Wound healing activity of Sida cordifolia Linn. in rats.

INTRODUCTION: The present study provides a scientific evaluation for the wound healing potential of ethanolic (EtOH) extract of Sida cordifolia Linn. (SCL) plant.
MATERIALS AND METHODS: Excision, incision and burn wounds were inflicted upon three groups of six rats each. Group I was assigned as control (ointment base). Group II was treated with 10% EtOH extract ointment. Group III was treated with standard silver sulfadiazine (0.01%) cream. The parameters observed were percentage of wound contraction, epithelialization period, hydroxyproline content, tensile strength including histopathological studies. RESULT: It was noted that the effect produced by the ethanolic extract of SCL ointment showed significant (P < 0.01) healing in all wound models when compared with the control group. All parameters such as wound contraction, epithelialization period, hydroxyproline content, tensile strength and histopathological studies showed significant (P < 0.01) changes when compared with the control.
CONCLUSION: The ethanolic extract ointment of SCL effectively stimulates wound contraction; increases tensile strength of excision, incision and burn wounds.

Introduction

Healing is a complex and intricate process initiated in response to injury that restores the function and integrity of damaged tissues. Healing process can be broadly categorized into three stages; inflammatory phase consisting the establishment of homeostasis and inflammation; proliferate phase (consisting of granulation, contraction and epithelization) a finally the remodeling phase, which ultimately determines the strength and appearance of the healed area.[1] Several natural products,[2] plant products, which are composed of active principles, such as alkaloids and flavonoids[3] and biomolecules[4] have been reported to promote the process of wound healing.

Sida cordifolia Linn. (SCL) (Malvaceae) syn. Country Mallow is a small, erect, downy shrub.[5] It is almost odorless with slightly bitter taste.[6] In Ayurveda practices, S. cordifolia had three common applications: Mashabaladi Kvatha, where the plant seeds were mixed with other ingredients to relieve muscular pain; Balataila, a process for the treatment of the nervous system complaints, stomach problems and as a cardiac tonic; and the crushed leaves of the plant as an astringent for the treatment and dressing of wounds or skin injuries. Traditionally, root juice is also used to promote the healing of wounds.[7] In view of these cited activities, observations and traditional uses of plant, the present study was undertaken to explore the wound healing potential of ethanolic extract of SCL in excision, incision and burn experimental models.

Materials and Methods

Plant Material

The whole plant of S. cordifolia were identified, and collected in the month of October from the forest of Pachmarhi region, dist. Hoshangabad. A voucher specimen no. No./HD/CHPY825 has been deposited in the Department of Botany, Jawaharlal Krishi Vishwavidhyala, Jabalpur. The plants were washed, shade dried, pulverized into moderately coarse powder, passed through a 40 mesh sieve and stored in an air tight container for further use.

Plant Drug Extraction

The powdered plant (60 g) of S. cordifolia was extracted with ethanol using the Soxhlet apparatus for 24 h until the extraction was completed. The solvent was removed under reduced pressure. The dried extract was weighed and percentage yield was obtained with respect to dry powdered material.

Preliminary Phytochemical Screening

The different identification tests were performed to detect the presence of metabolites in ethanolic extract of SCL.[89]

Preparation of Formulation and Standard Used

Simple ointment was prepared from the 10% ethanolic extract of SCL by trituration method in a ceramic pestle and mortar using White soft paraffin obtained from S.D. Fine Chemical, India.[10] About 10 g of semisolid extract was incorporated into the 100 g of simple ointment base B.P.[11] Simple ointment base was used as the control group and was applied twice per day. Extract ointment was used twice per day to treat different groups of animals. Silver sulfadiazine (0.01%) obtained from Rexin Pharmaceutical Pvt. Ltd. was used as standard drug for comparing the wound healing potential of extract in different animal models and was applied twice per day.

Animals

Wistar albino rats (150-180 g) of either sex were selected for the experiment. They were housed individually in standard laboratory environment for 7 days of period, fed with commercial pellets and water ad libitum. Animal study was performed in Division of Pharmacology, VNS Institute of Pharmacy, Bhopal. The procedures were reviewed and approved by the Institutional Animal Ethics Committee (Registration No.778/03/c/CPCSEA).

Wound Healing Activities

Grouping of Animals

For excision, incision and burn wound model, animals were divided into three groups in each model consisting of six animals as follows: group I-simple ointment base; Group II-10% ethanolic extract ointment of SCL and Group III-silver sulfadiazine (0.01%) cream was used as standard.

Excision wound model

Excision wound model as per the method described[12] was used. Three groups of animals containing six rats in each group were anesthetized by open mask method with anesthetic ether. The rats were depilated on the back and a predetermined area of 500 mm2 full thickness skins was excised in the dorsal interscapular region. Rats were left undressed to the open environment. The formulation ointment and standard drug were applied daily until the complete healing. In this model, wound contraction and epithelialization period was monitored. Wound contraction was measured as percent contraction in each 2 days after wound formation. From the healed wound, a specimen sample of tissue was collected from each rat for histopathological examination.[1314]

Incision wound model

In incision wound model, 6 cm long paravertebral incision were made through the full thickness of the skin on either side of the vertebral column of the rats, after all the animals of each group were anesthetized under light ether anesthesia.[15] No local or systemic antimicrobials were used throughout the experiment. All groups were treated same as in excision model, the both edges kept together and stitched with Black silk surgical thread (no. 000) and a curved needle (no. 11) was used for stitching. The continuous threads on both wound edges were tightened for good closure of the wound. After stitching, wound was left undressed then ointment base, standard ointment and extracts ointment were applied daily until 10 days; when wounds were cured thoroughly the sutures were removed on the day 10 and tensile strength of cured wound skin was measured using tensiometer.[16]

Burn wound model

Burn wounds were created on dorsal part of shaved rat's skin surface using concentrate sulfuric acid, exposed for 10 s. After 24 h, dead tissues were excised using sterile surgical blade through a template designed to produce a third degree burn.[17] All groups were treated same as in excision model. In this model, wound contraction and epithelialization period was monitored. A specimen sample from the healed wound of tissue was collected from each rat for histopathological examination.[18]

Wound Healing Evaluation Parameters

Measurement of Wound Contraction

An excision wound margin was traced after wound creation by using transparent paper and area measured by graph paper. Wound contraction was measured in each 2 days interval, until complete wound healing and expressed in percentage of healed wound area.[19] The evaluated surface area was then employed to calculate the percentage of wound contraction, taking initial size of wound, 300 mm2, as 100%, by using the following formula as:

Epithelialization period

It was evaluated by noting the number of days required for the Escher to fall off from the wound surface exclusive of leaving a raw wound behind.[20]

Measurement of tensile strength

The tensile strength of a wound represents the degree of wound healing. It indicates how much the repaired tissue resists to breaking under tension and may indicate in part the quality of repaired tissue. The sutures were removed on the 9th day after wounding and the tensile strength of removed tissue was measured on the 10th day with the help of tensiometer.[21] In this method, wound breaking strength was measured as the weight of water at the time of wound breaking per area of the specimen.

Hydroxyproline estimation

Wound tissues were analyzed for hydroxyproline content, a basic constituent of collagen. Tissues were dried in a hot air oven at 60-70°C to constant weight and hydrolyzed in 6 N HCl at 130°C for 4 h in sealed tubes. The hydrolysate was neutralized to pH 7 then subjected to chloramine-T oxidation for 20 min.[22] The reaction was terminated by the addition of 0.4 M perchloric acid and developed color with Ehlrich reagent at 60°C was read at 557 nm in ultraviolet (Cintra) spectrophotometer.

Histopathological studies

Wound tissue specimens from control, test and standard groups were taken after complete healing of excision, incision and dead burn wound and after usual processing 6-mm thick sections were cut and stained with hematoxylin and eosin.[23] Sections were qualitatively assessed under the light microscope and observed in respect of fibroblast proliferation, collagen formation, angiogenesis and epithelialization.

Statistical Analysis

All treated groups were compared with the control groups. The results were analyzed statistically using one-way analysis of variance (ANOVA). The result were found to be significantly at P < 0.01. All tests were conducted using Graph Pad Software, Inc., a privately held California corporation.

Result

Percentage yield was found to be 4.91 g (w/w). Preliminary phytochemical screening of ethanolic extract of SCL showed the presence of alkaloids, flavonoids and tannins.

Wound Contraction

A better healing pattern with complete wound closure was observed in standard and treated group of all models within 10 and 14 days respectively while it was about 22 days in control rats as shown in Tables 1 and 2.

Table 1

Effect of EtOH extract of Sida cordifolia and standard ointment on % wound contraction of excision wound models in rats

Table 2

Effect of EtOH extract of Sida cordifolia and standard ointment on % wound contraction of burn wound models in rats

Epithelialization Period

The epithelialization time was measured from the 1st day. The epithelialization time was found to be significantly (P < 0.01) reduced in Groups II and III as depicted in Tables 3 and 4 for incision and burn models. The treatments with ethanolic extract as well as conventional wound treatment cream were superior to control groups, which received ointment base.

Table 3

Effect of topical application of EtOH extract of Sida cordifolia on various wound parameters of incision wound model in rats

Table 4

Effect of topical application of EtOH extract of Sida cordifolia on various wound parameters of burn wound model in rats

Tensile Strength of Incision and Burn Wound Model

Tensile strength for the treated group on day 20 was found to be significant (P < 0.01) than the control group as shown in Tables 2 and 4.

Hydroxyproline Estimation

Treated group showed significant increased hydroxyproline level when compared with the control group (P < 0.01) [Tables 2 and 4].

Histopathological Studies

The histopathological studies of the tissue of the excision, incision and burn wound area was performed and histopathological characters of incision wound treated group with control, standard and extract ointment are shown in Figure 1a-c. Group I (control) showed reduced fibroblast cells, collagen fibers and blood vessels, while Group II (standard) showed increased in fibroblast cells, collagen fibers and blood vessels in both incision and burn wound models. Group III (extract ointment) also showed increased in fibroblast cells, collagen fibers and blood vessels, but not more than Group II in both incision and burn wound models. The histopathology examination showed that the original tissue regeneration was much greater on ethanolic extract and standard. Fibroblast cells, collagen fibers and blood vessels are prominently present in standard and extract treated group as compared with the control. While in control rats wounds have shown incomplete healing [Figure 1a].

Figure 1

(a) Histopathological characteristics of rat skin on 8th day by treatment with ointment base. Figure shows reduced fibroblast cells (F), collagen fibers (C) and blood vessels (B) in incision wound. (b) Histopathological characteristics of rat skin on 8th day by treatment with standard ointment. Figure shows increased fibroblast cells (F), collagen fibers (C) and blood vessels (B) in incision wound. (c) Histopathological characteristics of rat skin on 8th day by treatment with ethanolic extract ointment. Figure shows increased fibroblast cells (F), collagen fibers (C) and blood vessels (B) in incision wound

The results showed that ethanolic extract ointment possesses a definite prohealing action. This was demonstrated by a significant increase in the rate of wound contraction and by enhanced epithelialization. Significant increase (P < 0.01) in tensile strength and hydroxyproline content were observed, which was further supported by histopathological studies. Since incisional wound treated with ethanolic extract of SCL showed greater tensile strength, it might be inferred that the ethanolic extract of SCL not only increased collagen synthesis per cell, but also aided in cross-linking of the protein.

Discussion

The chemical constituents of SCL plants and their medicinal values have been reviewed. Its chemical constituents mainly consist of oils and fats, org. acids, flavonoids, triterpenes, steroids, sterols and proteins.[24] Biological activities in the skin are due to its interaction with various binding protein. In this tissue repair process inflammatory cells promote the migration and proliferation of endothelial cells, which synthesize extracellular matrices including collagen and of keratinocytes resulting to re-epithelialization of the wounded tissue. The wound healing action of SCL may probably be due to the phytoconstituents present in the plant or could be a function of either the individual or the additive effects of the phytoconstituents. The present result also indicated a significant decrease in wound area from day 8 onward indicating early healing. In incision wound, an increase in tensile strength of treated wounds was observed and this may be due to the increase in collagen concentration and stabilization of the fibers.[25]

The ethanolic extract of SCL increased cellular proliferation and collagen synthesis at the wound site as evidenced by the increase in total protein and total collagen contents reflected by hydroxyproline content of granulation tissues. In our study, hydroxyproline content was significantly increased (P < 0.01) when compared with the control. Since SCL extract has increased levels of these compounds considerably it is likely that the observed increase in tensile strength was not only due to increased collagen synthesis but also due to its proper deposition and alignment.

Collagen is the predominant extracellular protein in the granulation tissue of healing wound and there is a rapid granulation tissue of healing wound and there is wound rapid increase in the synthesis of this protein in the wound area soon after an injury which provides strength and integrity to tissue matrix. Measurement of this hydroxyproline, which comes from the breakdown of collagen, has been used as an index of collagen turnover. The hydroxyproline content of the granulation tissue indicates the presence of higher collagen content and its turnover leading to rapid healing with a concurrent increase in the tensile strength of the treated wounds.

Conclusion

The results of the study showed that the ethanolic extract ointment of SCL effectively stimulates wound contraction; increases tensile strength of excision, incision and burn wound as compared with the control group. These finding could justify the inclusion of this plant in the management of wound healing.