NewBioWorld A
Journal of Alumni Association of Biotechnology (2024) 6(1):33-41
RESEARCH
ARTICLE
A Study on Polyherbal Skin Ointment: Formulation and
Evaluation
Isha Agrawal1, Pratik Jangde2, Mayank
Bhardwaj3, Alka Saxena3, Bharti Sahu4, Andrea
Pereira4, Varaprasad Kolla5*
1Edward and Cynthia Institute of Public health, Mangalore
2Ameliorate Biotech, Bangalore
3Acube Labs, Lucknow
4Seth Phoolchand Agrawal Smriti Mahavidhyalay, Raipur
5Amity University Raipur
*Corresponding Author Email- annpereira@gmail.com
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ARTICLE INFORMATION
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ABSTRACT
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Article history:
Received
05 April 2024
Received in revised form
15 June 2024
Accepted
Keywords:
Polyherbal;
Phytochemicals; Antibacterial;
Ointment;
Staphylococcus aureus
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The skin is the protective
outermost covering of the human body. It is highly exposed to the external
environment making it prone to harsh changing environmental conditions, wide
range of organisms and hence infections. The long term usage of chemical
preparations to protect the skin can be very harmful. The use of plant parts/
herbs in medicine has been an traditional Indian practice. Phytochemicals
found in plant extracts demonstrate antimicrobial activity that help fight
infections and are long-term safe for use without any side effects. In this research
we have prepared and evaluated a polyherbal ointment. Water-based extracts of raw turmeric (Curcuma longa), nutmeg (Myristica fragrans), marigold (Calendula officinalis), neem (Azadirachta
indica), and orange (Citrus
sinensis) peel were prepared. Alkaloids, saponins, and tannins
were among the phytochemicals found in the extracts. Their susceptibility to antibiotics was examined against Staphylococcus aureus, with Ciprofloxacin as a positive control.
All extracts demonstrated antibacterial activity with a varying Zone of
inhibition. The herbal ointment was prepared using all the extracts after
determining their Minimum Inhibitory Concentrations. The evaluated herbal
ointment demonstrated antibacterial sensitivity against Staphylococcus aureus and Escherichia
coli. Further, the formulated polyherbal ointment demonstrated high
antibacterial sensitivity against skin bacteria when compared to a few
commercially available creams. Further, when compared to a few commercially
available creams the formulated polyherbal ointment demonstrated higher antibacterial
activity.
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Introduction
DOI: 10.52228/NBW-JAAB.2024-6-1-5
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Human skin is exposed to the external environment and harbors millions of
microorganisms. Skin is colonized with a wide range of microorganisms and
serves as a barrier to protect against invasion. Beneficial microorganisms
occupy the skin cavity and protect against pathogenic microorganisms. When skin
is damaged the balance is disturbed and results in skin disease. Generally, the
skin remains cool and acidic but different types of microbial habitats are
decided by thickness and folds of skin, hair follicle, and glands (Tagami 2008) Bacteria
are the major constituent microbe of skin among which Staphylococcus
aureus and Escherichia coli are the main cause of
skin infection (Handali et al. 2011). Numerous skin illnesses, including
impetigo, boils, carbuncles, cellulitis, and folliculitis, are brought on by
microorganisms (Sukumaran et al. 2016). Usually, gram-positive
strains such as S. aureus cause a majority of human skin infections (Tong et al. 2015). The economy and human mortality
are significantly impacted by multidrug-resistant microorganisms. Hospital
effluent and ambient wastewaters containing multi-drug resistance
Methicillin-resistant i (MRSA) are caused by the use of antibiotics (O’Neill 2016; Oladipo et al. 2019)
Since ancient times, plants have been utilized in
medicine. Drugs made from plant sources can
be substituted for those made from chemicals. Herbal medicines,
where different plant parts like leaves, bark, roots and seeds are used as
medicine is also known as a phytomedicine. Herbal medications are safer and more effective in
reducing symptoms than allopathic ones (Balkrishna et al. 2021). The combination
of two or more herbs to make medicine is known as a polyherbal phytomedicine.
Plants are rich in secondary
metabolites such as alkaloids, flavonoids, tannins,
amines, terpenoids and glycosides that possess antimicrobial and antioxidant
properties are promising elements used in the pharmaceutical industry (Sharanabasappa et al. 2007; Newman et al. 2016; Hosseinzadeh et al. 2015). Many
phytoconstituents such as
flavonoids and polyphenols
can heal wounds, in
addition to having
antioxidant, anti-inflammatory, and
antimicrobial actions that
contribute to wound healing
processes and are
generally easily accessible and have limited side effects (Rosas
Cruz et al. 2020).
One such
example of phytomedicine is an herbal ointment which is a semi-solid
preparation applied on skin surfaces to treat various skin-related infections
and inflammation. Various plants have been thoroughly examined for their medicinal
and antimicrobial properties. Barauna (Schinopsis brasiliensis), roselle
(Hibiscus sabdariffa), rosemary (Rosmarinus officinalis), clove (Syzygium
aromaticum), and thyme (Thymus vulgaris), tobacco (Nicotiana
tabacum), Rose myrtle (Rhodomyrtus tomentosa) are some
natural antimicrobials that inhibit Staphylococcus aureus and Escherichia
coli (Izabella et al. 2018; Gonelimali et al. 2018; Ameya et al. 2017; Mordmuang et al.
2015).
In this
article, a polyherbal ointment was formulated using peels of orange (Citrus
sinensis), flower of Marigold (Calendula officinalis), leaves of
Neem (Azadirachta indica), raw Turmeric (Curcuma longa), fruit of
Nutmeg (Myristica fragrans) against bacteria causing a skin
infection. Oranges
are abundant in vitamin C and tannins,
terpenoids, flavonoids, saponins are major phytochemicals present in Orange
peel (Gotmare and Gade 2018). The existence of phenolic substances in Citrus
species imparts anti-inflammatory, antioxidant, antiviral, antiallergenic
activities, antimicrobial properties (Mankar et al. 2017;
Escobedo-Avellaneda et al. 2014). Marigold contains a broad range of flavonoids,
carbohydrates, amino acids, saponins, phenolic substances, and tannins (Chakraborthy 2010). Marigold is studied for its application in medicine because
of its anti-inflammatory,
antibacterial activities, wound healing and anti-cancer
capabilities (Parente et al. 2012; Mahmoud 2013), it also
has coagulation property (Dasgupta 2016). Neem has gained recognition for
its wide range of medicinal properties. Different parts of the neem like its
leaves, fruit, bark, seeds, and roots exhibit different types of properties
like anti-inflammatory, anti-ulcer, anti-fungal, antiviral, antiseptic,
antipyretic, anti-cancer properties. The leaves of neem contain saponins,
tannins, protein, carbohydrate, alkaloid, and phenols (Subapriya R 2005; Sharma C i. 2014). Nutmeg
has alkaloids, steroids, tannins, flavonoids, phenolics, and glycosides (Saxena R 2012). Nutmeg oil
is effective against Staphylococcus aureus, Staphylococcus epidermis, Shigella dysenteriae, Salmonella
typhi, and possesses antimalarial properties against Plasmodium
falciparum (Nurjanah et al. 2017; Ibrahim et al. 2020). Turmeric
is being used in cosmetics for a long time. It has carbohydrates, alkaloids, coumarins,
flavonoids, steroids, phenols , tannins, saponins, and antimicrobial
activity against Lactobacillus, Staphylococcus aureus, Escherichia
coli, Salmonella typhi, and Candida albicans, it also
has anti-cancer and an anti-diabetic activity (Gupta et al. 2015; Gul et al. 2015; Kukula-Koch et al. 2018; Asadi et al. 2019; Panahi et al. 2017).
Plants
used for their therapeutic qualities
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SAMPLE
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PROPERTIES
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ORANGE
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Phenolic
compounds present in Citrus species impart anti-inflammatory, antioxidant,
antiviral, antiallergenic activities.
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RAW TURMERIC
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Anti-microbial,
anti-inflammatory, anticancer, antioxidant.
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Curcumin
when combined with antibiotics as sub-inhibitory dosage, it enhances its
anti-bacterial effect against S. aureus
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MARIGOLD
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Highly
effective against wound, acne, skin inflammation, burns, rashes, scars etc.,
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Marigold
flowers can be used to cure swelling caused by wasp or bee
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NEEM
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Anti-inflammatory,
anti-ulcer, anti-fungal, antiviral, antiseptic and antipyretic properties
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NUTMEG
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Ethanol
and acetone extract of nutmeg exhibits anti-bacterial property against
gram-positive bacteria like Staphylococcus aureus
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Acetone
extract of nutmeg seed has anti-oxidant property in addition to anti-microbial
property against Staphylococcus aureus and Aspergillus niger
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Materials
and Methods
a) Sample Collection and Extract preparation:
Leaves of
neem (Azadirachta indica), peels of
orange (Citrus sinensis), flower of
Marigold (Calendula officinalis),
were obtained, rinsed with water dried for 3-4 days at room temperature. Fruit
of Nutmeg (Myristica fragrans) and
Raw Turmeric (Curcuma longa) were not
dried their extracts were prepared by Maceration in 100 ml saline buffer. Extract of orange peel (10 gm) was obtained
by Soxhlet method in 100ml of saline buffer. Following that, extracts were concentrated
under concentrated under controlled temperature of 40-50°C and then preserved
at -20°C.
b)
Thin
layer chromatography (TLC)
TLC was used to check for phytochemicals in extracts
using silica gel as stationary phase and Isopropanol and acetone (2:1) as the
mobile phase.
c)
Phytochemical
tests
Test for Alkaloid
Wagner’s test: 1ml of plant extract was added to 1mL
of Wagner’s reagent. (2g of iodine and 6g of KI in 1000mL of water) The
presence of alkaloids is indicated by a reddish-brown precipitate.
Test for Saponin
To 1ml of plant extract, 5 - 10mL distilled water
was added and shaken. Froth formation indicates the presence of saponin.
Test for Tannin
Ferric chloride test: 2ml of 5% ferric chloride
solution was added to 1mL of plant extract and blue, black or dark green colour
indicates the presence of tannin.
Test for Flavonoids
2ml of 1% NaOH was added to 1ml of plant extract. Flavonoids
are indicated by the colour yellow.
Test for Starch
2 drops of iodine solution were added to 2mL of
plant extract. The presence of starch is indicated by a blue-black color.
Test for Carbohydrate
Fehling’s test: 0.5mL of Fehling A and Fehling B was
added to the 0.5mL of plant extract and the mixtures were then heated in a
water bath. Carbohydrates are present when a brick-red precipitate of cuprous
oxide forms.
Test for Fat
1mL of distilled water and few drops of ethanol were
added to 1mL of plant extract, formation of white precipitate indicates the
presence of fat.
d)
Anti-bacterial
test
Anti-bacterial test was carried out using the agar
well diffusion method. in nutrient agar media. 100µl extracts were tested
against Staphylococcus aureus, using ciprofloxacin as a positive
control. The Zone of inhibition was measured.
e)
Minimum
inhibitory concentration (MIC)
MIC was performed at 50µl, 75µl and 100µl
concentrations using S. aureus as bacterial test sample with agar well
diffusion method and the Zone of inhibition was calculated.
f)
Formulation
of polyherbal ointment
Polyherbal ointment was formulated by mixing the
constituents of aqueous and oil phase which was calculated for 20 ml (Table No.
1)
Constituents of both the phases were weighed, mixed,
heated separately at 70ºC and mixed together, heated again at 70ºC till the
ointment was formed, stored at 4 °C
g)
Evaluation
of formulated herbal ointment
Physical
parameters:
Formulated herbal ointment was examined for physical
characteristics like pH, odour, colour, appearance and texture.
Minimum
inhibitory concentration:
MIC was performed against Staphylococcus aureus,
Escherichia coli.
Comparative
study:
The efficiency of formulated ointment was contrasted
with the different commercially available market herbal creams i.e., Boroplus,
Ayur herbal cream, Avon herbal cream by agar well diffusion method against
isolated skin bacteria.
Table
No.1: Constituents of aqueous and oil phase for 20ml
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PHASE
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CONSTITUENTS
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% COMPOSITION
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WEIGHT AND VOLUME
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AQUEOUS PHASE
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Extracts
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5
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0.5 ml
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Glycerine
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6
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0.6 ml
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Water
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70
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7 ml
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OIL PHASE
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Stearic acid
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20
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2gm
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KOH
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0.5
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0.05 gm
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Sodium Carbonate
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0.5
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0.05 gm
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Results
Phytochemical analysis by TLC and Phytochemical
test
Extracts of orange and neem have demonstrated the
presence of alkaloid, saponin, flavonoids, tannin, fat, carbohydrate, protein,
starch while that of raw turmeric shows the presence of alkaloid, saponin, tannin,
fat and starch. Extract of nutmeg comprises of alkaloid, saponin, tannin,
carbohydrate, protein and fat and marigold have demonstrated the presence of
alkaloid, saponin, flavonoids, tannin, carbohydrate, protein and starch. (Table
No. 2 and 3)
Table
No. 2: RF value of extracted sample of Peels of orange (Citrus sinensis), Flower of Marigold (Calendula officinalis), Raw Turmeric (Curcuma longa), Fruit of Nutmeg (Myristica fragrans), Leaves of Neem (Azadirachta indica)
Table No. 3: Phytochemical
analysis of orange peel, Marigold, Raw Curcumin, Nutmeg, Neem
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EXTRACTS
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ALKALOID
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SAPONIN TEST
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FLAVANOID TEST
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TANNIN TEST
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CARBOHYDRATE TEST
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PROTEIN TEST
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FAT TEST
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STARCH TEST
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Orange Peel
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Positive
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Positive
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Positive
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Positive
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Positive
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Positive
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Positive
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Positive
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Marigold
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Positive (mildly)
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Positive
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Positive
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Positive
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Positive (mildly)
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Positive
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Negative
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Mildly positive
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Raw Curcumin
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Positive
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Positive
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Negative
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Positive
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Negative
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Negative
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Positive
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Positive
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Nutmeg
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Positive (mildly)
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Positive
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Negative
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Positive
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Positive
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Positive
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positive (mildly)
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Negative
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Neem
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Positive
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Positive
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Positive
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Positive
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Positive
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Positive
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Positive
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Positive
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Anti-bacterial test
The measured Zone of inhibition (ZOI) of 100µl of
extracts of orange peel, Neem leaves, Raw turmeric, nutmeg and marigold against
S. aureus was 10 mm, 12mm, 16mm, 6mm and 3mm respectively using
ciprofloxacin as positive control (17mm)(Table No. 4 Figure No. 1 a and 1 b).
Further the Minimum inhibitory concentration was estimated against S. aureus
with each of the extracts where MIC for orange was observed as 0.005g/µl,
Neem as 0.005375 g/µl, that of Raw turmeric as 0.005 g/µl, Nutmeg as 0.0125
g/µl and Marigold as 0.00765 g/µl. (Table
No.5, Figure No.2)
Table No. 4: Anti-microbial
test for Leaves of Neem (Azadirachta indica), Peels of orange (Citrus
sinensis), Raw Turmeric (Curcuma longa),
Fruit of Nutmeg (Myristica fragrans) and the Flower of Marigold (Calendula
officinalis) against S. aureus.
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EXTRACTS
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VOLUME (µL)
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ZOI (mm)
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Positive control
(Ciprofloxacin)
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10
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17
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Neem
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100
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12
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Orange peel
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100
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10
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Raw turmeric
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100
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16
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Nutmeg
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100
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6
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Marigold
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100
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3
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Table No.
5 : Minimum Inhibitory Concentration (MIC) test for peels of orange (Citrus sinensis), Flower of Marigold (Calendula officinalis), Raw
Turmeric (Curcuma
longa), Fruit of Nutmeg (Myristica fragrans), Leaves of Neem (Azadirachta
indica), against S. aureus
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SAMPLE
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CONCENTRATION
(µg/l) AT DIFFERENT VOLUME (µL)
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50
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ZOI
(mm)
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75
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ZOI
(mm)
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100
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ZOI
(mm)
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ORANGE PEEL
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0.005
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0.5
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0.0075
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0.7
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0.01
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2
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MARIGOLD
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0.0051
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0
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0.00765
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3
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0.0102
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6
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RAW CURCUMIN
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0.0050
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2
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0.0075
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4
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0.01
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7
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NUTMEG
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0.00625
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-
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0.009375
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-
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0.0125
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6
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NEEM
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0.005375
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5
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0.0080625
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10
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0.01075
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12
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Figure No. 1 (a) Anti-microbial test for Leaves
of Neem (Azadirachta
indica) Peels of orange (Citrus sinensis), Raw Turmeric (Curcuma longa) against S. aureus. (b)
Anti-microbial test for Flower of Marigold (Calendula officinalis), Fruit of Nutmeg (Myristica
fragrans) against S. aureus
Figure No. 2 MIC test for
(a) Fruit of Nutmeg (Myristica fragrans) (b) Leaves of Neem (Azadirachta indica), (c) Flower of Marigold
(Calendula
officinalis), (d) Raw Turmeric (Curcuma longa), (e) Peels of orange (Citrus
sinensis), against S. aureus
Evaluation
of formulated herbal ointment
Physical parameters
Different physical properties of formulated herbal
ointment was checked which indicates that the formulated herbal ointment was
viscous and alkaline with a of pH =9.51 (Table No. 6)
Table No. 6: Physical
properties of formulated herbal ointment
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S. No.
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PROPERTIES
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RESULT
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1.
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Colour
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Brown
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2.
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Odour
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Pungent
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3.
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pH
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9.51
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4.
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Texture
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Viscous
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5.
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Appearance
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Opaque
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Minimum
inhibitory volume (MIV)
ZOI at 50µl, 75µl and
100µl was measured against S. aureus
as 11, 13, 16 mm, E.coli-5, 7, 9 mm ,isolated Skin bacteria-11,12,14
mm using ciprofloxacin as positive control (Table No.7,8, Figure No. 3 a, b, c)
Table No.7 Minimum Inhibitory
Volume (MIV) of formulated herbal ointment against S.aureus at different
concentrations.
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VOLUME TAKEN (µl)
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ZOI (mm)
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10 (Positive control)
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17
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50
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11
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75
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13
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100
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16
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Table No. 8 Minimum Inhibitory
Volume (MIV) of formulated herbal ointment against E. coli at different
concentrations.
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VOLUME TAKEN (µl)
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ZOI (mm)
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10 (Positive control)
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12
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50
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5
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75
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7
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100
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9
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Figure No. 3 MIV test of
formulated herbal ointment against (a) S.
aureus, (b) Skin bacteria and (c) E. coli where zone was obtained with
each concentration.
Comparative study
The AST of the available market formulations of
Boroplus, Ayur herbal cream and Avon herbal cream were compared to the prepared
polyherbal formulation. The ZOI was measured as 4mm, 1mm and 9mm for Boroplus,
Ayur herbal cream and Avon herbal cream respectively against isolated skin bacteria
while the ZOI obtained by the formulated herbal ointment was measured as 10mm.
(Table No.9 Figure No. 4)
Table
No.9 depicting ZOI in comparative study of different
herbal creams with the formulated herbal ointment
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SAMPLE
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VOLUME (µL)
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ZOI (mm)
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Boroplus
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100
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4
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Avon herbal cream
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100
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9
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Ayur herbal cream
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100
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1
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Formulation
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100
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10
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Figure No. 4 Comparative study of different herbal creams
(Boroplus, Avon, Ayur) with formulated herbal ointment against Skin bacteria.
Conflict of interest Author
declares that there is no conflict of interest.
Funding
information not applicable.
Ethical
approval not applicable.
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