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Formulation and Evaluation of the Effectiveness of a Novel Hand Sanitizer

using Pleurotus ostreatus Oyster Mushroom Extract.

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International Journal of Pharma Research & Review, Jan 2017;6(1):7-15 ISSN: 2278-6074

Research Article

Formulation and Evaluation of the Effectiveness of a Novel Hand Sanitizer Using

Pleurotus ostreatus Oyster Mushroom Extract
C. N. Stanley1*, V. B. Alobari1, K. M. Ezealisiji2

1. Department of Pharmaceutical Microbiology and Biotechnology, University of Port Harcourt, Rivers

State, Nigeria
2. Department of Pharmaceutical and Medicinal Chemistry, University of Port Harcourt, Rivers State,
Nigeria
ABSTRACT
Background: Although hand sanitizers have been proven to be as effective as soap and water in reducing
bacterial load, there is still a need for more effective hand sanitizers to reduce the spread of infections.
The antimicrobial potency of Pleurotus ostreatus mushroom extract formulated into an alcohol based
liquid hand sanitizer was thus evaluated and the best alcohol concentration for the formulated hand
sanitizer determined. Methods: The chloroform, ethyl acetate and ethanol extracts were screened for
antimicrobial activity using standard methods with Pseudomonas aeruginosa, Escherichia coli,
Staphylococcus aureus and Candida albicans as test organisms. Broth dilution method was used to
determine the minimum inhibitory concentration (MIC) of the various extracts while the formulated and
commercial hand sanitizers were evaluated by agar pour method. Results: Only Staphylococcus aureus
was susceptible with a MIC of 6.25mg/ml and 12.5mg/ml respectively for ethyl acetate and ethanol
extracts of Pleurotus ostreatus mushroom. The various formulations of the Pleurotus ostreatus hand
sanitizer were compared with 3 commercial sanitizers namely Carex®, Dettol® and Health Smart® against
Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. The formulation of
the extract containing 70% alcohol was more effective than all others. Conclusion: The antimicrobial
activity of the mushroom, Pleurotus ostreatus, was potentiated by dimethyl sulfoxide (DMSO) which was
used to reconstitute the extract. The DMSO appeared to have oxidized the alcohol in the process of hand
sanitizer formulation probably yielding an aldehyde.

KEY WORDS: Evaluation, formulation, hand sanitizer, mushroom, Pleurotus ostreatus

Received 30 Oct 2016 Received in revised form 22 Dec 2016 Accepted 31 Dec 2016

*Correspondence Address:
Catherine N. Stanley,
Department of Pharmaceutical Microbiology and Biotechnology, University of Port Harcourt, Rivers State,
Nigeria.
Email: catherine.stanley@uniport.edu.ng

BACKGROUND bacterial load from the hands even in the

The skin is the part of the body most exposed
to environmental pollutants, sunlight as well
as pathogens. Some of the dangers of such
exposures include, but are not limited to, skin
disorders such as eczema (atopic dermatitis),
rashes, acne, psoriasis, warts and allergy.
Hence to protect the skin from harmful
microorganisms and prevent spreading of
skin infections, hand hygiene is an essential
precaution [1]. The hands play a very
important role in the transmission of
nosocomial as well as community acquired
infections [2].
Hand sanitizers have been proven to be as
effective as soap and water in reducing
presence of physical dirt, soil or grease [3].
Hand sanitizers are even more important in
places where there is no clean water, or
when a health care worker has so many
patients to attend to and does not have the
luxury of time to wash the hands after
attending to each patient [4]. The recent
outbreak of the Ebola virus disease in the
West African sub-region is a good example.
To facilitate microbial load reduction, hand
sanitizers can also be used after hand
washing [5]. It is therefore very important
that more effective and yet affordable hand
sanitizers are formulated to reduce the

Catherine N Stanley et.al, IJPRR 2017;6(1) 7

International Journal of Pharma Research & Review, Jan 2017;6(1):7-15
spread of infections particularly in resource
poor countries.
Formulating a hand sanitizer with edible
mushroom extract appears to have some
advantages compared to wholly synthetic
hand sanitizers. This is because mushrooms,
just like humans, consist of eukaryotic cells
and this may make hand sanitizers produced
from them milder and more tolerable to the
skin and probably more effective [6]. Hand
sanitizers made from mushroom if confirmed
to be effective, would be cheaper and more
affordable as the raw material abounds
locally. Moreover, interest in sourcing for
drugs from fungi appears to have been on the
increase lately [7].
The binomial name of oyster mushroom is
Pleurotus ostreatus (Jacq.ex.fr) P. Kumm. It is
widely grown in subtropical and tropical
regions and is easy to cultivate. It is used for
both culinary and non-culinary purposes
because it contains a variety of constituents,
nutrients and flavor. It is of the fungi
kingdom and belongs to the class
pleurotaceae [8, 9].
Phytochemical screening of wild oyster
mushroom had shown that Pleurotus ostreatus
contains varying amounts of carbohydrates,
saponins, alkaloids, polyphenols, tanins,
terpenoids and cardiac glycosides [10]. Some of
the components that may be responsible for its
antimicrobial activity include gallic acid, volatile
compounds, some phenols, free fatty acids and
their derivatives [11]. Pleurotus ostreatus has
been reported to have antimicrobial activity
from literatures [12 – 15].
A variety of plant materials and substances
have been incorporated into hand sanitizers
[16, 17]. However, to the best of our
knowledge and in spite of extensive search of
the literature, Pleurotus ostreatus (a
mushroom) has not been incorporated into
an alcohol based hand sanitizer.
Liquid hand disinfectants were reported to
have more virucidal and bactericidal action
than gel products [18]. This finding was
corroborated by Oke and his colleagues [19]
who found a liquid hand sanitizer containing
62% alcohol and glycerin more effective than
the gel products evaluated in their study.
This present study, therefore, determined to
formulate and evaluate the antimicrobial
efficacy of a novel hand sanitizer made from
Pleurotus ostreatus (a mushroom) extract.
Catherine N Stanley et.al, IJPRR 2017;6(1)
ISSN: 2278-6074
Figure 1: Pleurotus ostreatus mushroom
METHODS
Collection of mushroom
Fresh samples of the fruiting body of
cultivated Pleurotus ostreatus were collected
from the mushroom unit of the
demonstration farm of Faculty of Agriculture,
University of Port Harcourt, Rivers State.
Processing of mushroom
The fruiting body of the mushroom was cut
into smaller pieces and shade dried. The
dried mushroom was then pulverized into
powder.
Preparation of Pleurotus ostreatus extract
The powdered sample was passed through
successive extraction using chloroform, ethyl
acetate and ethanol respectively and
macerated with occasional shaking for 3
days. Filtration was done using Whatman No
1 filter paper and the filtrates obtained were
concentrated using a rotary evaporator at a
controlled temperature of 40 O C. The
concentrates were further dried using a
water bath at 50 OC, 60 OC and 70 OC for
chloroform, ethyl acetate and ethanol
extracts respectively.
Collection of test organisms
Clinical isolates of Pseudomonas aeruginosa,
Staphylococcus aureus, Escherichia coli and
Candida albicans were obtained from the
Medical Microbiology Laboratory of the
University of Port Harcourt Teaching
Hospital.
Identification of test organisms
The test organisms were sub-cultured on
their selective media; Staphylococcus aureus
on Mannitol salt agar (MSA), Escherichia coli
on MacConkey agar, Pseudomonas aeruginosa
8
International Journal of Pharma Research & Review, Jan 2017;6(1):7-15 ISSN: 2278-6074

on cetrimide agar and Candida albicans on 12.44%, 6.22%, 3.11% concentrations. The
Sabouraud Dextrose Agar (SDA). The different concentrations were tested against
morphological characteristics of their 0.1ml of a standardized Staphylococcus
colonies were observed. Gram staining and aureus suspension and incubated at 37oC. A
biochemical tests were carried out to confirm loop full of the mixture from each tube was
their identities. streaked on Mannitol Salt Agar after 24 hours
Antimicrobial susceptibility screening using to determine the MIC values which is the
agar diffusion (Kirby- Bauer) method. least concentration that inhibits the growth
A 0.1ml volume of the suspension of each of the microorganism as stated by Senekal
organism of 0.5 MacFarland turbidity was [21].
inoculated into 20ml of the molten agar, Formulation of hand sanitizer
poured into a sterile plate and allowed to Four liquid hand sanitizers were formulated
solidify. A cork borer of 6mm diameter was using the following composition shown in
used to make wells on the Muller Hinton agar (Table 1 to 4) below:
for bacteria and Sabouraud Dextrose Agar for
fungi. The wells were filled with 10mg/ml Table 1: Formulation 1
and 100mg/ml concentrations of each of the Ingredients Function Concentration
extracts to be tested against the organisms. (%v/v)
The plates were left for 30 minutes at room Alcohol Antimicrobial 70%
temperature to allow for diffusion of the Glycerol Humectant 3%
extracts and then incubated at 37oC for 24 Sterile water Vehicle 27%
hours. The resultant zones of inhibition
which serve as an indication of antimicrobial Table 2: Formulation 2
activity were measured. Ingredients Function Concentration
Antimicrobial screening using disc agar (%v/v)
diffusion method. Alcohol Antimicrobial 40%
The 10mg/ml and 100mg/ml concentrations Glycerol Humectant 3%
of each of the extract were re-tested against Sterile water Vehicle 37%
the test organisms using paper disc agar Mushroom Antimicrobial 20%
diffusion method. Sterile swab sticks were extract
soaked with the suspension of each test
organism. The swab sticks were then used to Table 3: Formulation 3
streak the surface of the Muller Hinton agar Ingredients Function Concentration
for bacteria and Sabouraud Dextrose Agar for (%v/v)
fungi. Sterile paper discs were impregnated Alcohol Antimicrobial 70%
with the different concentrations of the Glycerol Humectant 3%
different extract solutions and placed on the Sterile water Vehicle 7%
agar plates. The plates were incubated at Mushroom Antimicrobial 20%
37oC for 24 hours. The resultant zones of extract
inhibitions which serve as an indication of
antimicrobial activity were measured. Table 4: Formulation 4
Minimum Inhibitory Concentration (MIC) Ingredients Function Concentration
determination. (%v/v)
The MIC of the extract and DMSO against the Mushroom Antimicrobial 20%
susceptible organisms were determined extract
using broth dilution method as adapted from Glycerol Humectant 3%
Balouiri and co–workers [20] with some Sterile water Vehicle 77%
modifications. The stock solutions of ethyl
acetate extract and ethanol extract of The above ingredients for the respective
100mg/ml were serially diluted two-fold to formulations were measured and transferred
obtain concentrations of 50mg/ml, 25mg/ml, to a sample bottle. The mixture was shaken
12.5mg/ml, 6.25mg/ml and 3.125mg/ml. The vigorously to ensure homogeneity and
same two-fold serial dilution was also labelled accordingly.
applied to DMSO to obtain 49.75%, 24.88%,

Catherine N Stanley et.al, IJPRR 2017;6(1) 9

International Journal of Pharma Research & Review, Jan 2017;6(1):7-15 ISSN: 2278-6074

Evaluation of formulated hand sanitizer out confirms that ethanol extract had
The formulated hand sanitizers were antimicrobial activity as shown in (Fig. 8).
evaluated using agar pour method. A 0.1ml
suspension of the test organisms were
inoculated into molten agar. A 1ml volume of
the hand sanitizers were transferred to the
molten agar and mixed properly and poured
into sterile plates. The plates were incubated
at 37oC for 24 hours after which they were
observed for the degree of microbial growth.
Evaluation of commercial hand sanitizers
Three commercial hand sanitizers namely
Carex®, Dettol® and Health smart® were
evaluated using agar pour method. A 0.1ml Figure 2: Inhibition zone diameter of
volume of the suspension of test organisms Pleurotus ostreatus extract against four
was inoculated into molten agar. A 1ml microorganisms using agar diffusion
volume of the different hand sanitizers were method
transferred respectively to the molten agar
and mixed properly and poured into sterile
plates. The plates were incubated at 37oC for
24 hours after which they were observed for
the degree of microbial growth.
RESULTS AND DISCUSSION
Antimicrobial activity of Pleurotus ostreatus
The antimicrobial screening of the mushroom
extract against the test organisms using agar
well diffusion method revealed that the ethyl
Figure 3: Inhibition zone diameter of
acetate and ethanol extracts inhibited the
standard antibacterial drug, Cipro-
growth of Staphylococcus aureus only. The
floxacin against three microorganisms
chloroform extract did not inhibit growth of
using agar diffusion method
all the test organisms. This result was
obtained when the extracts were
reconstituted with DMSO. The results are
reported in (Fig. 2).
There may be concern why chloroform
extract did not show antimicrobial activity.
This could be attributed to the polarity of the
solvent used for extraction. Literatures have
suggested that the antimicrobial activity of
Pleurotus ostreatus may be due to its
antioxidant content which is more in polar
extracts. [22 -24] Figure 4: Inhibition zone diameter of
Paper disc diffusion method was also used to standard antifungal drug, Nystatin against
screen the extracts for antimicrobial activity. Candida albicans using agar diffusion
This was done to confirm the result obtained method
from the agar well diffusion method.
The paper disc diffusion method also yielded The sensitivity of the agar well diffusion
a similar result whereby ethyl acetate extract assay method over the disc diffusion assay
showed activity against Staphylococcus method as deduced in this study is in
aureus although the ethanol extract did not agreement with the findings of Smania [25].
show activity using this method. The result is Also, from the disparities observed when
reported in (Fig. 5). It can then be deduced comparing the results of the two assay
that the agar well diffusion method was more methods, the paper disc method was possibly
sensitive as the MIC determination carried

Catherine N Stanley et.al, IJPRR 2017;6(1) 10

International Journal of Pharma Research & Review, Jan 2017;6(1):7-15
affected by the poor absorption or release of
the test antimicrobial samples. This is also in
line with the findings of Smania [25].
Figure 5: Inhibition zone diameter of
Pleurotus ostreatus extract against four
microorganisms using paper disc assay
method
Figure 6: Inhibition zone diameter of
standard antibacterial drug, Cipro-
floxacin against three microorganisms
using paper disc diffusion method
Minimum inhibitory concentration (MIC)
determination
The MIC of ethyl acetate and ethanol was
assayed against Staphylococcus aureus. This
is because Staphylococcus aureus was the
only susceptible microorganism. Staphylococcus
aureus showed consistency in being the most
susceptible microorganism to the mushroom
extracts even with the different assay
methods used i.e. well agar diffusion and
paper agar diffusion test. This can be
observed in the results reported in (Fig. 1, 2,
4 and 5). In some literatures, Staphylococcus
aureus has been reported to be the most
susceptible microorganism to Pleurotus
ostreatus extract. [15, 26 – 27]
From the result shown in (Fig. 8) above, MIC
for Ethyl acetate extract is 6.25mg/ml while
the MIC for ethanol extract is 12.5mg/ml.
Catherine N Stanley et.al, IJPRR 2017;6(1)
ISSN: 2278-6074
The MIC result shows that ethyl acetate
extract exhibited more antimicrobial activity
than ethanol extract.
Figure 7: Inhibition zone diameter of
standard antifungal drug, Nystatin against
Candida albicans using paper disc
diffusion method
Figure 8: MIC of Pleurotus ostreatus
extracts reconstituted in DMSO against
Staphylococcus aureus
This may be due to the intermediate polarity
of ethyl acetate as compared to the other
solvents. This suggests that most of the
antioxidant compounds were extracted by
ethyl acetate before ethanol was used for
extraction. This is similar to the result of
Nehra and co-workers28 that showed that the
ethanol extract of Pleurotus ostreatus had
better antimicrobial activity than the extracts
of other solvents used. Similarly, ethanol was
also of intermediate polarity compared to
other solvents used.
Figure 9: Control test done alongside the
MIC of Pleurotus ostreatus extracts against
Staphylococcus aureus
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International Journal of Pharma Research & Review, Jan 2017;6(1):7-15
The results reported in (Fig. 3, 4 and 9)
show that the solvent DMSO used for
reconstitution of the extracts has some innate
antimicrobial activity. Literatures have also
reported that DMSO exhibits antimicrobial
activity [29, 30].
DMSO was chosen for reconstitution because
of its ability to properly dissolve extracts and
diffuse effectively into agar matrices [31, 32].
To determine any possible contribution of
DMSO to the antimicrobial activity seen, the
MIC of DMSO and ethanol extract reconstituted
in water was investigated respectively. (Fig.
10 and 11) report the results of the assay.
Figure 10: MIC of Pleurotus ostreatus
extracts reconstituted in water against
Staphylococcus aureus
(Fig. 10) shows that no MIC value for
Pleurotus ostreatus extract in water was
obtained while (Fig. 11) shows that the MIC
of DMSO is 24.88%.
From the results obtained, it can be
concluded that DMSO was potentiating the
innate antimicrobial activity of the
mushroom as literatures have also suggested
in respect of antifungal drugs [33, 34].
Figure 11: MIC of DMSO against Staphy-
lococcus aureus
This is because the ethyl acetate extract
reconstituted with DMSO had MIC of
Catherine N Stanley et.al, IJPRR 2017;6(1)
ISSN: 2278-6074
6.25mg/ml which is the fifth concentration of
the serial dilution. Also, ethanol extract
reconstituted with DMSO had MIC of
12.5mg/ml which is the fourth concentration
of the serial dilution. Meanwhile, the solvent
DMSO had MIC of 24.88% which is the third
concentration. This suggests that the MIC of
the extracts was not solely due to the DMSO
since the position of the concentrations in the
serial dilution varied. It is possible that the
combination of DMSO and the mushroom
extract exhibited a synergistic effect against
Staphylococcus aureus.
Antimicrobial examination of the formulated
vs commercial hand sanitizers
The extract reconstituted in DMSO was used
in the formulation of the hand sanitizer
because of the synergistic effect observed.
However, DMSO has been used in topical
products for therapeutic effects and as
penetration enhancer [35]. It was also
included in the formulation to serve as a
penetration enhancer to enable the
penetration of the mushroom extract and the
alcohol into the skin thereby promoting their
antimicrobial activity. This will enable the
extract to elicit its other beneficial effects
such as its antioxidant effect [10, 36].
The results of the evaluation of the
formulated hand sanitizers using the agar
pour method are reported in (Fig. 12).
Figure 12: Susceptibility of four different
microorganisms to the formulated hand
sanitizers
The result of the assay of the formulated
products showed that the formulation with
the best antimicrobial activity was
formulation 3 containing 70% alcohol,
extract, glycerol and water.
12
International Journal of Pharma Research & Review, Jan 2017;6(1):7-15
The formulation with the least antimicrobial
activity was formulation 4 containing the
mushroom extract, glycerol and water
without alcohol. Formulation 2 which
contains 40% alcohol, extract, water and
glycerol exhibited better activity than
formulation 1 which contains 70% alcohol,
glycerol and water without the extract.
The commercial hand sanitizers showed higher
antimicrobial activity against Pseudomonas
aeruginosa than the other test organisms.
The most resistant microorganism to the
commercial hand sanitizer was Staphylococcus
aureus which was also the most resistant to
the formulated hand sanitizers. The most
susceptible microorganism to the formulated
hand sanitizers was Candida albicans.
Figure 13: Susceptibility of four different
microorganisms to three commercial
hand sanitizers
Formulation 3 that had the best activity
amongst the formulated products was
observed to be better than all the commercial
sanitizers assayed irrespective of their high
alcohol content.
During the formulation, when the mushroom
extract reconstituted with DMSO was added
to the alcohol, a color change occurred and
the container gave up heat suggesting that
the reaction was exothermic. This may be an
oxidation reaction of the alcohol by DMSO to
yield aldehyde that might be further oxidized
to yield acetic acid. [37, 38]. This implies that
the antimicrobial activity of the formulated
hand sanitizer observed may have been due
to the aldehyde formed. It may also be the
reason why the formulations (2 and 3)
containing alcohol (40% and 70% respectively)
and the extract were more effective than the
formulation 1 containing 70% alcohol
without extract.
Catherine N Stanley et.al, IJPRR 2017;6(1)
ISSN: 2278-6074
CONCLUSION
The study established that cultivated
Pleurotus ostreatus extract alone did not
show significant antimicrobial activity. It had
also been shown that the hand sanitizer
formulated with 70% alcohol and mushroom
extract showed higher antimicrobial activity
than the other formulations. The novel hand
sanitizer was more effective than the
commercial hand sanitizers used in the study.
There is, however, need for further research
on the toxicity of aldehydes to the body to
determine if the hand sanitizer will be fit for
use.
ACKNOWLEDGEMENT
The authors acknowledge and appreciate the
technologists in the laboratory of the
Department of Pharmaceutical Microbiology
and Biotechnology for providing technical
assistance in the course of this study. The
authors did not receive any form of funding
in support of this research or in the
publication of this manuscript.
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