Research J.

Pharmacognosy and Phytochemistry 2011; 3(5):225-231

S. Debnath, et.al.

Formulation and Evaluation of Liquid Soap

Containing Herbal Antimicrobial Agent.
Subhashis Debnath*, M. Niranjan Babu, Mahesh Dega,
Bharathi. K, Jyothsna. T, Revathi. D and Kishore Kumar. T.S
Seven Hills College of Pharmacy, Tirupati-517561, Andhra Pradesh, India.

ABSTRACT:
ISSN 0975- 2331

Research Journal of Pharmacognosy

and Phytochemistry. 3(5): Sept.Oct.2011, 225-231

Research Article

Hand washing for hand hygiene is the art of cleaning the hands with or
without the use of water or another liquid, or with the use of soap, for the
purpose of removing soil, dirt, and/or microorganisms. The main medical
purpose of washing hands is to cleanse the hands from pathogens (including
bacteria or viruses) and chemicals which can cause personal harm or disease.
This is especially important for people who handle food or work in the
medical field, but it is also an important practice for the general public. In
response to this concern, we formulated the antibacterial Liquid soap.
Experiment was performed by selecting the herbs which were reported to
have antibacterial activity. They are Ocimum sanctum and Eugena
caryophyllus. Formulated liquid soaps were evaluated for physical
parameters like colour, fragrance and chemical parameters like pH, % Free
alkali, Test for chlorides, Foam height, Foam retention, Alcohol insoluble
matter, Microbial count etc. and the obtained results were in the acceptable
limits except foam height and foam retention as we are not using SLS.

KEYWORDS: Liquid Soap, infection, antibacterial, free alkali, microbial

count.

INTRODUCTION:

*Corresponding Author:

Subhashis Debnath,
Seven Hills College of Pharmacy,
Tirupati-517561, Andhra Pradesh,
India.
E-mail: subhashis_xyz@yahoo.com

Received on 06.05.2011
Accepted on 21.07.2011
A&V Publication all right reserved

Hand washing for hand hygiene is the art of cleaning the hands with or
without the use of water or another liquid, or with the use of soap, for the
purpose of removing soil, dirt, and/or microorganisms. Medical hand hygiene
pertains to the hygiene practices related to the administration of medicine and
medical care that prevents or minimizes disease and the spreading of disease.
The hands of Health Care Workers are the primary routes of transmission of
multidrug resistant pathogens and infection to patients. Antibacterial hand
soaps are drugs as defined by the Federal Food, Drug and Cosmetic Act
(FFDCA). They are categorized as drugs because they are intended and
labeled for topical antimicrobial use to prevent disease in humans. Therefore,
they are regulated by the Food and Drug Administration (FDA) as over-thecounter (OTC) drugs1-4.
Many of the chemical antiseptics are now available in market as alcohol
based sanitizers, chlorohexidine products, etc. these soaps or solutions help
reduce health care associated transmission of contagious diseases more
effectively but they have some shortcomings adverse effects. Their frequent
use can lead to skin irritation and also resistance among pathogens. Problems
arising with use of chemical anti-microbial agents are toxicity, local
irritancy, systemic toxicity, hypersensitivity, super infection etc. Organism
such as Staphylococcus aureus, Pseudomonas spp, Klebsiella pneumonia and
Proteus vulgaris are some of the causative agents of the skin infections.
Many medicinal plants have been found to possess active principles useful
for killing microorganisms. Plant drugs are frequently considered to be less
toxic and more free from side effects than synthetic chemicals.

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S. Debnath, et.al.

Because of above reason herbal anti- microbal agents are

used in the formulation of liquid soap. Experiment was
performed by selecting the herbs which were reported to
have antibacterial activity. They are Ocimum sanctum and
Eugena caryophyllus5,6.

MATERIALS AND METHODS:

Materials:
The following plant materials were selected for the present
study Ocimum sanctum (The Himalaya Drug Company,
Bangalore) and Eugena caryophyllus (Vyas Pharmaceuticls,
Indore). Other chemicals used were Potassium hydroxide
(Merck, Mumbai), coconut oil(collected locally), Olive
Oil(Central drug house Pvt Ltd, New delhi), ethyl alcohol
(Changshu Yangyuan Chemicals, China), phenolphthalein
(Fisher Scientific, Mumbai), calcium nitrate (Finar
Chemicals and Reagents, Ahmedabad), potassium nitrate
(Finar Chemicals and Reagents, Ahmedabad), silver nitrate
(S.D Fine Chemicals Ltd, Mumbai), potassium chromate
(Finar Chemicals and Reagents, Ahmedabad). Borax (Accord
Labs, Secunderabad), Amaranth (Merck Ltd, Mumbai)

5. Oils and lye were stirred together. Then, started

blasting it with the mechanical stirrer.
6. Depending on the type of oils, it will take a long time
to get to trace. Sometimes as much as 30 minutes.
7. Once the soap has reached trace it was stirred for one
more time and heated by placing a lid.
8. Soap was checked every 15-20 minutes for any
separation.
9. It was cooked for 3-4 hours. While cooking it was
transformed and go through several "stages" like,
Thick applesauce
Cooked custard with small bubbles
Watery mashed potatoes
Solid taffy
Chunky/creamy vaseline
Translucent Vaseline (Figure 1)

10. When the soap has softened and turned translucent, it

was checked to test whether it has cooked enough or not.
(Take 10 ml of boiling water and add 5 gm of soap paste.
Stir the soap, breaking it up and helping it dissolve in the
water. Once it's completely dissolved (several minutes)
Methods:
Selection of Method for Formulation of anti microbial check to see how clear it is. If it's just very lightly cloudy, it
will be satisfactory. It may be due to type of oil. Also, the
Liquid Soap:
soap
will "settle" after it's finished and get even clearer. But
Hot Melt Process:
The hot process method was selected for the formulation of if the dissolved soap mixture is milky or very cloudy, it
the liquid soap as it is more suitable for laboratory and shows that it was not cooked properly).
industrial preparation.
11. Sufficient amount of water was add to get the desire
consistency and mixed it well.
Formulation of antimicrobial liquid soap:7-9
12. After the soap paste has completely dissolved in the
Procedure:
1. Measured amount of oils for the formulation were kept water, it was neutralized using 33% borax solution.
13. Fragrance and color were added in this step.
in the beaker.
14. 10ml 4 % of Ocimum sanctum or 2.5 % Eugena
2. Oils were heated at 1600 C 100 C.
as per the
3. While the oils were heating up, lye-water (potassium caryophyllus oil and combination of both
10,-14
formulation
code
were
added
and
stirred
well
.
hydroxide solution) solution were prepared.
4. When the lye-water is completely mixed and clear, 15. It was then cooled and poured it into a suitable
dispenser and stored it in room temperature and used it for
slowly it was added into oils.
Amount of lye and water required to dissolve was the further evaluations.
calculated from the following formulas9.
of antimicrobial liquid soap
a. (Amount of Fat) (Saponification Value of the Fat) = Parameters for Evaluation
15,16
Physical
Parameters
(Amount of Lye)
b. (Amount of Lye) 0.3 = (Total Weight of Lye Water 1. Colour: Colour of the preparation was checked visually.
2. Fragrance: Liquid soap preparation was tested for good
Solution)
c. (Total Weight of Lye Water Solution) (Amount of fragrance.
Lye) = (Amount of Water)

Table 1: Composition of different formulations

Sr. No
Formulation
Composition
Code
Plant material
1
S1
Eugena caryophyllus
(2.5 % v/v, 10 ml)
2
S2
Ocimum sanctum
(4 % v/v, 10 ml)
3
S3
Eugena caryophyllus
(2.5 % v/v, 5 ml)
+ Ocimum sanctum
(4 % v/v, 5ml)

KOH (gm)
7.5

Coconut Oil (gm)

11.52

Olive Oil (gm)

27.03

7.5

11.52

27.03

7.5

11.52

27.03

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S. Debnath, et.al.

Fig1: Different Stages observed during the preparation of liquid

soap

e. Chunky Vaseline stage

a. Mixing of lye in oil

f. Final Formulation without colouring agent

b. Partial separation of the lye and oil

Chemical Parameters:15-18
1. pH of Liquid Soap:
1 gm of sample of liquid soap was taken and dissolved it
into 100 ml distilled water. The pH of solution was taken in
previously standardized pH meter.
2. % Free Alkali:
About 5 gm of sample was taken in a conical flask and
added to it into 50 ml of neutralized alcohol. Boiled it under
reflux on a water bath for 30 minutes. It was cooled and 1
ml of phenolphthalein solution was added. Then, it was
titrated immediately with 0.1N HCL.

c. Cooked custard

3. Foam Height:
1gm of sample of liquid soap was taken. Dispersed in 50
ml distilled water. Then, transferred it into 500 ml
measuring cylinder; volume was make up to 100 ml with
water. 25 strokes was given and stand till aqueous volume
measured upto100 ml and measured the foam height, above
the aqueous volume.
4. Foam Retention:
50 ml of the 1% liquid soap solution was taken into a 250ml
graduated measuring cylinder. The cylinder was covered
with hand and shaken 10 times. The volume of foam at 1minute intervals for 4 minutes was recorded.

d. Solid taffy stage

5. Alcohol Insoluble Matter:

5 gm of sample was taken in a conical flask. Added it to 50
ml of warm ethanol and shaken vigorously to dissolved.

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S. Debnath, et.al.

The solution was filtered through a tarred filter paper with The strains were confirmed for their purity and identified by
20 ml warm ethanol and dried it at 105C for 1 hour. The Grams staining method and their characteristic biochemical
weight of dried paper was taken.
reactions. The selected strains were preserved by sub
culturing them periodically on nutrient agar slants and
storing them under frozen condition. For the study, fresh 24
Formula:
Wt. of residue
hrs broth cultures were used.
% alcohol insoluble matter = ------------------------- 100
Wt. of sample
Working conditions:
The entire work was done using horizantal laminar flow
6. Test for Chlorides:
hood so as to provide aseptic conditions. Before
10 gm of sample of soap was dissolved in hot distilled commencement of the work air sampling was carried out
water and transferred to a 250 ml of narrow necked using a sterile nutrient agar plate and exposing it to the
graduated flask. Sufficient calcium nitrate was added to environment inside the hood. After incubation it was
precipitate the soap. The flask was filled to the 250ml mark checked for the growth of microorganism and absence of
with distilled water. The mixture was filtered and 100ml growth conformed aseptic working conditions.
taken in a conical flask, where it was titrated with standard
silver nitrate solution using little potassium chromate as an Preparation of inoculums:
indicator. Blank was also carried.
The inoculum for the experiment was prepared fresh in
Nutrient agar broth from preserved frozen slants. It was
7. High Temperature Stability:
incubated at 37C for 18-24 hrs and used
Liquid soap was allowed to stand at 50C for one week. The
stability of liquid soap was observed during this period .The Antibacterial screening by cup plate method:
sample which was homogeneous and stable liquid after Nutrient agar plates were prepared aseptically to get a
standing was indicated as stable and the sample in which thickness of 5-6 mm. The plate were allowed to solidify and
the crystals were roughened and the sample in which inverted to prevent the condensate falling on the agar
precipitation was caused; then liquid was said to be as surface. The plates were dried at 37C before inoculation.
unstable.
The organism as inoculated in the plates prepared earlier by
dipping a sterile swab in the previously standardized
inoculum, removing the excess of inoculum by pressing and
8. Antimicrobial study:15.16,18-20
rotating the swab firmly against the sides of the culture tube
Screening for Antibacterial Activity:
above the level of liquid and finally streaking the swab all
Media:
Medium was prepared by adding 2 % of agar.
over the surface of the medium three times, rotating the
plate through an angle 60C after each application. Finally
the swab was pressed round the edge of the agar surface. It
Ingredients:
was allowed to dry at room temperature, with the lid closed.
Peptic Digest of animal tissue :
5 g/l
Using the borer 0.5 cm well or cup for each plate were
Sodium Chloride
:
5 g/l
prepared. Add 0.15 ml of sample solution was introduced in
Beef extract
:
1.5 g/l
to
the well. After preparation of plates, kept it for 5min in
Yeast extract
:
1.5 g/l
freezer
for diffusion of sample solution. Plates were
Final pH at 25C
:
7.4 0.2
prepared in triplicate and they were then incubated for1824 hrs at 37C. Observation were made for zone of
Preparation:
inhibition
around the well. All the samples were tested for
The ingredients were dissolved in distilled water with the
antimicrobial
activity against gram positive and gram
aid of heat and pH was adjusted to 7.2 7.6 using alkali or
negative bacteria.
dilute acid if necessary.
Sterilization:
15-20 ml of agar medium was transferred to conical flask
and sealed with non-absorbent cotton.
It was then
autoclaved at a pressure of 15 psi (121C) for not less than
15 minutes.
Organisms used:
Staphylococcus aureus NCIM 3160, Escherichia coli
NCIM 443 and Bacillus subtilis NCIM 441 were procured
from National Collection of Industrial Microorganisms,
National Chemical Laboratory, Pune and stored in the
Pharmaceutical Biotechnology Laboratory, Seven Hills
College of Pharmacy, Trupati.

RESULT AND DISCUSSIONS:

Different combination of liquid soaps were prepared by hot

process and stored properly for the further evaluation of the
formulation. During formulation, fats were broken down
(hydrolyzed) yielding crude soap, with glycerol as a
byproduct. Different composition of liquid soaps were
prepared like S1, S2 and S3 which contain 2.5% Eugena
caryophyllus, 4 % Ocimum sanctum and combination of
both. The main ideology behind combining the plant
materials is to observe the additive effect of the active
constituents of different plants. The combination may
proves to be beneficial and hence it is used in the
preparation of the liquid soap Liquid soap prepared by each
composition were found to be smooth and stable.

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S. Debnath, et.al.

Table No. 2. Determination of chemical parameters

Sr. No.
Parameters
Tested Parameters of different formulation
S1
S2
S3
7.20
pH of solutions
1.
7.25
7.16
Foam Heights
130 ml
135ml
132ml
3.
% Alcohol insoluble matter
0.2 %
0.27%
0.34
4.
Chloride content
1.2%
1.18%
1.15%
5.
NLT = Not less then; NMT = Not more then

Acceptance Limit
8.5-10.5
NLT 400 ml
0.5 %
NMT 2%

4. Alcohol Insoluble Matter:

Alcohol insoluble matter were found to be in the range of
0.2-0.34%. Values were listed in the Table no 2(Fig no 4).

Physical Parameters:
1. Colour
Colour of all the formulated liquid soap were pale pink.
2. Fragrance
Mixed fruit
Chemical Parameters:
1. pH of Liquid Soap:
The pH of different composition of the liquid soaps were
measured and it was found to be in acceptable range (Table
No.2 and fig no 2) and it was non- irritating to skin. So it
can be used for routine hand washing.

Fig 4: Comparison of alcohol insoluble matter of different

formulations of the liquid soap

5. Test for Chlorides:

The chloride content of formulated liquid soaps were found
to be 1.2%, 1.18% and 1.75% for S1, S2 and S3
formulation respectively.(Table no 2 and figure no 5)
Fig 2: Comparison of pH of different formulations of the liquid
soap

2. % Free Alkali:
No colour change of solution after titration with 0.1 N
HCL. Hence, it shows absence of % free alkali as KOH in
different formulations of liquid soap.
3. Foam Height:
Foam height of liquid soap was found to be less than 150
ml as sodium lauryl sulphate was not added in the
formulations.Fig No 3 represents the foam height of the
different formulations.
Fig 5: Comparison of chloride content of different formulations of
the liquid soap

6. Foam Retention:
Foam retention time was checked for the different
formulation and it was found to be unstable for 5 minutes.

Fig 3: Comparison of Foam height of different formulations of the

liquid soap

Table 3: Determination
formulations.
Sr.
Time in minutes
no
1
Initial
2
1
3
2
4
3
5
4

229

of

foam

retention

Volume (ml)
S1
S2
2
2
1
1
1
1
0
0
0
0

of

different
S3
3
2
2
1
0

Research J. Pharmacognosy and Phytochemistry 2011; 3(5):225-231

S. Debnath, et.al.

Table 5: Anti microbial activity of the different formulations

7. High Temperature Stability:16
Sr.
Microorganism
Zone of inhibition (mm)
After standing the liquid soap at 50C for one week. The
no
S1
S2
S3
stability of liquid soap was observed during this period. The
1
Staphylococcus aureus
21
20
24
sample was homogeneous and stable liquid after standing. 2
Escherichia coli
20
22
23
Hence, it was indicated as stable sample. pH of all the
3
Bacillus subtilis
19
20
24
preparation were also measured on the final day and it was
found to be in the accepted range. Values were listed in
Table no 4. Fig no 6 shows the comparison of pH of
different formulations of the liquid soap before and after
stability study.
Table 4:Stability study
Formulation
Physical
code
Stability
S1
S2
S3

Stable
Stable
Stable

pH of the preparation after

exposing the liquid soap at 500
C for one week.
7.4
7.6
7.3

Fig. 8: Zone of inhibition of microorganism(Escherichia coli) after

24 hours

Fig 6: Comparison of pH of different formulations of the liquid

soap before and after stability study.

8. Anti microbial evaluation:

The results of zone of inhibition of the different formulation
were recorded in Table 5 and fig 7, 8 and 9. The result
shows that all the formulations have antibacterial activity
towards all tested phytopathogenic bacteria. The highest
antibacterial activity was found in S3 formulation which
contains 2.5% Eugena caryophyllus + 4 % of Ocimum
sanctum .
Fig. 9: Zone of inhibition of microorganism(Bacillus subtilis) after
24 hours

CONCLUSION:

Formulated liquid soaps were evaluated for physical

parameters like colour, fragrance and chemical parameters
like pH, % Free alkali, Test for chlorides, Foam height,
Foam retention, Alcohol insoluble matter, Microbial count ,
Skin irritation test etc. and the obtained results were in the
acceptable limits.
The formulated liquid soap containing tulsi and clove oil
was found to be easier and simpler, to produce stable liquid
soap. It is also having accepted range of antimicrobial
efficacy which may act as alternative of the liquid soap
Fig. 7: Zone of inhibition of microorganism(Staphylococcus aureus) containing synthetic antimicrobial agent.

after 24 hours

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S. Debnath, et.al.

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