JOURNAL OF ONE HEALTH

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ORIGINAL ARTICLE
Efficacy of Simarouba glauca Herbal Leaf Powder as a Hand-Wash Product with
Potential Antibacterial Activity against Pathogenic Bacteria
Arya R, Megha P.U., Asha K. and Prejit

Centre for One Health Education, Advocacy, Research and Training, Kerala Veterinary and Animal Sciences
University, Pookode, Wayanad, Kerala, India.

Abstract
Plants with diverse medicinal properties are found to be very
effective in the treatment of bacterial diseases. To prevent the entry of
bacterial pathogens to the most exposed parts of the body; especially skin
and hands, it is important to protect them using specific agents. Therefore,
herbal hand wash using leaf extracts of Simarouba glauca was formulated
using Sodium dodecyl sulfate (SDS) and distilled water. In this study, four
indicator organisms commonly attributable to cause diarrhea in humans
*
Corresponding Author: were selected to evaluate the anti-microbial efficacy of the formulated
herbal hand wash. Different concentrations of the products were prepared
Megha P.U and tested for the zone of inhibition which was compared with SDS control
Email: meghapu928@gmail.com and a commercial agent. The study revealed that the herbal formulation (20
µl concentration) was efficient in acting against Escherichia coli,
Received: 09/01/2020
Accepted: 21/02/2020
Staphylococcus aureus, Listeria monocytogenes and Salmonella
Typhimurium and the zone of lysis against these organisms were found to
be better when compared to commercial and control groups. Unlike the
tested herbal product, the commercially available antiseptic hand wash as
well as the control group did not show inhibition zone in any of the
concentrations against E. coli. Simarouba glauca herbal formulation thus
proved to be beneficial and had an excellent activity against all the tested
organisms. The study also showed a significant (p<0.01) result in inhibiting
Salmonella Typhimurium when compared to the other groups. Hence, it
shall be encouraging to use such herbal hand wash after further validation.

Keywords: Herbal handwash, Escherichia coli, Listeria monocytogenes,

Salmonella Typhimurium, Simarouba glauca and Staphylococcus aureus.

1. Introduction
Proper hand hygiene is the single most
important, simplest, and least expensive means of
preventing health care associated infections (Ravi et al.,
2005). Hand washing after handling contaminated
surfaces, after defecation and before handling food is
therefore a biologically plausible mechanism for
interrupting pathogen transmission. Hands can act as a
vector for transmission of faecal pathogens and
respiratory infections, either via direct person-to-person
transmission or by contaminating food that is later
consumed (Curtis and Cairncross, 2003). Interventions
promoting hand hygiene have been shown to reduce
gastrointestinal and respiratory illnesses by an average
of 31% and 21%, respectively (Aiello et al., 2008). The
hand wash products available in market are made from
synthetic chemical and may have side effect like
dryness of skin, rashes etc. Many plants have medicinal
properties and many reports have shown their
antibacterial activity against pathogenic skin bacteria
(White et al., 2003; Chaichompool and Thongwai,
2011; Kamonwannasit et al., 2013).
Simarouba glauca, otherwise known as Paradise
Tree or Bitter Wood, is an evergreen, small to medium-
sized tree growing up to 15 m in height, with a narrow
crown and well-developed root system. It is an
important medicinal as well as oil yielding plant,
commonly known as ‘Laxmitaru’ and it belongs to
family Simaroubaceae (Manasi and Gaikwad, 2011). It
is a native of South and Central America and
indigenous to the Amazon rain forest and other tropical
areas of South America like Mexico, Cuba, Haita,
Jamaica, Brazil, Bahamas, Puertorico, El-Salvador,
Guatemala, while it is exotic to India, Srilanka,
Phillippines and Mayanmar (Reddy et al., 2016). The
leaves and bark have a long history of medicinal use in
the tropics, particularly in the treatment of malaria,
fevers, dysentery, as an astringent, tonic, antibacterial,
antioxidant and antipyretic activity (Lakshmi et al.,
2014; Umesh, 2015). Research has discovered a range
of medically active compounds in the plant. The main
active compounds are a group of triterpenes called
quassinoids. Several of the quassinoids found in
Simarouba, such as ailanthinone, glaucarubinone, and

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© 2020 Jakraya
Arya et al...Efficacy of Simarouba glauca Herbal Leaf Powder as a Hand-Wash Product with Potential
Antibacterial Activity Against Pathogenic Bacteria
holacanthone, are considered the plant's main
therapeutic constituents and are the ones documented to
be antiprotozoal, anti-amoebic, antimalarial, and even
toxic to cancer and leukaemia cells (Rivero-Cruz et al.,
2005). Although there are few previous research works
on antibacterial activity of the plant, the authors have
not come across any work which has used Simarouba
glauca in the formulation of hand-wash product. Thus,
attempt has been made to formulate an herbal hand
wash product using S. glauca and to determine its
efficiency against certain pathogenic bacteria.
2. Materials and Methods
2.1 Collections of Plant Materials and
Preparation of Extracts
S. glauca plant was collected from the campus
of Kerala Veterinary and Animal Sciences University,
Pookode, Wayanad, India and other surrounding areas
(Fig 1). Collected leaves of plants were air dried and
powdered using mixer grinder. Ten grams of leaf
powder was added to methanol solution (9 parts
methanol: 1-part water). Further this mixture was
heated in water bath at 60oC at 60 minutes. The
contents were filtered and the filtrate was used as
methanol extract (Okogun, 2000).
Fig 1: Simarouba glauca plant leaf.
2.2 Bacterial Strains
Bacterial strains were obtained from the
repository of Department of Veterinary Public Health,
College of Veterinary and Animal Sciences, Pookode.
The test organisms used for the study were Escherichia
coli, Staphylococcus aureus, Listeria monocytogenes
and Salmonella Typhimurium. This organism was
selected as they are the indicator organisms of food
safety and food borne diseases.
2.3 Preparations of Herbal Hand Wash
The herbal hand wash was prepared by adding 4
ml of methanol extract of plant material in 6 ml of
distilled water. To the final volume of 10 ml, 3 gram of
Sodium dodecyl sulfate (SDS) was added as per the
requirement of standard procedure for preparation of
hand wash (Joshi et al., 2008). SDS is an organic
compound commonly used in molecular biology
laboratory. It is an anionic surfactant having
Journal of One Health | January-March, 2020 | Volume 08 | Issue 01 | Pages 10-14
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amphiphilic properties required for detergent. It is used
in many cleaning and hygiene products. This
formulation was further used to examine its
antibacterial activity at different concentration.
2.4 Antibacterial Activity of the Different Plant
Extracts and Herbal Hand Wash
The antimicrobial activity of individual extracts
was tested using disc diffusion technique (Jangale et
al., 2012). The combinations used for the study
included, control (C): 3 gram SDS + 4 ml methanol + 6
ml distilled water, commercially available antiseptic
hand wash (A) and formulated herbal (S. glauca) hand
wash (H): 3 gram SDS + 4 ml plant extract + 6 ml
distilled water. Sterile Whatman filter paper disc of 6
mm diameter was prepared and loaded with different
concentrations (5 µl, 10 µl, 15 µl and 20 µl) of above
combinations and retained for 2 hours. Muller Hinton
Agar (MHA) plates were spread with test organisms
viz., Escherichia coli, Staphylococcus aureus, Listeria
monocytogenes and Salmonella Typhimurium in
separate plates (bacterial suspension of OD was 0.6 at
600 nm) and further the antimicrobial activity in terms
of zone of inhibition (mm) was recorded after 24 hours
of incubation. The antagonistic action of each
combination was tested against test organism in
triplicates.
2.5 Statistical Analysis
Two-way analysis of variance (ANOVA) was
used to test for statistically significant differences, and
the factors were treatment and concentration. If
significant interaction was found, paired t-test was
performed to compare the outcome indicators before
and after the intervention. Data were analysed using
Statistical Package for the Social Science (SPSS)
version 24. The level of statistical significant was set at
0.01.
3. Results and Discussion
Plants with diverse medicinal properties are
found to be very effective in the treatment of bacterial
diseases (Raskin et al., 2002). Also, the increase in
antibiotic resistance in microorganisms and the side
effects of synthetic antibiotics has provoked researchers
to use medicinal plants in treating various bacterial
infections (Karuppiah and Rajaram, 2012). To prevent
the entry of bacterial pathogens to the most exposed
parts of the body; especially skin and hands, it is
important to protect those parts using specific agents.
Therefore, herbal hand wash using leaf extracts of
Simarouba glauca was formulated, which has already
proved to have no side effect and potential antibiotic
activity (Manasi and Gaikwad, 2011; Lakshmi et al.,
2014; Umesh, 2015). In this study Simarouba glauca
plant leaf was collected and extracts was prepared. Four
bacterial species which were the common diarrheal
pathogens as well as food safety indicator organisms
were selected to evaluate the anti-microbial activity -
Arya et al...Efficacy of Simarouba glauca Herbal Leaf Powder as a Hand-Wash Product with Potential
Antibacterial Activity Against Pathogenic Bacteria

Table 1: Antibacterial evaluation of commercial antiseptic hand wash

Bacteria tested Antiseptic hand wash (Commercial)

5(µl) 10(µl) 15(µl) 20(µl)
Salmonella typhimurium 11.3±1.53 12.0±0.00 12.3±0.58 14.0±0.00
Listeria monocytogenes 10.0±0.00 9.7±1.53 11.7±0.58 12.0±1.73
Staphylococcus aureus 10.3±0.58 10.7±0.58 11.3±1.53 13.0±1.73
Escherichia coli - - - -

Table 2: Antibacterial evaluation of Simarouba glauca herbal hand wash

Bacteria tested Simarouba glauca herbal hand wash

5(µl) 10(µl) 15(µl) 20(µl)
Salmonella Typhimurium 11.3±0.58 13.0±1.73 14.7±0.58 17.3±1.15
Listeria monocytogenes 10.7±1.15 10.7±0.58 13.7±1.15 14.7±0.58
Staphylococcus aureus 11.0±1.73 11.3±1.53 1.7±0.58 13.0±1.00
Escherichia coli 11.0±1.73 11.7±0.58 11.7±.58 13.0±1.73

Table 3: Antibacterial evaluation of control (SDS-Methanol combination)

Bacteria tested Control (SDS and methanol combination)

5(µl) 10(µl) 15(µl) 20(µl)
Salmonella Typhimurium 0.0±0.00 0.0±0.00 9.7±1.53 9.7±0.58
Listeria monocytogenes 11.0±1.73 10.7±0.58 10.7±0.58 13.0±1.73
Staphylococcus aureus - - 10.7±1.15 11.0±1.73
Escherichia coli - - - -

Table 4: Two-way ANOVA results for comparison of efficacy of preparations

Test organism Average Mean ± SD F value (p value=0.01)

Herbal Commercial SDS+Methanol Treatment Concentration Interaction
hand wash hand wash
Salmonella Typhimurium 14.08±0.72 12.41±0.35 4.83+1.47 338.74 91.78 22.01
Listeria monocytogene 12.42±0.58 10.83±0.42 11.33±4.32 6.02 12.56 1.62
Stapylococcus aureus 11.50±0.41 11.33±0.43 5.41+1.65 101.70 43.05 22.54
E.coli 11.58±0.41 - - 1.91 - -

of the selected formulations. The herbal hand wash was
prepared by adding methanol extract of plant material
in distilled water and Sodium dodecyl sulfate (SDS).
The antimicrobial activity of individual extracts was
tested using disc diffusion technique (Jangale et al.,
2012) against Escherichia coli, Staphylococcus aureus,
Listeria monocytogenes and Salmonella Typhimurium.
Different concentrations of the products were incubated
and observed for the zone of inhibition (Table 1-3). The
study revealed that the herbal formulation (at 20 µl
concentration) was efficient in acting against
Escherichia coli, Staphylococcus aureus, Listeria
monocytogenes and Salmonella Typhimurium. It was
also observed that, commercially available antiseptic
hand wash selected in this study did not provide
inhibition zone in any of the concentrations against E.
coli. Moreover, the zone of lysis detected in the case of
commercial antiseptic was found to be lesser when
compared to the formulated herbal hand wash (Table 1-
2). It implies that, the herbal formulation was efficient
in removing bacterial pathogens from biological
surfaces. Out of different concentrations (5 µl, 10 µl, 15
µl and 20 µl) of control, commercial and formulated
herbal hand wash evaluated, the zone of inhibition was
found to be maximum in 20 µl concentrations against
all bacterial species (Table 1-2; Fig 2). The control
(SDS methanol combination) could also show some
zone of inhibition against Salmonella spp., Listeria spp.
and Staphylococcus spp (Table 3). This can be
attributed to the presence of SDS which is an alcohol
detergent derived from Alcohol Sulfates. Toxicity of
SDS on gram-negative bacteria has been previously
evaluated by Belanger et al. (2006). Also increased
amount of SDS in the bacterial cytoplasm contributes to
misfolding of denatured protein which is toxic to the
cell with other toxic effects (Rajagopal et al., 2002).

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Arya et al...Efficacy of Simarouba glauca Herbal Leaf Powder as a Hand-Wash Product with Potential
Antibacterial Activity Against Pathogenic Bacteria
Fig 2: Effect on bacterial microflora after use of: a) herbal hand wash; b) commercial hand wash; c) SDS- Methanol.
Jangale et al. (2012) reported similar activity with
methanolic extracts of Simarouba glauca against S.
aureus and E. coli. Not much work has been reported
on the antibacterial effect of Simarouba glauca against
S. Typhimurium, and Listeria monocytogens. The
comparison of efficacy of different groups are shown in
Table 4. Herbal formulation proved to be beneficial and
had an excellent activity against all the tested
organisms. A highly significant (p<0.01) zone of
inhibition was obtained by herbal formulation against S.
Typhimurium when compared to the other treatment
methods used. Moreover, Herbal formulation could
provide inhibition zone in any of the tested
concentrations against E. coli when compared to the
commercial product and control. Hence, it was
encouraging to be used in the preparation of herbal
hand wash.
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4. Conclusion
In conclusion, based on the above findings it is
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