Assessment of In
vitro wound healing potential of Hibiscus leaf extract Emulgel
Deepa Amminbavi1, N
Prasanna Lakshmi2
1Assistant
Professor, Department of Pharmacognosy, Sri Venkateshwara College of Pharmacy,
Madhapur-Hyderabad-500081
2Assistant
Professor, Department of Pharmaceutical Chemistry, Sri Venkateshwara College of
Pharmacy, Madhapur-Hyderabad-500081
*Corresponding Author E-mail: deepa.amminbavi@gmail.com
ABSTRACT:
The lack of possible cure and
related foible in allopathic medicines has resulted in synoptic studies in
natural products as they have been discovered to be less adverse and cost
effective. The identification and evaluation of therapeutic potential in medicinal
products resulted to the discovery of innovative, cost-effective medicines for
treatment of several illnesses such as chronic wounds. In-vitro-cell scratch
testing is a cost-effective technique to understand the ability of plant
extracts to treat wounds. Hibiscus rosa sinensis (Malvaceae), has been used in
antifertility, as anti-inflammatory, as antiseptic and as a folk cure for the
treatments of skin diseases since ancient times. Considering the above claim,
an attempt was made to design emulgel formulations of the hibiscus leaf
methanolic extract (HL) and their impact was compared to the marketed
preparation commonly used for wound healing (WH) therapy. The emulsions were
produced using various oils, such as liquid paraffin (LEG), coconut oil (CEG),
olive oil (OEG) and varying Tween80 and Span80 concentrations. Using various
gelling agents such as Carbopol934 in varying proportions, the emulsion was
developed into emulgel. For their physicochemical parameters and percentage of
drug release, all formulations were assessed. Stability studies have been
carried out for three months in accordance with ICH guidelines. The % drug
release of OEG was found to be 82.52% within 5 hours and produced better
stability and promoted henceforth for in vitro WH studies. The
cytotoxicity of OEG on L929 cells was studied by MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and assay
revealed that OEG had no cytotoxic effect and the percentage viability of L929
cells at highest concentration (200μg/mL) was observed to be 74.5. In
vitro scratch assay was performed to evaluate the wound healing properties
of OEG and scratch assay showed significant concentration-dependent migration
and closed the gap by 24.16%, 51.66% and 86.72% in 12 hours, 24 hours and 48
hours respectively. These findings were compared to Standard (Cipladine), which
showed 9.35%, 49.6% and 100% of wound closures. Our research has shown OEG's
capacity to heal wounds in vitro. As a prospective source of wound
healing agents, OEG of HL can therefore be suggested.
KEYWORDS: Hibiscus rosa
sinensis, emulgel, in vitro wound healing, scratch wound assay.
INTRODUCTION:
The wound healing process continues to develop with an overwhelming
measure consisting of many molecular and cellular effects.1 In diabetic patients, neuropathy,
infection of the microbial tissue and low blood circulation compared with
ordinary patients, these deficient treatments are a serious procedure.2
India has been a major global player in its generic medicines and
pharmaceutical active ingredients.3 Modern dressings also aim at
transmitting therapeutic impacts like stimulation to reepithelialization and
synthesis of collagenand to promote angiogenesis within the injuries.4 Many
molecular biological developments have come to bear, for instance the creation
of skin tissue replacements, extremely advanced products such as wool dressings
and growth factor products, but they are not working well with all patients due
to absence of assistance for the natural healing procedures.5
For this reason,
the competent replacement of natural products has attracted researchers
worldwide as they are safe for the development of drugs.6 In
folklore medicine, medicinal plants have been widely used to facilitate wound
healing. The healing characteristics of the wound conducted their action
because of phyto-constituents.7 However, certain limits of herbal
extracts/plant activity such as high-acidic pH instability, liver metabolism
etc. resulted in drug concentrations below the therapeutic blood concentration
resulting in less or no therapeutic impact.8 In order to overcome
these, different drug delivery systems such as Emulgel, liposomes, niosomes,
microspheres and phytosomes have been formulated for the delivery of herbal
drugs. Herbal drugs also help boost solubility, improve pharmacological
activity, improve the macrophage distribution of tissues, maintain delivery and
protect them against physical and chemical degradation in the delivery system.9
This study seeks to find the wound healing power for herbal formulations.
Emulgel (Emulsion in gel) has appeared as one of the most helpful semi-solid
drug technologies as the stability of emulsion has been enhanced by integrating
a gel matrix.10 Leaf extract formulation of Hibiscus rosa sinensis
is presently being tested for its wound healing claim. The literature shows
that extensive work on leaf extracts of Hibiscus rosa sinensis was reported and
limited research was carried out in the formulation area. We are not aware of
any prior study about the in vitro wound treatment of HL emulgel
formulation, and for the first time we are reporting in this work. The healing
activities of Hibiscus rosa sinensis leaves in the aqueous and ethanol extracts
have been studied in rats before.11 We concentrate in this review on
in-vitro testing as it provides a quick, economic and ethical solution
compared to animal models.
Hibiscus rosa
sinensis (Malvaceae), also called as sorrel, red sordis and the Jamaican
sorrel. It is an ornamental plant cultivated all over India. The leaves are
used in Indian medicine for emollient, aperitif, anodyne, laxative and leaf
decoction and stem bark are used for abortion system. Due to the existence of
cholinergic and papaverine, alcoholic (50%) extract of aerial components showed
antispasmodic action on isolated smooth muscles-like substances, CNS depressive
and hypotensive action, while alcoholic leaf extract showed antipyretic,
analgesic and anti-inflammatory action. It is used to treat loss of appetite,
resentment, wound cure, treatment of damaged and irritated tissue and skin
rejuvenating.12 Leaves are easy ovate-lanceolate, leaves are whole
at the base and coarsely tooth on the apex, taste mucilaginous. Leaves comprise
β-sitosterols, stigmasterol, taraxeryl acetate and three cyclopropane
compounds with derivatives.13
Emulgel of HL
extract has been formulated using a range of gelling agents in the current
research. The prepared emulgel were evaluated for physicochemical and in
vitro wound healing activity.
MATERIALS AND METHODS:
Collection of plant material:
Hibiscus rosa sinensis
(Malvaceae) leaves were collected from Tirupati. The plant was authenticated by
Botanist Dr K Madhava Chetty working in Sri Venkateshwara University, Tirupati,
with voucher 1251.
Preparation of extract:
The leaves of Hibiscus rosa
sinensis were shade-dried seven days and then powdered. The powder was sieved
through Sieve no12 and then stored in air tight container. The leaf powder was
then extracted with ethanol in round bottomed flask and reflux for 7 days at
room temperature, to obtain ethanolic extract of HR. Fresh solvent is added and
all the extracts were combined by using Rotavap to get the HL extract.14
Chemicals and reagents:
All the chemicals used were of
Analytical grade and Methyl paraben, propyl paraben and Triethanolamine were
purchased from SD Fine chemicals Ltd. L929 Mouse Normal Fibroblast Cell lines
were procured from National Centre for Cell Science (NCCS), Pune. Cell culture
medium(#AL111), Fetal Bovine Serum (#RM10432), MTT (3-(4,5-Dimethyl
thiazol-2-yl)2,5-tetrazolium bromide) Reagent and D-PBS (#TL1006) were
purchased from Hi media Laboratories. Dimethyl Sulfoxide DMSO (#PHR1309) and
Camptothecin (#C9911, Sigma) were purchased from Sigma.
Formulation of emulgel:
Emulsion has been prepared by
adding the oil phase to the aqueous phase. By dissolving span 80 in light
liquid paraffin the oil phase was produced and the aqueous phase was by
dissolving Tween 80 in purified water. The extract was then dissolved in water
by sonication technique. Methyl paraben and propyl paraben were dissolved in
propylene glycol. Both systems were heated to 70-750c individually.
The oil phase was introduced with constant stirring to the aqueous phase and
then cooled at room temperature. The prepared emulsions were mixed in 0.5% to
4% concentrations of polymer (Carbopol 934) with constant stirring in
homogeniser for the formulation of Emulgel. (Table 1) and pH of the emulgel
were adjusted using triethanolamine.15,16,17
Evaluation of emulgel:
Physical evaluation:
Prepared gels of Hibiscus are
evaluated for physicochemical parameters like colour, consistency and odour.
They appear light brown, free from gritty particles.
Homogeneity:
All emulgel are tested for the
presence of any aggregates visually and homogeneity is justified.18
pH:
pH of the emulgel were
measured using Digital pH meter. 1 gm gel was dissolved in 100 ml distilled
water and kept for 2 hours. Average pH measurements of each formulation were
calculated in triplicate.19
Table 1: Formulation of herbal
emulgel
INGREDIENTS |
LEG |
CEG |
OEG |
Drug(mg) |
250 |
250 |
250 |
Light liquid paraffin(ml) |
1.5 |
|
- |
Coconut oil(ml) |
- |
1.0 |
- |
Olive oil(ml) |
- |
- |
1.5 |
Tween80(ml) |
0.4 |
0.2 |
0.3 |
Span80(ml) |
0.2 |
0.3 |
0.3 |
Methyl paraben |
0.002 |
0.002 |
0.002 |
Propyl paraben |
0.003 |
0.003 |
0.003 |
Propylene glycol(ml) |
1.5 |
1.5 |
1.5 |
Triethanolamine (q.s) |
q.s |
q.s |
q.s |
Caropol934(gm) |
0.3 |
0.3 |
0.3 |
Water up to20ml |
q.s |
q.s |
q.s |
Spreadabilty:
A measure of the spread
diameter of 1 g Emulgel between 20 x 20 cm glass plate after 1 min was
necessary for determining the diffusion of the prepared formulations. The plate
weight was standardized at 125 gm. A shorter interval indicated better
spreadability.
Spreadability was measured
using the formula,
S=M.L/T
S= Spreadability
M= Weight in the pan (tied to
upper slide)
L= Length moved by the glass
slide
T= Time (in sec) taken to
separate from the upper slide to the ground slide. 20
Viscosity:
The viscosity of the ready
formulations was measured using the Brookfield Synchroeletro Viscometer, (LV D
V Pro-II) spindle S64 (small cell adapters) by increasing the angular velocity
5, 10, 25, 50, 100 rpm and the readings were noted.21
Drug Content:
Spectrometrically, the drug
content was estimated by dissolving 1g of emulgel in 100ml phosphate buffer pH
7.4 and retained for 1-2 hours. The solution was filtered with the
Whatman filter paper no.42 and appropriate dilutions were made, if necessary
and the drug content was spectrophotometrically measured at 278 nm against
phosphate buffer pH 7.4.22
Stability Studies:
In accordance with ICH
guidelines, the physical stability of the prepared Emulgel were achieved by
storing the emulgel over three months at two distinct temperatures, i.e.
cooling (2-8±20C) and at room temperature (25±20C) for a
three-month period and studied for appearance, pH and spreadability.23
Invitro Diffusion Studies:
The studies of drug release
were conducted by modified diffusion device which consists of the beaker with a
100 ml phosphate buffer of pH 7.4 was put on magnetic stirrer and the medium is
stabilised at 37±0.50c. Emulgel was then applied on one end of the
dialysis membrane and the other end was sealed and suspended in the medium. For
a period of 8 hours, the emulgel samples (5 ml) were removed for every 1 hour,
filtered and replaced with a fresh medium at once. The drug content of the
sample was analysed by UV Spectrophotometer at 278nm using phosphate buffer as
blank. The cumulative drug release was calculated.24,25
Invitro wound Healing
Activity:
Culture 200μl
L929 cell suspension in a 96-well plate at required cell density (20,000 cells
per well), and allow the cells to grow for about 24 hours. Different
concentrations of the HL OEG emulgel (12.5,25,50,100 and 200μg/ml) are
treated with cell cultures and were incubated the plate for 24 hrs at 37°C in a
5% CO2 atmosphere. After the incubation period, the plates were
removed from incubator, add MTT reagent to a 20 μL of 0.5mg/mL of total
volume and wrap the plate with aluminium foil to avoid exposure to light.
Return the plates to the incubator and incubate for 3 hours. Remove the MTT
reagent and then add 100 μl of DMSO solution. Gentle stirring in a
gyratory shaker will enhance dissolution. Occasionally, pipetting up and down
may be required to completely dissolve the MTT formazan crystals especially in
dense cultures.
Scratch Wound Assay:
Cell lines L929 (mouse normal
fibroblast cell lines) were procured from National Centre for Cell science
(NCCS), Pune. Cells were grown in DMEM (Dulbecco's Modified Eagle's Medium-high glucose) in 10% FBS
(Fetal Bovine Serum) until cells reached with confluency. Cells were seeded
into six well tissue culture plate at a density of 0.5millin cells per well so
that after 24 hours, they should grow as to reach Approximately 80-100% the tip
of the pipette should always be at the bottom of the well. The resulting gap
distance should be therefore equivalent to the outer diameter of the tip. The
gap distance could be adjusted using different tips of the pipette. The line
would be scratched in one direction. Another line was scratched perpendicular
to the first line to obtain a cross in the well. After scratching, the well was
washed gently twice with the medium to remove the detached cells. The wells
were replenished with the fresh medium along with the test reagents at the
required concentrations. The cells were grown for additional 24 hours. Image
were captured at intervals of 0, 12, 24, 48 hours. The microscopes were
configured at the same position when capturing picture at the different views
of the monolayer. The gap distance was evaluated by using software Image J. The
multiple views of the wells were documented to avoid variable results with
repeated multiple times of experiment.26
Cell Culture and Cytotoxicity:
IC50 value of
emulgel were determined by (i) Medium control (medium without cells) (ii)
Negative control (medium with cells but without the experimental drug/compound)
(iii) Positive control (medium with cells and 25µM of Camptothecin). Frozen
cell lines L929 procured from National Centre for Cell science, Pune were
defrosted by placing in water bath at 370c. Cells (200µl) were
seeded into 96well plate at a density 2X104 cells per well at 370c
in 5% CO2 atmosphere along with OEG of HL of different
concentrations. After the incubation period of 24hours the medium was replaced
with DMEM along with emulgel and then the plates were incubated for further
24hours. Then 0.5mg/ml of MTT {3-(4,5-Dimethyl thiazol-2-yl)2,5-tetrazolium
bromide} reagent was added to each well and the incubated for 3hours at 370c.
Then 100µl of DMSO (Dimethyl sulfoxide) was added for the complete dissolution
and stirred gently for the dissolving MTT formazan crystals. The absorbance was
determined spectrophotometrically at 570nm taking 630nm as reference standard.
The IC50 value was determined by using linear
regression equation.26
RESULTS AND DISCUSSION:
The methanolic and
hydroalcoholic leaf extracts of HR were prepared and methanolic were used for
the emulgel preparation. An absorption maximum of 278nm was determined by UV
spectroscopy, and the drug solution is prepared in phosphate buffer pH 7.4.
(Fig.1). Different Emulgel were formulated (LEG, CEG, OEG) using polymer
Carbopol in different concentrations (Table 1). The IR spectrum of the Hibiscus
extract was compared with IR spectrum of combination of Hibiscus extract and
excipient and it was found that there were no specific interactions between the
drug and excipient. (Table 2). HR extract and Drug –Excipient compatibility
studies were carried out by Fourier Transform Infrared Spectroscopy analysis
(Shimadzu 8400S). Fig 2,3.
Figure1: UV spectra
of methanolic leaf extract of HR at 278nm.
Figure 2: FT-IR of
HR extract
Table 2: The FT IR
of Hibiscus leaf extract and the OEG emulgel
Functional group |
Observed functional group in drug |
Observed functional group in emulgel |
C=C |
1629.9 |
1629.9 |
S=O |
1045.5 |
1041.6 |
O-H |
3421.8 |
3421.8 |
N-H |
3500.2 |
3500.2 |
Figure 3: FT-IR of
drug-excipient.
The emulgel
formulations (OEG, LEG, CEG) were light brown in colour, homogenous and opaque
in appearance (Table 3). pH of the emulgel were found in the range of 6.01-7.4
which is not irritant to skin (Table 3). All the formulated emulgel were
homogenous in nature, were spreadable easily with the normal shear. The
viscosity of the prepared emulgel were measured using Brookfield viscometer
(LV-DV Pro II), Spindle S64 (Small sample adapter) and the angular velocity
increased from 5, 10, 50, and 100rpm and values were noted and represented in
the (Table 4). The drug content was found to be 87.4-97.4% indicating the
uniform release of drug shown in the (Table 5). The in vitro drug
release of all the formulations were impressive, and the drug release was
observed to be 82% (Table 6).
Table 3: Physical
evaluation, pH, homogeneity and spreadability of Emulgel formulations
Formulation |
Colour |
Apperarence |
Homogeneity |
PH |
Spreadability (gm.cm/sec) |
LEG |
Light brown |
opaque |
++ |
6.9±0.1 |
3.1 ±0.3 |
CEG |
Light brown |
opaque |
++ |
6.9 ±0.18 |
2.1±0.12 |
OEG |
Light brown |
opaque |
++ |
7.2± 0.2 |
3.2± 0.13 |
Table 4: Viscosity
of Emulgel formulations
Emulgel |
Viscosity at 5rpm |
Viscosity at 10rpm |
Viscosity at 15rpm |
Viscosity at 20rpm |
LEG |
6953 |
4183 |
1172 |
693 |
CEG |
5136 |
3648 |
1254 |
462 |
OEG |
9842 |
5962 |
1892 |
1081 |
Table 5: Drug
content of Emulgel formulations
Days |
%Drug content at room temperature conditions (25±20C/60+5%RH) |
%Drug content at room temperature conditions (40±20C/75+5%RH) |
0 |
98.68 |
98.21 |
30 |
96.18 |
96.62 |
60 |
95.41 |
95.48 |
90 |
97.16 |
97.42 |
Table 6: Drug
release of Emulgel formulations
Time (hrs) |
% Drug release of leg |
% Drug release of ceg |
% Drug release of oeg |
0 |
0 |
0 |
0 |
30 |
5.85 |
6.72 |
8.82 |
45 |
10.61 |
12.88 |
13.58 |
1 |
17.85 |
19.36 |
28.78 |
2 |
34.68 |
31.18 |
41.18 |
3 |
46.41 |
42.06 |
52.18 |
4 |
58.21 |
59.89 |
71.18 |
5 |
71.18 |
64.21 |
84.28 |
The impact of
cytotoxic effect of different concentrations (12.5μg/ml to 200μg/ml)
of OEG on L929 cell viability after incubation for 48hrs were determined using
MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} reduction
assay. The concentrations of OEG used for the treatment and their percentage
cell viability and represented in (Fig 3). The IC50 falls
within the range of 12.5μg/ml to 200μg/ml. The percentage cell
viability of L929 cells for OEG at the highest concentration of 200μg/ml
was found to be 74.5%. (Fig 4)
Fig 3: The effect of
OEG on L929 cell line viability MTT assay method.
Scratch wound assay test:
Using scratch
assay, response of cell migration to an artificial wound was made to observe
the effects of OEG on migration process under the inverted biologic microscope
and the migration rate of cells tested for 48hrs are shown in Fig 4. Percentage
wound closure at different time intervals in control, OEG treated and standard
drug-treated cells have been represented in Fig 4. The rate of migration of
cells was significant at the concentration 100 μg/ml and closed the gap by
86% in 48 hrs compared to standard.
Fig 4: Percentage of
cells migrated towards the wound and involved in wound closure.
CONCLUSION:
The quest for
virtuous topical preparations continues, given the availability of many
formulations. This research is currently being done to formulate and evaluate
topical emulgel containing Hibiscus leaf extract. The findings of the present
study showed that the formulated emulgel has no cytotoxic effect and is capable
of effectively completing the wound closure. Thus, Hibiscus emulgel (OEG) can
found to be a possible source of natural wound healing compounds.
AKNOWLEDGEMENT:
We are grateful for
the support we have received from Dr. M. Bhagavan Raju to carry out the
research project. We thank Surabhi Educational Society for providing laboratory
facilities of Sri Venkateshwara College of Pharmacy.
CONFLICT OF INTEREST:
The authors declare no
conflict of interest.
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Received on 19.10.2019
Modified on 25.11.2019
Accepted on 29.12.2019 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Res. 2020; 10(2):67-72.
DOI: 10.5958/2231-5691.2020.00013.1