Topical grape (Vitis vinifera) seed extract promotes repair of full thickness wound in rabbit

Abstract

In recent years, oxidative stress and free radicals have been implicated in impaired wound healing. Grape (Vitis vinifera) seed extract (GSE) possesses anti‐inflammatory and antioxidant properties. The present study was undertaken to assess the potential activity of grape seed hydroalcoholic extract in wound healing in rabbits. Rabbits of either sex were subjected to a 20 × 20 mm square excision made over the skin of the back. The animals were randomly divided into seven experimental groups, as negative and positive control, eucerin and treatments. Negative control group did not receive any treatment. Positive control and eucerin groups received phenytoin cream (1%) and topical eucerin, respectively, twice a day from the beginning of experiments to complete wound closure. Treatment groups were treated topically by cream of GSE (2, 5, 10 and 70% w/w) in eucerin base, twice daily. For evaluation of the percentage of wound healing, area of the wound was measured daily. Histological studies were performed on the 7th and 15th days of treatments. After complete healing, hydroxyproline content and tensile strength measurement of tissue samples were done. Results showed that there were statistically significant differences between GSE treatments groups and eucerin animals (P < 0·05) in most of the days. Rabbits treated with 2% GSE had best results (completed healing in 13 days, higher hydroxyproline content and higher tissue resistance). We concluded that the extract of 2% GSE administered topically has a good potential to promote wound healing in wound model of rabbits.

INTRODUCTION

Hemmati AA, Aghel N, Rashidi I, Gholampur‐Aghdami A. Topical grape (Vitis vinifera) seed extract promotes repair of full thickness wound in rabbit.

Wound healing is the process of repair following an injury to the skin and other soft tissues. It is fundamentally a connective tissue response. Wound healing occurs in three stages: inflammation, proliferation and remodelling. Inflammation is the earliest stage which starts immediately after injury associated with the recruitment of inflammatory cells, for example monocytes and macrophages. The proliferative phase is characterised by angiogenesis, collagen deposition, granulation tissue formation, epithelialisation and wound contraction. In angiogenesis, new blood vessels grow from endothelial cells. In fibroplasia and granulation tissue formation, fibroblasts grow and form a new, provisional extracellular matrix by excreting collagen and fibronectin. Collagen is the most abundant protein in the animal kingdom and constitutes 30% of human total proteins. It is the major component of the extracellular matrix and provides strength, integrity and structure. Collagen has an important role in wound healing also. It contains substantial amounts of hydroxyproline, which has been used as a biochemical marker for tissue collagen (1). In epithelialisation, epithelial cells proliferate and spread across the wound surface. Wound contraction occurs as the myofibroblasts contract. Platelets release growth factors and other cytokines (2). Remodelling phase is a long process which helps to restore the tissue integrity and convert the repaired area similar to the normal tissue.

Chronic wounds are wounds that fail to heal despite adequate and appropriate care. Such wounds are difficult and frustrating to manage (3). Current methods used to treat chronic wounds include debridement, irrigation, antibiotics, tissue grafts and proteolytic enzymes, which possess major drawbacks and unwanted side effects.

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, haemorrhagic shock, atherosclerosis, advancing age, ischaemia and reperfusion injury of many organs, Alzheimer and Parkinson's disease, gastrointestinal dysfunctions, tumour promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes (4). A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. Proanthocyanidins, condensed tannins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress (5).

Grape (Vitis vinifera Linn.) (Family: Vitaceae) seed was reported to be rich in polyphenols that range from the monomeric flavanols: catechin, epicatechin and epigallocatechin to dimers, trimers and polymers with seven or more flavanol units: the proanthocyanidins (6).

The biological, pharmacological and medicinal properties of the bioflavonoids and proanthocyanidins have been extensively reviewed 7, 8, 9, 10. Besides the free radical scavenging and antioxidant activity, proanthocyanidins exhibit vasodilatory, anticarcinogenic, anti‐allergic, anti‐inflammatory, antibacterial, cardioprotective, immune‐stimulating, anti‐viral and estrogenic activities, as well as being inhibitors of the enzymes phospholipase A2, cyclooxygenase and lipooxygenase 8, 9. Proanthocyanidins and other tannins are known to facilitate wound healing 11, 12, 13. The aim of the present study was to investigate the wound healing effect of the hydroalcoholic extract of seed grape (V. vinifera) in rabbit model.

MATERIALS AND METHODS

Plant material

The ripened grapes were collected from Takestan, province of Ghazvin, north of Iran, in the summer season. The plant was identified as V. vinifera (agricultural name of careh rooyieh) by Ghazvin Agricultural Research Center, Ghazvin, Iran. A voucher specimen was kept in the herbarium at the Department of Phamacognosy, Faculty of Pharmacy, Joundishapour Medical Sciences University, Ahwaz, Iran. Grape seeds were separated from the pulp and dried in the shade at room temperature. The seeds then were finely powdered using a grinder. The powdered seeds (100 g) were macerated in 80% ethanol for 72 hours in laboratory temperature. The filtrated extract was evaporated under vacuum below 40°C to give a final yield of 11·5 g.

Animals

Healthy Iranian rabbits of either sexes weighing 1500–2000 g were used for the study. They were individually housed in stainless steel cages at a 12‐hour cycle of light and dark, room temperature was kept at 24 ± 2°C and humidity maintained at 50%. Standard food, vegetables and water was provided ad libitum. The rabbits were used after acclimatisation to the laboratory environment for a 7‐day period.

Wounding procedure

The dorsal fur of the rabbits was shaved with an electric clipper and the desired area of the wound to be created was outlined on the back of the animals. Marked area was locally anaesthetised using lidocaine 2%. Full thickness excision wound of 20 × 20 mm was created along the markings using scalp blade and forceps.

Wound area was outlined using a transparent plastic sheet every day. Wound surface was measured by retracing the outline of wound on a millimetre scale graph paper. Area of the first day was 100 and the degree of healing every day was calculated as compared with the first day (14).

Experimental groups

The animals were randomly divided into seven groups of six animals each.

• 1Negative control: did not receive any treatment.
• 2Positive control: treated with phenytoin 1% twice daily till the wounds completely healed.
• 3Vehicle control: treated with eucerin base twice daily till the wounds completely healed.
• 4Test 1: treated with 2% GSE in eucerin twice daily until the wounds completely healed.
• 5Test 2: treated with 5% GSE in eucerin twice daily until the wounds completely healed.
• 6Test 3: treated with 10% GSE in eucerin twice daily until the wounds completely healed.
• 7Test 4: treated with 70% GSE in eucerin twice daily until the wounds completely healed.

Histological study

Histological studies were performed on the 7th and 15th days of treatment. Skin specimens from treated and untreated rabbits were collected in 10% buffered formalin and after the usual processing, 5‐µm‐thick sections were cut and stained with haematoxyline and eosin. Sections were qualitatively assessed under the light microscope.

Estimation of hydroxyproline

After complete healing, a piece of skin (100 mg) from the healed wound area was collected and analysed for hydroxyproline content, which is a basic constituent of collagen. Hydroxyproline was measured using the method of O’Brien and colleague. (15). Tissues were hydrolysed in 6 M HCl for 18 hours at 110°C. Samples were dried on a hot plate and then washed three times with distilled water. The acid‐free samples were reconstituted in 2·0 ml of acetate–citrate buffer [1·2% sodium acetate trihydrate, 5% citric acid, 12% sodium acetate, 3·4% sodium hydroxide (pH 6·0)]. 500 ml of 0·05 M chloramine‐T was added to 1 ml of sample, after which samples were incubated for 20 minutes at room temperature, followed by the addition of 0·5 ml of 15% perchloric acid and 15% 4‐dimethyl amino‐benzaldehyde in 1‐propanol. After incubation for 15 minutes at 60°C, each sample was transferred to a microtitre plate and absorbance read at 550 nm using UV/Vis spectrophotometer. Hydroxyproline concentrations of the unknown samples were calculated from a linear standard curve and presented as µg/mg dry tissue weight (Table 1).

Table 1.

Comparison of hydroxyproline content of wound tissue sample after treatment period

Groups	Hydroxyproline content (µg/g tissue)
No treatment	868·3 ± 25
Eucerin treatment	878 ± 18
Phenytoin 1%	1239 ± 38 *
GSE 2%	1074 ± 33 *
GSE 5%	933·4 ± 21 *
GSE 10%	935 ± 28 *
GSE 70%	930 ± 29 *

GSE, grape seed extract.

^* P < 0·001 (significant differences from no‐treatment or eucerin‐treated groups).

Tensile strength

The tensile strength of a wound represents the degree of tissue integrity. The tensile strength increment indicates better wound healing stimulation by the applied drug (Table 2). At the end of healing period, a strip of repaired tissue measuring 20 × 5 mm was isolated and the tensile strength was measured with tensiometer. Tensile strength was calculated using the following formula (16):

Table 2.

Comparison of tensile strength of wound tissue sample after treatment period

Groups	Tensile strength (g/cm2)
No treatment	980 ± 36
Eucerin treatment	987 ± 33
Phenytoin 1%	1229 ± 60 *
GSE 2%	1170 ± 34 *
GSE 5%	1079 ± 35
GSE 10%	1068 ± 34
GSE 70%	1048 ± 26

GSE, grape seed extract.

^* P < 0·001 (significant differences from no‐treatment or eucerin‐treated groups).

Animal ethical committee approval

The study was carried out with prior approval from the animals committee of Ahwaz Jundishapur University of Medical Sciences.

Statistical analysis

All treated groups were compared with the control group and the results were analysed statistically using one‐way ANOVA and followed by Dennett's test to identify the differences between treated groups and control. The data were considered significant at P < 0·05 (Table 2).

RESULTS

Period of healing in non‐treatment and eucerin‐treatment groups was equal to 20 days. Trend of healing in these groups was very similar without any significant difference (Figure 1). Healing in group treated with phenyion (1%) was completed within 15 days (Figure 2). Percentage of healing in phenyion group was significantly more than no‐treatment or eucerin‐treated groups along the period of treatment. Wound closure of animals treated with GSE 2, 5, 10 and 70% was completed within 13, 15, 15 and 16 days, respectively (Figure 3). GSE cream of 2% produced the best healing rate and better than phenytoin. Significant differences (P < 0·05) between GSE 2% and no‐treatment or eucerin‐treated group was observed from initial day of treatment until the complete healing. Thirteen days after treatment with GSE 2% cream, the wound compeletly closed and healing was evaluated as 100%. The wound healing pecentage of GSE 2% cream was more pronounced (P < 0·05 and P < 0·01) compared with phenytoin except on days 4–6 (Figure 4). GSE 2% could show a better profile than other GSE groups (5, 10 and 70%). In most days of treatment there is significant differences (P < 0·05) between GSE 2% and other GSE groups. Application of GSE 70% had no accelarating effect on wound healing in fact its impact was similar to eucerin‐treated or no‐treatment one (Figure 5).

Figure 1.

Comparison of the wound healing in no‐treatment and eucerin‐treated groups. Data are expressed as mean ± SEM. There is no significant difference between the two groups.

Figure 2.

Comparison of the wound healing in no‐treatment, eucerin‐ and phenytoin‐treated groups. Data are expressed as mean ± SEM. Values significantly different from eucerin‐treated or no‐treatment are indicated as *(P < 0·01).

Figure 3.

Comparison of the wound healing in grape seed extract‐ (2, 5, 10 and 70%) and phenytoin‐treated groups.

Figure 4.

Comparison of the wound healing in phenytoin‐ and GSE 2% treated groups. Data are expressed as mean ± SEM. Values significantly different from phenytoin treatment are indicated as *(P < 0·01).

Figure 5.

Comparison of the wound healing in eucerin‐treated, no‐treatment and GSE 70% treated groups. There is no significant difference between the three groups.

Promotion of wound healing by GSE was confirmed by histological investigation, such improvement was quicker with GSE 2%. On the 7th day of treatment with GSE, in most samples, granulation tissue and reconstruction of epidermis was initiated whereas in no‐treatment or eucerin‐treated groups no sign of repair was evident and still necrosis was observed (6, 7). In the first week, existence of inflammatory cells in the dermal area was observable in most groups. On the Day 13 in which the wound closure was completed for GSE 2% the histological studies of wound section confirmed the formation of epidermis, associated with less inflammatory cells in the dermis layer. In GSE 2%, the epidermis was thinner but close to the normal structure and it was similar to phenytoin group (8, 9).

Figure 6.

Photomicrograph of normal skin section of rabbit. E, epidermis layer and D, dermis (H&E ×100).

Figure 7.

Photomicrograph of skin wound section of rabbit on the 7th day of treatment with eucerin. Necrosis and inflammation are still evident (H&E ×200).

Figure 8.

Photomicrograph of skin wound section of rabbit on 15th day of treatment with grape seed extract (2%). Recently formed epidermis layer and dermis with normal structure and less inflammatory cells are seen (H&E ×100).

Figure 9.

Photomicrograph of skin wound section of rabbit 15 days after treatment with phenytoin 1% (H&E ×100).

DISCUSSION

In recent years much attention has been focused on the use of GSE effects in different diseases. There are published reports on the use of GSE in cardiovascular diseases, cancers and inflammation. In the process of wound healing, inflammation is an important part of the acute response, results in a coordinated recruitment of neutrophils at the wound site. These cells are able to produce active oxygen radical, which is critical for defence against bacteria and other invasive pathogens (17). Macrophages are also involved in the production of oxidants at the wound site which builds up a ‘long‐term’ response to injured cells following the acute response. Wound‐related non‐phagocytic cells also generate O₂•⁻ by a non‐phagocytic NAD(P)H oxidase or Nox‐dependent mechanism (18).

Superoxide anion radical is rapidly converted to membrane permeable form, H₂O₂, by superoxide dismutase activity or even spontaneously. Release of H₂O₂ may promote formation of other oxidant species, including hypochloric acid, chloramines, aldehydes, etc. Taken together, this suggests that the wound site is rich in both oxygen‐ and nitrogen‐centred reactive species along with their derivatives (e.g. H₂O₂, O₂•⁻, •NO, peroxynitrite, HOCl and chloramine) mostly contributed by neutrophils and macrophages (17).

GSE contains proanthocyanidins or condensed tannins. These are a group of biologically active polyphenolic bioflavonoids that are synthesised by many plants. Proanthocyanidins and other tannins are known to facilitate wound healing 12, 13. The mode of action, however, remained unclear. Grape seed proanthocyanidin extract has been reported to have various clinically relevant redox‐active properties 5, 19 We have previously shown that other types of inflammation such as silica or bleomycin‐induced pulmonary fibrosis and rat paw oedema may be prevented by GSE 20, 21.

Oxidants present at the wound site are thought to support wound repair (22). Few studies suggest that GSE treatment was associated with enhanced tissue oxidation at the wound site. While it is known that under certain conditions GPE may demonstrate potent antioxidant properties, it must be considered that oxidative modification of antioxidants may result in the formation of potent oxidants. Tannins and tannic acid are an integral component of grape seed 23, 24. Tannic acid is known to be capable of generating hydroxyl radicals. The radical structures obtained after oxidation of flavon(ol)s and proanthocyanidins have been characterised 25, 26.

The current study supports the idea that the topical application of GSE represents a feasible and productive approach to enhance dermal wound healing. In the present study, excision wound model for contraction and epithelisation was employed. There was no similar study of GSE on wound healing. Therefore, according to our previous studies on herbal extracts, we decided to test 2, 5 and 10% GSE creams to find the most effective concentration. The aim of the use of 70% GSE was to observe the effect of pure extract. In this work, optimum effect was obtained by 2% GSE. Results of these work verified that topical GSE 2% markedly promotes wound contraction or closure and the period of healing significantly reduced from 20 days (for no‐treatment or eucerin‐treated) to 13 days. The effect for GSE 2% was distinctly visible from 1 day after wounding. The time course results of GSE were reproducible because they were tested in a group of at least six animals. The macroscopic study of the GSE 2% group was consistent with the results from hydroxyproline assay or tensile strength values of this group (Tables 1 and 2).

GSE 2% was more potent than phenytoin which is commercially available for clinical uses. In this study, we observed that GSE 2% has the best effect whereas highest concentration of GSE (70%) had no promotive effect. This means that for maximal promotion of wound healing only a small amount of GSE can modulate the wound environment for faster healing. The present study supports the idea that topical application of GSE 2% represents a feasible and productive approach to enhance dermal wound healing. However, in order to elucidate the exact mechanism of GSE in wound healing further investigations need to be performed.

ACKNOWLEDGEMENTS

This article is a part of the PharmD thesis which was carried out in the School of Pharmacy. It was financially supported by vice chancellor of research (grant number: 85U118) Ahwaz Jundishapur University of Medical Sciences. We thank Mr M Nokhbeh for technical assistance.