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Ranjit Singh et al. / International Journal of Advances in Pharmaceutical Research
IJAPR
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Research Paper
ISSN: 2230 – 7583
FORMULATION OF HERBAL LIPOSOMES CONTAINING GREEN TEA AND
GAULTHERIA PROCUMBENS FOR ANTI ACNE ACTIVITY
Ranjit Singh 1,* , C.Sankar 1
1 KMCH College of Pharmacy, Department of Pharmaceutics, Kovai estate, Kalapatti Road, Coimbatore–641035,
Tamil Nadu (INDIA).
Received on 14 – 08 - 2012 Revised on 17 – 09- 2012 Accepted on 24– 09 – 2012
ABSTRACT
Novel drug delivery carriers such as liposomes are very versatile to suit the delivery of various drug molecules.
Clindamycin is generally considered the most effective antibiotic in treatment of acne. Topical clindamycin are
used less often due to lower efficacy or increased side effects. Green tea and Gaultheria procumbens are the best
herbal remedies known to treat acne because of its antibacterial properties. Thus, herbal liposomes have been
selected for the present work assuming that incorporation of green tea and Gaultheria procumbens extract into
liposomes may reduce the side effects associated with synthetic drug. Liposomes with green tea and Gaultheria
procumbens extract were prepared using lipid film hydration method and the optimum ratios of the components
were determined. Herbal liposomes were characterized for their vesicle size, shape, encapsulation efficiency, drug
content and in-vitro drug release study. Highest encapsulation efficiency (70.0%) and in-vitro drug release (95.2%)
was achieved with formulation F1. Liposomal formulations have been incorporated into carbopol gel base and
found to be more superior against Micrococcus luteus.
Keywords: Herbal liposome; anti-acne; green tea; Gaultheria procumbens; Micrococcus leutus; novel drug delivery
system.
INTRODUCTION
The leaves and flowers of Gaultheria procumbens
have a mildly perfume scented aroma 1 . This plant
can survive very cold winters with annual averages
as low as -40° Fahrenheit. A few of its uses include:
as an anti-inflammatory, as an antiseptic, as a
digestive tonic and as an antirheumatic 2 . Green tea is
the best herbal remedies known to treat acne because
of its antibacterial properties 3 . Acne vulgaris is a
disorder of the pilosebaceous unit resulting in the
formation of comedones, inflammatory papules,
pustules, nodules, cysts and scars 4 . It affects the face
and trunk, most commonly presenting in puberty.
Address for corresponding author
Ranjit Singh
KMCH College of Pharmacy,
Department of Pharmaceutics,
Kovai estate, Kalapatti Road,
Coimbatore–641035,
Tamil Nadu (INDIA).
ranjitsingh08685@gmail.com
Mobile No: 09042978206
Liposomes can transport drugs to target sites and
maintain a higher drug concentration than
conventional dosage forms. As a result, the
therapeutic effectiveness of liposomal drugs can be
enhanced for several folds. The study on liposomes
for targeting drugs into the pilosebaceous units has
suggested that liposomes are potent drug delivery
systems for treating hair follicle-associated disorders
such as acne 5 . Acne skin-care preparations
containing antibiotics, which are useful for treatment
of mild to moderate inflammatory acne, partially
exert their beneficial effects by decreasing the
follicular population of P. acnes. However, the
widespread use of antibiotics in dermatological
treatments has led to the development of drugresistant
P. acnes strains. To overcome the potential
risk of adverse effects and antibiotic resistance from
prescription medications, green tea and Gaultheria
procumbens have been used 2, 3 .
MATERIALS AND METHODS
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Ranjit Singh et al. / International Journal of Advances in Pharmaceutical Research
The chemicals used are Cholesterol , Soya Lecithin ,
Chloroform , Triton X- 100, Methyl hydroxyl
Benzoate , Triethanol amine (SD fine Chemicals,
Mumbai ), Electronic balance BL-220H (Shimadzu
Corporation Japan), Centrifuge apparatus C- 30BL
(Remi equipment, Mumbai), Orbital Shaker CIS
24BL (Remi equipment, Mumbai), Incubator HIS
TC 102 (Remi equipment, Mumbai), Microscope
BM- 800(Olympus, Japan).
Collection of plant material
The leaves of plant belonging to the genus Camellia
and Gaultheria were collected from Korakundah,
Nilgiri estate (Tamil Nadu) and were authentificated.
The leaves were air dried under shade and coarsely
powdered. The powdered leaves were used for the
extraction and anti acne activity.
Extraction from collected materials
The collected materials were extracted by
continuous hot percolation (soxhletation). 50g
of powdered leaves of green tea and Gaultheria
procumbens were defatted using petroleum
ether. The marc obtained from both the
powdered leaves was successfully extracted
separately with 250 ml of methanol by using
soxhlet apparatus. The extraction was carried
out for 24 hours. After extraction, the solvents
were distilled out; the concentrated residues
were analyzed for anti acne activity 6 .
Formulation of liposomes containing green tea
and Gaultheria procumbens
Five liposomal formulations were prepared by thin
film hydration method using extract, soya lecithin,
cholesterol. The soya lecithin: cholesterol: leaves
extract were used in a ratio of 50:50:20 (F1),
60:40:20 (F2), 70:30:20 (F3), 80:20:20 (F4) and
90:10:20 (F5) as shown in the Table 1. Extract was
used in each preparation. Both extract being watersoluble
was incorporated along with aqueous phase.
A 10 ml solution of chloroform containing
cholesterol and soya lecithin was dissolved and kept
for evaporation until depositing a thin layer of the
solid admixture on the walls of the flask. The dried
lipid film was hydrated with 10 ml of distilled water
containing drug by shaking it in vibrator for about 1
hour to disperse the film in form of poly dispersed
liposomes 7, 8, 9, 10 .
PREPARATION OF LIPOSOMAL GELS
Formula
Carbopol 940 : 2%
w/w
Triethanolamine : q.s.
Methyl hydroxy benzoate : 0.15 % w/w
Distilled water : 95.8 % w/w
Preparation of Carbopol 940 Gel Base
Carbopol 940 was sprinkled slowly to 5 ml of water
as medium and the medium were continuously
stirred to get a uniform dispersion of carbopol. The
methyl hydroxy benzoates were pre dissolved in
separate portion of water (5 ml) and added to
carbopol dispersion. Final volumes were adjusted
with water and pH brought to neutral by using the
triethanolamine. This preparation was kept
overnight, till the carbopol becomes uniform in
texture and appearance and the air bubbles are
removed 11 .
Incorporation of Liposomes into Gel Base
The drug loaded liposomes were incorporated into
gel base (carbopol 940) in such a way that final
formulation contained 1 % w/w (0.2 g/10ml
liposomal suspension was levigated with 10g of gel
base) drug 11 .
CHARACTERISATION OF LIPOSOMES
CONTAINING LEAVES EXTRACT
Average size and size distribution
Average size and size distribution of herbal
liposomes were determined by microscopic method.
Shape
A drop of herbal liposomal formulation were placed
on the microscopic slide and covered using a cover
slip and observed under microscope 9 .
Drug content
The herbal liposomal suspension were centrifuged
and washed three times with distilled water. Then the
herbal liposomal suspension were lysed using 1%
v/v solution of triton X-100 (0.2 ml in 2 ml of
liposomal suspension), Then it was centrifuged for 1
hour at 2000 rpm and 0.5 ml supernatant were
separated and analyzed for drug content 9 .
Encapsulation efficiency
A 2ml of herbal liposomal suspensions were
centrifuged at 2000 rpm for 1 hour. Clear
supernatant were separated and after decantation of
clear supernatant, remaining residues which are in
the herbal liposomal form were lysed using 1% v/v
solution of triton X- 100. Then it was centrifuged for
1 hour at 2000 rpm and 0.5 ml supernatants were
separated for analysis of encapsulation 9 .
Stability
Stability of the selected formulation was carried out
at
25⁰C ± 2⁰C / 60% RH ± 5% RH for 3 months.
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Ranjit Singh et al. / International Journal of Advances in Pharmaceutical Research
Effects of temperature and RH on the vesicle size
and drug content were studied 9 .
In-vitro drug release study
In-vitro drug release study were done using egg
membrane by using a diffusion cell. The release
studies were done for continuous 8 hours 10 .
Determination of Minimum Inhibitory
Concentration (MIC)
The minimum inhibitory concentration has been
determined using organism Micrococcus luteus by
tube dilution method 10 .
RESULTS AND DISCUSSION
Average size and size distribution
The average particle size of liposome was carried out
by using the microscopic method. The results are
given in fig 2. Prepared herbal liposomes were
viewed under microscope to study the lamellarity
and shape as shown in photomicrograph in figures 3.
Drug content
Drug content of liposomal formulation was
determined, the results are given in the table 2.
Encapsulation efficiency
The amount of drug entrapped in the liposomes was
determined by complete vesicle disruption followed
by extraction with 1% v/v solution of triton X- 100.
The percentage of entrapped drug in the liposomal
formulations is shown in fig 4 and table 3.
In-vitro drug release study
The in-vitro drug release study was carried out using
egg membrane. The release studies were done for
continuous 8 hours and then last sample was
withdrawn at 24 hours to determine whether drug
release was still continued. The results are shown in
fig 1.
Stability study of liposomal suspension
The effect on vesicle size and drug content during
stability studies was done at 25⁰C±2⁰C / 60%RH ±
5%RH and results are given in the table 2.
Determination of Minimum Inhibitory
Concentration (MIC)
MIC of extract and formulated liposomes was found
to be 0.1 mg/ml and 0.2 mg/ml. Liposomes was
effective against Micrococcus luteus. Data of MIC
for antibacterial activity is given in the table 4 and 5.
Table. 1. Formulation of liposomes containing Extracts
F 1
50:50:20
F 2
60:40:20
Extract
F 3
70:30:20
F 4
80:20:20
F 5
90:10:20
Table. 2. Effect on vesicle size and drug content during stability
Parameter
Vesicle size
Initial
Liposomal suspension
Final
4.26±0.012 4.75±0.016
Drug content (%) 69.9±0.39 68.2±0.29
*n=3
Table. 3. Table showing the drug content and Encapsulation Efficiency
Formulatio
n
Drug content (%)
Encapsulation efficiency
F1
71.2±0.71 69.5±0.98
F2 64.2±0.23 56.4±0.37

F3 57.8±0.65 52.6±0.58
F4 53.5±0.77 48.2±0.41
F5 49.3±0.47 45.7±0.95
*Value represented mean± S.D, n = 3.
Table. 4. MIC of test compound against tested organism
MIC values
Test compounds
Micrococcus luteus (μg/ml)
Extract 100
Formulated Green Tea liposomes 200
Dilutions (µg/ml)
Table. 5. Data of MIC for antibacterial activity
Presence or absence of growth
Micrococcus luteus
1 2 3 4 5 6
Extract
-
-
-
-
+
+
Formulated herbal
liposomes
-
-
-
+
+
+
Figure. 1. In-vitro drug release of formulated liposomes
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Ranjit Singh et al. / International Journal of Advances in Pharmaceutical Research
Figure. 2. Size distribution of formulated liposomes
Figure. 3. Shape of formulated liposomes
Figure. 4. Encapsulation efficiency of formulated liposomes
CONCLUSION
Herbal liposomes containing green tea and
Gaultheria procumbens extract was prepared using
different ratio of lipid and cholesterol by thin film
hydration method in order to achieve a controlled
and prolonged release of drug, when administered by
topical route. The formulation containing lipids and
cholesterol with 1:1 ratio is found to be better when
it’s characterized for various pharmaceutical
characters. Herbal liposomes showed prolonged
release even upto 24 hours. In conclusion herbal
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Ranjit Singh et al. / International Journal of Advances in Pharmaceutical Research
liposomes have real potential for treatment of Acne
vulgari
.
ACKNOWLEDGEMENT
The authors are thankful to The Principal Dr. A.
Rajasekaran, KMCH College of Pharmacy for
providing the necessary facilities in the College,
Sincerely thanks to Dr.K.S.G Arulkumaran,
Department of Pharmacuetics, KMCH College of
Pharmacy, Coimbatore -35, Tamil Nadu (INDIA) for
his valuable support. We would also like to thank
our colleagues, lab assistants for their support.
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