undertaken to study the effect of
antidandruff activity in PHO that was prepared by using the extracts of six
medicinal plants namely Albizia amara (Mimosaceae), Achyranthes aspera
(Amaranthaceae), Cassia fistula (Caesalpiniaceae), Cassia auriculata
(Caesalpiniaceae), Datura stramoniuum (Solonaceae) and Azadirachta indica
(Meliaceae). Candida sp is also a suspected causative agent for dandruff [2].
These organisms are widely considered to be the commensal flora of the scalp
and skin region [3]. The reason for, why this commensal organisms turns to be
pathogens is not clearly understood [4]. It is believed that, P.ovale converts the
sebum lipid into fatty acids and triglycerides. These fatty acids may presumably
accelerate hyper proliferation of keratinocytes [5] PHO is reported and known
to have activity against P.ovale in in-vitro studies [6, 7]. Preclinical trials were
conducted in human volunteers. Upon using the PHO continuously for 8 days,
symptomatic relief in severity, reduced level of scaling from ‘severe to mild’ in
all the 10 volunteers was observed. Complete elimination ‘traces to nil’ scaling
was also observed in all the volunteers after 10 days use of the formulated PHO.
MATERIALS AND METHODS
Plant materials
Six medicinal plants were selected for antidandruff studies namely Albizia amara
(Mimosaceae), Achyranthes aspera (Amaranthaceae) , Cassia fistula
(Caesalpiniaceae), Cassia auriculata (Caesalpiniaceae), Datura stramoniuum
(Solonaceae) and Azadirachta indica (Meliaceae).The plant specimen were
Collected from medicinal garden of Anna University of Technology and authen-
ticated and identified by Botanical Survey of India, Coimbatore.
Culture
Clinical isolates of Pityrosporum ovale (No.1374 and No.637) and Candida
albicans was procured from Institute of Microbial Technology, Chandigarh,
India, for the in-vitro study. All the isolates obtained were maintained in Sabouraud’s
agar medium at 37 °C until use .
Phytochemical screening
The six plant extract were the presence of subjected to different tests for
*Corresponding author.
P.Suresh Kumar
Department of Biotechnology
Anna University of Technology
Tiruchirappalli - 620024, Tamil Nadu, India
Tel.: + 91-9786075353
E-mail:sureshbiotech2003@yahoo.co.in
detecting various phytochemical constituents by using various organic solvents.
The total alkaloid content was determined by performing Drangendroff’s test
and similarly, the total terpenoid and coumarin content was performed by
Wagner’s test [8].
Preparation of Polyherbal Hair Oil
Methanolic leaf extract of Albizia amara, Datura stramonium, Achyranthes
aspera, Cassia fistula, Cassia auriculata and aqueous extract of Azadirachta
indica was prepared. 1g of each extract was weighed and mixed together with 50
ml of pure coconut oil and was boiled for 3 hours with intermittent shaking.
Thereafter, the oil was cooled, filtered and tested for its potency as a novel anti-
dandruff agent.
Determination of Minimum Inhibitory Concentration (MIC)
MIC was determined by incorporating different concentrations of PHO (0.5 mg/
ml to 10 mg/ml) in 10 ml Sabouraud’s medium [9]. The medium was prepared and
mixed with oil and then emulsified thoroughly with Tween 20 and allowed to
solidify at room temperature [10]. Then the organism at 10 3 density was inocu-
lated in each plate that was formerly incorporated with different concentrations
of PHO. Further, it was incubated at 37 °C for 48 hours. The medium without
PHO was used as control. The lowest concentration of the PHO that inhibited
the growth of test organisms was compared with that of control
Zone of Inhibition Studies
Diffusion dependent antimicrobial activity of the PHO was studied by the zone
of inhibition method. The organism was uniformly inoculated on the surface of
the Sabouraud’s medium. A well was made in the center of the medium and the
known concentration of the oil and Tween 20 was loaded in the well. The plate
was incubated at 37 °C for 2 days. The zone of inhibition was measured.
Methylene blue reductase test
This test was performed to confirm the antimicrobial effect of PHO on P.ovale
and C.albicans at sub-MIC level. The PHO was incorporated into 10 ml of the
nutrient medium at its sub-MIC level. The organisms were scooped from the
surface of the medium, stained with Methylene blue stain and examined under
microscope. The percentage of dead cells was calculated [10].
Clinical studies of polyherbal oil for antidandruff activity
Ten volunteers between the age group of 18-25 years studying in our university,
having severe dandruff were included in this study. PHO (10-15 ml/day) was used
every day instead of commercial hair oil. During PHO usage, all the volunteers
were requested to abstain from the use of other antidandruff shampoos/hair
cream or any other antifungal medicament. They were examined every alter-
nate day up to 21 days.
Scalp scrapings were collected from the volunteers every alternate day, prior and
after oil application. The scrapings were divided into two parts, one used for

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 P.Suresh Kumar et al. / Journal of Pharmacy Research 2010, 3(12),2956-2958
Table 1.Phytochemical analysis of selected medicinal plants
S.No Plants Phytochemical Analysis
Alkaloids Terpenoids Coumarin Saponins

1. A. amara 6.12 + 0.18 % - - -

2. A. indica 7.64 + 0.24 % - - significant
3. D. stramoniuum 6.84 + 0.16 % - - -
4. A. aspera - - 5.26 + 0.12 % -
5. C. fistula - significant - -
6. C. auriculata - significant - -
The experiments were conducted thrice and each value was expressed in mean
±standard deviation
Fig. 4. Microscopic examination of dead cells of P.ovale after PHO treat-
Table 2.Effect of PHO on P.ovale burden in the scalp ment
Pityrosporum ovale
No of volunteers Isolation of
Pre use Day 2 Day 4 Day 6 Day 8 Day 10

10 TNTC 2000 500 124 12 No colonies

TNTC:Too numerous to be counted

Table 3.Culture study of P.ovale in the scalp of volunteers after PHO

use
Number of Isolation of P.ovale
Volunteers Pre use Day2 Day4 Day6 Day8 Day 10

10 100+ 80 20 No colonies No colonies No colonies

Fig. 5. Dead cells of P.ovale stained using Methylene Blue, after treat-
ment with the PHO

direct microscopical examination and the other for culture evaluation. The
scarped material was then digested with few drops of 10 % potassium hydroxide
and observed under microscope. The total spore burden on the scalp before and
after the use of PHO was assessed. The scarped materials was inoculated in
Sabouraud’s dextrose agar medium and incubated at 37º C for 3 days. The sever-
ity of the scaling was determined before and after PHO use and the reduction of
scaling subsequent to ‘severe, moderate, mild and traces to nil’ was also assessed.

RESULTS AND DISCUSSION

The results of this study showed that PHO inhibited the growth of P.ovale at 1.0
mg/ml concentration (Fig.1). The MIC of C.albicans was 5.0 mg/ml which is
highly effective when compared to commercially available formulated hair oil
Dano [7].The zone of inhibition of PHO was 15 mm diameter for P.ovale and 8
Fig.1 P.ovale cells isolated from the volunteer before the treatment. mm for C.albicans (Data not shown). In the case of control, the death of
Control P.ovale cells could not be seen (Fig.2).The crude extracts of Albizia amara,
Datura stramonium and Azadirachta indica showed distinct orange bands which
confirm the presence of alkaloids [8]. The total alkaloid content was found to be
6.12 + 0.18 % in Albizia amara , 6.84 + 0.16 % in Datura stramonium and 7.64
+ 0.24% in Azadirachta indica. The crude extracts of Achyranthes aspera
contain coumarin (5.26 + 0.12 %) whereas Cassia fistula and Cassia auriculata
showed the presence of terpenoids (Table.1). The phytochemically analyzed
compounds of the six medicinal plants extract were subjected to antidandruff
studies by performing MIC and zone of inhibition studies. In the clinical studies,
P.ovule isolated from the human volunteer served as a control (Fig.3). Out of 10
volunteers treated, 3 had severe scaling, while 7 had moderate scaling before
PHO use. After 8 days of treatment with PHO, there was reduction in the
scaling from “severe to mild” and “traces to nil” in all the 10 volunteers
(Table.2). Complete elimination of scaling was observed in all the volunteers
Fig.2 Inhibition of P.ovale at 1.0 mg of the PHO. after 10 days application of PHO (Fig .4 & Fig.5). After 6 days usage of PHO,
the isolation of P.ovale was not possible in all the volunteers (Table.3), whereas
in Dano, this was possible after 8 days only [9] . PHO is found to be very effective
against P.ovale in vitro. There was clear symptomatic relief from dandruff in all
the volunteers after 10 days of use. Further, the isolation of P.ovale, the caus-
ative organism of the dandruff was not found in culture after the use of PHO . In
the methylene blue reductase test 90% of the P.ovale cells appeared blue in
colour taking up the stain which indicates that the cells were dead. (Fig.5).

CONCLUSION
The antifungal activity coupled with keratinocyte proliferation inhibition ac-
tivity of A.amara in PHO will make the oil very effective in the management
of dandruff. Similar observations were made by using the extracts of Azadirachta
Fig. 3. P.ovale cells (Untreated, Control) indica. (11) Further, the localized immune elicitation activity of bitter fraction of

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 P.Suresh Kumar et al. / Journal of Pharmacy Research 2010, 3(12),2956-2958
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of ‘Antidandruff shampoo’ in the treatment of dandruff. The Antiseptic, 2004 (201): 5-8.
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Solution for dandruff. African J. Biotechnology, 2006 (5): 960-962.
8. Mammen A, Daniel H, Methods in Plant Chemistry &Economic Botany, Kalyani Publishers,
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Source of support: Nil, Conflict of interest: None Declared

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