-
www.wjpps.com
674
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
DEVELOPMENT AND VALIDATION OF HERBAL ANTISEPTIC
TOPICAL FORMULATION
Dhanashri B. Nagulwar*1, Pravin K. Bhoyar 2, Jagdish R. Baheti
3, Dinesh M. Biyani 5,
Dharmendra R. Mundhada4 , Prasad P. Kathade 2
1. Department of Pharmaceutics, Sharad Pawar College of
Pharmacy, Dist: Nagpur,
Maharashtra, India.
2. Siddhivinayak College of Pharmacy,Warora, Dist-Chandrapur,
Maharashtra, India.
3. Shriman Suresh Dada Jain College of Pharmacy, Chandwad,
Nasik, Maharashtra, India
4. Department of Pharmaceutics, Agnihotri College of Pharmacy,
Wardha, Maharashtra,
India
5. Department of Pharmaceutical Sciences, Birla Institute of
Technology, Mesra, Dist- Ranchi, Jharkhand, India.
ABSTRACT
For skin infections, topical treatments applied directly to the
skin are
safest. Present work was decided to carry out antimicrobial
activity
and antiseptic effect of formulated cream. Oil of J. regia oil
of K.
galanga, methanolic extract of C. tora seeds and T. arjuna bark
were
successfully extracted and phytochemically screened. Thin
layer
chromatography of above extracts showed that it contains
different
active constituents. The result of phytochemical screening
reveals that
the major constituents of extracts were coumarins, flavonoids,
steroids
and triterpenoids. The in- vitro antimicrobial activity showed
that the
oil, methanolic extract and formulated cream posses
prominent
activity compared with that of standard. Zone of inhibition were
found
to be prominent in concentration 5g / ml of oil and 155ug / ml
for C.
tora seeds extract. In antimicrobial activity of cream, the
formulation
containing maximum percent of oils i.e. 5% and 155ug/ml of C.
Tora
methanolic extract showed highest zone of inhibition. Validation
of cream was done and was
found within the limits. The pH of cream was found to be in
range of 6.5 7.0 which is good
WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD
PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS
VVoolluummee 11,, IIssssuuee 22,, 667744--669922..
RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 4357
Article Received on 02 June 2012, Revised on 28 June 2012,
Accepted on 13 July 2012
*Correspondence for
Author:
* Dhanashri B. Nagulwar, Department of Pharmaceutics,
Sharad Pawar College of
Pharmacy, Dist: Nagpur,
Maharashtra, India.
[email protected]
om
-
www.wjpps.com
675
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
for skin pH. The consistency of cream was in the range of 230 to
290 1/10mm. The
formulated cream was used for antiseptic action. Patient trial
was carried out and successful
results were seen. For this purpose cream was applied topically.
In human study, trials were
conducted on 10 patients suffering from burn infection, tinea
pedis and acne. Signs of the
infection were healed in the period of 2 week.
Key Words: Skin, C. tora, tinea pedis, acne.
INTRODUCTION
India has an ancient heritage of traditional medicine. Materia
medica of India provides lots of
information on traditional aspects of therapeutically important
natural products. The
evaluation of these drugs is mainly based on phytochemical,
pharmacological and allied
approaches including various techniques like chromatography and
microscopy. [1] Herbals are
being used, in medicine from time immemorial because they have
fitted the immediate
personal need, they are accessible and inexpensive. It is widely
accepted that the use of
herbal medicine is well established and safe. [2]
For skin infections, topical treatments applied directly to the
skin are safest. Drugs taken
orally affect both disease and normal tissues, thus increasing
the chance of side effects.
Conventional skin disease treatments such as the drugs
ketoconazole, ciclopirox, naftifine
and tolnaftate can irritate the skin, causing stinging, itching,
redness, drying or allergic
reactions. Therefore treatment of many herbs can be used safely
for superficial skin
infections.
Herbal medicine, now a days are gaining importance for treating
many diseases due to their
significant effect and lesser side effects as compared to
allopathic medicines.[3] Plants like
Clove, Creosote, Garlic, Chamomile, Goldenseal, Tea tree oil,
Echinacea, Sassafras has
antiseptic and wound healing action. From literature survey, it
was revealed that
phytochemical analysis of C. tora, j. regia, T. arjuna, k
galangal confirmed the presence of
flavonoids, steroids, triterpenoids in methanolic extract and
these extract exhibited highest
antimicrobial activity against S. aureus, B. subtilis, P.
bacteria. Plants can be used for
antiseptic action therefore decided to use of these plants for
further study.
From market survey, it was indicated that no cream is available
containing combination of
these plants therefore decided to formulate antiseptic herbal
cream for microbial infection.
-
www.wjpps.com
676
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
In present project, methnolic extract of C. tora seeds and
n-hexane extract (oil) of j. regia
and k. galanga will be used for antiseptic action of cream.
extracted oil are soluble in oily
phase and C. tora methanolic extract is soluble in aq. phase
therefore it is expected that
extract should be compatible with cream base therefore decided
to use o/w cream base for
development of herbal antiseptic topical formulation. o/w creams
also cause less dryness and
burning, nonstaining and water miscible. Creams are well
absorbed and tolerated by many
people. [4]
In addition to this combination of plant extracts formulation
will be enriched with Aloe-vera
extract as emollient for the skin. Therefore these formulations
will be expected to exhibit
better efficacy as compared in the marketed antiseptic
formulation.
In present work main objective is development and validation of
Herbal antiseptic cream, in
vitro evaluation of antimicrobial activity, and in vivo study,
assessment of antiseptic activity
of developed cream on patients through various skin infections
will be expected to exhibit
better antiseptic effect as compared in the marketed antiseptic
formulation.
MATERIAL AND METHODS
Collection and authentication of plant material
The T. arjuna bark, J. rgia seeds, K. galanga rhizome, C. tora
seeds were collected from the
Shri Shail Medi Pharma of Nagpur district. The plant specimen
was dried and its herbarium
sheet was prepared and it is available in Department of Botany,
Nagpur University, Nagpur.
Procurement of Material and Extraction
These parts of the above mentioned plants subjected to size
reduction to get coarse powder.
Such powdered material was charged into the Soxhlet apparatus
and extraction was carried
out using n-hexane as solvent for J. regia seeds and for K.
galanga rhizomes whereas
petroleum ether (60-80o) and methanol (GR) for T.arjuna bark and
C. tora seeds. Various
Phytochemical screening tests are carried out such as tests for
sterol like salkowaski test,
liebermanns test, liebermann-burchard test.[5] Tests for
alkaloids like dragendorffs reagent,
mayers reagent, wagners reagent, hagers reagent. Test for
saponins and test for tannins like
ferric chloride test, lead acetate test, potassium dichromate
test, gelatin solution test. Test for
flavonoids (Shinoda test) and Test for proteins like biuret
test, xanthoproteic test, millon's test
(Mercury nitrate solution) and test for fixed oils are carried
out.
-
www.wjpps.com
677
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Thin Layer Chromatography of different extracts [6-7]
Chromatographic pattern of active extract of T. arjuna bark, C.
tora seeds j. regia seeds and
K. galanga rhizome was studied by thin layer chromatography to
find out the number of
phytoconstituents present in them. The adsorbent material used
for the thin layer
chromatography was silica gel G.
Analysis of oil [8]
The various physical constants like acid value, saponification
value and ester value were
determined by using standard methods of Indian Pharmacopoeia
(1996).
Acid value
Oil sample (2 g) was accurately weighed and dissolved in 50ml of
mixture of ethanol (95%)
and ether (1:1), which was previously neutralized with 0.1M
potassium hydroxide solution.
The flask was connected with a reflux condenser and warmed
slowly with frequent shaking
until the sample was dissolved; 1ml of phenolphthalein solution
was added and titrated with
0.1M potassium hydroxide until the solution remained faint pink
after shaking for 30sec. The
acid value was calculated from the expression.
Acid Value = 5.61 n / m
Where,
n = ml of KOH required.
w = weight in gram of sample.
Saponification value
Accurately weighed 2g of the oil was added in 250ml flask of
borosilicate glass fitted with
reflux condenser. 25ml of 0.5M ethanolic potassium hydroxide was
added and boiled under
reflux on water bath for 30min, Phenolphthalein solution (1 ml)
was added and titrated with
0.5M hydrochloric acid (ml) and repeated the same procedure for
blank (bml).
Saponification value = 28.05
Where,
w = weight in g of the sample
Ester value
Acid value and saponification value were determined as per the
Indian pharmacopoeia
procedures and the ester value was calculated from the
expression,
-
www.wjpps.com
678
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Ester Value = Saponification Value Acid Value
Anti-microbial activity [9-11]
The antimicrobial activity of K. galanga oil, J. regia oil ,
methanolic exrtract of C. tora seeds
and T. arjuna bark were studied against Gram-positive S. aureus
and B. substilis, P. bacteria
and C. albicans and A. niger fungi by agar diffusion method. For
antibacterial activity
streptomycin while for antifungal activity griseofulvin was used
as the standard.
Screening of antimicrobial activity was carried out using the
cup plate agar diffusion method.
This method depends on the diffusion of drug through the
solidified agar layer of a petridish
to an extent such that growth of the inoculated microorganism
prevented entirely in a circular
area zone around the cup containing the solution of the compound
under test. One loopful
of the stock culture was inoculated at 10 ml of agar slant
previously in sterilized test tubes,
and incubated at 37oC for 24 h and 20oC for 48 h respectively
for bacteria and fungi. About
3ml of distilled water was added to the test tube and a
suspension of the culture was obtained
by shaking for few minutes. The solutions were made by
dissolving the test oil compound in
minimum amount of DMSO (dimethyl sulphooxide), and solid
compound in minimum
amount distilled water, volume was made with sterilized water to
produce a concentration of
100g/ml.
Respective sterile medium was melted on water bath and kept at
45oC. In each sterile
petridish 25ml of molten medium was added and 107/ml of sub
cultured organism under
study was inoculated.
Formulation of cream [12-13]
For the convenient application of oil and other methanolic
extract and by considering the
antimicrobial activity, cream was selected as a suitable dosage
for topical application. Creams
are nonstaining and water miscible. Creams are well absorbed and
tolerated by many people.
Cream base formula was selected for the development of herbal
antiseptic cream as shown in
table 1.
-
www.wjpps.com
679
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Table 1: formula for development of cream base
Sr. No Ingredients % w/ w
Oil phase
1. Stearic acid 26.56%
2. Cetyl alcohol 1.0%
3. Propyl paraben 0.01%
4. Butylated hydroxyl toluene 0.02%
Water phase
1. Propylene glycol 5.0 %
2. Glycerine 5.0 %
3. Triethanol amine 1. 0 %
5. Methyl paraben 0.01%
6. Water 61.42%
Method of preparation
Oil soluble components were dissolved in oil phase and heated to
75C. Other water soluble
components were dissolved in aqueous phase and heated to 75C.
After heating, oil phase
was added in aqueous phase with continuous stirring till a thick
viscous cream was obtained.
Formulation development of herbal creams of botanical
extract.
Topical herbal creams of mentioned botanical extracts were
prepared as per the following
way on trial and error basis.
Batch I- In this batch, a single botanical extract (C. tora
seeds methanolic extract ) was
added which was compatible with above base.
Batch II- In this batch, cassia tora seeds methanolic extract
and Juglans regia oil were added
which were also compatible and more emollient than the
pervious.
Batch III- In this batch, all mentioned extracts were added, it
was noted that all extracts were
compatible with the base formula and cream texture and color was
more acceptable than the
-
www.wjpps.com
680
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
previous batch. Cream formulated in trial batch III was taken as
the master formula and
subjected for further evaluation study. Formulas for each trial
batch cream as shown in table
2.
Table 2: Formula for each batch
Ingredients Batch I Batch II Batch III
Stearic acid 26.56% 26.56% 26.56%
Cetyl alcohol 1% 1% 1%
Butylated hydroxy toluene
0.02% 0.02% 0.02%
Propyl paraben 0.01% 0.01% 0.01%
J. regia oil - 5.0% 5.0%
K. galangal oil - - 5.0%
Glycerine 5% 5% 5%
Propyleneglycol 5% 5% 5%
Triethanol amine 1% 1% 1%
Methyl paraben 0.01% 0.01% 0.01%
C. tora seeds methanol extract
1.55%
1.55%
1.55%
Aloe vera extract - - 2.0%
Water 59.85% 54.15% 54.15%
Evaluation of cream
As per the requirements for skin creams specified in Indian
standards (6608: 2004), following
parameters were used for evaluation of cream.
Thermal stability
For thermal stability testing, a humidity chamber and clear
glass container of around 30ml
capacities with screw cap were used. With the help of spatula
cream was inserted in the glass
containers and tapped to settle to the bottom and plug was
inserted and tightens the cap. The
filled bottle was kept inside the incubator at 45 10C for 48h.
On removal from the
-
www.wjpps.com
681
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
incubator, it was noted that no oil separation or any other
phase separation was not observed,
formulated cream was stable at 450C.
Determination of pH of formulated Cream
The digital pH meter was calibrated using buffer solution of pH
4.01, 7.0 and 9.2. Cream was
taken in a beaker and the pH of the cream was determined.
Determination of total fatty substance content
About 2g of the accurately weighed formulated cream was taken
into a conical flask, Dilute
Hydrochloric acid (25ml) was added and a reflux condenser was
fitted into the flask and the
solution was boiled until it perfectly cleared. Then contents of
the flask were poured into a
300ml-separating funnel and was cooled to room temperature. In
portion of 10ml the conical
flask was rinsed with 50 ml of petroleum ether and poured into
the separating funnel,
separating funnel was then shaked well and left until the layers
were separated. An aqueous
phase was separated and all ether extract was then washed with
water. This petroleum ether
extract was then filtered through a filter paper and dried the
material in the flask at a
temperature 90 20C. of to constant mass.
Formula:
Total fatty substance = 100 M1/ M2
Where,
M1= mass (g) of the residue
M2 = mass (g) of the cream
Determination of residue
About 5g of the cream was taken in a weighed, clean and dry
squat form weighing bottle and
dried to constant mass at 105 10 C. Cooled in a desiccator and
weighted.
Formula: Residue = 100 M1/ M2
Where,
M1 = mass (gm) of the residue
M2 = mass (gm) of the cream
Test for lead
Standard lead solution was prepared by using 1.600gm of the lead
nitrate taken in water and
the solution was made to 1000ml. solution (10ml) was Pipette out
and diluted to 1000ml with
water. One mm of this solution contains 0.01mg of lead (Pb).
About 2.00gm of cream was
-
www.wjpps.com
682
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
taken in a crucible and heated on a hot plate and then taken in
a muffle furnace to ignite it at
6000 C to constant mass. Dilute hydrochloric acid 3ml (5N) was
added and warmed and
volume was made to 100ml. solution was then filtered. In the
second Nessler' s cylinder, 2 ml
of dilute acetic acid (1N) was added, volume was made with water
to 25ml. standard
hydrogen sulphide(10ml) solution was added to each Nesslers
cylinder and volume was
made with water (50ml) mixed and allowed to stand for 10min and
the colour produced in
two Nesslers cylinders was compared. The colored produced with
hydrogen sulphide was
matched against that obtained with standard lead solution.
Spreadability
Spreadability of cream was measured with the glass slide
apparatus, excess of cream was
placed between two slides and 1 kg weight was placed on slide
for 5 min. to compress the
sample to uniform thickness, time in seconds to separate two
slides was taken as measure of
spreadability.
S = w l / t
where,
S = spreadability (g cm/sec)
w = weight on upper slide (g)
l = length of Slide (cm)
t = time taken in sec (sec)
Homogeneity
The developed cream was tested for homogeneity by visual
inspection, after the cream have
been set in the container, spread on the glass slide for the
appearance, tested for the presence
of any lumps, flocculates or aggregates.
Skin irritation test
The skin irritation was carried out on human volunteers. For
formulated cream, five
volunteers were selected and 1.0g of formulated cream was
applied on an area of two square
inch to the back of the hand. The volunteers were observed for
lesions or irritation.
Consistency
Consistency of the formulation was determined by penetrometer
model no. 2. Associated
instrument Pvt. Ltd. Culcutta.
-
www.wjpps.com
683
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Microbial evaluation [14]
Microbial evaluation of herbal formulations is essential to
check the limits of microbial
contamination and extents of pathogencity. This evaluation has
direct correlation with the
quality of products. For the evaluation of total microbial count
details of different count
media were used, nutrient agar medium used for the growth of
bacteria and Potato dextrose
agar medium was used for the growth of fungi. [15]
In aseptic conditions cream equivalent to one gram was dissolved
in 10ml of sterile water and
was serially diluted. The medium and apparatus required for
experimental were sterilized in
an autoclave at 1210C for 15min. In aseptic conditions, 1ml of
test sample was transferred to
petridish containing melted agar medium at about 420C and mixed
well by rotating the
petridish. It was allowed to solidify and then incubated at 370C
for 24h for detection of
bacteria. After incubation period, colonies were counted.
Formula:
No. of colonies dilution factor
No. of microorganisms =
Volume of sample
Stability testing of formulated cream.
For assessing the stability of formulated creams following
parameters were taken into
consideration like Thermal stability testing, pH, Total fatty
substance content, Total residue,
General test for lead, Consistency, Spreadability. [16] These
studies are essential to ensure that
product is stable over its designated shelf life. The stability
study was carried out for three
months as per ICH norms, at three different temperatures such as
at room temperature, 450C
and 8 to 100C. Results of stability testing of formulated cream
are given in table.
Patients study
Realising the importance of patient acceptability the
formulation was filled into 20gm
capacity container and dispensed to the patients suffering from
acne, Tinea pedis, burn, in the
dermatology department of government Ayurvedic hospital Nagpur.
The study was
supervised under the Dr. Kabra and Dr. Deepak Madavi and others
doctors.
Each Polyherbal cream (containing combination of methanolic
extract of Cassia tora seeds,
Juglans regia oil, Kaemferia galanga oil) was prescribed to the
patients suffering from skin
diseases. A retrospective analysis of their record revels that
the cream was well tolerated by
-
www.wjpps.com
684
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
the patients. Consent form was signed by the patients before
prescribing cream. The
treatment was given for two weeks to the patients suffering from
burn infection, Tinea pedis
and acne.
Patient study for Burn infection
Three patients suffering from burn infection were taken for
patients study. Damage in
superficial burns was not deeper than papillary epidermis.[17]
Clinical features were oedema
redness, bleb formation and pain due to preserved nerve endings.
Compliance of patients was
good against formulated cream than the marketed preparation.
Patient study for acne
Acne is an inflammatory skin disorder of the skins sebaceous
glands and hair follicles and
occurs when the cells lining the sebum canals are injured or
when follicles or pores are
blocked or clogged by sebum (oil) and dead cells mixed
together.
Patient of either sex, age between 21- 45 yrs. were selected,
two types of pigmentation were
observed having black coloured and white coloured pigmentation,
while giving the treatment
it was observed that pimple infection did not spread further,
cream act effectively on P.
bacteria, sebum irritation decreases, pores did not clog
further.
Patients study for Tinea pedis
Tinea pedis is the fungal infection of fact mostly involving in
between the toes. It is usually
associated with prolonged exposure to moisture. It involved,
skin appears blanched with
cracks and associated itching. Treatment involves the
application of cream base, formulated
cream and marketed preparation (skina cream).
RESULT AND DISCUSSION
All the extracts of plant material were subjected to preliminary
phytochemical screening for
the detection of various plant constituents. Phytochemical
screening of methanolic extract of
T. arjuna bark, C. tora seeds showed presence of sterols,
saponins, tannins, flavonoids, sugar,
proteins and coumarin. Hexane extracts of J. regia seeds and K.
galanga rhizome showed
positive test for sterols, flavonoid and coumarin hence both the
extracts were used for further
study.
Chromatographic pattern of above mentioned plant extracts were
studied by thin layer
chromatography to find out the number of constituents present in
them. Stationary phase used
-
www.wjpps.com
685
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
was Silica gel G and for mobile phase different combinations of
the solvents were tried for
the development of the solvent system for different
extracts.
Mobile phase with Benzene: Ethyl acetate (8:2) showed maximum
resolution and
reproductive results. After spraying with 50 % H2SO4 and in
iodine vapour gives 3 spots with
Rf values 0.34, 0.61, 0.87. of petrolium ether extract T. arjuna
bark and gives 3 spots with Rf
values 0.34, 0.54, 0.74 C. of petrolium ether extract of C. tora
seeds.
Mobile phase with Butanol; Acetic acid; Water (6:2:2) showed
maximum resolution and
reproductive results. After spraying with 50 % H2SO4 and in
iodine vapour gives 2 spots with
Rf values 0.48, 0.76 methanol extract of. T. arjuna bark and 6
spots with Rf values 0.32, 0.45,
0.57, 0.74, 0.81, 0.96 of methanol extract of C. tora seeds.
Mobile phase with Benzene: Ethyl acetate: Acetone (8:1:0.5)
showed maximum resolution.
After spraying with 10 % vanniline sulphuric acid and in iodine
vapour gives 4 spots with Rf
values 0.32, 0.68, 0.82.0.94 of J. Regia oil and 6 spots with Rf
values 0.38, 0.46, 0.52, 0.64,
0.77, 0.84 of K. galanga oil.
As shown in Table 11, acid value, saponification value and ester
value of J. regia seeds oil
were found to be 15.54, 134.5, and 118.7 and of K. galanga oil
were found to be 1.2, 105.4
and 121.7 respectively as shown in table 3.
Table 3: Evaluation parameters of J. regia and K. galanga
oils
It was indicated that methanolic extract of C. tora seeds, K.
galanga oil and J. regia oil had
maximum antimicrobial activity whereas methanolic extract of T.
arjuna bark had not
maximum antimicrobial activity as compared to that of other
plants extract so T. arjuna bark
extract was not used in formulation of cream as shown in table
4, 5, 6 and 7.
Parameter J. regia Oil
K. galanga oil
Acid value 15.54 1.2
Saponification Value 134.5 105.4
Ester value 118.7 121.7
-
www.wjpps.com
686
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Table 4: Antibacterial activity of K. galanga oil.
Table 5: Antibacterial activity of J. regia oil.
Microorganism Zone of Inhibition in mm
J. regia oil Streptomycin
(5g/ml) 1g/ml 2g/ml 3g/ml 4g/ml 5g/ml
P. bacteria 9 11.2 12.5 13.3 14.1 18.2
B. subtilis 10.2 11.1 12.2 13.2 16.7 21.1
S. aureus 7 9 13 15 18.7 23.2
Table 6: Antibacterial activity of methanolic extract of C. tora
seeds and T. arjuna bark extract
Extracts Zone Of Inhibition in mm
B. Subtilis S. aureus P. bacteria C. tora seeds
methanolic extract (155ug/ml)
14.3 16.4 13.2
T. arjuna methanolic extract
(170ug/ml) 3 2 -
Streptomycin 5ug/ml 18.2 21.1 23.2
Table 7: Antimicrobial activity of all combined extracts
Microorganism Zone of Inhibition in mm K. galangal
Streptomycin
(5g/ml) 1g/ml 2g/ml 3g/ml 4g/ml 5g/ml P. bacteria 4 7 10 13 14
18.2 B. subtilis 7 10 14 15 17 21.1 S. aureus 7 9 13 17 19 23.2
Microorganism
Zone of Inhibition in mm K. galanga
oil +
5 g/ml
J. regia oil +
5g/ml
C. tora seeds + methanolic
extract 155 ug/ml
Streptomycin (5g/ml)
P. bacteria 16.7
19.5
21.9
18.2
B. subtilis 21.1
S. aureus 23.2
-
www.wjpps.com
687
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Cream formulated in trial batch III was taken as the master
formula and subjected for further
evaluation study. It was indicated that evaluation parameter of
formulated cream are within
the limits as per the requirements for skin creams specified in
Indian standards as shown in
table 8.
Table 8: Evaluation of Cream
The results for determination of total plate count of bacteria
and fungi are shown in following
table 9 and 10.
Sr. No Parameter Cream base
Cream base +
C. tora methanolic
extract
Cream base + C. tora
methanolic extract +
J. regia oil
Cream base +
C. tora methanolic
extract +
J. regia oil +
K. galanga oil
1 Thermal stability testing No phase separation No phase
separation
No phase separation
No phase separation
2 pH 6.4 6.6 6.7 6.8
3 Total fatty substance content, (%/gm) 26.32%/gm 27.56%/gm
32.97%/gm 37.78%/gm
4 General test for lead Passes the test Passes the test Passes
the test Passes the test
5 Total residue, (%/gm) 37.26%/gm 38.81%/gm 42.57%/gm
46.94%/gm
6 Homogeneity No lumps No lumps No lumps No lumps
7 Spreadability 10.32gm.cm/sec 10.30gm.cm/se
c 12.59gm.cm/sec 13.01gm.cm/sec
8 Skin irritation test No irritation No irritation No irritation
No irritation
9
Consistency at room temperature
237 233 239 243
At 450C 1/10 mm 276 273 179 283
At 8-10 0C 1/10 mm 151 149 159 165
-
www.wjpps.com
688
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Table 9: Total number of viable bacteria present in formulated
cream.
Table 10: Total number of viable fungi present in formulated
cream.
Formulated cream
Types of microorganism
Fungi (C.F.U./ml)
Cream base Cream base + C. tora methanolic extract
Cream base + C. tora methanolic extract + J. regia oil
Cream base + C. tora methanolic extract + J. regia oil + K.
galanga oil
3.29 102 2.42 102 2.21 102 1.15 102
From stability study of cream, it was indicated that cream was
stable at three different
temperatures such as at room temperature 450C and 8 to 100C.
From ANOVA, it was indicated that there were no significant
difference between the
consistency, pH, spreadability, total fatty substance content,
total residue content values of
the different batches of formulated cream. Calculated F ratio
were less than table F value,
therefore accepted hypothesis that there were no difference
between the batches at 5% level
of significance.
Three batches of cream were formulated, F1, F2 and F3 and their
antimicrobial activity was
carried out and the results of zone of inhibition are as shown
in table 11, 12 and figure 1 and
2.
Formulated cream
Types of microorganism Bacteria (C.F.U./ml)
Cream base
Cream base +
C. tora methanolic extract
Cream base +
C. tora methanolic extract
+ J. regia oil
Cream base +
C. tora methanolic extract
+ J. regia oil
+ K. galanga oil
3.12 102 2.57 102 2.39 102 1.21102
-
www.wjpps.com
689
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Table11: Antibacterial activity of cream
Microorganism
Zone of Inhibition in mm Formulated cream Streptomycin
(5g/ml) F1 F2 F3 P. bacteria 17.2 17.4 17.4 18.2 B. subtilis
19.7 19.7 19.7 21.1 S. aurbeus 22.2 22.3 22.3 23.2
Figure 1: Antibacterial activity of cream
Table 11: Antifungal activity of cream
Microorg
anism Zone of Inhibition in mm
Formulated cream 5ug/ml grisevofulvin
(5g/ml) F1 F2 F3
C. albicans
23.4 23.5 23.7 25.2
A. niger 20.6 20.9 20.9 21.7
Thus, maximum zone of inhibition is found in cream containing
all polyherbal extract which
is more or less equal to that of standard.
Figure 2: Antifungal activity of of formulated cream
0
5
10
15
20
25
1 2 3
F1F2F3Sreptomycin
zone of inhibition in mm
Antibacterial activit
1 . B. subtilis2. S. aureus3. P. bacteria
0
10
20
30
1 2
F1F2F3grisevofulvin
zone of inhibition
in mm
Bacterial
Antifungal activity
1.B. subtilis2. S. aureus3. P. bacteria
-
www.wjpps.com
690
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
From patients study it was indicated that all the parameters and
signs of the disease infection
were healed in the period of 2 week.
Following photo shows the effect of base, cream and marketed
preparation on patients
suffering from Tinea pedis, burn injury and acne as shown in
figure 3, 4, 5, 6 and 7.
Figure 3: Effect of marketed cream on tinea pedis patients.
Before treatment after one week after two week
Figure 4: Effect of formulated cream on tinea pedis patients
Before treatment after one week after two week
Figure 5: Effect of marketed cream on acne patients
Before treatment after one week after two week
Figure 6: Effect of formulated cream on acne patients
-
www.wjpps.com
691
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Before treatment after one week after two week
Figure 7: Effect of formulated cream on burn patients.
REFERENECES
1. Kumar V, Parmar NS. Herbs: A Potential Source for the
Development of New
Phytomedicinals. The Pharma Review. 2003; 1: 56.
2. Mukherjee PK. Quality Control of Herbal Drugs. 1st ed., New
Delhi; Business Horizon
Pharmaceutical Publishers: 2002.
3. World Health Organization. Research Guidelines for Evaluating
the Safety and Efficacy
of Herbal Medicines; 1993.p. 397.
4. Khan MR, Khihana M, Omoloso AD. Antimicrobial activity of
Michelia champaca.
Fitoteripia, 2003; 73: 744.
5. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 1st ed.,
Mumbai; Nirali Prakashan:
1990.
6. Stalhl E. Thin Layer Chromatography. 2nd ed., Berlin;
Springer- Verlay Berlin
Before treatment After one week After two week
Before treatment After one week After two week
Before treatment After one week After two week After two
week
-
www.wjpps.com
692
Dhanashri et al. World Journal of Pharmacy and Pharmaceutical
Sciences
Publisher:2005.
7.Wagne H, Bladt S. Plant Drug Analysis a TLC Atlas. 2nd
ed.,
New Delhi; CBS Publisher and Distributors: 1995.
8. The Indian Pharmacopoeia. Govt. of India, Ministry of Health
and Family Welfare, The
Controller of Publication, New Delhi 1996; A- 52.
9. Copland A, Nahar AT, Tomlinson CM. Antibacterial and free
radical scavenging activity
of the seeds of Argimonia eupatoria. Fitoteripia, 2003; 73:
133.
10.Khan MR, Khihana M, Omoloso AD. Antimicrobial activity of
Michelia Champaca.
Fitoteripia, 2003; 70: 659.
11. Pistelli L, Betroli AT, Lepori DH. Antimicrobial and
antifungal activity of crude extracts
and isolated saponins from Astragalus verrucosus. Fitoteripia,
2002; 72: 340-4.
12.Pharmacopoeial standards for Ayurvedic Formulation. Central
council
for research in Airbed and Sided; 1987.p. 65.
13. Ansel CA, Howard BG. Introduction to Pharmaceutical Dosage
Form. 5th ed., New
Delhi; B.I. Publication: 1999.
14. The Indian Pharmacopoeia. Govt. of India, Ministry of Health
and Family Welfare, The
Controller of Publication, New Delhi 1999; A- 57.
15. Bhaskaran S. Physical Pharmaceutics. 1st ed., New Delhi;
Birla Publication Pvt. Ltd:
2007.
16. Vadas EB. Stability of Pharmaceutical Products. 20th ed.,
London; Lippincott Williams
and Wilkins publishers: 2002.
17. Goodman and Gilmans, The Pharmacology Basis of Therapeutics,
10th ed., London;
McGraw-Hill Publication: 2001.