Evaluation of Wound Healing Activity of Polyherbal Formulation in Rats

Krishna Murti^1*, Vijay Lambole ^1, Mayank Panchal ¹, Megha Shah¹, Vipul Gajera¹

Dept. of Pharmacology, Vidyabharti Trust College of Pharmacy, Surat, Gujarat, India.

ABSTRACT:

Rungia pectinata Linn, Rubia cordifolia Linn and Scoparia dulcis Linn. are reported to possess wound healing, anti-inflammatory, analgesic and anti-bacterial activities. The formulations containing extracts of the above mentioned herbs were formulated and their wound healing activity was studied on experimentally induced open wounds in albino rats through topical route. The wound healing property of polyherbal formulation appears to be due to the presence of its active principles, which accelerates the healing process and confers breaking strength to the healed wound. It may be attributed to the synergistic action of constituent of plants present in the polyherbal formulation. Povidine iodine 5% was used as Standard drug.

KEYWORDS: Rungia pectinata, Rubia cordifolia, Scoparia dulcis, wound healing, Polyherbal formulation

INTRODUCTION:

A wound may be defined as a break in the epithelial integrity of the skin or may also be defined as a loss or breaking of cellular and anatomic or functional continuity of living tissue.¹. Wound healing studies are mainly aim to detect various means and factor influencing healing process, so they could be either used or avoid in clinical practice to favorably alter the healing process ². The vast literature on wound healing is focused mainly on skin, which is the most susceptible organ in the body that interacts with the environment and, therefore, receives constant insult and damage. Wound healing involves a complex interaction between epidermal and dermal cells, the extra cellular matrix, controlled angiogenesis and plasma-derived proteins all coordinated by an array of cytokines and growth factors. This dynamic process is classically divided into three overlapping phases “Inflammation, Proliferation and Remodeling” ³.

The growing popularity of natural and herbal medications, easy availability of raw materials, cost-effectiveness prompted us to formulate a polyherbal topical preparation and assess its wound healing ability. The combination is used in order to enhance the wound healing activity.This Polyherbal formulation was selected on a huge literature survey and there was no scientific data available.

The polyherbal formulation contains three plants namely: Rungia pectinata, Rubia cordifolia, and Scoparia dulcis.

Rubia cordifolia Linn (Rubiaceae) is a slender, branched; climbing plant, with very long cylindrical roots, widely distributed in China, India and tropical Australia. It contains Anti –inflammatory, hepatoprotective and anticonvulsant property.^4-6

Rungia pectinata ( Acanthaceae ) is a much branched, procumbent or erect herb found as a weed throughout the warmer parts of India^7,8. The juice of the leaves is considered cooling and aperient, and is given to children suffering from smallpox. Bruised leaves are applied to contusions to relieve pain and reduce swelling. In Bihar, the roots are used as a febrifuge by the tribal population^{.9, 10}

Scoparia dulcis (Scrophulariaceae), commonly known as sweet broomweed is a perennial herb widely distributed in tropical and subtropical regions. In these regions, fresh or dried S. dulcis plants have been traditionally used as remedies for stomach troubles¹¹, hypertension¹², diabetes ¹³, bronchitis¹⁴, and as analgesic and antipyretic agents¹⁵.

MATERIALS AND METHODS:

The plants were selected on the basis of their wide medicinal uses in the traditional literatures. Roots of Rungia pectinata, Rubia cordifolia, and Scoparia dulcis were collected and authenticated by the Associate Professor Dr. M. K. Saxena, Department of Botany, M.P.G.College campus, Modinagar, C.C.S University, Meerut (U.P), by carrying out macroscopic and microscopic evaluation.

Extraction: Root of the all the plants were dried in shade individually. The dried roots were powdered (3 kg each), defatted with petroleum ether (60–80 ◦C), and soaked in ethanol (95%) and kept aside for 4 days. After 4 days, the ethanolic layer was decanted off. The process was repeated for four times. The solvent from the total extract was distilled off.

Preliminary phytochemical studies: The preliminary phytochemical studies were performed for testing different chemical groups present in ethanolic extract of each plant (16). The freshly prepared root extract of Rungia pectinata, Rubia cordifolia, and Scoparia dulcis were qualitatively tested for the presence of chemical constituents. Phytochemical screening of the extract was performed for alkaloids, flavonoids, phenolic compounds, tannins, carbohydrates, proteins and amino acids. Saponins was also tested .These were identified by characteristic color changes using standard procedures.

Preparation of topical formulation: A topical formulation was prepared. An ointment with water soluble base was of first choice due to their ease of preparation and also eases of cleaning after application. Polyethylene Glycol (PEG) Ointment base (17), a mixture of PEG 4000 and PEG 600 found to have sufficient consistency in ratio 3:7 respectively, thus suitable for ointment preparation with concentration of 10 % w/v of extract. The prepared formulations was then evaluated by various parameters e.g. consistency, stability etc.

Animals: Wistar albino rats of either sex weighing between 180 and 200 g were obtained from Nitin Scientific Biologicals, New Delhi. The study was approved by the Institutional Ethics Committee for animal experimentation NKBR College of Pharmacy, Meerut (NKBR/ IAEC/2009/03) and all the procedures on animals were carried out as per CPCSEA guidelines, India. These animals were used for the wound healing activity studies. The animals were stabilized for 1 week. They were maintained in standard conditions at room temperature, 60±5% relative humidity and 12 h light dark cycle. They had been given standard pellet diet and water ad libitum throughout the course of the study.

Excision wound model: Under light ether anesthesia an impression of 500 sq mm was made on the shaved back of the rat as described in (18). The skin of the impressed area was excised carefully. Animals were kept in separate cages. The day on which wound was made consider as ay‘0’ (Zero). Animals divided into four groups of each with 5 animals. Group A considered as control and treated with simple ointment (eg. Bees wax, Cetosteryl alcohol etc.), group B considered as standard and treated with 5% w/w Povidine iodine ointment, group C were Polyherbal Preparation treated group and applied ointment 10%(w/v) respectively. The percentage of wound closure was recorded on day 4,8,12 and 16. Wound area was traced and measured planimetrically with the help of sq mm graph paper. Number of days required for falling of the eschar without any residual raw wound gave the period of epithelization.

Incision wound model: The rats were anesthetized by administering ketamine (0.5 ml/kg b. w. i.p.). Incision wounds of about 6 cm in length and 2mm in depth were made with sterile scalpel on the shaved back of the rats 30 min later the administration of ketamine injection. The parted skin was kept together and stitched with black silk at 0.5cm intervals. Surgical thread (no. 000) and a curved needle (no. 9) were used for stitching. The continuous thread on both wound edges were tightened for good closure of the wounds. The wounds of animals in the different groups were treated with topical application of the Ointments as described above, for the period of 10 days. The wounding day was considered as day 0. When wounds were cured thoroughly, the sutures were removed on the 8th post-wounding day and the tensile strength of the skin that is the weight in grams required to break open the wound/skin was measured by tensiometer on the 10^th dayreported (19).

Tensile strength: Tensile strength was calculated using the following formula:

Tensile strength = Breaking strength (g)

Cross-sectional area of skin (mm²)

Estimation of biochemical marker: Circular wound with approximate area of 500 mm² was created using the procedure described in excision wound model. The wounds were treated with topical application of ointments as described above for 10 days. The scab was removed on 11th day and dried in oven at 110 ⁰C.

The hydroxyproline content in dried scab was determined by extracting hydroxyproline from scab using concentrated Hydrochloric acid followed by reaction between amino groups of hydroxyproline with p-dimetylaminobenzaldeyde to develop red colour. The red colour thus measured on Spectrophotometer at 558nm using the method described by Bergman I and Loxley R. (20)

Statistical analysis: The mean value ± SEM was calculated for each parameter. Results were statistically analyzed by one-way-analysis of-variance (ANOVA) followed by dunnet’s test. P < 0.05 was considered as significant.

RESULTS:

Phytochemical analysis: Qualitative phytochemical analysis revealed presence of tannins especially hydrolysable tannins and alkaloids and saponins. The presence of alkaloid was confirmed by performing TLC and spraying with Dragendorff’s reagent.

Excision wound model: Topical application of Polyherbal Preparation increased the percentage of wound contraction and completed wound healing by 16th day, which indicates rapid epithelization and collagenization. In fact, topical administration of Polyherbal Preparation extract accelerated the progression of wound healing by 12th day, i.e. (87.71 ± 0.56**) p < 0.001 compared with control (57.88 ± 1.47) with 10% extract. It also reduced the epithelization time from 24.33±0.33 to 18.97±0.06** p < 0.01 compared with control. Povidine iodine also showed significant effect, (79.79± 0.98**) on 12^th days i.e. p < 0.01 as compared with control (Table 1) (Fig.1)

Incision wound model: The breaking strength of the incision wounds was increased in drug treated groups to significant extent, i.e. 271.7 ± 4.37. in control was increased up to 497.9 ± 6.28** (p < 0.01) with 10% extract. The results are also comparable to standard drug Povidine iodine. (Table 2) (Fig. 2)

Table 2: Effect of 10% extracts ointment of Polyherbal Preparation on breaking strength(g) in incision wounds.

Groups	Breaking Strength(g)
A Control	271.7 ± 4.37
B Standard Povidine iodine 5% w/w	577 ±6.73***
C Polyherbal Preparation 10%	497.9 ± 6.28**

The values are expressed as Mean ± SEM, n=5 in each group. If * P<0.05, **P<0.01 and ***P<0.001 vs. control.

Biochemical marker estimation: The results indicated that the animals treated with ointment containing 10% extract have good wound healing activity, hence, the biochemical marker such as hydroxyproline content in the scab of excision wound created in the animals treated with stated extracts was determined on the 11th day. The animals treated (Group C) with ointment containing 10% extract indicated significantly high (P < 0.001) levels of hydroxyproline (19.45 ± 0.33**µg /500mg) as compared to control (09.10 ± 0.55 µg/500mg). (Table 3) (Fig-3)

Table 3: Effect of 10% extracts ointment of Polyherbal Preparation on Hydroxyproline content in the scab of excision wound.

Groups	Hydroxyproline (µg/500mg)
A Control	09.10 ± 0.55
B Standard Povidine iodine 5% w/w	32.37 ± 0.63***
C Polyherbal Preparation 10%	19.45 ± 0.33**

• The values are expressed as Mean ± SEM, n=5 in each group. If * P<0.05, **P<0.01 and ***P<0.001 vs control.

CONCLUSION AND DISCUSSION:

Wound healing, a complex sequence of events, is initiated by the stimulus of injury to the tissues. A positive stimulus may result from the release of some factors by wounding of tissues. Cutaneous wound repair is accompanied by an ordered and definable sequence of biological events starting with wound closure and progressing to the repair and remodeling of damaged tissue. (21). From the above result of excision wound model it is evident that on the day 4^th to 8^th day, there was no significant increase in wound contraction in both the groups compared to control groups. However, the wound contraction was increased significantly (P < 0.01) on the day 12^th onwards.

Hydroxyproline is one of the biomarkers indicating wound healing process, as the content of the same is increased on 10th day. The increased hydroxyproline content in the scab of the animals treated with 10% extracts supported the wound healing process.

The tensile strength with incision model showed maximum activity for wound healing and the result was significant (P < 0.01), i.e.; 497.9 ± 6.28** with 10% extract in comparision to control 271.7 ± 4.37. The standard drug povidine iodine was also comparable and significant. (577 ±6.73***, P < 0.001). Several phytoconstituents like alkaloids (22) and saponins (23) are known to promote wound healing process due to their antioxidant and antimicrobial activities. The study reveals that 10% extracts treated groups possesses good wound healing properties which may be attributed to the individual or combined action of phytoconstituents like, alkaloids, saponins and tannins present in it.

It can also be concluded that the maximum wound healing exerted by the extract of three plants may be due to their synergistic effect.

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Received on 25.01.2011

Accepted on 28.02.2011

Research Journal of Pharmacognosy and Phytochemistry. 3(3): May- June 2011, 112-115

Groups	% Wound contraction				Epithelialization Time (Days)
Groups	4^th Day	8^th Day	12^th Day	16^th Day	Epithelialization Time (Days)
A Control	20.84 ± 2.28	40.89 ± 1.33	57.88 ± 1.47	83.51±0.91	24.33±0.33
B Standard Povidine iodine 5% (w/w)	31.09 ± 1.06	54.48 ± 1.20*	79.79± 0.98**	98.03±0.53***	19.16±0.31**
C Polyherbal Preparation 10%	24.20 ± 2.15	63.69 ± 3.22*	87.71 ± 0.56**	99.02±0.43***	18.97±0.06**