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