DOI:

J. Res. Trad. Medicine | May - Jun 2016 | Volume 2 | Issue 3 10.21276/jrtm.2016/136

Original Article - Pharmaceutical Research QR CODE

Evaluation of Fungicidal Activity of Herbal Hand

Sanitizer
Harsha MR*, Baidyanath Mishra1, Chaithra CS2 , Vivekananda Ramana3
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*Research Ofcer, 1Managing Director & Chief Scientic Ofcer, 2Research Associate, R & D Centre, Website:
lnno Vision Healthcare Ltd. No. P 6(B), 1st oor, 1st cross, 1st stage, Peenya Industrial Estate, Bengaluru – www.tmjournal.org
560058, Karnataka, India, 3President and CEO, Inno Vision Therapeutics Inc., 700 Lavaca, Suite PMB 2457,
Austin –78701, Texas, USA.
Received:28/05/2016 Revised: 21/06/2016 Accepted: 01/07/2016

Abstract
Background: Keeping hands clean is one of the safest ways to control the transmittance of germs and
microbes that can cause several types of diseases. Hand sanitizers are effective cleansing agents that
provide alternate and effective means to maintain hand hygiene and are considered as better choices
over hand washing agents due to the ease and convenience they offer. In this regard, apart from
keeping the hands free from germs herbal ingredient based hand-sanitizers also provide added
advantage of keeping the skin nourished and moisturised. Aim: The primary objective of the current
study was evaluating the fungicidal activities of an herbal based hand sanitizer - Clean Hand Gel, a
combination containing extracts of herbs, by time kill assay. Materials & Methods: Standard operating
procedures prepared based on European standard guidelines for evaluation of antimicrobial efcacy
(EN 1276 and EN 1650) was used in the present study on the species C. albicans and A. niger. Results:
The study material at a concentration of 20% when tested for 120 seconds of contact time against
specied fungi resulted in logarithmic reduction of 0.629 and 0.108 in the viable counts of C. albicans
and A. niger, respectively. Conclusion: The present study concluded that Clean Hand Gel possessed
fungicidal effects on the specied fungi, C. albicans and A. niger, and there is potential for it to be used as
an effective herbal hand sanitizer in preventing the spread of microbes (specied fungi) from hand to
hand and thus can be implicated in controlling the transmission of related communicable diseases.
Keywords: C. albicans, A.niger, Fungicidal Activity, Herbal Hand Sanitizer
Introduction

N
osocomial infections (NI) / Hospital
acquired infections, encompass a wide
range of disease causing infections that the
susceptible patients could harbor from other
personnel and/or objects present in hospital/health
centre environment, given that the patient did not
carry the infectious agent in the rst place at the time
[1]
of admittance to the hospital . It has been observed
that potentially 10 to 30% of individuals admitted to
hospitals/health centers are more than likely to pick
up such kind of infection. Intensive care units seem to
have the highest frequency in terms of occurrence of
NI. NI is highly prevalent and hundreds of millions
of patients are affected worldwide and the incidence
and frequency of NI have risen alarmingly and is
attributed with severe consequences of morbidity
[2]
and mortality, especially in developing countries .
The most common sites through which NI occur
include surgical site, respiratory, urinary,
bloodstream sites and through vascular catheter
routes. The different modes of pathogen transmission
resulting in NI could happen either directly or
indirectly through – i) physical contact; ii) common
vehicles such as food; iii) airborne; iv) vector borne,
such as, through ies etc [3]. The frequent pathogens
that primarily account for NI are - gram
positive/negative bacteria, including species of fungi
such as Aspergillus niger and Candida albicans [4,5].
Corresponding address:
Mr. Harsha M R,
Research Ofcer, Research & Development Centre,
lnno Vision Healthcare Ltd.
No. P 6(B),1st oor,1st cross, 1st stage, Peenya Industrial
Estate, Bengaluru – 560058, Karnataka , India.
Email: bioresearch@innovisionhealth.com

Harsha, et al.: Fungicidal Activity of Herbal Hand Sanitizer 70

J. Res. Trad. Medicine | May - Jun 2016 | Volume 2 | Issue 3
Though there are many different routes and modes of
NI acquisition and its overall epidemiology, and it is
a tough job to singly pinpoint as to what is the exact
source of NI, a gross estimation has been made that at
any given time, especially in developing countries,
more than 80 % infectious diseases could result due
to direct hand-to-hand contacted transmission of
infectious microbial agents. To compliment this
statement, several studies have ascertained that our
hands are possibly main carriers of potential
pathogenic bacteria that can cause a sequence of
[6, 7]
infectious diseases .
In such a scenario, hand hygiene becomes a
signicant control measure against NI and
contributes signicantly towards keeping diseases
and infections at bay. Thus, hand hygiene plays out
as a single, extremely important, economical, easy
and one of the most effective means of abating NI and
preventing diseases [8, 9]. But unfortunately poor hand
hygiene compliance amongst health care personnel
and also the lack of appropriate scientic knowledge
about the signicance of hand hygiene in the relative
reduction of NI - are major factors promoting the
transmission and outbreak of health care associated
pathogenic microbes. However, a methodical
appraisal about hand hygiene procedures, its key
advantages in preventing NI and its essential
implementation at various levels has been
apparently proven to be a very effective strategy in
reducing the transmission of various diseases
[9, 10]
causing bacteria and fungi .
Presently, there are two main types of hand
sanitation techniques which are fundamentally
different from each other – conventional soap-water
based hand washing and; waterless alcohol based
hand-rub-in (hand sanitizers). The principle of hand
washing involves rather tedious steps of applying
the anti-bacterial soap on the hands, gathering lather
with soap using rigorous friction/rubbing of hands,
then rinsing off thoroughly under water and drying
hands using a towel. This method only physically
eliminates mostly the transient microbial ora on the
hands by washing them off, but does not kill them.
The hand-rub technique involves dispensing a small
volume (2-3) ml of liquid gel onto the palm and
gently rubbing in for 20-30s or until the substance
evaporates. The advantage and effectiveness of this
type over hand washing technique is that it kills the
resident and transient microorganisms rather than
just washing those away from the hands.
Harsha, et al.: Fungicidal Activity of Herbal Hand Sanitizer
Today, infection control experts in Northern Europe
and many western countries recommend hand-
sanitizers for maintaining routine hand hygiene. The
surveillance of NI infection in India and as well as the
Asia-Pacic countries show similar trends that
demand tackling of NI primarily by maintaining
proper hand hygiene. Also, in a fast paced
mechanistic life, the ease of use and its availability
carrying hand-sanitizers as portable cleansing agents
should explain the consumer's preference in choosing
a ready hand-sanitizer over hand-washing practices.
Though alcohol based hand-sanitizers which are
exclusively chemical in nature are highly effective,
they can dry-out the skin with regular indiscriminate
usage. In light of this, herbal based hand-sanitizers are
handy and come with added benets of their anti-
microbial effects and they also contain natural and
safe ingredients that moisturize and nourish the skin
without leaving them dry and itchy [11-13].
Materials and Methods
The current study deals with the evaluation of anti-
fungal activity of herbal hand sanitizer Clean Hand
Gel containing a combination of herbal extracts of,
Karpura (Cinnamomum camphor) – 0.5 mg; Svetajiraka
(Cuminum cyminum) Fruit – 0.5 mg; Ushira (Velveria
zizaniodes) Root – 0.7 mg; Nimbu (Citrus limon) Fruit
peel – 0.5 mg; Nimba (Azadirachta indica) - 10 mg;
Base-IPA and perfume in each gram of the gel.
Details of the materials and methods that are specied
in the subsequent sections of this study plan are
contained in the appropriate Radiant Research
standard operating procedures prepared based on
European standard guidelines for evaluation of
antimicrobial efcacy (EN 1276 and EN 1650).
Microbial cultures: Candida albicans ATCC 10231 and
Aspergillus niger ATCC 16404 were purchased from
American Type Collection Center (ATCC), United
State of America (USA).
Media and Chemicals: Bovine Serum Albumin
(BSA), Tween-80, Lecithin, Tryptone, Soyabean
Casein Digest Medium (SCDM) and Sabouraud
Dextrose Agar (SDA) were purchased from HiMedia.
Experiment: In 0.6 ml of sterile water, 0.2 ml of clean
hand sanitizer was directly added to a vial containing
0.1ml of BSA and 0.1 ml of fungal inoculum.
Subculture of A. niger, grown on SDA plate and
incubated for 7 days at 28˚C was used for the
experiment.
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J. Res. Trad. Medicine | May - Jun 2016 | Volume 2 | Issue 3

Preparation of fungal inoculum 8. After 24 hours of incubation, CFU/ml of both test

In 10 ml of sterile diluents, a loopful of working and control plates were calculated.
culture of C. albicans was inoculated. Inoculum was 9. Logarithmic (Log) reduction in viable count of test
adjusted to 107 cells/ml by counting number of cells organism was calculated by comparing CFU/ml of
under hemocytometer. the test with CFU/ml of the control.

Inoculum of A. niger was prepared using sterile Results

cotton bud. Conidiospores taken from working
culture were dissolved in 10 ml of sterile saline
containing 0.05% Tween 80. The number of spores in
inoculum was adjusted to 107 spores/ml by counting
them under hemocytometer.
Time kill assay of fungi
1. Prior testing all reagents were equilibrated to the
test temperature of 20°C.
2. To the test tube containing 0.1 ml of 0.03% BSA
solution, 0.1 ml of fungal inoculum was added and
tube was incubated at 20˚C for 2 minutes.
3. After incubation, 0.2 ml of sample dissolved in 0.6
ml of hard water (0.03% CaCO3) was added to the
above solution. Test tube was further incubated at
20˚C for 2 minutes.
4. Immediately after 2 minutes, 0.1 ml of test mixture
was transformed to a test tube containing 0.8mL of
neutralizer and 0.1 ml of sterile water.
5. After 5 minutes of incubation, test solution was
serially diluted from 10-0 to 10-5 dilutions and 0.1 ml
test mixture from each dilution was spread on SDA
plates.
6. Plates inoculated with C. albicans were incubated
at 36˚C for 48 hours and plates inoculated with A.
niger were incubated for 7 days at 36˚C.
7. Control experiment was prepared by replacing the
test sample with 0.2 ml of sterile water.
Calculation
The results of the fungicidal activities using time kill
assay are expressed as a logarithmic reduction in the
density of viable count of tested organisms. Table 1
shows that the clean hand sanitizer tested at a
concentration of 20% for 2 minutes contact time
demonstrated 0.629 and 0.108 logarithmic reduction
in viable counts of C. albicans and A. niger,
respectively, and the substantiating macroscopic
pictures of the same are presented in Figures 1 and 2.
Discussion
Although the history of hand-sanitizers dates back to
more than half a century ago when the rst alcohol-
based sanitizer was created, the best thing happened
in the last decade with the world-wide pandemic
caused by H1NI swine inuenza when people became
highly conscious about personal hygiene including
[14]
hand hygiene practices . Also, multiple scientic
studies have ascertained the effectiveness of ethanol
based hand-sanitizers in the removal of a series of
detectable microbes, including multi-drug resistant
pathogens from hands and the prevention of
[13]
infectious diseases . The effectiveness is touted to be
approximately 100 times more than other types of
hand-hygiene maintaining practices such as, plain
soap-based hand washing, etc. In addition, several
authentic healthcare institutions of world repute,
such as, the WHO, have cited the important roles of
hand-hygiene and hand-sanitizers in disease
[10, 11]
prevention .

Colony Forming Unit (CFU/mL) = Average number of colonies * Dilution factor

Amount of sample plates

Log reduction = Log (CFU/mL of Growth Control) – Log (CFU/mL of Test Sample)

Table 1: Time kill assay of clean hand sanitizer tested under clean conditions

Log reduction
Test organism Conc. of product
Colony forming units/mL in clean condition
(0.3% BSA)
Control (fungal) Test (fungal)
Log reduction
Suspension Suspension
C. albicans 20 % 2.3 x 107 5.5 x 108 0.629
A. niger 20 % 1.7 x 107 1.3 x 108 0.108

Harsha, et al.: Fungicidal Activity of Herbal Hand Sanitizer 72

J. Res. Trad. Medicine | May - Jun 2016 | Volume 2 | Issue 3
Fig 1: Fungicidal activity of Clean Hand Gel against C. albicans
Control
Fig 2: Fungicidal activity of Clean Hand Gel against A. niger
Control
In order to minimize the mild adverse effects of skin
drying and itching caused by the regular application
of alcohol-based hand-sanitizers. Other potential
antimicrobial with herbal ingredients are being made
use in the formulation of a much more elegant,
effective, improved, more importantly safe and
nontoxic hand-rub with skin-friendly moisturizers
[15]
and emollients . Such herbal based sanitizers have
proven to be microbiologically more effective both in
vitro and in vivo.
The trial product of this study, Clean Hand Gel, an
herbal sanitizer, showed considerable antimicrobial
activity against C. albians and A. niger. It is
noteworthy here that fungal species such as C.
albicans and A. niger have accounted for the highest
frequency of opportunistic fungal infections in the
scope of NI. Since it is generally well known that
herbal/phytoconstituents exhibit antimicrobial
(anti-fungal) activity, the synergistic action of the
different essential oils, secondary plant metabolites
such as alkaloids, ovonoids and other phenolic
bioactives optimally present in the composition of
Clean Hand Gel might have provided the observed
fungicidal effects against the tested organisms. Also,
the native herbal ingredients in the composition of
this product made of Cinnamomum camphor,
Cuminum cyminum, Velveria zizaniodes, Citrus
Limon, Azadirachta indica with all of their
constituent bioactives with proven antimicrobial and
Harsha, et al.: Fungicidal Activity of Herbal Hand Sanitizer
Test
Test
several other biological effects make this product an
apt source of hand sanitizer [16-22].
Conclusion
Several clinical studies have demonstrated the usage
of herbal based hand sanitizers to be more efcacious
and safe without any harsh effects on skin, than soap-
based hand wash. In view of this, it may be concluded
that Clean Hand Gel can be used as an effective herbal
hand-sanitizer with its tested fungicidal properties.
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A v a i l a b l e f r o m : Source of Support: Inno Vision Healthcare Ltd.
http://indianmedicine.eldoc.ub.rug.nl/root/D/
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the online materials, does not assume any responsibility or liability for any
18. T.Subburaju, Sachin K Singh, Devprakash*,
consequences arising out of the usage of the contents in the published
Prashant Singh, K.K.Srinivasan,. Antifungal articles. The contents presented in the articles are purely the opinion of the
activity of alcoholic and aqueous extracts of contributing authors and not necessarily of the Journal.

Harsha, et al.: Fungicidal Activity of Herbal Hand Sanitizer 74