Pharmaceutical Sciences Asia Pharm Sci Asia 2021; 48(6), 523-534

DOI:10.29090/psa.2021.06.21.031

Research Article

Antimicrobial activity of a combination of three natural

plant extracts and development of a herbal soap
Nirupama Wijayawardhana1, Dhanushi Cooray1, Banukie Jayasuriya1*
Inoka Uluwaduge2, Farah Meedin3, Menuka Arawwawala4
1 Department of Pharmacy and Pharmaceutical Sciences, Faculty of Allied Health Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka
2 Department of Basic Sciences, Faculty of Allied Health Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka
3 Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, University of Sri Jayewardenepura, Nugegoda , Sri Lanka
4 Industrial Technology Institute, Colombo 07, Sri Lanka

ABSTRACT

Azadirachta indica, Cassia fistula and Nelumbo nucifera are medicinal plants that are frequently found
in Sri Lanka. Although the antimicrobial activity of these plants has been investigated individually, the
effectiveness of a combined extract has not been determined in previous studies. This study aimed to evaluate
the antimicrobial activity of A. indica, C. fistula, N. nucifera and their combined extract to formulate an antimi-
crobial herbal soap. Different concentrations of aqueous and ethanol extracts of powdered leaves of A. indica,
C. fistula and flowers of N. nucifera were prepared separately. Each extract was tested against Staphylococcus
aureus, Pseudomonas aeruginosa and Candida albicans using the agar well diffusion method. Combined extract
was prepared using selected extracts of each plant with the highest antimicrobial activity. Combined extract
formulated from ethanol extract of C. fistula and aqueous extracts of A. indica and N. nucifera was incorporated
into a herbal soap. The antimicrobial activity of the combined extract and the herbal soap was determined using
the agar well diffusion method. Combined extract and formulated soap exhibited antimicrobial activity against
tested organisms with the highest activity against S. aureus. The physical and chemical parameters of the formulated
herbal soap were determined. The pH of the formulated soap at 28℃ was 9.11, the percentage of alcohol insoluble
matter and free alkali were 24.6% and 1.6% respectively, which were within the accepted range. In conclusion,
compared to the results of individual extracts, the soap has demonstrated enhanced antimicrobial activity against
tested organisms. Further development of herbal soap as a value added product would be beneficial.

Keywords:
Azadirachta indica, Antimicrobial activity, Cassia fistula, Combined extract, Herbal soap, Nelumbo nucifera

1. INTRODUCTION synthetic compounds and availability. Recent studies

have revealed that natural constituents of higher plants
Medicinal plants have been used in traditional include new sources of antimicrobial agents2. Plants
medicine predominantly in Asian countries and they are possess antimicrobial properties due to their secondary
considered as the root of traditional medicine. Many metabolites. Tannins, flavonoids, phenolic compounds
recent research studies showed that most of the medicinal and alkaloids have demonstrated in vitro antimicrobial
plants possess diverse biological activities such as activity3.
antibacterial, antifungal, antiviral, antioxidant and anti- Sri Lanka has a diverse medicinal plant system.
inflammatory effects, etc1. As one of the best sources of Medicinal herbs used in Ayurveda medicine are reported
obtaining antimicrobial agents, medicinal plants are to possess antimicrobial activity. According to Hewage
gaining attention in the present world today. That is et al.,4 in Sri Lanka, there are about 3300 flowering plants
mainly due to increased resistance to synthetic com- in which 830 species (25%) are endemic to Sri Lanka.
pounds, inexpensive, fewer side effects compared to There are a vast number of conditions that can

*Corresponding author:
*Banukie Jayasuriya banukie@sjp.ac.lk

Pharmaceutical Sciences Asia © 2021 by

Faculty of Pharmacy, Mahidol University, Thailand is licensed under CC BY-NC-ND 4.0. To view a copy of this license, visit
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N. Wijayawardhana et al. Pharm Sci Asia 2021; 48(6), 523-534

affect skin diseases. Skin infections are of several types the plant13. Mahmoodin, margolone, mergolonone and
that differ from the type of pathogen which causing isomargolonone which have been isolated from A.
infection such as fungal infections and bacterial indica show antibacterial activity whereas cyclic
infections5. Recently there is a surge of using various trisulphide and cyclic tetrasulphides show antifungal
antiseptics, including commercially available synthetic activity14. The plant is used in Ayurveda, Unani and
soaps and alcohol based sanitizers. Even though these homeopathy for the treatment of many infectious,
formulations reduce the transmission of contagious metabolic or cancer diseases9. The leaves are bitter,
diseases, these are produced on a large scale with harsh astringent, acrid, depurative, demulcent and refrige-
chemicals, synthetic ingredients such as parabens, rant15 . A strong decoction of the fresh leaves has
phthalates, petrochemicals, cheap fragrance oils and antiseptic properties and is used for cleaning wounds,
artificial colors all of which may lead to skin irritation ulcers and leprosy16. Leaves poultice is used for boils
and resistance among pathogens. Hence there is an and sores treatment whereas the infusion is reported to
urgent need to develop herbal soaps and other disinfec- be used in the treatment of malarial and intermittent
tants and evaluate their efficacy. fevers. Leaf juice is given in worms, jaundice and skin
Cassia fistula belongs to the family of Fabaceae diseases11,16,17. In Sri Lanka, the juice of the fresh leaves
and is commonly known as a golden shower plant. It is given with rock salt for intestinal worms and with
has greenish-grey bark and has compound leaves. The honey for jaundice and skin diseases10. The leaf paste
conspicuous feature of this plant is golden yellow color has been used externally for small pox treatments
hanging flower bunches about 40 cm in length6. Recent whereas the tincture is applied locally to treat bruises
research findings have demonstrated that C. fistula and sprains. Leaf ash mixed with ghee is useful in
possesses antioxidant, anti-mutagenic, antitumor, anti- psoriasis16. Moreover, A. indica is useful in leucoderma,
microbial and anti-inflammatory activities7. C. fistula pruritus, ophthalmopathy, dyspepsia, tuberculosis,
consists of various constituents such as flavonoids, burning sensation and eczema and exhibits wound
phenols and proanthocyanidins. The bark, leaves and healing properties11,15.
flowers of the plant were evaluated for potential Nelumbo nucifera is an aquatic plant, belonging
constituents with antimicrobial activity7, C. fistula is to the family of Nelumbonaceae. This is a rhizomatous
considered as one of the abundantly used medicinal plant consisting of a creeping, elongated stem with
plants in Unani and Ayurvedic medical systems. Fruit nodal roots. It is having cup-shaped aerial leaves and
pulp, bark, flowers, pods, leaves and roots of the plant flat-shaped floating leaves. The colour of the flower
are used8. Preparations like infusions, decoctions or varies from white to reddish-pink and has a pleasant
powders which are either alone or in combination with fragrance. The flower of N. nucifera demonstrated hypo-
other medicinal plants are used in traditional medicine9. glycemic, antipyretic, antioxidant, antimicrobial and
Mainly the tender leaves are used as a purgative while antihypertensive abilities. Furthermore, it also acts as
the older ones together with the bark are ground into a anti acne due to its antibacterial properties17. A variety
paste and applied during ringworm infections, insect of chemical constituents with different therapeutic
bites, rheumatism, facial paralysis, leprosy, chronic activities were isolated from the flowers of N.nucifera18.
eczema and psoriasis10. In traditional system of medi- The flower is used in Ayurveda, Siddha, Unani and other
cine, the plant is used against skin diseases, diabetes, medicinal systems9 and reported to be astringent refri-
haematemesis, worm and microbial infections, abdomi- gerant and cardiotonic14. The stamens of the flowers are
nal diseases, abdominal tumours, heart diseases, during used for bleeding piles and debility and weakness in
retention of urine, abdominal colic, heamostatic, due to children10. Flowers with stamens and juice of the flower
its antipyretic and analgesic nature and to reduce stalk are used for diarrhea, cholera, fever, liver complaints
oedema and prevent burning sensation8,11. Chakramardha and gastric ulcers in Ayurveda10. The dose of the flower
tailamu, is a compound ayurvedic oil prepared using C. extract used in decoctions is 12-24 g and has been
fistula which is beneficial in eczema, ringworm and claimed in the treatment of skin diseases9,11. Syrup made
other skin diseases. The plant is used in preparations such from flower is recommended for coughs, dysentery and
as Maha-marichayadi taila, aragvadhadi-kwatha etc9. haemorrhages and the ground petals are assumed to be
Azadirachta indica, which is often known as effective in rectifying syphilis in Malaya10. A scanty
Neem, belongs to the family of Meliaceae. Leaves of literature was available on formulation of herbal soaps
the plant are compound and each carry 5-15 leaflets. and evaluating their antimicrobial activity.
According to several studies conducted, it has been Moreover, the available literature did not support
demonstrated that different plant parts of A. indica evidences on any herbal formulations based on com-
possess diverse medicinal properties such as antibac- bined extracts of C. fistula, A. indica and N. nucifera.
terial, antifungal, antiviral, anti-oxidant, anti-malarial, Therefore, the purpose of this study was to formulate a
etc12. Azadirachtin, nimbin, nimbidol and nimbolinin herbal soap using the extracts of C. fistula, A. indica and
are important compounds that have been isolated from N. nucifera and to investigate the antimicrobial activity

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of the combined extract and the herbal soap against Wells were cut by using a sterile dropper with a 6 mm
common microorganisms. Furthermore, this study was diameter. A drop of molten agar was added to each well
aimed to evaluate the physicochemical parameters of to seal the bottom of the well. Stock solutions were
the prepared herbal formulation. prepared for each extract with a concentration of 1000
mg/ml in 1% dimethyl sulphoxide (DMSO). Each stock
2. MATERIALS AND METHODS solution was diluted using 1% DMSO and a concentra-
tion series ranging from 31.25-1000 mg/ml was prepared.
2.1. Study setting From each concentration, 50µl were added to the wells.
Bacterial cultures and fungal cultures were
The study was conducted at the Department of incubated at 37℃ for 24 hrs. and 48 hrs. respectively.
Pharmacy and Pharmaceutical Sciences and Depart- 0.03% of gentamycin and 1% of clotrimazole were used
ment of Medical Laboratory Sciences, Faculty of Allied as a positive control for bacteria and fungi respectively,
Health Sciences, University of Sri Jayewardenepura, Sri and 1% DMSO was used as the negative control. Each
Lanka and Industrial Technology Institute, Colombo 07, concentration includes triplicates. Antimicrobial activity
Sri Lanka. was determined by measuring the diameter of the zone
of inhibition around the well against each microorga-
2.2. Collection of plant materials and authentication nism20,21.

Fresh leaves of A. indica, white flowers of N. 2.5. Determination of the effective extract of each plant
nucifera, and fresh leaves of C. fistula were collected and effective concentration of selected plant extracts
from Southern Province and Western Province, Sri against tested microorganisms to formulate a combined
Lanka. Plants were authenticated at the National Her- extract
barium, Botanical Gardens, Peradeniya, Sri Lanka and
the voucher specimens were deposited under reference According to the results of the antimicrobial
numbers, A Z Pharm .01 (A. indica), N N Pharm .01 (N. activity of each extract of the three plants, dose-response
nucifera) and C F Pharm .01 (C. fistula). curves were graphed separately. The suitable types of
extracts were selected from each dose-response curve
2.3. Preparation of extracts by considering the IC50 value of each plant extract to
incorporate into the combined extract. Effective con-
The collected plant parts (leaves and flowers) centrations of extracts selected were determined. Dose-
were washed, air dried for three days and dried in a hot response curves of selected plant extracts were graphed
air oven at 40℃ until constant weight was obtained and separately against each microorganism tested. Effective
powdered to a coarse powder, and stored in air tight concentrations were selected at the highest point of the
bottles for the studies. A sample (50 g) of each plant was linear range. A common effective concentration for all
added to a round bottom flask containing 150 ml of tested organisms was selected from the linear range of
ethanol or distilled water and boiled for four hours. Then the graph.
the extract was filtered and the filtrate was concentrated
using a rotary evaporator. Water extract was freeze- 2.6. Formulation of herbal soap using the combined
dried. The extracts were stored at 4℃ until used19. extract

2.4. Determination of antimicrobial activity of selected Glycerin soap base was weighed and melted in
plant extracts a water bath. Previously determined concentrations of
plant extracts were prepared by dissolving the relevant
The antimicrobial activities of the selected plant solvents (Assuming that the active ingredient should be
extracts were determined by the agar well diffusion about 5% of the total weight of soap). The plant extracts
method20. Isolates of Candida albicans, Staphylococcus were added to the melted soap base. Stearic acid was
aureus ATCC 25923 and Pseudomonas aeruginosa dissolved in a small amount of hot water and added to the
ATCC 27853 were obtained from the Department of mixture. As the fragrance enhancer, the volatile oil of
Microbiology, Faculty of Medical Sciences, University of N. nucifera was added. The ultimate mixture was stirred
Sri Jayewardenepura. Subcultured organisms were used using a magnetic stirrer. The melted mixture was poured
to prepare microbial suspensions. The turbidity of the into separate moulds and left until solidified22.
suspensions was adjusted to 0.5 McFarland standard
(1×108 CFU/ml inoculum). The prepared inoculum was 2.7. Determination of the antimicrobial activity of the
streaked by using a sterile cotton swab on the surface combined plant extract and formulated herbal soap
of Mueller Hinton Agar (MHA) using the spread plate
method. Then the plates were allowed to dry for 5 min. The antimicrobial activity of the combined

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extract was determined by the agar well diffusion makes up to 50 ml. Finally, the height of the foam was
method20. According to the proportions determined, the measured above the 50 ml mark of the measuring
selected concentration of each plant extract was cylinder22.
introduced into the wells of the agar plates that were
previously streaked with S. aureus, P. aeruginosa and 2.8.5. Foam retention
C. albicans. A percentage of 0.03% gentamycin and 1%
clotrimazole were used as the positive control for Formulated soap solution (1%) was prepared by
bacteria and fungi respectively. 1% DMSO was used as dissolving the soap in distilled water. A volume of 25
the negative control. The plates were incubated at 37˚C ml of soap mixture was placed in a 100 ml measuring
for 24 hrs. (48 hrs. for fungi) and the diameters of the cylinder and shaken it for 10 times. The volume of foam
zone of inhibition were measured. Six concentrations of was recorded for 10 min at 1-min intervals22.
formulated soap were prepared by dissolving it in 1%
DMSO. Antimicrobial activity of each solution was 2.8.6. Alcohol insoluble matter
tested by agar well diffusion method22.
Formulated soap (5 g) was dissolved in 50 ml of
2.8. Evaluation of physicochemical parameters of the warm ethanol vigorously. The resulting solution was
formulated herbal soap filtered through a tarred filter paper with 20 ml of
additional warm ethanol. Then the filter paper was kept
2.8.1. Physical characteristics in the oven for 1 hour at 105˚C. Finally, the weight of the
residue was weighed22.
Odour, colour and appearance of the
formulated soap were observed22. 2.8.7. Moisture / Volatile matter

2.8.2. Determination of pH Formulated soap (5 g) was placed in an oven for

2 hrs at a temperature of 105℃ and repeated until a
Formulated soap was dissolved in 100 ml of constant weight was obtained and the % moisture was
distilled water and stored for 2 hours. Then pH was calculated22.
measured by a calibrated pH meter22.
2.9. Statistical analysis
2.8.3. Determination of percentage free alkali
The data were presented as mean±SD (Standard
Formulated soap (5 g) was dissolved in 50 ml of Deviation) of three replicates. One-way analysis of
neutralized alcohol. The mixture was refluxed for 30 variance (ANOVA) was performed by using IBM SPSS
minutes. The resulted mixture was cooled and titrated Statistics 25.0 software to determine significant group
with 0.1 N HCl using phenolphthalein as the indicator22. differences and means were considered as statistically
significant if p<0.05.
2.8.4. Foam height
3. RESULTS
Formulated soap (0.5 g) was dissolved in 25 ml
of distilled water and it was transferred into a 100 ml 3.1. Determination of the zones of inhibition of different
measuring cylinder. Volume of soap mixture was made concentrations of C. fistula leaf extracts against selected
up to 50 ml with water. Then the cylinder was shaken microorganisms
25 times and allowed to stand until the aqueous volume

Table 1. Diameter of the zones of inhibition of different concentrations of leaf extracts of C. fistula against selected microorganisms.

Concentration of C. fistula leaf extract The diameter of zone of inhibition (mm) of leaf extracts of C.fistula
(mg/ml) Ethanol extract Aqueous extract
S.aureus P.aeruginosa C. albicans S. aureus P.aeruginosa C. albicans
1000 21.0±0.7* 10.0±0.5* 0.0±0.0 18.0±0.7* 12.0±0.5* 0.0±0.0
500 18.0±0.7* 8.0±0.5* 0.0±0.0 16.0±0.7* 7.0±0.5* 0.0±0.0
250 15.0±0.7* 0.0±0.0 0.0±0.0 11.0±0.7* 0.0±0.0 0.0±0.0
125 13.0±0.7* 0.0±0.0 0.0±0.0 9.0±0.7* 0.0±0.0 0.0±0.0
62.5 9.0±0.7* 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
31.25 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Positive control 37.0±1.7* 32.0±1.7* 25.0±1.7* 33.0±1.7* 31.0±1.7* 31.0±1.7*
Negative control 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Data presented as Mean±SD
*Significant when compared with the negative control; p<0.05

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Table 2. Diameters of the zone of inhibition of different concentrations of white flower extracts of N. nucifera against selected
microorganisms.

Concentration The diameter of zone of inhibition (mm) of white flower extracts of N. nucifera
(mg/ml) of N. nucifera flower extract Ethanol extract Aqueous extract
S.aureus P.aeruginosa C. albicans S. aureus P.aeruginosa C. albicans
1000 19.0±0.5* 10.0±0.3* 0.0±0.0 20.0±0.5* 10.0±0.3* 10.0±0.3*
* * * *
500 8.0±0.5 9.0±0.3 0.0±0.0 10.0±0.5 7.0±0.3 8.0±0.3*
250 12.0±0.5* 7.0±0.3* 0.0±0.0 16.0±0.5* 0.0±0.0 7.0±0.3*
* *
125 8.0±0.5 0.0±0.0 0.0±0.0 15.0±0.5 0.0±0.0 0.0±0.0
62.5 0.0±0.0 0.0±0.0 0.0±0.0 7.0±0.5* 0.0±0.0 0.0±0.0
31.25 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Positive control 31.0±0.5* 31.0±0.3* 25.0±0.3* 30.0±0.5* 30.0±0.3* 31.0±0.3*
Negative control 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Data presented as Mean±SD
*Significant when compared with the negative control; p<0.05

Table 3. Diameters of the zone of inhibition of different concentrations of leaf extracts of A. indica against selected microorganisms.

Concentration The diameter of zone of inhibition(mm) of leaf extracts of A. indica

(mg/ml) of A. indica leaf extracts Ethanol extract Aqueous extract
S.aureus P.aeruginosa C. albicans S. aureus P.aeruginosa C. albicans
1000 16.0±0.5* 0.0±0.0 0.0±0.0 21.0±0.5* 0.0±0.0 0.0±0.0
500 13.0±0.5* 0.0±0.0 0.0±0.0 18.0±0.5* 0.0±0.0 0.0±0.0
250 12.0±0.5* 0.0±0.0 0.0±0.0 15.0±0.5* 0.0±0.0 0.0±0.0
125 8.0±0.5* 0.0±0.0 0.0±0.0 13.0±0.5* 0.0±0.0 0.0±0.0
62.5 7.0±0.5* 0.0±0.0 0.0±0.0 9.0±0.5* 0.0±0.0 0.0±0.0
31.25 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Positive control 34.0±0.5* 32.0±0.3* 25.0±0.3* 31.0±0.5* 30.0±0.3* 25.0±0.3
Negative control 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Data presented as Mean±SD
*Significant when compared with the negative control; p<0.05

Figure 1. Dose-response curves of ethanol and aqueous extracts of C. fistula, A. indica and N. nucifera against S. aureus. A: C. fistula, B: A.
indica and C: N. nucifera.

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Figure 2. Dose-response curves of ethanol extract of C. fistula, aqueous extract of A. indica and aqueous extract of N. nucifera against tested
organisms.
A: Ethanol extract of C. fistula against S. aureus and P. aeruginosa
B: Aqueous extract of A. indica against S. aureus
C: Aqueous extract of N. nucifera against S. aureus, P. aeruginosa and C. albicans

The antibacterial activity of different concen- that the different concentrations of aqueous extract of
trations of ethanol and aqueous extracts of C. fistula is white flower of N. nucifera showed antimicrobial
represented in Table 1. The results indicated that the activity at variable degrees against all three selected
plant extracts showed antibacterial activity at variable organisms. The extract showed the highest antimi-
degrees against S. aureus and P. aeruginosa. The highest crobial activity against S. aureus. However, different
antibacterial activity was exerted at 1000 mg/ml of both concentrations of ethanol extract of white flowers of
extracts against S. aureus and P. aeruginosa,. However, N. nucifera showed antimicrobial activity against S.
both extracts did not show antifungal activity against C. aureus and P. aeruginosa except for C. albicans. When
albicans. Both extracts showed moderate antibacterial consider the both extracts, the aqueous extract of the
activity against P. aeruginosa at the concentrations of white flower of N. nucifera showed the highest activity
500 and 1000 mg/ml. When consider the both extracts, against S. aureus.
the ethanol extract of leaves of C. fistula at 1000 mg/ml
showed the highest activity against S. aureus. 3.3. Determination of the zones of inhibition of
different concentrations of leaf extracts of A. indica
3.2. Determination of the zones of inhibition of against selected microorganisms
different concentrations of white flower extracts of
N. nucifera against selected microorganisms The antimicrobial activity of different concen-
trations of ethanol and aqueous extracts of A. indica
The antibacterial activity of different concen- leaves is represented in Table 3. The results indicated that
trations of ethanol and aqueous extracts of N. nucifera the ethanol and aqueous extracts of A. indica leaves
flowers is represented in Table 2. The results indicated showed antibacterial activity against S. aureus only. All

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the tested concentrations of both extracts were exhibited aureus, P. aerogenosa and C. albicans.
antibacterial activity against S. aureus and the aqueous According to the effective concentrations
extract showed the highest activity against S. aureus. selected from each extract, the proportions of the extract
Furthermore, both extracts did not show antifungal in the combination that incorporated into herbal soap
activity against C. albicans. were determined as follows.
C. fistula : N. nucifera : A. indica
3.4. Determination of the effective extract of each plant 250 mg/ml : 250 mg/ml : 1000 mg/ml
and effective concentration of selected plant extracts
against tested microorganisms to formulate a combined 3.5. Determination of the antimicrobial activity of the
extract combined plant extract and formulated herbal soap

Ethanol and aqueous extracts of all three plants Table 4 represents the antimicrobial activity of
of C. fistula, A. indica and N. nucifera at concentrations the combined plant extract. Combined extract showed
ranging from 62.5-1000 mg/ml exhibited maximum the highest activity against S. aureus. Combined extract
antimicrobial activity against S. aureus with zones of showed enhanced activity compared to individual
inhibition ranging from 7.0 to 21.0 mm. Hence, the extracts against the tested microorganisms.
half-maximal inhibitory concentration (IC50) values of The antimicrobial activity of the formulated
ethanol and aqueous extracts of all three plants of herbal soap was investigated using the agar well
C. fistula, A. indica and N. nucifera were determined diffusion method. Figure 3 and Table 5 represents the
against S. aureus. diameter of the zone of inhibition for S. aureus, P.
Figure 1 shows the IC50 values of ethanol and aeruginosa and C. albicans at different concentrations
aqueous extracts of C. fistula, A. indica and N. nucifera of herbal soap. The diameter of the zone of inhibition
against S. aureus. The values of IC50 of ethanol extracts exhibited by gentamycin for S. aureus and P. aeruginosa
of C. fistula, A. indica and N. nucifera were 87 mg/ml, were 31.0±0.5 mm and 30±0.4 mm respectively and of
119.9 mg/ml and 221 mg/ml respectively. The value of clotrimazole for C. albicans was 26.0±0.4 mm.
IC50 of aqueous extract of C. fistula was 166 mg/ml. The The extracts that exhibited maximal activity
values of IC50 of aqueous extracts of both plants of A. were ethanol extract of C. fistula and the aqueous
indica and N. nucifera were determined as 87 mg/ml. extracts of A. indica and N. nucifera. Ethanol extract of
Since the ethanol extract of C. fistula and the aqueous C. fistula exhibited zones of inhibition ranging from 9.0
extracts of A. indica and N. nucifera were demonstrated to 21.0 mm and 8.0 to 10.0 mm against S. aureus and P.
the maximum inhibition effect, these extracts were aeruginosa respectively. Aqueous extract of A. indica
selected as the suitable extracts to incorporate into the exhibited zones of inhibition ranging from 9.0 to 21.0
combined extract. mm against S. aureus whereas aqueous extract of N.
Effective concentrations of ethanol extract of nucifera exhibited zones of inhibition ranging from 7.0
C. fistula, aqueous extract of A. indica and aqueous to 20.0 mm, 7.0 to 10.0 mm and 7.0 to 10.0 mm against
extract of N. nucifera were determined against the tested S. aureus, P. aeruginosa and C. albicans respectively.
microorganisms. Figure 2 represents the dose-response According to the Table 5, formulated herbal soap
curves of the ethanol extract of C. fistula, aqueous exhibited zones of inhibition ranging from 15.0 to 28.0
extract of A. indica and aqueous extract of N. nucifera mm, 15.0 to 25.0 mm and 15.0 to 25.0 against S. aureus,
against tested microorganisms. P. aeruginosa and C. albicans respectively at a similar
According to the dose-response curve of the range of concentrations. Hence, the formulated herbal
ethanol extract of C. fistula, the effective concentration soap showed enhanced antimicrobial activity compared
ranges for S. aureus and P. aerogenosa were 30-257 to individual extracts.
mg/ml and 173-933 mg/ml respectively. Hence, the
final effective concentration of ethanol extract of C. 3.6. Evaluation of physical and chemical parameters
fistula was selected as 250 mg/ml for both microorga- of formulated soap
nisms. Effective concentration range of aqueous extract
of A. indica for S. aureus was range from 47.44 mg/ml The physical and chemical parameters of the
to 1233.00 mg/ml. Hence 1000 mg/ml was selected as the formulated herbal soap were determined. Parameters
effective concentration of aqueous extract of A. indica such as color, odor, appearance, pH, etc. were tested.
for S. aureus. By considering the dose-response curves Table 6 represents the physical and chemical parameters
of aqueous extract of N. nucifera, the effective concen- of the formulated soap. The formulation exhibited good
tration ranges for S. aureus, P. aerogenosa and C. appearance characteristics as well as the pH was 9.1.
albicans were 35-281 mg/ml, 36-309 mg/ml and 170-776 Other parameters such as percentage free alkali, foam
mg/ml. Hence, 250 mg/ml was selected as the effective height, foam retention, alcohol insoluble matter and
concentration of aqueous extract of N. nucifera for S. percentage of moisture were determined.

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Table 4. Diameters of zone of inhibition of combined extract for selected microorganisms.

Microorganism The diameter of the zone of The diameter of the zone of The diameter of the zone of
inhibition of combined extract inhibition for the positive inhibition for negative control
(mm) control (mm) (mm)
S. aureus 30.0±1.0 37.0±1.0 00.0±0.0
P. aeruginosa 26.0±1.0 32.0±1.0 0.0±0.00
C. albicans 16.0±1.0 25.0±1.0 0.0±0.00
Data presented as Mean±SD

Table 5. Diameters of zone of inhibition of formulated soap for selected microorganisms.

Concentration (mg/ml) The diameter of zone of inhibition (mm) of formulated herbal soap
S. aureus P. aeruginosa C. albicans
1000 28.0±0.5* 25.0±0.4* 25.0±0.4*
500 24.0±0.5* 23.0±0.4* 25.0±0.4*
250 22.0±0.5* 20.0±0.4* 20.0±0.4*
125 20.0±0.5* 19.0±0.4* 19.0±0.4*
62.5 18.0±0.5* 17.0±0.4* 18.0±0.4*
* *
31.25 15.0±0.5 15.0±0.4 15.0±0.4*
Positive control 31.0±0.5* 30.0±0.4* 26.0±0.4*
Negative control 0.0±0.0 0.0±0.0 0.0±0.0
Data presented as Mean±SD
*Significant when compared with the negative control; p<0.05

Table 6. Physical and chemical parameters of the formulated herbal soap.

Color Odour Appearance pH % free Foam height Foam Alcohol Moisture/Volatile

alkali (cm) retention insoluble Matter (%)
(min) matter (%)
Dark
Fragrant Good 9.11 1.6 9.5 7 24.6 15.85
brown

Figure 3. Dose-response curve for formulated soap against S. aureus, P. aeruginosa and C. albicans.

4. DISCUSSION Vast arrays of plants have the potential for

antimicrobial activity. Mahato and Sharma23 have
Herbal soaps are products which are incorpo- reviewed and summarized the antimicrobial properties
rated various plant extracts into the soap base. Now- of some medicinal plants.
adays, herbal soaps are gaining much attention due to The present study has formulated a herbal soap
the various advantages over synthetic soaps. Synthetic using the extracts of C. fistula, A. indica and N. nucifera
ingredients and artificial colors that are incorporated in and the antimicrobial activity of the individual extracts,
soaps may lead to skin irritations. the combined extract and the herbal soap was evaluated

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against the common microorganisms. Furthermore, the inhibition against S. aureus and P. aeruginosa. A simi-
physicochemical parameters of the herbal soap were lar type of finding was observed in the study conducted
evaluated. Local availability and feasibility were con- by Bhalodia and Shukla24. However, there was a growth
sidered in selecting plants possessing antimicrobial inhibition against C. albicans by hydroalcoholic extract
activity. of leaves of C .fistula in the study conducted by Bhalodia
According to the literature, aqueous extract of and Shukla24 whereas no antifungal activity was detected
leaves of A. Indica has shown antimicrobial activity in the present study against C. albicans by both aqueous
against human pathogenic bacteria (S. aureus, Entero- and ethanol extracts of leaves of C. fistula.
coccus feacalis, Proteus mirabilis and P. aeuroginosa) Even though Reddy et al20. have confirmed the
and fungi (Aspergillus fumigatus and C. albicans)20. antimicrobial activity of aqueous extract of leaves of
Hydro-alcohol extract of white flowers N. nucifera A. indica against S. aureus, P. aeuroginosa and C.
has shown antimicrobial activity against S. aureus, P. albicans, the present study did not show any growth
aeruginosa, Escherichia coli, Klebsiella pneumonia, inhibition against P. aeruginosa and C. albicans.
Bacillus subtilis and A. niger21. Hydro-alcohol extracts A limited literature was available on the anti-
of the leaves of C. fistula possessed antimicrobial microbial activity of the white flower of N. nucifera.
activity against human pathogenic bacteria (S. aureus, According to the literature hydroethanolic extract of N.
Streptococcus pyogenes, E. coli, P. aeruginosa) and nucifera white flowers showed antimicrobial activity
fungi (A. niger, A. clavatus and C. albicans)24. C. fistula, against S. aureus and P. aeruginosa21.In this study, the
A. indica and N. nucifera are considered as important aqueous extract of N. nucifera white flower has exhibited
herbs used in various traditional systems of medicine antimicrobial activity against S. aureus, P. aeruginosa
and all parts of the plants are used in traditional medi- and C. albicans. However, Brindha and Arthi21 have not
cine. The selected plants are used in the treatment of evaluated the antifungal activity against C. albicans.
skin diseases, worm and microbial infections, treatment When considering the antimicrobial activity of
of wounds, sores and ulcers etc8,10,11,18. individual plant extracts of this study, the highest activity
Selected plant extracts in the present study was detected against S. aureus, mild and poor activities
possessed antibacterial activity against both Gram-positive were detected against P. aeruginosa and C. albicans
and Gram-negative bacterial isolates and antifungal respectively.
activity against fungal isolates. Antimicrobial activity was characterized by the
To establish the previous findings on Sri IC50 value. The IC50 is used to assess and compare the
Lankan plants, we have evaluated the antimicrobial effectiveness of inhibitory compounds26. Ethanol or
activity of aqueous and ethanolic extracts of leaves of aqueous extract of each plant was selected to formulate
A. indica, white flowers of N. nucifera and leaves of C. the combined extract. The most effective extract of each
fistula against S. aureus (Gram-positive), P. aeruginosa plant was selected by comparing the IC50 values obtained
(Gram-negative) and C. albicans (fungi). Further the from the log dose vs the response curve of each extract
findings of the study supported traditional claims in the against S. aureus. Bhat et al.,27 have used IC50 values to
literature. determine the antimicrobial effectiveness of different
Very few studies were reported about the formu- extracts of Acacia powder. Ethanol extract of C. fistula,
lation of herbal soaps and evaluating their antimicrobial aqueous extracts of A. indica and N. nucifera were
activity. Polyherbal soap formulation prepared using selected based on IC50 values against S. aureus to
coconut oil, castor oil, neem oil, mentha oil and rose petal incorporate into the soap formulation.
extracts has exhibited antimicrobial activity against E. From all three types of plant extracts selected,
coli25. Afsar and Khanam, has formulated a herbal soap a combination of extract was formulated. Proportions
incorporating extracts of C. fistula, Milletia pinnata of each extract in the combination were determined by
and Ficus religiosa and the antimicrobial activity was using the log dose vs. response curve for each extract
evaluated against E. coli (MTCC-1698), S. aureus (MTCC- considering all three organisms. (Concentration that
1143) and P. aeruginosa (MTCC-2453)22. However, there lies within the range for all organisms was selected).
are no any formulations based on combined extracts of Combined extract was included with all three extracts at
C. fistula, A. indica and N. nucifera found in literature. a ratio of 1:1:4 (C. fistula: N. nucifera: A. indica).
Authenticated plants were collected and basic In this study, the antimicrobial activity of the
operations such as pre-washing, drying and grinding combined extract was determined. Results gained for the
were done to obtain a homogenous sample to facilitate growth inhibition (diameter of the zone of inhibition)
extraction. Aqueous and ethanol extracts of selected were comparatively higher than the results of individual
medicinal plants were evaluated for antimicrobial extracts against the S. aureus, C. albicans and P. aruge-
activity by agar well diffusion method. According to the nosa. Enhanced antimicrobial activity of the combined
findings of the present study, aqueous and ethanol plant extract in the present study can be attributed to
extracts of leaves of C .fistula have exhibited growth the synergistic effect produced after the combination of

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N. Wijayawardhana et al. Pharm Sci Asia 2021; 48(6), 523-534

individual extracts. A similar phenomenon has been The herbal soap incorporated seed oil of A. indica
discovered by other scientists by combining different reported 0.06% of free alkali38, whereas the said value
plant extracts 28,29. However, there was no reported of a herbal soap formulated incorporating extracts of
literature on the combination of selected extracts of C. fistula was 0.27%17. The free caustic alkali amount of
C. fistula, A. indica and N. nucifera and evaluation of its the formulated herbal soap under this study was 1.6%
antimicrobial activity. which is much higher than the reported values. However,
Antimicrobial herbal soap was formulated according to SLS 1220, the percentage of free alkali
considering its microbiological, physical and chemical should be higher than 0.006%. 0.05% is the maximum
properties. The selected herbal extracts as active ingre- amount declared by ISO standards39. Therefore, the soap
dients and several additives have been incorporated into does not comply with the ISO standards regarding the
the glycerin soap base which has been used as the vehicle percentage of free alkali.
for the formulation. Herbal soaps in 50 g in weight were One of the parameters that use to detect the
formed by adding plant extracts, distilled water, stearic purity of the soap is matter insoluble in alcohol (MIA).
acid and natural volatile oil into the glycerin soap base. This parameter is used to determine the non-soap
However, the active ingredients and added excipients ingredients known as builders or fillers such as sodium
should be compatible with the vehicle30. carbonate, sodium silicate and minor compounds such
Physicochemical characteristics of soap include as whitening agents, bleachers in the final product40. The
moisture content, total fat matter, pH, free caustic additives and foreign matter are known as ethanol-
alkalinity and percentage chloride. These characteristics insoluble matter in soap. Additive/s is not a part of soap
depend mainly on the strength and purity of alkali, the and includes colour additives, fragrance, herbs, silicon
kind of oil used and the completeness of saponification31. dioxide, etc. Inorganic matter such as carbonates, borates,
Appearance, color and odour were observed as physical perborates, chlorides and organic matter such as starches,
properties of the final product. The dark brown color of dextrins, caseins, sugars, cellulose derivatives consider
the soap arises due to the plant extractions. Most of the as foreign matter in soap. Main principle based for MIA
herbal soaps exhibit a brown color due to the dominant is that separation of builders from active detergent and
color coming from various plant extracts33. Fragrant that is achieved by the use of alcohol. The standard pro-
odor was the result of adding the natural volatile oil of cedure for analyzing commercial synthetic detergents
N. nucifera. consists in the separation of alcohol-soluble and insoluble
Healthy skin has a pH range of 4 to 632. The pH fractions by extraction with ethanol37. The higher MIA
of the skin products is expected to maintain closer to value indicates that it contains a high level of impurities
this range as much as possible to reduce irritation 33. As which may cause the level of impurities of alkali used
soap is alkaline (pH~10) in an aqueous solution because for the soap31. The standard percentage of the MIA range
the soap base is the salt of a weak acid (fatty acid) and is between 36-77%41. The value of MIA for formulated
strong base (NaOH). The alkalinity favours detergency34. herbal soap was 24% and it was in an acceptable range.
The pH of the formulated herbal soap at 28˚C was 9.11. However, the herbal soap formulated by Afsar and
According to the SLS 1220 standard (Sri Lanka accredi- Khanam22, incorporating extracts of C. fistula was 18%.
tation board for conformity assessment), the pH of the Moisture content is the major factor affecting
soap should be in the range of 4-10. The pH of the the deterioration of formulated soap. It is a parameter
formulated soap was in an acceptable range and safe to that measures the shelf life. High moisture content leads
use. The pH of a soap prepared from A. indica seed oil to the hydrolysis of soap on storage and facilitates
was 10.434 and the pH of a herbal soap formulated microbial growth33. The standard range for the moisture
incorporating extracts of C. fistula, Milletia pinnata and content is between 10-20%41. Moisture content of the
Ficus religiosa was reported as 7.027. The increased pH formulated soap was 15.85% which falls in the acceptable
of the soap produces a significant increase in microbial range. Therefore, it has the potential to prevent deterio-
growth35. Korting et al. examined the effect of different ration that causes due to high moisture content. The
skin cleansing methods in healthy individuals aiming herbal soap incorporating seed oil of A. indica reported
on the density of bacterial flora and the pH of the skin 12.6% of moisture content34, which is lower than the
surface. After soap washing, the count of propionibac- product formulated under this study.
teria elevated strongly and dropped after application of Another important physicochemical characte-
syndet which is more acidic in nature36. ristic is the percentage chloride levels. Excess chloride
The total alkali means the presence of total levels may cause soaps to crack34. However, the percentage
alkaline components in the finished soap. The value of of chloride levels was not determined under this study.
free caustic alkali measures the abrasiveness of any given Glycerin is the most effective humectant avail-
soap. Free caustic alkali results from improper or incom- able to increase stratum corneum hydration42. Soap base
plete saponification. High free alkali amount makes the used in the present study was composed of glycerin which
soap more abrasive and it may cause harm to the skin37. promotes the moisturizing ability of the skin. This is a

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