Effect of Topical Application and/or Systemic Use of Red Ginseng Extract on Wound Healing in Rats With Experimentally Induced Diabetes

Red ginseng (Rg) is traditional medicine that has been used for many years in Asian and European countries, especially Korea, China, and Japan.¹ The major components of Rg are ginsenosides, Rg1, Rb1, Rb2,and Rb3, each with its own pharmacological effect.² In vivo studies have been conducted to evaluate the hepatoprotective effect of Rg in chronic liver disease and its vasoprotective effect in heart disease.^3,4 In addition, an in vitro study revealed that Rg extract may stimulate wound healing by increasing growth factors on fibroblast obtained from diabetic patients.⁵ A similar effect of Rg extract has been observed on full-thickness skin wounds in rats.⁶ In another study conducted in patients with colorectal cancer, oral intake of Rg was shown to have no effect on blood cytokine levels and biochemical parameters, but oral Rg used in combination with chemotherapy regimen was found to reduce cancer-related fatigue.⁷

The biological mechanism of Rg on wound healing has been investigated in several in vitro studies. Rg extract has been shown to stimulate angiogenesis with phosphoinositide 3-kinase (PI3K)/Akt-dependent extracellular endothelial nitric oxide synthase activation and induction of vascular endothelial growth factor (VEGF). ⁸ It has also been shown to stimulate collagen synthesis by increasing human dermal fibroblast proliferation, suggesting that Rg can be used in areas with ischemic changes and tissue repair, especially in wound healing.⁹ Another in vitro study investigated the effects of Rg on diabetes mellitus (DM). Results of this study showed Rg has a protective effect against ß-cell damage caused by streptozotocin (STZ) and thus could be used to prevent progression of type 1 DM.¹⁰ A randomized, double-blind, placebo-controlled trial demonstrated that daily oral intake of Rg improved plasma glucose levels and increased insulin levels after meal tolerance testing and therefore may be therapeutic for patients with type 2 DM.¹¹ It is known that the immune system is weakened in patients with DM.¹² It is also known that CD4⁺ and CD8⁺ lymphocytes play a crucial role in moderating the immune response.¹³ However, to the best of the authors’ knowledge, there are no studies investigating either the CD4⁺ and CD8⁺ lymphocytes in patients with DM or the effects of Rg on these lymphocytes.

To date, researchers have explored the effect of Rg when applied locally^3,5 and used systemically^4,7,14 alone and combined with other therapies.⁶ However, no adequate evidence has been presented to delineate which usage method is optimal for Rg. In addition, to the authors’ knowledge, there have been no studies of the effect of Rg on wound healing in rats with experimentally induced DM. The present study in rats with induced DM compared the effects of Rg extract on wound healing when used systemically, topically, and combination. Expression of VEGF, an important growth factor, and CD4⁺ and CD8⁺ cell counts, which are indicators of immunity, were compared across the groups.

Ethical considerations. The experimental procedures and techniques in this study were performed in accordance with the Guidelines on the Care and Use of Laboratory Animals. Institutional Review Board (IRB) approval was obtained from Ankara Training and Research Hospital (November 26, 2020; IRB number: 645). Ethical rules and principles were followed for all procedures.

Materials. Korean Rg extract, produced from 6-year-old Korean plant roots, was obtained from Dae Han Red Ginseng Promotion Co. Ltd. The product was confirmed to be made in the Republic of Korea’s manufacturing facilities (lot no: 200416-02). Each 1 g of Rg extract contained 4 mg of ginsenosides Rg1+ Rb1+ Rg3. Streptozotocin was purchased from Sigma-Aldrich Corp.

Animals. A total of 60 Wistar albino female rats weighing 235 ± 25 g and aged 5 to 6 months were used in the study. Rats were kept in wire cages with a 12-hour light/dark cycle at a permanent temperature of 21°C ± 2°C for a 1-week adaptation period. A standard laboratory food diet (Bil-yem) and drinking water were provided ad libitum. Animals fasted for 12 hours before anesthesia but could drink water freely until 2 hours before.

Induction of DM. After a 1-night fast, a single dose of 40 mg/kg streptozotocin dissolved in 0.05 mol/L sodium citrate buffer (pH 4.5) was administered intraperitoneally to all rats. Food was provided 4 hours after streptozotocin administration to avoid the expected hypoglycemic shock. Blood glucose was measured with a VivaChek Eco blood glucometer (VivaChek Biotech) from blood samples taken from the tail vein on days 3 and 7 after streptozotocin administration. Rats with fasting blood glucose levels greater than 200 mg/dL were considered to have DM and were assigned to a study group.

Study groups. A total of 60 rats in which DM was created with streptozotocin induction were randomly divided into 4 groups of 15 rats. Surgery and anesthesia were performed under sterile conditions by the same team for all rats. All animals were anesthetized by intramuscular injection of 50 mg/kg of ketamine hydrochloride (Ketalar; Parke-Davis) and 5 mg/kg of xylazine (Rompun; Bayer). The surgical area was shaved and disinfected with povidone-iodine. A 2 cm × 1 cm rectangular incision was made on the back of each rat, centered on the midline, and then a standard full-thickness skin defect, including panniculus carnosus, was created.

The open wound was not sutured. The following treatment protocols were carried out daily for 14 days, and the dressings were also changed in each application. The saline-moistened sponges were held in place by mesh bandages that were dressed on the rats.

Control group. The wound was cleaned with saline and covered with a saline-moistened dressing.

Local red ginseng (LRg) group. For each rat, 100 µg/mL of Rg extract was applied locally to cover the wound completely and then covered with a saline-moistened sponge.

Systemic red ginseng (SRg) group. For each rat, 100 mg/kg of Rg extract was administered through an orogastric tube. The wound was cleaned and covered with a saline-moistened dressing.

Combined red ginseng (CRg) group. For each rat, 100 µg/mL of Rg extract was applied locally to cover the wound completely, and 100 mg/kg of Rg extract was administered systemically through an orogastric tube. The wound was covered with a saline-moistened sponge.

Wound areas of all rats were drawn on an acetate sheet on day 0 (at the time of wound creation and before the start of the treatment protocol) and day 14. The outlined wound areas were photographed with a digital camera. No complications developed at the wound sites, and no rats died during the study. Rats were killed with an anesthetic overdose on day 14, and tissue samples were taken from the wound areas for histopathological evaluation. The measurement of wound areas and the histopathological evaluation of tissue samples were performed by the researchers who were blinded to the study groups.

Evaluation of wound contraction. After the drawings from days 0 and 14 were transferred to digital media, the wound area was calculated in square millimeters using a scientific image processing program (Image J, version 1.52; National Institutes of Health). Wound contraction was calculated as a percentage by using the change in wound area from day 0 to day 14.

Histopathologic evaluation. The histopathologic examinations consisting of light microscope analyses were carried out by the hospital pathology department. The samples were fixed in a 10% neutral buffered formalin solution. Tissues were washed in running water and dehydrated with increasing ethanol concentrations (50%, 75%, 96%, and 100%). After dehydration, specimens were placed in xylene to obtain transparency and then embedded in paraffin. The embedded tissues were cut into 4-µm sections and stained with hematoxylin and eosin (H&E) and trichrome for histopathological examination.

The H&E staining was used to score inflammation and epithelialization. Both H&E and trichrome stains were used to evaluate fibrosis. Inflammation, epithelialization, and fibrosis were scored using a semiquantitative system as follows:

Inflammation scoring system. A 4-point scale was used: 0 = no inflammation; 1 = giant cells, lymphocytes, and plasma cells; 2 = giant cells, plasma cells, eosinophils, and neutrophils; and 3 = inflammatory cell infiltration and micro-abscess formation.

Epithelialization scoring system. A 3-point scale was used: 0 = no epithelialization, superficial ulcers; 1 = incomplete re-epithelialization, focal epidermal hyperplasia; and 2 = complete re-epithelialization.

Fibrosis scoring system. A 4-point scale was used: 0 = no fibrosis; 1 = mild; 2 = moderate; and 3 = severe.

The immunohistochemistry (IHC) stain used monoclonal antibodies for CD4⁺ (Dako monoclonal mouse antibody, clone 4B12; Agilent Technologies), CD8⁺ (Dako monoclonal mouse antibody, clone C8/144B; Agilent Technologies), and VEGF (Dako monoclonal mouse antibody clone VG-1; Agilent Technologies). The staining procedure was performed on a Dako Omnis autostainer (Agilent). All tissue sections were examined using an Olympus CX41 light microscope (Olympus). Images were taken using a digital camera mounted on an Olympus CX41 microscope to evaluate CD4⁺ and CD8⁺ cells. The Olympus software was used to measure the area at 40×, and the number of stained cells was counted. A total of 10 areas were evaluated to analyze a minimum of 1 mm² for each tissue sample. Results were recorded as numbers of cells/mm².

Statistical analysis. Categorical variables were expressed as raw numbers and percentages. The Kolmogorov-Smirnov test was used to determine the normal distribution of continuous variables. Quantitative variables were expressed as median (min-max) for non-normalized variables and mean ± SD for normal distributions. Comparisons of CD4⁺, CD8⁺, CD4⁺:CD8⁺ ratio, and VEGF parameters across the four groups were performed using the ANOVA test with post hoc analysis. Comparisons of wound area and contraction rate across the four groups were performed using a Kruskal-Wallis test with post hoc Dunn’s test. A chi-squaretest was performed to compare categorical variables (ie, inflammation, fibrosis, and epithelialization scores) as appropriate. P values < .05 were accepted as statistically significant. Statistical analysis was performed with SPSS v23.0 (IBM).

According to visual evaluation, no inflammation or other problem occurred during the wound healing process due to local or oral Rg extract administration.

Wound contraction. The measurements of the wound areas (mm²) on day 0 (just after the excision) were 213.6, 233.7, 195.6, and 212.8 in the control, LRg, SRg, and CRg groups, respectively; on day 14, the measurements were 86.4, 41.4, 22.1, and 28.3 in the control, LRg, SRg, and CRg groups, respectively. There was no significant difference among the groups for wound area on day 0 (P = .11); however, there were significant differences among the groups for wound area on day 14 (P < .001). Wound contraction measurements (%) were 63.8, 80.5, 88.5 and 86.6 in the control, LRg, SRg and CRg groups, respectively. There were significant differences among the groups for wound contraction (P < .001) (Table 1). In all study groups, the wounds were almost completely closed on day 14; however, recovery was slower in the control group (Figure 1). Wound contraction was highest in the SRg group and lowest in the control group (Figure 2). In pairwise comparisons, the wound areas on day 14 were significantly smaller in the LRg, SRg, and CRg groups than in the control group (P = .080, P < .001, P < .001, respectively). The wound areas of the SRg and CRg groups were significantly smaller than that of the LRg group (P = .013, P = .008, respectively). There was no difference between the SRg and CRg groups (P = 1.000). Comparison of wound contractions showed significant differences between all treated groups and the control group but no difference among the treated groups (Table 2).

Histopathological results. The mean values ​​of CD4⁺ and CD8⁺ (lymphocyte count/mm²), CD4⁺:CD8⁺ ratio, and VEGF (number of vessels positively stained with antibody/mm²) in the tissue samples are shown in Table 3. The highest CD4⁺and CD8⁺ numbers and CD4⁺:CD8⁺ ratios were observed in the control group (32.40 ± 2.44, 15.73 ± 2.28, and 2.10 ± 0.39, respectively). The lowest CD4⁺ and CD8⁺ numbers and CD4⁺:CD8⁺ ratios were observed in the CRg group (16.00 ± 1.51, 10.53 ± 1.35, and 1.52 ± 0.21, respectively). There were significant differences between the control group and all treated groups in terms of CD4⁺ numbers. There was no significant difference between LRg and SRg groups (P = .210); however, comparisons of CRg with SRg and LRg revealed a significant difference (P < .001). There were significant differences between the control group and all treated groups in terms of CD8⁺ numbers. While there was no significant difference between the LRg and SRg groups (P = 1.000), the CRg group was significantly different than the SRg and LRg groups (P < .001). There were significant differences between the control group and LRg, SRg, and CRg groups in terms of CD4⁺:CD8⁺ ratio (P = .029, P = .001, P < .001, respectively). While there was no significant difference between LRg and SRg groups and between SRg and CRg groups (P = .668, P = .306, respectively), there was a significant difference between the LRg and CRg groups (P = .027). VEGF-positive vessel/mm² was observed 4.00 ± 0.75, 5.93 ± 0.70, 5.93 ± 1.93, 7.93 ± 0.70 in the control, LRg, SRg, and CRg groups, respectively. There was no difference between LRg and SRg (P = 1.000) in terms of VEGF expression, but there were significant differences for all other groups compared with each other (Table 4). The histological images for the control and CRg groups are shown in Figure 3 to depict the IHC marker changes, and the box plot graphic for all groups is shown in Figure 4.

The histopathological (epithelialization, fibrosis, and inflammation) scores are shown in Table 5. The highest and lowest mean inflammation scores were in the control group (2.46 ± 0.49) and in the CRg group (0.4 ± 0.48), respectively. In contrast, the highest mean fibrosis and epithelialization scores were in the CRg group, while the lowest were in the control group (2.20 ± 0.40 vs 0.8 ± 0.65 and 1.53 ± 0.49 vs 0.26 ± 0.44, respectively). There were significant differences in all intergroup comparisons for epithelialization, fibrosis, and inflammation scores (P < .001). The histogram graph of the scores is shown in Figure 5.

Korean Rg, used in traditional medicine for many years, has antiaging, antioxidant, neuroprotective, anticancer, immunomodulatory, high vascular regeneration, and antidiabetic effects.^4,7,8,10 Due to its beneficial pharmacological effects, studies investigating its action on wound healing have also gained momentum.^6,14 Wound healing in mammals begins with the cessation of bleeding in the tissue, continues with the reformation of the damaged tissue, and ends with the formation of a barrier against microorganisms. Wound healing in the skin occurs in 3 phases, including inflammatory, re-epithelialization, and remodeling processes.¹⁵ In patients with DM, one or more of the steps in this process may be interrupted, resulting in delayed wound healing or incomplete healing.^13,16 In the present study, Rg was found to decrease inflammation and increase epithelialization-fibrosis compared with the control group.

There have been many studies that evaluated wound healing using the histopathological scoring system in the literature. In a study investigating the topical effect of curcumin (medicinal plant) on wound healing in rats with induced DM, epithelialization scores and wound maturity were found to be significantly higher whereas inflammation score was found to be significantly lower than those of the control group.¹⁷Omma et al¹⁸ examined topical and systemic effects of N-acetyl-cysteine (an antioxidant agent) on wound healing in rats with induced DM. The results were parallel to the present study in terms of histopathological scoring. It was observed that N-acetyl-cysteine improved wound healing, and also combined (topical + systemic) usage was more effective. In addition, the researchers emphasized that all application methods reduced oxidative stress factors in tissue, while systemic and combined application extended this effect to plasma. 

The largest component of Korean Rg, Rg1, has been reported to have an antidiabetic effect by increasing glucose-stimulated insulin secretion.¹⁹ When the pharmacokinetics of Rg extract were investigated in a study performed with rats with DM, Rg extract used together with metformin was reported to have an additive effect in glycemic control by not affecting such biochemical parameters as aspartate aminotransferase, alanine aminotransferase, and cholesterol.²⁰ In another in vitro study, researchers showed that Rg protects against ßß-cell damage caused by streptozotocin by downregulating cyclooxygenase-2 and tumor necrosis factor-alpha and by blocking nuclear factor kappa B, suggesting that Rg may be useful in preventing progression of type 1 DM.¹⁰ Furthermore, another study investigating the effect of Rg on type 2 DM emphasized that postprandial glucose levels improved in a group administered Rg, and as such, it could be used in the treatment of type 2 DM.¹¹

The effects of Rg extract on wound healing and DM have been shown separately in many studies, but its effect on wounds in rats with induced DM has not yet been investigated. The present study used a DM-induction model to complicate the wound healing process and investigated the effectiveness of Rg extract on the tissue.

To date, researchers have explored the effect of Rg when applied locally^3,5 and used systemically^4,7,14 alone and combined with other therapies.⁶ Park et al⁶ investigated a powder form of Rg was given orally, and the extract form was applied topically to full-thickness skin wounds in Sprague-Dawley rats.Placebo was used as the oral comparator, and the topical extract was compared with petroleum jelly. While skin moisture, lipid content, and elasticity were significantly higher in the oral Rg group than in the placebo group, no difference was found between the topical Rg extract and petroleum jelly groups. Compared with the control group, wound healing was faster, and expression of transforming growth factor-ß, VEGF, and matrix metalloproteinase was higher in Rg-treated groups. Although there was a significant difference in wound contractions in the Rg groups compared with the control group, this difference emerged in the last days of healing. Overall, this study reported that 2 different forms of Rg are effective in wound healing; however, no comparison was made regarding which is more effective.⁶

Another similar study reported a significant difference for wound contractions between a group of mice treated topically with Rg extract and a control group, with 85% and 65% contraction, respectively. The investigators indicated that Rg extract showed an effect similar to mouse growth factor.²¹ In the present study, wound contraction in the local, systemic, and combined groups were significantly higher than in the control group, coinciding with the literature. Wound contractions were similar in the systemic and combined groups and slightly lower in the local group, but this difference was not significant. Macroscopically, Rg appears effective in wound healing with no difference between the methods of use assessed.

It has been shown in vivo and in vitro that Rg extract stimulates angiogenesis by increasing VEGF through activation of PI3K/Akt-dependent extracellular endothelial nitric oxide synthase.⁸ In a study in which Rg extract was applied topically and its effect on ischemic flap healing examined, VEGF expression, CD31⁺ vessels, and live flap area measurements on days 3, 7, and 10 were significantly higher in the Rg extract group than in the control group. The study emphasized that Rg extract could be used topically in ischemic flap situations.¹⁴ In another study investigating the effect of Rg root on burn injuries, Rb1 was shown to be the most effective ginsenoside for burn treatment. Moreover, Rg was reported to increase VEGF and keratinocytes significantly more than was seen in a control group.²²

In a study investigating the effect of different doses of Rg on wound healing, the most effective topical application dose was reported to be 100 µg/mL; however, healing factors were not examined in the study, and only wound contraction and tension were measured.²³ Since this was the only study in the literature known to compare topical doses, the most effective dose (100 µg/mL) presented was used for the current study; for systemic administration, the common dose of 100 mg/kg was used.¹⁴ VEGF expression in each of the Rg groups in the current study was higher than in the control group.

The fact that Rg extract escalates wound healing by increasing VEGF independent of the application method is compatible with the literature. While there was no significant difference between LRg and SRg, VEGF was highest in the CRg group, and this difference was significant compared with both the LRg and the SRg groups. These data show that, at the cellular level, wound healing increases in direct proportion to the dose of Rg. Therefore, the combined method with the highest dose was most effective in wound healing, but this difference was not reflected in the visual observations of area contraction.

In a study investigating the effect of daily administration of Rg on immune response in healthy adults, the numbers of T lymphocytes (CD4⁺ and CD8⁺) were reported to increase in the Rg group after 8 weeks compared with the control group.²⁴ In another study investigating the effect of Rg on immunity in HIV-infected patients, Rg intake was found to correlate with a decrease in CD8⁺ numbers.²⁵ The results of these studies suggest that Rg increases the systemic immune response, but its effect on T lymphocytes in the wound has not been studied yet.

Lymphocytes are found in the entire wound area a few days after injury and are known to affect fibroblasts and collagen synthesis through the lymphokines they secrete.²⁶ T lymphocyte migration to the wound peaked on day 7 in a study by Fishel et al²⁷ and on day 10 in a study by Agaiby and Dyson.²⁸ The CD4⁺:CD8⁺ ratio was approximately 2:1 in both studies. Boyce et al²⁹ reported that this ratio decreased as healing progressed toward covering the wound. In the present study, the CD4⁺:CD8⁺ ratio in the control group was close to the ratio for the normal healing process reported in the literature. The authors attribute the lower ratio in the treated groups to the fact that Rg heals and covers the wound faster. CD8⁺ cytotoxic lymphocytes are proinflammatory, and their elevation in tissue causes impaired wound healing.²⁶ In a study investigating the wound healing effect of an herbal product, the number of CD8⁺ cells in the tissue on day 15 was significantly lower in treated groups than in the control group, and the CD8⁺ level was reported to affect wound healing positively.³⁰ In the present study, the number of CD8⁺ cells measured on day 14 was lowest in the combined group in which Rg was administered at the highest dose. The authors consider that the combined usage of Rg protects against inflammation by decreasing CD8⁺ cells more than in the other groups and that it stimulates the transition to re-epithelialization and granulation by decreasing the CD4⁺:CD8⁺ ratio earlier. The fact that the highest rate of epithelialization was observed in the CRg group whereas the highest rate of inflammation was seen in the control group (according to the histopathological scoring) supports this opinion.

The study is limited by the fact that only 60 rats were included, and only a limited number of parameters for wound healing were examined. More research is needed before these findings can be applied to treatment of wounds in patients with DM.

Rg extract can be used for healing full-thickness wounds in rats with induced DM. All usage methods of Rg extract increased the wound contraction, and differences were observed versus the control group. However, the authors conclude that the combined usage is more effective due to higher VEGF expression levels and a lower CD4⁺:CD8⁺ ratio. Further experimental studies should evaluate other growth factors (eg, transforming growth factor-ß, insulin-like growth factor-1) and other immune system cells (eg, B lymphocytes, natural killer cells) to clarify the role of Rg in wound healing in DM.

Mehmet Esat Duymus, MD¹; Hulya Ayik Aydin, MD²; Abdullah Bulgurcu, MD³; Zeynep Bayramoglu, MD⁴; Abdullah Durhan, MD⁵;
Salih Tuncal, MD⁵; Mevlut Recep Pekcici, MD⁵; and Kemal Kismet, MD⁶

¹Department of General Surgery, Hatay Mustafa Kemal University, Hatay, Turkey
²Department of Gynecology and Obstetrics, Private Saglik Hospital, Denizli, Turkey
³Department of General Surgery, Hatay Training and Researcher Hospital, Hatay, Turkey
⁴Department of Pathology, Konya Training and Researcher Hospital, Konya, Turkey
⁵Department of General Surgery, Ankara Training and Researcher Hospital, Ankara, Turkey
⁶Department of Nursing, Selcuk University, Konya, Turkey

Address all correspondence to: Mehmet Esat Duymus, MD, Hatay Mustafa Kemal University, Hatay, Turkey; email: esatduymus@hotmail.com

None disclosed