B.Satpathy et al., Int. J. Res. Pharm. Sci.

, 2(3), 2011, 373-378

ISSN: 0975-7538
Research Article
www.ijrps.pharmascope.org

Formulation and evaluation of herbal gel containing essential oils of piper betle
against skin infecting pathogens
B.Satpathy*, M.Sahoo, P.Sahoo, S.R.Patra
Dadhichi College of Pharmacy, Sundargram, Cuttack, Orissa, India
ABSTRACT
The aim of the present study was to investigate the antimicrobial potency of essential oil of piper betle against
different skin infecting pathogens and to formulate and evaluate suitable dermatological preparation containing
betle oil as the active ingredient. The evaluation of antimicrobial potency of betle oil was studied prior to gel for-
mulation to compare the changes in activity after incorporation in polymer gel. The assay was done using agar
well disc diffusion method for zone of inhibition and broth dilution method for MIC (Minimum Inhibitory Concen-
tration). Zone of inhibition was found to be maximum that was 26mm at 15µl for S. aureus followed by 19mm at
25 µl for P.aeruginosa, 20 mm at 20 µl for S.epidermidis, 22mm at 18 µl for E. coli, 21.6mm at 18 µl for M.luteus
.Among the fungus C.albicans showed maximum sensitivity to the oil that was 24mm at 20 µl and 20mm at 20 µl
for A. niger. Gels were formulated by using different polymers like hydroxy propyl methyl cellulos e (HPMC), Car-
bopol 934, sodium carboxy methyl cellulose (sodium CMC), sodium alginate and evaluated for various physico-
chemical parameters like pH, viscosity, consistency, homogeneity, spreadability, skin irritation test and stability
study. MIC for all the tested pathogens was below 60 µg/ml. Dimethyl sulfoxide (DMSO) as the co‐solvent for the
essential oil and Carbopol 934 (1%), HPMC (5%) as gelling agent showed the best results in final formulations.
Zone of inhibition of oil was not much affected by incorporation in gel which was confirmed from the antimicro-
bial results of final formulations. The gel showed promising antibacterial and antifungal activity against other
strains used for the study. The gel was stable at room temperature.
Keywords: Antimicrobial activity; minimum inhibitory concentration (MIC); Sabroud dextrose agar (SDA); Sabroud
dextrose broth (SDB); Nutrient broth (NB); Nutrient agar (NA); Gel.
Introduction The antimicrobial potency of oil was evaluated by agar
well disc diffusion method. The zone of inhibition of oil
Bacterial and fungal skin infections are very common in
was measured and compared with standard antibiotics.
population. Though numbers of antibiotics are avail-
The MIC of betle oil was also determined by broth dilu-
able but development of multi drug resistance by many
tion method. The oil showed profound activity against
skins infecting pathogens and severe side effects asso-
all tested pathogens (bacteria: S.aureus, P.aruginosa,
ciated with traditional antibiotics have now drawn the
S.epidermidis, M.luteus, E.coli and fungus: C.albicans,
attention of researchers towards herbal medicine sys-
A.niger). These pathogens are very harmful and re-
tem (Abhijeet Pandey et al., 2011). Betle (Piper betle
sponsible for severe human skin infections including
belonging to the family Piperaceae), the neglected
pimples, impetigo, boils (furuncles), cellulitis, follicu-
green gold of India has been used as mouth wash, den-
litis, carbuncles, scalded skin syndrome, abscesses,
tal medicine, cough medicine, astringent, tonic, carmi-
acne and candidiasis (M. A. Ekpo et al., 2009). The re-
native, stimulant, antiseptic and others Several re-
sults of antimicrobial assay were highly promising
searchers also reported that betle extract and betle oil
which prompted us to formulate a suitable gel formu-
showed antimicrobial and antioxidant activities in
lation of betle oil against these severe skin infecting
model systems (Panuwat Suppakul et al.,2006). How-
pathogens as gels are becoming more popular due to
ever a suitable dermatological formulation of piper
ease of application and better percutaneous absorp-
betle against skin infecting pathogens is yet to be
tion, than other semisolid preparation.
evaluated and validated.
Gels were formulated with different polymers. Sixteen
different formulations were prepared having betle oil
* Corresponding Author 1% in each formulation. All the prepared formulations
Email: happy.bss07@gmail.com were evaluated for their physico-chemical properties
Contact: +91-9777215886 like homogeneity, consistency, spreadability, pH, pri-
Received on: 05-04-2011 mary skin irritation test, stability study etc. and finally
Revised on: 21-05-2011 the suitable formulation was selected. The selected gel
Accepted on: 24-05-2011 formulations were again evaluated for their antimi-

©JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 373

crobial activity. The results showed that activity of es-
sential oil was not affected after incorporation into
polymer gel. Finally the suitable gel formulation was
selected which can be a better alternative for skin in-
fection in place of traditional harmful antibiotics.
MATERIAL AND METHODS
Chemicals and reagents
Carbopol 934, HPMC, Sodium CMC and PEG, DMSO
were obtained from Burgoyne & Burbidge’s co., Mum-
bai. Methyl paraben, Propyl paraben and glycerol were
obtained from Merck pvt. ltd, Mumbai. Sodium algi-
nate was obtained from Loba Chemie Pvt.ltd, Mumbai.
Extraction of essential oil
The essential oil from betle leaf was extracted by using
a Clevenger’s apparatus. Around 200 gm. of fresh
leaves were washed by tap water to remove all the
dirty materials and cut into small pieces. Sliced fresh
leaves were mixed with distilled water. A round bot-
tom flask containing the homogenate was heated up to
6-10 hr. The vapour condensed and separated
throughout an auto-oil/water separator. The oil pre-
sent at the upper most layer was collected in the test
tube. The volume of oil was measured and finally the %
of oil present in the sample was calculated. The total
amount of oil in the sample was calculated by following
method,
Leaf oil (%) yield (v/w) (fresh wt.) = Vol. of essential oil
(ml.)/ Wt. of raw materials *100
Antimicrobial activity assay of essential oil
The test organisms used in this study were Staphylo-
coccus aureus, Pseudomonas aeruginosa, Staphylococ-
cus epidermidis, E.coli, Micrococcus luteus, Candida
albicans, Aspergillus niger purchased from the micro-
bial type culture collection, Chandigarh India. All the
recommended media were purchased from Hi media.
Preparation of inoculums
The stock culture of micro-organisms used in the assay
was maintained on plate count agar slants for bacteria
& on SDA slants for fungi at refrigerated temperature
(4°C). Working bacterial culture and fungal culture
0
were grown from stock culture at 37 C for 24 hr on
0
nutrient agar and at 30 C for 48 hr on SDA, respec-
tively. The fresh sub-cultures of micro-organisms were
prepared by inoculation of each bacterial strain into
10ml. of nutrient broth & fungal strain into SD broth.
Incubation was performed at 37°C for 24 hr for bacte-
0
ria and 30 C for 48hr for fungi. Cell densities of ap-
6 -1
proximately 1×10 CFU ml were calculated and pre-
pared from cultures.
Antimicrobial assay of Betle oil Vs standard antibiotic
The essential oil of Piper betle was investigated for its
antimicrobial activity by modified agar well diffusion
technique & the diameter of zone of inhibition was
374 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences
B.Satpathy et al., Int. J. Res. Pharm. Sci., 2(3), 2011, 373-378
compared with that of standard antibiotic. In this
method, pre-sterilized petridish contained 15 ml of
Muller Hilton agar (MHA) for bacteria or Sabroud dex-
trose agar (SDA) for fungi, were aseptically introduced
with 0.2 ml of 24 hr broth culture of microbial strains
and evenly spread using a bent sterile glass rod. A ster-
ile cork borer was used to cut four wells of 6mm di-
ameter in each of plates. Measured quantity of betle
oil diluted with DMSO at 1:1, was carefully placed in
o
the well. The plates were incubated at 37 C for 24 hr
o
and at 30 C for 48 hr, for bacteria and fungi respec-
tively. Antimicrobial activity was evaluated by measur-
ing the inhibition zones. Inhibition zones were re-
corded as the diameter of growth free zone, including
the diameter of the well, in millimetres of the incuba-
tion period. Oil was classified as active when the di-
ameter of the inhibition zone was equal to or larger
than 6 mm simultaneously standard antibiotic gen-
tamicin for bacteria and ketoconazole for fungi was
used for comparison of zone of inhibition with that of
the oil. DMSO blank was used as control. The sample
was tested in triplicate.
Determination of minimum inhibitory concentration
(MIC)
MIC is defined as the lowest concentration where no
visible turbidity is observed in the test tube. In this
method, the broth dilution technique was utilized
where oil was prepared to the highest concentration of
50µl/ml. (stock concentration) in sterile DMSO and
serially diluted to a working concentration ranging
from 10µl/ml to 50µl/ml using NB for bacteria and SD
broth for fungi and later inoculated with 1ml suspen-
sion of the test organisms. After 24 hours of incuba-
tion, the test tubes were observed for turbidity. The
least concentration where no turbidity observed was
determined and noted as MIC.
Preparation of gel formulation
For all the formulation, methyl and propyl paraben
were dissolved in distilled water prior to the addition
of thegelling agents. Carbopol 934 gels were prepared
by soaking Carbopol 934 in water and then neutralizing
it with triethanolamine. HPMC and sodium carboxy
methyl cellulose gels were prepared by dispersing the
polymers in distilled water and soak it overnight to get
the gel. Gels containing sodium alginate were prepared
by triturating and then soaking it in distilled water.
Weighed quantities of polyethylene glycol and DMSO
were taken in separate test tube to which accurately
measured amount of betle oil was dissolved with con-
tinuous shaking. Thus the mixtures obtained were fi-
nally mixed to different polymers with continuous stir-
ring taking care to avoid entrapment of air. Then re-
quired amount of glycerine was added to all the formu-
lation and finally remaining quantity of purified water
was added with stirring to get the required weight.
Total sixteen type gel formulations were prepared us-
ing four different polymers and two different permea-
 B.Satpathy et al., Int. J. Res. Pharm. Sci., 2(3), 2011, 373-378

Table 1: Composition of gel formulation

Dislled
Sodium Sodium Methyl Propyl
Batch Oil Carbopol HPMC Glycerin DMSO PEG Water
CMC Alginate Paraben Paraben
No. (ml) 934 (gm) (gm) (ml) (ml) (ml) (ml)
(gm) (gm) (gm) (gm)
Upto
F1 0.5 0.5 - - - 5 0.18 0.02 0.5 - 100
F2 0.5 1 - - - 5 0.18 0.02 0.5 - 100
F3 0.5 - 5 - - 5 0.18 0.02 0.5 - 100
F4 0.5 - 7 - - 5 0.18 0.02 0.5 - 100
F5 0.5 - - 1.5 - 5 0.18 0.02 0.5 - 100
F6 0.5 - - 2 - 5 0.18 0.02 0.5 - 100
F7 0.5 - - - 9 5 0.18 0.02 0.5 - 100
F8 0.5 - - - 10 5 0.18 0.02 0.5 - 100
F9 0.5 0.5 - - - 5 0.18 0.02 - 0.5 100
F10 0.5 1 - - - 5 0.18 0.02 - 0.5 100
F11 0.5 - 5 - - 5 0.18 0.02 - 0.5 100
F12 0.5 - 7 - - 5 0.18 0.02 - 0.5 100
F13 0.5 - - 1.5 - 5 0.18 0.02 - 0.5 100
F14 0.5 - - 2 - 5 0.18 0.02 - 0.5 100
F15 0.5 - - - 9 5 0.18 0.02 - 0.5 100
F16 0.5 - - - 10 5 0.18 0.02 - 0.5 100

Table 2: Assay of Antimicrobial Activity of Betle Oil

ZONE OF INHIBITION OF OIL VS ANTIBIOTIC
MIC of
Micro- Vol. of oil Applied Mean IZD of oil Conc. of standard IZD of antibiotic
oil
organisms with DMSO (µl) (1:1) (mm) ± SD antibiotic (µg/ml) (mm)± SD
(µl/ml)
S.aureus 15 26±1 4 26.3±0.68 25
P.aeruginosa 25 19±0.57 4 21±1 >50
S.epidermidis 20 20±1 4 20.6±0.72 35
E. coli 18 22±1 4 23.6±0.23 < 30
M. luteus 18 21.6±0.5 4 22±0.57 40
C. albicans 20 24±0.57 6 24.6±0.34 30
A.niger 20 22.3±0.4 6 23±0.57 35
IZD-inhibition zone diameter, MIC-minimum inhibitory concentration, SD-standard deviation

tion enhancers i.e. Dimethyl sulfoxide (DMSO), Poly Determination of pH

ethylene glycol (PEG). All the prepared gels were then
subjected to various evaluation tests in order to select
the best formulation. The compositions of different gel
formations are listed in Table.1.
Precipitation or turbidity occurs in some of the batches
which could be due to the incompatibility in the system
due to presence of glycerin or polyethylene glycol.
Hence, these batches were discarded and remaining
batches (F2, F3, F5, F7, F10, F11, F13 and F15) were
considered for further study.
Evaluation of gel formulations
Evaluation of the prepared anti-microbial gels include
the assessment of parameters like Spreadability, Vis-
cosity, pH, Homogeneity, consistency, Primary skin
irritation test, stability studies and finally antimicrobial
assay.
The pH of the gels was determined using a digital pH
meter by dipping the glass electrode completely into
the gel system so as to cover the electrode.
Determination of viscosity
The viscosities of formulated gels were determined
using the Book field DV – II Programmable viscometer
using the spindle no. 4 at 1.5 rpm. About 100g.of gel
was taken for measurement. Temperature was main-
tained at 20°C.
Determination of Consistency
The measurement of consistency of the prepared gels
was done by dropping a cone attached to a holding rod
from a fix distance of 10cm in such way that it should
fall on the centre of the glass cup filled with the gel.
The penetration by the cone was measured from the
surface of the gel to the tip of the cone inside the gel.

©JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 375
 B.Satpathy et al., Int. J. Res. Pharm. Sci., 2(3), 2011, 373-378

Figure 1: Bar diagram of comparative antimicrobial assay of oil Vs antibiotic

Table 3: Characteristics of gel formulations

Viscosity Spreadability Consistency

Batch no. pH Skin irritation test Homogeneity
Cps gm.cm/sec (mm)
F2 6.7 52,000 16.25 Nil Very Good 6
F3 6.8 53,000 14.44 Nil Very Good 5
F5 6.8 56,000 13.00 Nil Good 4.5
F7 6.9 57,000 12.57 Nil Good 4
F10 6.7 52,000 16.25 Nil Very Good 6
F11 6.8 53,000 14.44 Nil Very Good 5
F13 6.8 56,000 13.00 Nil Good 4.5
F15 6.9 58,000 12.57 Nil Good 4

The distance travelled by cone was noted down after Determination of Homogeneity
10sec.
All developed gels were tested for homogeneity by
Determination of spreadability visual inspection after the gels have been set in the
container. They were tested for their appearance and
Spreadability of formulations was determined by an
presence of any aggregates.
apparatus, which was fabricated in laboratory and used
for study. The apparatus consist of a wooden block, Primary Skin irritation test
with a fixed glass slide and movable glass slide with
Test for irritation was performed on human volunteers.
one end tied to weight pan rolled on the pulley, which
For each gel, five volunteers were selected and 1.0g of
was in horizontal level with fixed slide.
formulated gel was applied on an area of 2 square inch
Spreadability was then calculated by using the formula, to the back of hand, covered with cotton and secured
S = M.L / T firmly in adhesive plaster. This was allowed to remain
in close contact with the skin for over 24 hours, after
Where, S is the spreadability, M is the weight tide to
which the site of application was examined for any
upper slide, L is the length of glass slide, T is the time
signs of lesions or irritation.
taken to separate the slide completely from each
other.

376 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences
 B.Satpathy et al., Int. J. Res. Pharm. Sci., 2(3), 2011, 373-378

Table 4: Stability studies of gel formulation having carbopol 934 and HPMC

Batch No. Month Consistency pH Homogeneity

0 6 6.7 Excellent
F2 1 6 6.7 Excellent
2 6 6.7 Excellent
3 5.9 6.6 Good
0 5 6.8 Excellent
1 5 6.8 Excellent
F3
2 5 6.8 Good
3 4.8 6.7 Good
0 6 6.8 Good
1 6 6.8 Good
F10
2 5.9 6.7 Good
3 5.8 6.7 Fair
0 5 6.9 Good
1 5 6.9 Good
F11
2 4.9 6.8 Fair
3 4.6 6.7 Fair
Table 5: Antimicrobial activity of gel formulations (F2, F3)
Micro-organisms Mean IZD of HPMC gel (F3) (mm) Mean IZD of cabopol 934 gel (F2) (mm)
Staphylococcus aureus 25±0.57 25.3 ±0.17
Pseudomonas aeruginosa 18.3±0.51 19 ±0.57
Micrococus luteus 19.3±0.17 20.3±0.17
E.coli 21.6 ±0.50 22 ±0.57
Staphylococcus epidermidis 20.3±0.17 22±0.57
Candida albicans 23±0.57 24.6 ±0.34
Aspergillus niger 22.3±0.68 23±0.57

Stability study of gel formulations
Stability studies of gel were done according to the In-
ternational Conference on Harmonization (ICH) har-
monized guidelines .All the selected formulations were
subjected to a stability testing for three months as per
ICH norms at a temperature of 40°C ± 2°C. All selected
formulations were analyzed for the change in appear-
ance, pH by procedure stated earlier. (ICH guidelines
8th, 2003)
Antimicrobial assay of gels
The gels showing well accepted physicochemical prop-
erties were finally selected for antimicrobial assay in
order to confirm whether any significant changes in the
activity occurred after formulation. 0.5 mg gel was ap-
plied on sterilized filter paper discs on agar media and
zone of inhibition was measured. The zone of inhibition
of gels was also compared with that of standard antibi-
otics. DMSO was used as blank.
RESULTS AND DISCUSSION
Determination of Antibacterial and Antifungal activity
Betle oil showed antimicrobial activity against various
skin infecting microorganisms.The antimicrobial activ-
ity of betle oil was compared with the standard antibi-
otics i.e. gentamicin for bacterial strain and ketocona-
zole for fungal strain (Fig.1).The results were shown in
Table.2
Zone of inhibition was found to be maximum that was
26mm at 15µl for S. aureus followed by 19mm at 25 µl
for P.aeruginosa, 20 mm at 20 µl for S.epidermidis,
22mm at 18 µl for E. coli, and 21.6mm at 18 µl for
M.luteus .The oil showed cidal activity against all tested
bacteria except P.aeruginosa where it showed static
effect at relatively higher concentration. Among the
tested fungus C.albicans showed maximum sensitivity
to the oil that is 24mm at 20 µl followed by 20mm at
20 µl for A. niger. MIC for all the tested pathogens was
below 60 µg/ml. Since these pathogens are severe skin
infecting microbes, the excellent activity of essential oil
of Piper betle against these microorganisms provide a
hope for the development of suitable dermatological
formulation.
Evaluation of gel formulations
Table.3 shows the results of homogeneity, consistency,
pH, viscosity, spreadability carried out on gels. pH of all
formulation lies in between 6.7‐7, which is compatible
to normal pH range of skin. Spreadability denotes the
extent of area to which the gel readily spreads on ap-
plication to skin or the affected part.
The bioavailability efficiency of a gel formulation also
depends on its spreading value. Maximum spreading

©JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 377
 B.Satpathy et al., Int. J. Res. Pharm. Sci., 2(3), 2011, 373-378

value was found for formulation with 1% Carbopol 934
and 5% HPMC with DMSO as permeation enhancer.
The skin irritation studies of selected gels were carried
out on human volunteers and no redness or irritation
was found on the applied surface which confirmed that
the prepared formulation was purely safe for applica-
tion on human skin.
From the selected formulations, F2, F3, F10 and F11
showed well physicochemical properties as compared
to other formulations. Hence these formulations were
further selected for stability studies.
Table 4 showed the stability studies of the selected gel
formulations which confirmed that batch F2, F3 exhib-
ited excellent stability profile. The formulation were
much cleared and having good Spreadability as com-
pared to other formulations. Considering above stabil-
ity studies and physicochemical properties (Table.3),
batch F2, F3 were selected for antimicrobial activity
studies. The result was shown in Table.5
CONCLUSION
The micro-organisms used in our study are the major
cause of most human skin infections. In the present
study essential oil of piper betle was investigated for its
activity against Pseudomonas aeruginosa, Staphylococ-
cus aureus, E coli, Micrococcus luteus, staphylococcus
epidermidis, Aspergillus niger and Candida albicans.
The result obtained from the antimicrobial assay was
highly promising which prompted us to formulate a
suitable dermatological gel preparation against these
skin infecting pathogens.
Formulation F2 with 1% carbopol 934 and F3 with 5%
HPMC was the best formulation, having good invitro
activity; the formulated gels were evaluated for gross
visual appearance, pH, and, spreadability and antim-
icrobial activity and almost no side effects can be ex-
pected from this formulation as the oil is natural ingre-
dient. We can conclude that industrial manufacturing
of this product can be taken up after conducting clini-
cal trials on human volunteers.
ACKNOWLEDGEMENT
Authors are very thankful to respected Principal and
management of Dadhichi college of Pharmacy for their
support.
REFERENCES
A R. Fathilah, R. Sujata, Antiproliferative activity of
aqueous extract of Piper betle L. and Psidium gua-
java L. on KB and HeLa cell lines, J. Med. Plant. Res,
Vol. 4,no.11, 2010 pp. 987-990.
Abhijeet Pandey, Jui V. Jagtap, S.A.Polshettiwar, For-
mulation and evaluation of invitro antimicrobial ac-
tivity of gel containing essential oils and effect of
polymer on their antimicrobial activity, Int J Pharm
Pharm Sci, Vol 3, No1, 2011 pp.234-237.
Adeltrudes B. Caburian, Marina O. Osi , Characterization
and Evaluation of Antimicrobial Activity of the Essen-
tial Oil from the Leaves of Piper betle L. E-
International Scientific Research Journal ,Vol. 2, no.
1, 2010 pp.2094-1749.
Bozena Michniak-Kohn, Priya Batheja, Topical drug
delivery by a polymeric nanosphere gel: Formulation
optimization and in vitro and in vivo skin distribution
studies, Journal of Controlled Release ,vol. 149, 2011
pp.159–167.
British Pharmacopoeia (1980) Vol.11, pp. A77, A104.
ICH Harmonized Tripartite Guidelines, Stability Testing
of New Drug Substances and Products, ICH Commit-
tee, 8, 2003.
M. A. Ekpo and P. C. Etim, Antimicrobial activity of
ethanolic and aqueous extracts of Sida acuta on mi-
croorganisms from skin infections, Journal of Me-
dicinal Plants Research Vol. 3,no.9,2009 pp. 621-624.
Nikhil Kumar, Pragya Misra, Piper Betle Linn. A Ma-
ligned Pan-Asiatic Plant with an array of
Pal Mahesh and Chandrasekhar K., Mosquito Repellent
Activity of Piper Betle Linn. Int. Jour. of Pharma. &
Life Sc. Vol.1, No.6, 2010 pp.313-315.
Pharmacological Activities and Prospects for Drug Dis-
covery, Current Science, vol. 99, no. 7, 2010 pp.922-
932.
S Bhattacharya, D Banerjee, AK Bauri, Healing property
of the Piper betle phenol, allylpyrocatechol against
indomethacin-induced stomach ulceration and
mechanism of action, World J Gastroenterol
,vol.13,no.27, 2007 pp.3705-3713.
T. Nalina and Z.H.A. Rahim, The crude aqueous extract
of Piper Betle L. and its antibacterial effect towards
Streptococcus mutans, Am. J. Biochem. & Biotech.,
vol.3,no.1,2007 pp.10-15.
U. D. Shivhare, Formulation Development And Evalua-
tion Of Diclofenac Sodium Gel Using Water Soluble
Polyacrylamide Polymer, Digest J. of Nanomaterials
and Biostructures., vol.4, no.2,2009 pp. 285 – 290.
Vivek Kumar R and Satish Kumar, Formulation and
evaluation of Mimosa Pudica gel, Int J Pharm Pharm
Sci, vol 3, no.1, 2011 pp.55-57.

378 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences