Medicinal Chemistry Research

, Volume 19, Issue 8, pp 936–947

Evaluation of wound healing potential of Calotropis gigantea latex studied on excision wounds in experimental rats

Authors

  • V. Saratha
    • Department of BiochemistryUniversity of Madras
    • Department of BiochemistryUniversity of Madras
  • S. Sivakumar
    • Department of BiochemistryUniversity of Madras
Original Research

DOI: 10.1007/s00044-009-9240-6

Cite this article as:
Saratha, V., Subramanian, S. & Sivakumar, S. Med Chem Res (2010) 19: 936. doi:10.1007/s00044-009-9240-6

Abstract

The present study was aimed at evaluating the wound healing potential of Calotropis gigantea latex on excision wound induced in rats. Topical application of Calotropis gigantea latex extract ointment (10% w/w in petroleum gel) for 14 days significantly improved wound contraction when compared with control group of rats. The levels of hydroxyproline (collagen), hexosamine, uronic acid, DNA, RNA, and protein in the wound matrix revealed the pro-healing effects of Calotropis gigantea latex. The results obtained were comparable to those of nitrofurazone, a standard drug widely used for wound healing. The pro-wound-healing activity of the latex extract may be due to its high content of glycosides, flavonoids, phenolic compounds, and triterpenoids with antimicrobial and antioxidant properties. From the results obtained, it may be concluded that Calotropis gigantea latex has the potential to be developed into new therapeutic agent for wound healing.

Keywords

Wound healingCalotropis giganteaLatexExcision woundHydroxyproline

Introduction

Wounds cause discomfort and are more prone to infection and other troublesome complications (Meyer-Ingold, 1993). Impaired wound healing leads to significant patient morbidity and mortality. With a rapidly increasing aging population that has multiple comorbidities that predispose to wound development and decreased healing efficiency, the cost of wound healing is expected to escalate proportionally. Wound healing is frequently a therapeutic challenge. It involves a number of measures including dressing, bandaging, and administration of painkillers and anti-inflammatory drugs. The wound healing process involves a highly coordinated cascade of cellular responses encompassing the interaction of many cell types over long periods of time. It comprises three complementary and overlapping phases: an inflammatory phase, proliferative phase, and remodeling phase. The smooth progression of these events leads to successful completion of wound closure.

Many of the synthetic drugs currently used for treatment of wounds are not only expensive but also pose problems such as allergy and drug resistance, and this situation has forced scientists to seek alternative drugs (Subramanian et al., 2006). Hence, efforts are being made all over the world to discover efficacious pro-healing agents that could obviate prolonged treatment, decrease cost, and save the patient from amputation or other severe secondary complications. More than 80% of the world’s population still depends upon traditional medicines for treatment of their ailments (WHO, 2000), especially for wound management (Purna and Babu, 2000) as they provide a moist environment to encourage the establishment of suitable conditions for wound healing.

India is a tropical country blessed with vast natural resources and ancient knowledge for judicious utilization. However, in order to make these remedies acceptable to modern medicine, there is a need to evaluate them scientifically in order to identify their active principles and understand their mechanism of action. Calotropis gigantea R. Br. (Asclepiadaceae), commonly known as milkweed or swallow-wort, is a medicinal plant widely used as a folk medicine in India as a rich source of biologically active compounds capable of promoting diverse benefits such as control of dermal fungal infections, antimicrobial action, and pain relief, among other useful properties (Rahman and Wilcock, 1991). Being native to India, it grows wild throughout the country on a variety of soils and in different climates, sometimes where nothing else grows (Shilpkar et al., 2007). The plant is popularly known because it produces a large quantity of latex which is easily collected from its green parts when the plant is wounded. This latex is often applied to soften the outer skin portion while removing thorns. It is also used on fresh cuts to stop bleeding. Several tribal peoples used this latex for easy delivery, abortion, and for other ailments (Mascolo et al., 1988).

The latex has been described as exhibiting potentially pharmacological properties (Rasik et al., 1999; Kareem et al., 2003). Nevertheless, its properties are accompanied by toxic effects following injections, oral administration or dermal contact of the latex to animals (Badwi et al., 1998; Singh et al., 2000). The potential of the latex as a pharmaceutical therefore depends on separating the curative from the toxic properties. Fractionation of the latex into its rubber and rubber-free fractions prior to analysis affords better insight into its potential and limitation. The rubber-free fraction of the latex is rich in soluble proteins and is responsible for most of its medicinal properties (Ramos et al., 2006).

In the absence of systematic studies in the literature on the wound healing properties of Calotropis gigantea latex, the present study was carried out to scientifically validate its use as a world health agent in the light of claims of traditional healers and small-ruminant farmers.

Materials and methods

Plant material and latex collection

Fresh latex of Calotropis gigantea was collected from aerial parts of healthy plants as described by Aworh et al., (1994) in plastic tubes containing distilled water to give a dilution rate of 1:2 v/v. The plant exsiccate was deposited at the herbarium of the Centre of Advanced Studies in Botany, University of Madras, where the plant was identified by a taxonomist. The mixture was gently handled to maintain homogeneity during transport to the laboratory, where it was kept overnight at 4°C (Kareem et al., 2003). The supernatant was decanted and centrifuged at 12,000×g for 20 min at 25°C. The clear supernatant devoid of rubber was decanted carefully and subjected to exhaustive dialysis using a membrane of 8,000 Da molecular weight cutoff against distilled water at 25°C. Finally the samples were centrifuged as previously described and the clear soluble supernatant was collected and lyophilized.

Preparation of Calotropis gigantea latex ointment

The aqueous extract of Calotropis gigantea latex was prepared as an ointment using petroleum jelly (melting point 60–65°C) at a concentration of 10% (w/w) using a Silverson homogenizer at 1,500 rpm for 45 min, and the ointment was kept in a sterile glass container, properly sealed, and preserved at 4°C for topical application to wounds. The stability of ointment was evaluated in terms of physical changes such as phase separation and changes in objectionable color and odor, and consistency of the formulation. Samples of the ointment formulation were kept at different temperature conditions such as 37°C, 40°C, and room temperature for 45 days. They were periodically observed for physical changes. The other parameters analyzed include chemical stability, accelerated deterioration, if any, during centrifugation, and spreadability using standard protocols. The acute dermal toxicity study was performed in adult male Wistar rats by “fix-dose” method following Organization for Economic Cooperation and Development (OECD) guidelines. Lyophilized latex was applied at dose of 1,000 mg/kg body weight.

Animals

Male Wistar rats weighing about 150–170 g were used in the present study. They were individually housed and maintained in a laboratory environment in a 12 h dark–light cycle. All animals were fed with standard pellet diet and water ad libitum. The experiments were conducted under protocols approved by the Institutional Animal Ethics Committee (IAEC No. 01/015/08).

Wound creation

The animals were fasted overnight and anesthetized with 1 ml intravenous thiopentone sodium (40 mg/kg b.w.) (Perez Gutierrez and Vargas, 2006). A wide area of the dorsum of each rat was depilated using toothed forceps, sterile pointed scissors, and a scalpel blade. The area was then cleaned with 70% ethanol to maintain aseptic conditions. An excision wound was created according to the method of Morton and Malone (1972). A full-thickness excision wound of circular area 300 mm and 2 mm depth was inflicted on either side of the depilated dorsum of each rat. Excess bleeding was mopped using sterile gauze. The entire wound was left open throughout the experiment (Diwan et al., 1982). Animals were closely observed for any infection and those that showed signs of infection were separated and excluded from the study. Animals were euthanized after completion of the study.

Experimental design

The animals were divided into three groups as follows. Each group comprised a minimum of six rats:
  • Group 1: excision wound induced rats treated with petroleum jelly, considered as control

  • Group 2: excision wound induced rats treated with Calotropis gigantea latex ointment (10% w/w) at a dose of 50 μg/wound

  • Group 3: excision wound induced rats treated with standard ointment (0.2% w/w nitrofurazone ointment) (Harish et al., 2008)

The treatment schedule was twice daily with topical application of the formulated ointment as well as the standard ointment, while the control group was dressed with ointment base containing the same quantity of petroleum gel. Sterile cotton swabs were used for uniform application of ointments. Wounds were traced on 1 mm2 graph paper on the day of wounding and subsequently on alternate days, until healing was complete. Changes in wound area were calculated, giving an indication of the rate of wound contraction. The period of epithelization was calculated as the number of days required for dead tissue remnants to fall off without any residual raw wound. The percentage reduction in wound size was calculated using the equation
$$ {\text{wound size reduction }}\left( \% \right) \, = \, \left[ {A_{0} \, - \, A_{\text{t}} } \right]/A_{0} \times 100, $$
where A0 and At are the initial wound area and wound area after time interval t, respectively. The distance from right wound margin to left wound margin was measured. The length of newly generated epithelium across the surface of the wound was determined as the sum of the new epidermis growing from right and left margins of the wound (Balakrishnan et al., 2006).

Biochemical estimations

On the 7th and 14th postoperative day, an appreciable amount of granulation tissue formed on the wound, which was excised and its weight recorded. The tissues were dried in an oven at 60°C for 72 h, and the dry weight was again noted. The dried tissue was added to 5 ml 6 N HCl and kept at 110°C for 24 h in sealed tubes. The hydrolysate was neutralized to pH 7.0. The neutralized acid hydrolysate of the dry tissue was used for determination of collagen by estimation of hydroxyproline as described by Woessner (1961). The collagen content can be calculated by multiplying the hydroxyproline content by the factor 7.46 (Neuman and Logan, 1950). Hexosamine content was estimated according to the method of Adamsons et al. (1964). Uronic acid content was estimated by the method of Bitter and Muir (1962). The DNA and RNA contents were assayed by the methods of Burton (1966) and Almog and Shirey (1978), respectively. The protein content in the tissue extract was estimated by the method of Lowry et al. (1951).

Statistical analysis

All experiments were performed in triplicate. Data analysis was done by an investigator who was blinded to the treatment. Results, expressed as mean ± standard error of the mean (SEM), were evaluated by one-way analysis of variance (ANOVA) using SPSS (version 15.0) program followed by least significant difference (LSD). Values were considered statistically significant when p < 0.05.

Results and discussion

Although there have been some advances in the wound healing processes, the best treatment remains undecided and the duration could not be shortened. The aim of this study was to evaluate the wound healing potential of Calotropis gigantea latex extract at the wound site of excision wounds in experimental rats. The extract in the form of an ointment was topically applied and the efficacy was compared with a standard commercial formulation. The parameters analyzed include wound area reduction, hydroxyproline (collagen), hexosamine, uronic acid, DNA, RNA, and protein.

Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers in damaged tissue as closely as possible to its normal state. Wound contracture is a process that occurs throughout the healing process and it mainly depends on the extent of tissue damage, repairing ability, and general state of health of the tissue. The wound healing process involves a highly coordinated cascade of cellular responses encompassing the interaction of many cell types over long periods of time.

Studies on acute wounds in animal models show that wounds heal in four phases: hemostasis, inflammation, granulation, and maturation (Kerstein, 1997). The normal healing cascade begins with an orderly process of hemostasis, which leads to an inflammatory cell cascade (Broughton et al., 2006). Hemostasis occurs within minutes of the initial injury unless there are underlying clotting disorders. Platelets seals off the damaged blood vessels by secreting vasoconstrictive substances. They also secrete factors which interact with and stimulate the intrinsic clotting cascade through the production of thrombin, which in turn initiates the formation of fibrin from fibrinogen. The fibrin mesh strengthens the platelet aggregate into a stable hemostatic plug. Finally, platelets also secrete cytokines such as platelet-derived growth factor (PDGF), which is recognized as one of the first factors secreted in initiating subsequent steps.

Inflammatory cells invade the wound site within a few hours after injury. Neutrophils arrive first, followed by monocytes, macrophages, fibroblasts, and lymphocytes (Li et al., 2007). This stage usually lasts up to 4 days post injury. The inflammatory response causes the blood vessels to become leaky, releasing plasma and polymorphonucleocytes into the surrounding tissue. The neutrophils with the aid of mast cells phagocytize the debris and microorganisms and provide the first line of defense against infection. Macrophages are able to phagocytize bacteria and provide a second line of defense. They also secrete a variety of chemotactic and growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), and interleukins-I (IL-1), which initiates the formation of granulation tissue (Broughton et al., 2006).

The granulation phase is characterized clinically by the presence of pebbled red tissue in the wound base and involves replacement of dermal tissues and sometimes subdermal tissues in deeper wounds as well as contraction of the wound. It usually lasts until day 21 in acute wounds, depending on the size of the wound. As the granulation phase progresses, the predominant cells in the wound site are reparatory cells such as fibroblasts, endothelial cells, pericytes, and keratinocytes, which are responsible for the formation of new matrix needed for structure and function repair of injured tissue (Diegelmann and Evans, 2004; Whitney, 2005). In the remodeling or maturation phase, the fibroblasts promote tensile strength, which sometimes may take up to 2 years after wounding.

The results of the present study are in line with the above data. It was observed that the control rats had hard and crusty wounds. By comparison, wounds treated with the Calotropis gigantea latex ointment as well as standard ointment were clean with healthy granulation tissue. Granulation tissue is a temporary yet densely cellular matrix which rapidly fills an excision dermal wound and involutes as the wound closes by contraction (Xu and Clark, 1996). Excision wounds treated with Calotropis gigantea latex ointment showed conspicuous granulation tissue and the formation of a stratified epithelium. Migrating epithelium from wound margins was evident at different points. Wounds treated with Calotropis gigantea latex ointment showed better healing than in wounds treated with standard ointment. In control wounds, epithelial reorganization was very slow.

The percentage wound contraction was nearly 90% for both ointment-treated groups of rats in 14 days. The hydroxyproline content in the granulation tissue was significantly (p < 0.05) increased in Calotropis gigantea latex ointment treated group of rats when compared with control. Similarly, uronic acid level also increased significantly (p < 0.05) when compared with control. Hexosamine level was significantly (p < 0.05) increased in groups treated with both Calotropis gigantea latex ointment as well as standard ointment when compared with control.

Higher concentration of hydroxyproline, an amino acid found only in collagen, in the granulation tissues indicates faster rate of wound healing. Significantly (p < 0.05) elevated levels of hydroxyproline observed in both Calotropis gigantea latex ointment as well as standard ointment treated groups of rats indicate the wound healing potential of Calotropis gigantea latex ointment (Fig. 2). This confirms the observation in the case of wound contraction (Fig. 1). The quantitative measurement of hydroxyproline is directly related to the formation of collagen, and its estimation helps to understand clinically the progress rate at which the healing process is occurring in the wound tissue (Gardner, 1967).
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Fig. 1

Effect of Calotropis gigantea latex extract on the level of percentage wound contraction in excision wound model. Values are mean ± SEM; n = 6 in each group. * Significant at p < 0.05 is compared to control group of rats

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Fig. 2

Effect of Calotropis gigantea latex extract on the level of hydroxyproline in excision wound model. Values are mean ± SEM; n = 6 in each group. * Significant at p < 0.05 is compared to control group of rats

Hexosamine and uronic acid, the ground substratum for collagen synthesis, are significantly increased during early stages of wound healing, and their degree of elevation is decreased thereafter. The relative decrease in hexosamine content was connected with a concomitant increase in collagen content (Dunphy et al., 1956). However, highest synthesis of hexosamine was noticed on 7th postoperative day in all groups; thereafter, the degree of elevation was decreased at 14th postoperative day when compared with the control group (Figs. 3 and 4).
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Fig. 3

Effect of Calotropis gigantea latex extract on the level of hexosamine in excision wound model. Values are mean ± SEM; n = 6 in each group. * Significant at p < 0.05 is compared to control group of rats

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Fig. 4

Effect of Calotropis gigantea latex extract on the level of uronic acid in excision wound model. Values are mean ± SEM; n = 6 in each group. * Significant at p < 0.05 is compared to control group of rats

The increase in DNA and RNA content in wounds treated with Calotropis gigantea latex ointment as well as with standard drug indicates cellular hyperplasia (Figs. 5 and 6). Concomitant increase in total protein content indicates active synthesis and deposition of matrix proteins in granulation tissues (Fig. 7). The healing process depends, to a large extent, on the regulated biosynthesis and deposition of new collagen and their subsequent maturation (Dunphy and Udupa, 1955). Assessment of collagen content in granulation tissues of control and experimental groups of rats clearly suggested that Calotropis gigantea latex ointment enhances collagen synthesis and deposition. The amount of collagen may be increased in total cell number as a result of increased cell division.
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Fig. 5

Effect of Calotropis gigantea latex extract on the level of DNA in excision wound model. Values are mean ± SEM; n = 6 in each group. * Significant at p < 0.05 is compared to control group of rats

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Fig. 6

Effect of Calotropis gigantea latex extract on the level of RNA in excision wound model. Values are mean ± SEM; n = 6 in each group. * Significant at p < 0.05 is compared to control group of rats

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Fig. 7

Effect of Calotropis gigantea latex extract on the level of protein in excision wound model. Values are mean ± SEM; n = 6 in each group. * Significant at p < 0.05 is compared to control group of rats

The importance of collagen in wound healing has been appreciated for a long time for the simple reason that the ultimate result of most repairs in the higher vertebrates is the formation of scar tissue composed of collagenous fibers (Shoshan and Gross, 1974; Bardsley et al., 1995). The observed increase in collagen, an important constituent of extracellular matrix in the Calotropis gigantea latex ointment treated rats, confirmed that the extract had a positive effect on cellular proliferation, granulation tissue formation, and epithelization. The increase in collagen content in the Calotropis gigantea latex ointment treated group agrees with the increase in the levels of DNA, RNA, and protein content, which is predominantly due to enhanced collagen synthesis.

Exposed subcutaneous tissues often provide a favorable substratum for a wide variety of microorganisms to contaminate and colonize. Wound contaminants are likely to originate from three main sources, namely the environment, the surrounding skin, and endogenous sources involving mucous membranes (Duerden, 1994). A literature survey of Calotropis species revealed that the plant latex contains mainly cardenolides and triterpenes with antibacterial activity against both Gram-positive and Gram-negative bacteria (Alam et al., 2008). Furthermore, calotropin, a proteolytic enzyme, and flavonoids in the latex were reported to possess marked antiinflammatory and antioxidant properties, respectively (Atal and Sethi, 1962; Sen et al., 1992).

The results in the healing and sealing of wounds makes Calotropis gigantea latex an important natural product for assistance in the healing of cuts, scraps, and even skin ulcers, which may be due to the synergistic actions of biologically active ingredients present in Calotropis gigantea latex. It is possible that the active principles in Calotropis gigantea play an initial role in wound healing and, as healing progresses, they adopt a more regulatory role in the wound maturation process.

Thus, it may be concluded that Calotropis gigantea latex has the potential to satisfy all the requirements for an ideal topical ointment in that it provides an environment at the surface of the wound in which healing takes place at the maximum rate consistent with the formation of granulation tissue. The present study also provides a rationale for the use of Calotropis gigantea latex in the traditional system of medicine to promote wound healing. Further studies are in progress to isolate, characterize, and identify the specific active compounds responsible for wound healing activity.

Copyright information

© Birkhäuser Boston 2009