Effect of Asiaticoside, Collagenase, and Alpha-chymotrypsin on Wound Healing in Rabbits

Wound dressing materials such as asiaticoside, collagenase, and alpha-chymotrypsin are often used for effective wound healing activity. Objectives. In this study, the effects of asiaticoside, collagenase, and alpha-chymotrypsin were studied in rabbit models with open wounds with tissue loss and with full-thickness flank excisions for a period of 21 days. Materials and Methods. Three groups of 4 rabbits were examined during trial periods of 7, 14, and 21 days. Four circular wounds measuring 1.5 cm in diameter were made on the dorsal sides of the animals: 2 on the right and 2 the left. Asiaticoside, collagenase, and alpha-chymotrypsin were applied to wounds daily for a period of 7, 14, and 21 days, while 1 gauzed wound served as the control. All biopsy specimens were histopathologically evaluated for recovery. On day 7, microscopic review showed no differences in wound healing between groups. Results. By day 14, alpha-chymotrypsin showed the quickest reepithelialization (P < 0.05); and by day 21 asiaticoside and collagenase (P < 0.01) showed effective recovery, due to the completion of wound healing for all animals in both groups. Conclusion. Alpha-chymotrypsin is more effective than the other 2 groups for only 14 days. The effectiveness of asiaticoside and collagenase displayed a more rapid improvement in comparison to alpha-chymotrypsin for healing open wounds with tissue loss for a period of 21 days. 

Injury is defined as any damage to a biological organism that can be classified according to various criteria.  A wound is a kind of injury in which the skin is torn, cut, or punctured (ie, an open wound) or in which blunt force trauma has caused a contusion (ie, a closed wound). In pathology, an open wound specifically refers to a severe injury that damages the dermis. 

Upon receiving a wound, an organism’s natural response is to heal the wound as quickly as possible and to reestablish the continuity of normal structures. This process is called wound healing. The organism’s first response to injury begins the process of local inflammation, followed by reparation, regeneration, and the formation of a scar. Regeneration and reparation are the 2 processes of reestablishing tissues and cells for integrity. During regeneration, the formation of new tissue seeks to reproduce the original structure; this phase includes the epithelial regeneration of skin wounds. At the same time, reparation aims to ensure the continuity of tissue while forming new connective tissue.^1-2 

With better understanding of pathophysiology, researchers have developed new principles and methods for treating open wounds. Current treatments now include closing the wound surface, preventing fluid-electrolyte loss and secondary infections, accelerating the formation of epithelial and connective tissue, and clearing all necrotic debris as soon as possible to ensure cleanliness.^3-9 

Open wound treatments also use asiaticoside (C48H78O19), which is obtained from the plant Centella asiatica (CA). This plant contains several active constituents, the most important of which are triterpenoid saponins, including asiaticoside, centelloside, madecassoside, and asiatic acid. Centella asiatica also contains volatile oils, flavonoids, tannins, phytosterols, amino acids, and sugars. As such, CA extracts (CAE) have traditionally been used to heal wounds, and research has increasingly confirmed the effectiveness of such usage. When asiaticoside is applied to tissues, its histological and biochemical aspects have been reported to be topically effective and nontoxic, and it has been shown to stimulate the synthesis of collagen in wounds.^10-11 

Sunilkumar et al¹² reported that different formulations — cream, gel, and ointment — of an aqueous CAE applied to open wounds in rats culminated in increased collagen synthesis and cellular proliferation at the wound site, as shown by increased collagen content and tensile strength. Those authors¹² also found CAE-treated wounds epithelialized faster, the rate of wound contraction was higher than in untreated control wounds, and healing was more prominent with the gel product. Specifically, CAE gel is believed to be effective in keratinization, which aids in thickening the skin in areas of infection.¹³ At the same time, CA with alcoholic extracts applied to wounds in rats has been shown to initiate collagen synthesis, increase cell proliferation, hasten and improve the maturation of the granulation tissue, and increase the rate of epithelialization and wound contraction.^12,14 Furthermore, CA reportedly stimulates collagen synthesis even in low-dose applications.¹¹ Different CA concentrations are also known to be effective for epidermal wounds and possibly increase keratinization, as has been histologically shown.¹³ Madecassol cream (Dangkook Pharmaceutical Co Ltd, Seoul, South Korea), an extract of CA ointment, and tetrandrine, a bisenzylisoquinoline alkaloid, have each decreased acute radiation dermatitis caused by radiotherapy in rats, as well as been shown to induce an anti-inflammatory effect.¹⁵ According to Yasan et al,¹⁶ asiaticoside is more effective than nitrofurazone and clostridiopeptidase-A for intranasal histological application in rabbits.

Collagenase cleans necrotic tissue from the wound area by the enzymatic method, as well as facilitates granulation tissue formation and wound reepithelialization.¹⁷ Collagenase-containing gel formations are also reportedly effective in improving necrotic wounds in pigs.¹⁸ According to Taçyildiz et al,⁵ using collagenase to treat open wounds to accelerate wound healing in rats provided clean granulation tissue, whereas increased fibroblastic and myofibroblastic activation resulted in a regular collagen increase. It has also been indicated that collagenase is quite effective in improving burn injuries in rats¹⁹ and applying collagenase is better than surgical intervention for burn injuries and chemical necrosis in infants.²⁰ 

Alpha-chymotrypsin, a preparation naturally obtained from bovine pancreases that activates plasminogen and collagenase, has a proteolytic effect in relieving inflammation. It is used to remove necrotic tissue in skin and corneal wounds, as well as to prevent the formation of inflammatory edema and hematoma, partially by inhibiting subacute and chronic fibrin formation. Proteolytic enzymes are used to effectively treat tissue loss, delayed cicatrization, and split, purulent, chronic, and necrotic wounds. Alpha-chymotrypsin is also useful for preventing inflammation as well as edema and hematoma reabsorption in addition to quickly removing local circulatory disorders. Its effects increase when used with antibiotics and, according to the literature, does not affect healthy tissues.^21-25 It has also been reported that administering alpha-chymotrypsin subconjunctivally can benefit cats and dogs with corneal ulcers, as well as accelerate reepithelialization and recovery better than classical methods.²⁵ 

RaviKumar et al²⁶ shows chymotrypsin applications reduced edema and cytokines and accelerated epithelialization, which resulted in earlier wound healing and good scar tissue formation in burn wounds. Their study also showed treatments often use wound dressing materials and pomades containing chymotrypsin to accelerate the wound healing process, as these substances clean the area of necrotic debris and stimulate epithelial regeneration and granulation tissue formation.²⁶ 

In the present study, the effects of wound covering materials asiaticoside (extract of CA ointment), compared to frequently used enzymatic debriding in practice such as collagenase (Novuxol, Smith & Nephew, Seoul, South Korea) and alpha-chymotrypsin (Alphachymocutan, Strathmann GmbH & Co. KG, Germany) on wound healing were investigated in rabbits with experimental open wounds showing tissue loss by comparing the effects of the healing process.

Materials and experimental design
After the ethical committee of the University of Selcuk (Konya, Turkey) approved this study, 12 rabbits were kept for 10 days in suitable cages before experiments commenced. The rabbits were equally divided into 3 groups for experiment periods lasting 7, 14, and 21 days. All rabbits were anesthetized with 10 mg/kg IM xylazine (Rompun, BayerDVM, Shawnee Mission, KS) and 50 mg/kg IM ketamine hydrochloride (Alfamine, Ege Vet, Izmir, Turkey). The surgical area on the right and left sides of the spine on each rabbit was shaved, cleaned, and disinfected with preoperative routine cleaning and disinfection procedures after which 4 full-thickness wounds, each measuring 1.5 cm in diameter, were made on the skin: 2 on the right flank and 2 on the left. Wounds on each side were set 3 cm apart so the wounds would not affect the others. In each group, ointment of asiaticoside, collagenase, and alphachymotrypsin was applied once daily to the wounds for 7, 14, or 21 days to completely close the wound surface. On each rabbit, 1 wound served as a control group. An analgesic, flunixin meglumine (1.1 mg/kg SC) was applied to all rabbits to relieve pain during the postoperative period. The skin behind the neck was pinched gently between thumb and forefinger and the skin was gently pulled. The needle was angled at 45 degrees with the skin.  

Histopathological analysis
Using regular anesthesia, biopsy samples 2 cm x 1 cm were taken from wound sites in all animals on days 7, 14, and 21. Tissues were fixed in 10% formalin and embedded in paraffin wax blocks. Sections 5 µm thick were cut with a microtome and received hematoxylin and eosin (H&E) and Van Gieson (vG) stains.²⁷ All sections were evaluated with a light microscope. The length of the wound epithelium, epithelial thickness, and the size of the wound openings were measured with an ocular micrometer. The size of the wound opening was measured at the distance between 2 epithelium (facing each other) as wound size with ocular micrometer in the histological section taken from the middle of the wound.

Statistical analysis
An analysis of variance and Duncan tests were performed using SPSS version 10.0 (SPSS Inc, Chicago, IL). Results were expressed as mean ± SD; P < 0.05 was considered to be the acceptable probability of a type 1 error.

Clinical findings
Macroscopically, on day 14, wound contraction and early reduction in wound area were better in the group that received collagenase and alpha-chymotrypsin, while on day 21 the groups that received asiaticoside and collagenase showed closed wounds for all animals. Though scarring was light in the collagenase group, it was also very light in the asiaticoside group (Figure 1). 

Histopathological findings
The Table shows the distribution of histopathological findings for each group. For histopathological examination, the wound epithelium was measured by length, epithelial thickness, and wound size (Figure 2). On day 7, reepithelialization commenced in all groups, and the wound area began to fill with young granulation tissue. Necrosis and bleeding were found in some wound areas. Reepithelialization on the wound edges was most advanced in the collagenase group, followed by the control and alpha-chymotrypsin groups. Though there was no statistically significant difference in epithelial thickness, asiaticoside showed the best results, followed by alpha-chymotrypsin and collagenase. All experimental groups showed thicker epithelium than the control group. In terms of connective tissue, there was no difference among groups (Figure 3). Though abscess formation was generally not observed by day 7, 1 animal in the asiaticoside group did exhibit an abscess.

On day 14, reepithelialization and granulation tissue formation remained most advanced in the alpha-chymotrypsin group, followed by the collagenase and control groups, while the asiaticoside group showed the least epithelization (Figure 4). The edges of the wound epithelium formed by reepithelialization were thickest in the collagenase group, followed by the alpha-chymotrypsin, asiaticoside, and control groups. The alpha-chymotrypsin group showed the longest epithelial proliferation, followed by the asiaticoside, control, and collagenase groups. Abscesses were observed in 1 rabbit of both the alpha-chymotrypsin and collagenase groups, while 2 rabbits in the asiaticoside group and 3 rabbits in the control group showed abscess formation. With vG staining, collagen appeared more often in the alpha-chymotrypsin and collagenase groups, while fibroblasts were more intense in the asiaticoside and control groups. 

Reepithelialization was complete on day 21, and the wounds had closed in the asiaticoside and collagenase groups. However, 1 animal in both the alpha-chymotrypsin and control groups exhibited incompletely healed wounds due to abscess formation. According to epithelial thickness, the control group ranked first, followed by the collagenase, alpha-chymotrypsin, and asiaticoside groups. Van Gieson staining showed granulation tissue rich in collagen in the collagenase, asiaticoside, and control groups. Fibroblasts were more intense in regions of incomplete reepithelialization in the rabbits of the alpha-chymotrypsin group (Figure 5).

The process of wound healing is an organism’s response to ensure wound closure as soon as possible. In this process, physicians can help by protecting wounds from external influences and microorganisms and by facilitating epithelialization and granulation tissue formation. To these ends, a wide variety of wound dressing materials can aid epithelial formation. In the present study, those materials reputedly active in the formation of granulation tissue and enzyme preparations past to present were tested in wound healing in rabbits with open wounds showing tissue loss. Results generally showed the effects of asiaticoside increased the formation of granulation tissue, collagenase triggered reepithelialization, and alpha-chymotrypsin prevented the formation of inflammation, as well as cleaned the wound’s necrotic debris.

Infections have been reported to induce adverse effects on the development of collagen and fibroblastic activation in wounds.⁵ On day 7 of the present study, reepithelialization was best in the collagenase group, though by day 14 the alpha-chymotrypsin group showed the best reepithelialization. Abscess development was considered to primarily cause this difference. On day 21, the completion of wound healing was observed in all animals in the asiaticoside and collagenase groups. Two studies have reported that asiaticoside facilitates a quick, better maturation of granulation tissue and enhances reepithelialization, as well as the rate of wound contraction increases collagen synthesis and cell proliferation. In another study,⁵ collagenase was found to stimulate fibroblastic and myofibroblastic activation and to accelerate wound healing with an increase of collagen fibers in both regular and intensive patterns. Meanwhile, several studies^6,21-23,25  have shown the intense effects of alpha-chymotrypsin on chronic, necrotic wounds by removing necrotic waste. In the current study, which focused on treating new wounds and scars, the effectiveness of alpha-chymotrypsin was less than in other groups in this study. Asiaticoside accelerates wound healing and prevents keloid formation by reducing the thickening of the connective tissue.¹² In the present study, the formation of scar tissue was macroscopically more advanced in the asiaticoside group, in which scars were nearly invisible, than in the control and collagenase groups (Figure 1).

Asiaticoside functions by stimulating the synthesis of collagen in wounds,^10-11 while collagenase enhances both regular and intensive collagen by increasing the amount of fibroblasts.⁵ On day 14, collagen showed regular formation in the collagenase group, while fibroblasts were more intense in the asiaticoside group. These results suggest the infection in 2 animals in the asiaticoside group delayed the development of granulation tissue formation.

Considering epithelial thickness, there was no difference among the groups. However, in comparing groups by period, a significant increase in epithelial thickness was seen (P < 0.01) in the collagenase group on day 14, which may be due to this drug’s ability to stimulate reepithelialization.¹⁸ 

When wound size was considered on days 14 and 21, the control and asiaticoside (P < 0.01) groups showed significant differences, while significant differences were found in the collagenase (P < 0.01) and alpha-chymotrypsin (P < 0.03) groups on days 7, 14, and 21 (Figure 2). By day 14, the wound apertures had closed more quickly in the collagenase group. This finding aligns with those observed by Tacyildiz et al,⁵ who reported that collagenase activates fibroblasts as well as myofibroblasts, which then boost wound contraction. By day 14, the asiaticoside group was not as advanced in the healing process as the collagenase group, which could have been caused by infection in 2 rabbits in the group. Tacyildiz et al⁵ has also reported that infection delayed wound healing. In the present study, since recovery was complete in both groups by day 21, asiaticoside and collagenase were concluded to be very effective in postoperative wound healing. ¹⁶

According to microscopic findings on day 7, no significant difference in healing was detected among the groups. When reepithelization speed is considered, on day 14 alpha-chymotrypsin had outperformed the other groups, though on day 21 the asiaticoside and collagenase groups showed the best results, as all animals in both groups achieved complete wound healing. The authors therefore concluded that the effectiveness of asiaticoside and collagenase is better than alpha-chymotrypsin for open wounds showing tissue loss. 

Affiliation: Faculty of Veterinary Medicine, Department of Pathology, University of Selcuk, Konya, Turkey

Correspondence:
Ozgur Ozdemir, PhD, DVM
Faculty of Veterinary Medicine
Department of Pathology
University of Selcuk
Konya, Turkey
oozdemir@selcuk.edu.tr

Disclosure: The authors disclose no financial or other conflicts of interest.