Ganesan V. et al. / Asian Journal of Research in Pharmaceutical Sciences and Biotechnology. 2(2), 2014, 47 - 54.

Research Article ISSN: 2349 – 7114

Asian Journal of Research in

Pharmaceutical Sciences and
Biotechnology
Journal home page: www.ajrpsb.com

FORMULATION AND IN-VITRO ANTIMICROBIAL EVALUATION OF HERBOSOMAL

GELS OF EXTRACTS OF QUERCUS INFECTORIA AND ACORUS CALAMUS

V. Ganesan*1, Sreekanth. S. Kaithavalappil1, M. Kannappan1, Deepa. T. Vasudevan1

*1
Department of Pharmaceutics, Research Scholar, Karpagam University, Coimbatore, Tamilnadu, India.

ABSTRACT .
The emerging technology of drug delivery is being applied to phyto‐pharmaceuticals to improve the
bioavailability of herbal extracts for medicinal applications. Plant extracts can be standardized accordingly
and may be formulated as phytosomes for systematic investigation for any improved potential use.
Phytosomal herbal formulations are better absorbed, and as a result produce better bioavailability and
actions than the conventional phytomolecules or botanical extracts. The term “Phyto” means plant while
“some” means cell-like. It is also often known as herbosomes. Phytosomes are produced by a process
whereby the standardized plant extract or its constituents are bound to phospholipids, mainly
phosphatidylcholine producing a lipid compatible molecular complex. This phyto-phospholipid complex
i.e. phytosome resembles a little cell. Phytosomes exhibit better pharmacokinetic and pharmacodynamic
profile than conventional herbal extracts. These drug-phospholipid complexes can be formulated in the
form of solution, suspension, emulsion, syrup, lotion, gel, cream, aqueous micro dispersion, pill, capsule,
powder, granules and chewable tablet.

KEY WORDS
Herbosomal gel, Bioavailability and Herbal extracts.

INTRODUCTION
It is widely accepted that the use of herbal medicine
Author for correspondence: is well established and safe1. Herbal medicine, now-
V. Ganesan, a-days are gaining importance for treating many
Department of Pharmaceutics, diseases due to their significant effect and lesser side
Erode College of Pharmacy, effects as compared to allopathic medicines2. The
Veppampalayam, Erode, Tamilnadu, India. goal of topical antimicrobial therapy in skin
infections is to control microbial colonization and
Email: [email protected]. subsequent proliferation thus promoting the healing
of the wounds3. In this research, Quercus infectoria
is a small tree or a shrub belonging to the Fagaceae
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(Quercaceae) family and here it was studied in order distillation apparatus. In order to obtain a completely
to investigate its antimicrobial properties. Gall of dry extract, the resultant extracts were transferred to
Quercus infectoria (QI), or better known as glass dishes, and were left in 50 Cº ovens for 24
Manjakani, is originated from Western Asia and hours. Then, they were left14 at 4 Cº.
Southern Europe. Galls are irregular plant growth, Formulation of Phytosomal Gel
which is stimulated by the reaction between plant Herbosomes are prepared by reacting the herbal
hormones and powerful growth regulating chemicals extract and soy lecithin and cholesterol in a ratio of
produced by insects or mites4. The QI galls are 1:1 and dissolving them in ethyl acetate. After
produced by the insect, Cynipsquercufolii, for solubilization has completed, the complex
depositing its eggs5. The chemical constituents of the compounds are removed by solvent evaporation
galls have been reported to comprise a large amount technique. Gel was prepared by using Carbopol 940
of tannins and small amounts of free gallic acids, as the gelling agent. Then the prepared herbosomes
ellagic acid and synergic acid6. Acorus calamus were incorporated into the gel and thus the
Linn. belongs to the family Acoraceae, commonly herbosomal gel was obtained.
known as “sweet flag” or “calamus”, is a Physicochemical Evaluation of Phytosomal Gel
semiaquatic, perennial, aromatic herb with creeping Physical Evaluation
rhizomes. The plant is found in the northern Preliminary evaluation was carried out as follows:-
temperate and subtropical regions of Asia. Acorus pH
Calamus (AC) has been used in traditional Indian The pH meter was calibrated using standard buffer
prescriptions for its beneficial effects on solution such as pH 4.0 and 7.0. About 0.5g of the
antiproliferative7, 13antidiarrhoeal, antioxidant8 and gel was weighed and dissolved in 50 ml of distilled
hypolipidemic activity9. The aim of the present study water and its pH was measured.
was to screen for the aqueous and ethanolic extracts Viscosity
of AC that could be useful for the development of Viscosity of the formulation was determined by
new tools as antibacterial agents for the control of Brookfield Viscometer at 100 rpm, using spindle no
infectious diseases. The herbosome process has been 7.
applied to many popular herbal extracts, including Homogeneity
Milk thistle, Ginkgo biloba, Grape seed, Green tea, The formulations were tested for the homogeneity by
Hawthorn, Ginseng etc. The herbesome technology, visual appearance and by touch.
markedly enhances the bioavailability of select Spread ability
phytomedicines, by incorporating phospholipids in Two glass slides of 20 x 20 cm were selected. A
to standardized extracts and so vastly improve their small amount of sample was sandwiched between
absorption and utilization10. the two glass slides. A 100g weight was placed upon
the upper slide so that the gel between the two slides
MATERIALS AND METHOD was pressed uniformly to form a thin layer. The
Preparation of extracts weight was removed and then fixed to a stand
The galls of Quercus infectoria and rhizomes of without slightest disturbance in such a way that only
Acorus calamus used in this study were collected the upper slide, slides off freely, to the force of
and washed with distilled water, and dried in air. The weight tied to it. The time taken for the upper slide
galls were crushed in mechanical mortar. Ethanol to separate away from the lower one was noted using
extracts were prepared by the following method. 50 a stop clock. This parallel plate method is the most
gm of gall and rhizome powders were used with 300 widely used method for determining and quantifying
ml of solvents, seperately with an extraction period the Spread ability of semisolid preparations.
24-72 hours. The extracts were filtered using filter S = M x L/T
paper and the solvents were evaporated using rotary

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Where, S – Spread ability; M - Weight tied to the RESULTS AND DISCUSSION

upper slide; L - Length of the glass; T - Time taken Herbal medicines are the important tool for many
in seconds. diseases because of their less side effects, compared
Extrudability to allopathic medicines.
It is an empirical test to measure the force required So medicinal plants extracts with antibacterial and
for the gel to extrude out from the tube. The prepared antioxidant potential were utilized for the
cream was filled into a collapsible tube and it was development of herbosomal gel formulation and
sealed and the weight of the tube was recorded. assessment of in-vivo skin permeation was the main
Placed a 500g weight on the tube and the amount of approach.
gel that extruded out was collected and weighed. Formulation and Evaluation of Herbosomal Gel
Then the percent of gel extruded was calculated. The Herbosomal gels were prepared by using carbopol
packing of gel have gained a considerable 940as gelling agent. Physical evaluation like pH,
importance in the delivery of desired quantity of Viscosity, Homogenity, Spreadability, Extrudability,
cream, therefore measurement of Extrudability have were determined as shown in Table No.1.
become an important criteria for creams. Physical Evaluation of Phytosomal Gel
Characterization of Herbosomal Gel The ethanolic extracts of were formulated into a
The behavior of Herbosomes in both physical and herbosomal gel and all studied formulations did not
biological system is governed by the factors such as show a considerable change in characters like color,
physical size, shape, stability and its distribution. odor and consistency with the viscosity of 78, 000-
Visualization 98, 000cPs and homogenous. The values are
Visualization of Herbosomes can be achieved by represented in the Table No.1. This showed that in
using scanning electron microscopy (SEM). the case of a gel, the consistency depended on the
Vesicle size and Zeta potential ratio of the solid fraction, which produced the
The particle size and zeta potential can be structure of the liquid fraction. The spreadability of
determined by Dynamic Light Scattering (Nicomp the formulation is recorded in Table No.1. The
380 DLS) using a computerized inspection system spreadability of the formulations was acceptable.
and photon correlation spectroscopy. The extrusion of the topical formulation from the
Vesicle stability tube is important during the application, as also
The stability of vesicles can be determined by patient acceptance. The study showed that the
assessing the size of the vesicles overtime. Mean size extrudability of the gel was comparatively better
is measured by DLS (Nicomp 380 DLS). than the ointment.
Stability studies Characterization of Herbosomal Gel
Stability studies were performed according to the Visual Inspection
ICH (International Conference on Harmonization) The prepared Herbosomal gel was visually inspected
guidelines. The optimized formulation was kept at and it showed that the formulation was homogeneous
two different temperatures, i.e. 30 ± 20c and 4 ± 20c without any gritty particles and was of optimum
for 90 days. consistency.
Comparison of the Optimized Herbosomal gel Globule Size Determination
with marketed Anti-microbial Gel Microscopic Evaluation
The prepared antimicrobial Herbosomal gel was The prepared antimicrobial Herbosomal gel was
compared with a standard drug Gentamycin (1µg/ml) observed under optical microscope at 100x and
and Nystatin (1µg/ml) as antibacterial and antifungal observed that the formed vesicles were of uniform
standard drug. size.

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Size and Size distribution Scanning Electron Microscopy

The vesicular size and size distribution was From the Figure 6, it is clear that the particle size of
evaluated by using dynamic light scattering, the the optimized formulation was confirmed to be 52-
results showed that increase in extract phospholipid 115nm. This was in accordance with the particle size
concentration increases the mean vesicular size. The of Herbosomes in the literature.
vesicular size was between 52nm-115nm. The Stability Studies
polydispersity index was found to be low, shows that The best formulation of the Herbosomal gel was kept
the particles were of low value shows that at varying conditions of temperature. The system
Herbosomes formed by hydration was of uniform was stable at 250C. There were no significant
size. changes in the formulation when kept at room
Zeta Potential Determination temperature (30 ±20C) and also at refrigerated
The magnitude of zeta potential gives a potential temperature (4 ±20C). Not much change of pH,
stability of the colloidal dispersion. If the particles viscosity, homogeneity, Spreadability, Extrudability
have, large positive or negative charge reveals that and degradation of the samples were observed during
they repeal each other and there is dispersion 45 days period. Table 2shows the datas for stability
stability. The zeta potential of the optimized studies for antimicrobial Herbosomal gel F2. There
formulation showed that the sample is sample is was no much change in the zeta potential of the
highly stable. It was found as -22.21, and hence this sample and this proves that the Herbosomal gel
indicates that the prepared formulation is stable. system remains stable.

Table No.1: Physical parameters of the formulation

S.No Formulation Code pH Viscosity Homogeneity Spreadability Extrudability
(cp) (gm/sec)
1 F1 6.0 26155 Homogeneous 38.1 94.85
2 F2 60.4 39481 Homogeneous 36.9 95.21
3 F3 60.6 37182 Homogeneous 21.3 93.29
4 F4 60.4 31094 Homogeneous 23.8 96.77
5 F5 60.4 27165 Homogeneous 27.7 94.28
6 F6 60.4 28920 Homogeneous 33.9 98.65
7 F7 60.6 22371 Homogeneous 27.5 93.43
8 F8 60.7 59172 Homogeneous 26.4 92.48
9 F9 60.5 56193 Homogeneous 24.3 91.22

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Table No.2: Size Distribution and Polydispersity of Formulations

S.No Formulation Code Vesicular size(nm)* Polydispersity**
1 F1 52.44±31.45nm 0.145
2 F2 53.23±36.79nm 0.1453
3 F3 98.28±48.36nm 0.1621
4 F4 65.45±25.46nm 0.08335
5 F5 76.56±46.79nm 0.1478
6 F6 107.89±36.57nm 0.1759
7 F7 103.28±21.45nm 0.1109
8 F8 115.14±26.79nm 0.1982
9 F9 110.23±21.34nm 0.1050
*
Data obtained from Nicomp 380 DLS, **PI=Standard deviation/mean vesicular size

Table No.3: Stability Studies of Herbosomal Gel, F2

S.No Months Temperature Formulation Parameters
pH Viscosity Homogeneity Spreadability Extrudability
0 RT 300c±20c 2 6.4±0.2 27634± Homogeneous 35.7±0.2 90.9±0.9
1 0 0
4 c±2 c 2 6.5±0.1 26889± Homogeneous 25.4±1.1 93.1±1.0
1 RT 300c±20c 2 6.5±0.1 31279± Homogeneous 36.1±1.1 90.3±1.2
2
40c±20c 2 6.3±0.1 27736± Homogeneous 24.3±1.1 92.5±1.0
0 0
3 2 RT 30 c±2 c 2 6.3±0.2 39127± Homogeneous 37.1±1.1 93.5±0.9
40c±20c 2 6.2±0.1 48193± Homogeneous 26.7±1.1 92.5±0.9
0 0
3 RT 30 c±2 c 2 6.1±0.1 37162± Homogeneous 34.2±1.3 91.5±1.2
4 0 0
4 c±2 c 2 6.8±0.1 51092± Homogeneous 31.5±0.8 93.2±0.8
Parameters are derived using mean ± SD

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Table No.4: Comparison Antimicrobial Activity (Agar Well Diffusion Method)

S.No Formulation INHIBITION ZONE (Mean± SD,n=3)
Staphylococus Escherichia Pseudomonas Candida
aureus coli aeruginosa albicans
(ATCC29737) (ATCC2068) (ATCC9027) (ATCC10231)
1 Phytosomal Gel 14.0±0. 15.0±0. 15±0. 20.0±0.
2 Positive Gentamycin (1µg/ml)
µg/ml) 22±0.0 21±0.0 23±0.0 NT
Control Nystatin (1 µg/ml) NT NT NT 16±0.0
3 Negative AQ, ET, PT ------ ------ ------ ------
control

Figure No.1: Vesicular Size of Herbosome F1-F2

Figure No.2: Vesicular Size of Herbosome F3-F4

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Biotechnology 2(2), 2014, 47 - 54.

Figure No.3: Vesicular Size of Herbosome F5-F6

Figure No.4: Vesicular Size of Herbosome F7-F8

Figure No.5: Vesicular Size of Herbosome F9

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Figure No.6: Scanning Electron Microscopy of Optimized Formulation

CONCLUSION 5. Phytosomes: A technical revolution in

The above article gives an outline about the phytomedicine.Available at: http://
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of polar nature exhibiting remarkable therapeutic Yadav V S R, Pandey V S and Singh V K. An
efficacy. Herbosomal Gels containing Quercus overview on traditional uses and
infectoria and Acorus calamus were successfully pharmacological profile of Acorus calamus Linn.
prepared. Physical evaluation, Characterization and (Sweet flag) and other Acorus species,
Stability studies of the prepared gels were done and International Immunopharmacology, 3, 2003,
compared with the marketed preparation. 53-61.
8. Manikandan S, Srikumar R, Jeya Parthasarathy
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