Wound Healing Effects of Quercus Brantii and Pelargonium Graveolens Extracts in Male Wistar Rats

Introduction. Wound healing comprises a complex pathophysiological process. Recent studies suggest plants play a major role as medications in wound healing. Quercus brantii and Pelargonium graveolens are 2 herbs used in Iran as a traditional remedy in speeding up the wound healing process. Materials and Methods. Forty male Wistar rats were divided into 4 groups of 10. Two distinct groups were reserved for the 2 herb extracts and 2 groups were allocated for positive and negative controls. The animals were anesthetized with ketamine and xylazine. After shaving their dorsal skin, a circular excision with an area approximation of 150 mm² and 2 mm deep was created. Each rat received an application of 200 mg/kg/day of its allocated treatment. On days 3, 6, 9, 12, 15, 18, and 21, the wound areas were traced and photographed. On days 6, 12, and 18, biopsies were taken from the active peripheries. The data were collected and analyzed by statistical analysis software (SPSS for Windows Version 6, IBM, Armonk, NY). Results. The results were representative of the significant effects of Q. brantii extract on the enhancement of wound contraction velocity in comparison to nitrofurazone (on day 12, P = 0.001). According to histopathological assessments, healing in group A was significantly faster than the 3 other groups, even the positive control group (P < 0.01). Lesser inflammatory cells and fibrins in addition to more reepithelialization in group A in comparison to even positive control group, led to the potential of Q. brantii in treating wounds. Conclusion. The primary faster healing of P. graveolens plus the overall advantage of Q. brantii over nitrofurazone were confirmatory evidences of the traditional use of these herbs in the wound healing process.

The wound healing process comprises complex pathophysiological stages, including inflammation, angiogenesis, collagen deposition and etc.¹ Persistent inflammation and inadequate vessel formation are the most important causes of delayed wound healing.^1-3 On the other hand, wound fibrosis or abnormal accumulation of collagen in the wound can lead to an unpleasant scar. 

Recent studies^1,2 show plants and herbal products play an important role in the wound healing process. Different products are utilized in different regions and cultures.^3,4 Approximately one-third of all traditional medications are for wound remedy and skin disorders, compared to only 1% to 3% of modern drugs.^5,6 In addition, about 80% of the global population prefer herbal plants for treating skin problems^7,8; and the medical use of traditional plants is a focus of attention in scientific communities. There are various scientific studies^9-12 indicating the role of medicinal plants in different wound healing phases such as inflammation, coagulation, fibroplasia, wound contraction, epithelialization, and collagenation to improve healing and prevent wound infections.

Quercus brantii, which is called Iranian oak, is also known as Q. globularis, Q. persica, Q. baneica, Q. ungeri, and Q. saii in herbal pharmacopeia. This plant is a well-known traditional herb used in tribal medicines of Iran and is locally known as “Maazouj” in the western side of this country. Some studies approved great antimicrobial effect of the Q. brantii fruit on Escherichia coli, Helicobacter pylori, and Streptococcus pyogenes, which is due to tannins and phenolic components in this herb. Tannin is responsible for contracting and disinfective effects of this herb.¹³ According to previous studies^3-5,13 about traditional experiences and easy accessibility and effects of Q. brantii, the authors decided to investigate the effects of this plant on wound healing processes in Wistar rats. 

Pelargonium graveolens, a family in the Pelargonium genus, essentially contains citronellol, geraniol, and 10-epi-γ-eudesmol.¹⁴P. graveolens—also known as geranium, Rose pelargonium, and “Shamdani e atri” in traditional documents — has plenty of pharmaceutical virtues. It has antiseptic effects, a balancing effect on the nervous system (especially the adrenal cortex), and relieves depression and anxiety. With respect to its natural homeostatic and pain-killing effects as well as its consumption as a cure for burns in folkloric medicine, the authors also selected this herb for their investigation.¹⁵

Herbs and extract preparation
Q. brantii fruits were picked in the Zagros Mountains (more than 2500 m height), District of Kohkilouyeh va Boyer-Ahmad, Iran. P. graveolens herbs were collected in the meadows in the District of Kurdistan, Iran. The identities of the plants were authenticated by Professor M. Rafieian, Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran.

The maceration method was used to prepare the extract. For this purpose, about 100 g of Q. brantii fruit powder and grinded flower of P. graveolens (Herbarium No. 183 & 215, Medical Plants Research Center, SKUMS) were transported into an Erlenmeyer flask and 1 L ethanol, 70% (Nasr Alcohol Cooperation, Lorestan, Iran) was added, and the solution was left at laboratory temperature. Forty-eight hours later, the extract was filtrated through a paper filter, and the pulps were squeezed to discharge. Then, the extract was concentrated by a rotary evaporator,¹ dried, and mixed with pure generic petroleum jelly (Ehsan Chemi Estahban Co, Tehran, Iran) to make a petroleum jelly-based 0.6% ointment.³ 

Animals
Forty male Wistar rats (weighing 200-250 g) of 3-4 months of age were divided into 4 groups of 10; 1 group for each herb and 2 for positive and negative controls.

The animals were left for 3 days at room temperature for acclimatization, then were housed in the same standard environmental condition with a temperature of 20°C to 22°C, humidity of 60% to 65% and a 12-hour light/dark cycle. Their diet during the experiment was standard pellet (Pasteur Institute of Iran, Tehran, Iran) and water ad libitum. The study was performed according to the international rules considering the animal experiments and biodiversity right of Shahrekord University of Medical Sciences number 91-12-1.

Wound incisions
Animals were anesthetized by a combination of 2 mg/kg of ketamine 10% (Alfasan Co, Woerden, The Netherlands) and xylazine 2% (Alfasan Co), administered intramuscularly.¹⁶ The dorsal skin was disinfected with Betadine 10% (Tolid Daru Co., Tehran, Iran), and the hair of the allocated area of the skin was completely shaved with a razor blade. 

A circular excisional wound with a surface area of approximately 150 mm² and 2 mm deep was created by a circular metal model, toothed forceps, and a sterilized surgical blade, 15 mm aside from the spinal column.¹⁶ Bleeding was controlled by a sterile cotton tipped applicator (Medline Industries, Mundelein, IL), and the wounds were left open.^16,17

Experiment designation
The animals were randomly divided into 4 groups of 10 rats each. Group A was treated with  200 mg/kg/day of Q. brantii ointment, group B was treated with 200 mg/kg/day of P. graveolens ointment, group C+ received 200 mg/kg/day of nitrofurazone ointment as a positive control; and group C- was treated with 200 mg/kg/day of a simple petroleum jelly-based ointment as a negative control.

On days 3, 6, 9, 12, 15, 18, and 21 of the experiment, the wound area was traced manually and photographed in a bright environment with the same position, distance, and camera (Canon SX30, Tokyo, Japan) the same as day zero. The wound area was calculated by AutoCAD R14 software (Autodesk, San Rafael, CA) and recorded separately for each rat. 

In order to eliminate the bias of primary wound size differences, the wound contraction percentage (WCP) in each time section was calculated using the following formula, in which (n is the number of the day):

[(Wound Area on Day #0 - Wound Area on Day #n) / Wound Area on Day #0] * 100 = % of Wound Closure on Day #n

On the determined days, the mean variance of WCP of each group was calculated by paired two-sample t test to compare remedial progression between groups. 

At days 6, 12, and 18, the experiment was temporarily stagnated and a small piece of tissue was removed from the active edge of each animal’s wound for pathological and histological examinations. These specimens were dissected and put into a solution of formaldehyde 10% for transferring to the lab.¹⁸ The skin tissues were embedded in paraffin, cut into 4-micrometered sections by using a microtome (Olympus Cut 4055, Olympus, Center Valley, PA) and then stained by hematoxylin and eosin, which were then evaluated by light microscope (Olympus BX51, Olympus, Shinjuku, Tokyo, Japan). In a pathological assessment, each sample was evaluated according to the existence of Inflammatory Cells (IC), Collagen Fibrins (CF), Reepithelialization (RE), Collagen Organization (CO), Necrosis (N), and Fibrin (F).  Each were classified separately as -, +, ++, or +++.

Statistical analysis
Data were collected and analyzed by SPSS for Windows Version 16 (IBM, Armonk, NY) using survival analysis, t-test and one-way analysis of variance for macroscopic analysis, and Mann-Whitney U and chi-square tests for histopathology analysis. P < 0.05 was considered as statistically significant. 

The results were representative of the significant effects of the extract of Q. brantii on speeding up the wound contraction velocity (WCV) in comparison to nitrofurazone (positive control group). With respect to Table 1, during the first 6 days, the WCP of the C+ group was significantly more than group A; however, surprisingly, after that the WCV increases in this group (day 12, P = 0.001) and is almost completely healed by day 12 — significantly faster than the 3 other groups (Figure 1). This process is interpreted in Table 1 and Table 2. 

Table 1 also shows a paradoxical effect of P. graveolens extract (group B). During the first 6 days after wound induction, the WCV in group B was significantly more than in groups A and C+ (Table 2). But from day 9, the authors encountered an unpredictable WCV reduction in group B (in day 12 between groups C+ and B, P = 0.0008). As a result of that abatement, group B rats achieved complete wound healing after day 18, while groups A and C+ were completely healed at days 12 and 15, respectively. 

According to histopathological assessments, the WCV and the wound healing process,  in group A were significantly faster than the other 3 groups, even the positive control group (P value < 0.01). Table 3 shows the histopathological results. Figure 2 shows fewer inflammatory cells and fibrins as well as higher amount of reepithelialization in group A in comparison to the positive control group in the second biopsy, which confirmed more efficacy of Q. brantii than nitrofurazone in wound healing process. 

Also in Figure 3 which is related to the C– group, persistent inflammation and incomplete reepithelialization are shown.

Wound contraction is the process of shrinking the wound surface, while wound healing is a process in which the damaged tissue is restored to as close to its normal state as possible.¹² It essentially depends on the type and size of the wound, general health condition of the patient, and the natural potential for repair of the injured tissue.¹⁶ Wound healing is also a complicated process in which interruption could lead to delayed healing, an increased possibility of infection, excessive fibrosis formation, inappropriate recovery, and unpleasant scar formation.¹ 

Despite the usage of Q. brantii and P. graveolens extracts in Iranian traditional medicine, to the best of the authors’ knowledge no scientific evidence proving the wound healing efficacy of these herbs exists in the literature. However, Shellie and Marriott¹⁴ showed Pelargonium in a mixture with Aloe vera and Lavandula stoechas have a significant effect in healing second-degree burn wounds compared to silver sulfadiazine; though, this study could not be of that value for the present study, due to the distinctive difference between extracting methods and utilization of combination therapy, which may have been leading to synergistic effects. Sabzghabaee et al¹⁹ have proven the antifungal effect of Pelargonium and its treatment potential for denture stomatitis, which indicates the anti-inflammatory effect of this herb by some substances like phenolic structures. 

These phytochemicals ordinarily react with some compounds such as oxygen-free radicals and other macromolecules to neutralize free radicals and/or initiate biological effects.¹ 

The antibacterial effect of Pelargonium on E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans infections shows additional support for its wound healing potenitial.^20,21 In addition to these features, the authors declare that wound contraction, especially in the premier week of remedy, may be in response to the anti-infection effect of Pelargonium extract in the wound site. 

According to Pirbalouti et al,²¹Q. brantii does not show strong antifungal effects but has proven potential as an antidiarrheal substance and tonic balm in ointment form. It is also beneficial in gastric ulcer treatment, which those healing effects achieved by polyphenolic-rich components in Q. brantii extract.^21,22

 Although the gastric environment epithelium is completely different from skin, but because of similarity in wound structure and healing process of both wounds, polyphenolic components could be responsible for increasing the wound healing velocity in both studies.

The WCV of the rats in group A was significantly faster than the other 3 groups, and the WCV occurred during the first week of injury when the tissue shrinkage was considerably slower than group C+ and B. Notwithstanding the results of group A, inflammatory cells were ++ during the first week, which is significantly more than group B and group C+. These cells may slow down the healing velocity after the first week.

There are many herbs in Iranian folkloric medicine that are consumed for their polyphenolic and anti-oxidant components,^23-29 among which Q. brantii and P. graveolens are 2 well-known ones. Their anesthetic and antiseptic effects, as well as mood-stabilizing potential, have been proven in literature^3-5,13-15 and prompted the authors to investigate what additional effects these substances might have on the wound healing process, and found they demonstrated remarkable results. As a result of a lower amount of fibrous tissue formation and deformed scars in Q. brantii group and its higher wound contracture velocity in comparison to nitrofurazone group, the authors suggest further studies on Q. brantii extract. These results show pharmacological evidence confirming the folkloric use of Q. brantii on wound healing. Although P. graveolens increases the WCV during the first few days of its use on a wound, the overall results are not as beneficial as treatment with nitrofurazone. An experiment assessing the topical influence of this mixture on the wounds may contribute greatly to salient outcomes. 

The authors would like to thank Islamic Azad University, Shahrekord branch, Shahrekord, Iran, Isfahan University of Medical Sciences, Isfahan, Iran, and Shahrekord University of Medical Sciences, Shahrekord, Iran, for their support.

Affiliations: Medical Education Research Center, Isfahan University of Medical Sciences, Isfahan, Iran; Department of Veterinary Sciences, University of Turin, Grugliasco, Italy; Department Of Surgery, Shahrekord University of Medical Sciences, Shahrekord, Iran; and Department of Veterinary Clinical Science, Science and Research Branch, Islamic Azad University, Tehran, Iran; Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

Correspondence:
Pouya Parsaei, DVM, PhD
Young Researchers and Elite Club, 
Shahrekord Branch, Islamic Azad University
Shahrekord, Iran
pouyaparsaei@yahoo.com

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