Effects of Hypericum perforatum on an Experimentally Induced Diabetic Wound in a Rat Model

Objective. The aim of this study was to investigate the probable effects of Hypericum perforatum (HP) on wound healing in diabetic rats.Materials and Methods. Thirty-five male Wistar rats were divided evenly into 5 groups. Diabetes formation was induced by intraperitoneal streptozotocin (60 mg/kg) administration for groups 1 (HP extract in olive oil), 2 (HP extract in ethanol), 3 (povidone-iodine application), and 4 (diabetic rats without any applied medication); group 5 was the control. Dorsal dermoepidermal incision was performed on each rat after 48 hours. The aforementioned solutions were applied only to groups 1, 2, and 3; groups 4 and 5 did not receive solution applications. At the end of the 7-day period, the cutaneous tissue was resected from the center of the incised and sutured region and divided into 3 pieces for biomechanical, biochemical, and histopathological assessments. Results. Ultimate stress and toughness significantly decreased in groups 3, 4, and 5 compared to group 1. There was a significant difference between groups 2 and 3 for the same parameters (P < .05). Compared with group 4, tissue malondialdehyde levels were found to be lower in the HP groups (P < .05). Histopathological evaluation revealed the fibroblast count was reduced considerably in the HP-applied rats compared with other groups (P < .05). Conclusion. Application of HP may be recommended as effective on wound healing in diabetic rats, but further investigation is needed to adapt the findings for clinical use.

Diabetes mellitus (DM) is a major, pandemic endocrine/metabolic disorder in the 21st century.¹ Due to its comorbidities, DM increases the need for surgical or operative procedures.² Unfortunately, impaired wound healing is one of the important complications of DM.³ Moreover, DM is one of the major causes for chronic, nonhealing wounds.^4,5 The reasons for impaired wound healing include delayed cellular infiltration, granulation tissue formation, decreased collagen organization, diminished blood flow, increased blood viscosity, and reduced angiogenesis.^6–8 Appropriate wound healing through all its phases (ie, hemostasis, inflammation, proliferation, and remodelling) is an indicator of well-coordinated reparative response to traumatic damage or surgical intervention.

 The World Health Organization defines traditional medicine as “the health practices, approaches, knowledge, and beliefs incorporating plant, animal, and mineral based medicines, spiritual therapies, manual techniques, and exercises applied singularly or in combination to treat, diagnose, and prevent illness or maintain well-being.”⁹Hypericum perforatum (HP), also known as St. John’s wort, has a long history of use in traditional medicine. It is a yellow-flowered, perennial weed common in North and South America, Europe, and Asia. Galen, Dioscorides, Pliny, and Hippocrates advised the use of HP as a diuretic, a wound healing herb, a treatment for menstrual disorders, and a cure for intestinal worms and snakebites.¹⁰ St. John’s wort has been used to treat a number of diseases such as urogenital inflammations, DM, neuralgia, heart diseases, gastritis, hemorrhoids, peptic ulcers, lung ailments, melancholy, and madness in European and Turkish traditional medicines.^11–13 Moreover and above, olive oil macerate of HP is a popular home remedy used to accelerate the healing process of cuts and burns.^12,14

Phloroglucinol derivatives, naphtodianthrones, flavonoids, procyanidins, xanthones, essential oils, and other constituents are the active components of HP.¹⁵ Phloroglucinol derivatives (around 0.2%–4%) are mainly hyperforin, and its homologues are adhyperforin and furanohyperforin. Hypericin, isohypericin, and pseudohypericin are naphtodianthrones derivatives. Hypericin content varies between 0.1% to 0.15%. Rutin, hyperoside, and isoquercitrin are flavonoids with concentrations reported as 1.6%, 0.9%, and 0.3%, respectively. Content of tannins and essential oils have been reported as 8% to 9% and 0.05% to 0.9%, respectively.¹⁵

Recent research¹⁶ has focused on the use of inexpensive and extensive herbal extracts in wound healing. There is a dearth of experimental investigations of HP on diabetic animals. Yadollah-Damavandi et al¹⁷ demonstrated that HP improves tissue regeneration by enhancing fibroblast proliferation, collagen bundle synthesis, and revascularization in diabetic skin injuries based on stereological analysis.

 To the best of the authors’ knowledge, there is no study investigating the effect of HP on wound healing in a diabetic rat model by using biochemical, biomechanical, and histopathological techniques. The aim of this study is to evaluate the effects of HP extract on the wound healing process in the streptozotocin (STZ)-induced diabetic rat model.

Animals. Adult male Wistar rats (N = 35), weighing 200 g to 250 g, were obtained from the Experimental Animal Research Laboratory of Mersin University (Mersin,Turkey). The animals were given a standard laboratory diet (commercial nutrition product with 2600 kcal/kg metabolic energy and 16-mm diameter) and water ad libitum. They were subjected to a 12-hour light/dark cycle, 45% to 55% relative humidity, and a temperature of 22°C to 25°C. All animals were acclimatized to laboratory conditions for at least one week before any manipulation was made; a period of 48–72 hours is suggested for acclimation. Each animal was subjected to the experimental procedure once. Principles of laboratory animal care (NIH publication number 85-23, revised 1985) guidelines were followed.

The animals were divided into 5 groups each consisting of 7 rats: group 1, diabetic rats treated with olive oil HP extract; group 2, diabetic rats treated with ethanol HP extract; group 3, diabetic rats treated with povidone-iodine solution; group 4, diabetic rats without any applied medication; and group 5, control (nondiabetic wounded animals without any applied medication). 

Induction of DM. Experimental DM was induced by STZ in the rats.¹⁸ All animals, except for those in group 5, were fasted for 12 hours overnight before receiving an injection of STZ. Diabetes was induced by an immediate single intraperitoneal injection after the preparation of STZ (60 mg/kg) in 0.1 M citrate buffer (pH 4.5). The blood glucose levels of animals were measured following tail tip amputation by glucometer 48 hours after STZ administration. Rats with fasting blood glucose levels > 200 mg/dL were considered diabetic.

Plant extracts and administration. Aerial parts of HP were collected in Çamlıyayla, Mersin, Turkey, in July 2011. Olive oil and ethanol extracts were prepared from the plant material. Hypericum perforatum extracts were prepared according to the methods of Süntar et al.¹⁹ 

Preparation of the olive oil extract. Fresh, crushed HP flower (50 g) was placed in a glass jar containing 500 mL olive oil. The jar was kept under sunlight for 12 hours in air temperatures between 25˚C to 38˚C for 4 weeks during the summer season according to the protocol.²⁰ The oil extract became brilliant red with an orange-red fluorescence at the end of this period. 

Preparation of ethanol extracts. Stems and leaves of HP (above-ground section of the plant, 100 g) were broken and crushed into pieces. To extract the HP, 2000 mL of 96% ethanol solution was used 3 times at room temperature. The mixture was filtered with Whatman filter paper: grade 1 (GE Healthcare, Little Chalfont, UK). After filtration, the solvent from the extract was removed by using a rotary evaporator at 40˚C; dry extract was obtained (yield 17.5%). The extract was mixed thoroughly in a mortar with a mixture of glycol stearate, propylene glycol, and liquid paraffin (3:6:1) into an ointment form. The obtained 10% HP concentration in the ointment is in accordance as the optimal percentage in the related literature.^21,22 The ethanol extract was topically applied to the wounded area of the animals.

Excision wound model and wound treatment. After 48 hours post STZ injection, the rats were anaesthetized by an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). The dorsal surface of each animal was shaved with a razor. Dorsal dermoepidermal 3-cm long incisions were made with a sterile surgical blade to the skin on the shaved back of the rats. The wounding day was considered as day 0. The following were applied topically twice daily for 7 days: HP extract in olive oil to group 1, HP extract in ethanol to group 2, and povidone-iodine (10% solution in water yielding 1% available iodine) to group 3 animals. After the first 7-day period, the cutaneous tissue was resected with the incised and sutured region at the center. The tissue was divided into 3 pieces for biomechanical, biochemical, and histopathological assessments.

Determination of skin biomechanics. The biomechanical properties of the skin were investigated using a tensile testing machine (Commat Ltd, Ankara, Turkey) equipped with a 50-kg load cell. The tensile loading speed in all tests was 1 mm per minute. Data were transferred to the computers translating to the numerical signals by a 16-bit A/D converter for the offline analysis. The sampling rate was chosen as 1000 sample per second. Each specimen was subjected to a small initial preload (0.1 N) before the actual testing. The load-displacement data were recorded using the BIOPAC MP 100 Acquisition System Version 3.5.7 (Santa Barbara, CA). The load-displacement recordings were normalized by cross-sectional area, and this curve was converted to a stress-strain curve. Stress-strain curves for each specimen were generated and ultimate stress, ultimate strain, and toughness were determined. 

The ultimate stress was calculated from the following equation²³: 

σ = F/A,

where σ is the ultimate stress (MPa), F is the failure load (N), and A is the cortical area of the specimen (m²). 

The ultimate strain was calculated from the following equation²⁴: 

ε = ΔL/L₀,

where ε is the strain, ΔL is the change in the length (mm), and L₀ is the original length. The area under the stress-strain curve is defined as the toughness (MPa). 

Toughness was calculated by the following equation: 

T = 1/2(σ*ε).

Histological assessment of wound healing. Postoperatively, the authors evaluated the wound healing process with inflammation, fibrosis, vessel proliferation, and thickness of the wound according to the modified assessment system by Lee et al.²⁴ The skin wounds were harvested, including the epidermis, dermis, and subcutaneous loose tissue, with the surrounding healthy tissue. The removed tissue samples were fixed in formalin, processed, and then embedded in paraffin. The paraffin blocks were cut into 4-µm to 6-µm thick slices, and the slices were stained with hematoxylin-eosin and Masson’s trichrome for histological examination. To evaluate the degree of inflammation in all groups, the number of inflammatory cells, including the neutrophils, plasma cells, and lymphocytes, were counted by a pathologist on highpower fields (×400) 3 times with Olympus BX 50 microscope (Olympus, Tokyo, Japan ). The degree of fibrosis was analyzed by counting all fibroblasts in 10 randomly selected highpower fields (×400). To evaluate the blood vessel proliferation, the authors counted the number of blood vessels on the highpower field (×400) 3 times and compared the mean number of blood vessels in each group. The thickness of the healing wound was determined by measuring the distance between the basal layer of the epidermis and the deepest point of inflammation of the fibrosis under microscopic examination. 

Determination of lipid peroxidation. Lipid peroxidation was assayed by measuring the level of malondialdehyde (MDA). As an index of lipid peroxidation, the MDA level was determined by thiobarbituric acid reaction according to Yagi.²⁵

Determination of protein content. Protein content was determined by using bovine serum albumin as standard following the method by Lowry et al.²⁶

Statistical analysis. The commercial statistical program SPSS version 11.5.0 for Windows (IBM, Armonk, NY) was used for the statistical evaluation of the data. The significance level was set to P = .05. Kruskal Wallis, analysis of variance, and Conover honest significant-difference tests were performed to determine the relationship between groups.

Skin biomechanics. The parameters investigated for the biomechanical properties were ultimate stress, ultimate strain, and toughness values. Their mean values and standard deviations are represented in Table 1. Ultimate stress and toughness significantly decreased in groups 3, 4, and 5 compared to group 1 (P < .05). There was a significant difference between stress and toughness values of group 2 and group 3 (P < .05). There was no significant difference in stress, strain, and toughness values between group 1 and group 2 (P > .05).  

Biochemical results. Mean MDA levels are represented in Table 2. Tissue MDA levels were found to be lower in the HP groups (groups 1 and 2) compared to group 4 (P < .05). 

Wound healing activity. Figure 1 displays the response of the skin wound, which was treated topically with HP in olive oil (group 1). The staining results of other groups are illustrated in Figure 2 (group 2), Figure 3 (group 3), Figure 4 (group 4), and Figure 5 (group 5). The wound healing activity of all groups is summarized in Table 3. There was no significant difference in inflammation and wound thickness parameters (P > .05). Vessel proliferation was found to be lower in group 4 compared with group 5 (P < .05). The histological evaluation revealed less fibrosis in group 1 and group 2 compared with group 4 and group 5 (P < .05). 

The present study revealed an increase in the biomechanical properties and the quality of the skin wound healing on diabetic rats with the administration of HP. 

St. John’s wort has a long history of medicinal use and has been used in traditional medicine both orally and topically for centuries worldwide, mainly for wound healing, ulcers, and inflammation.^10-13 There are numerous studies regarding the effect of HP treatment on wound healing. Süntar et al²⁷ showed the olive oil extract in HP has a significant effect on the healing process of excision and circular incision wound models. Further investigations were conducted regarding the effects of various HP species for wound healing assessment on NIH3T3 fibroblasts; the results indicated that even the 2 different subspecies of HP had different wound healing effects pertaining to fibroblast migration and stimulation of collagen synthesis.²⁸ Similarly, investigations of HP on cultured chicken embryonic fibroblasts indicated mainly an increase in the stimulation of fibroblast collagen production and the activation of polygonal-shaped fibroblast cells, which played a role in wound repair by closing the damaged area.²⁹ As studied for various body regions, Samadi et al³⁰ reported the beneficial effects of topical application of HP on post-cesarean wounds.

In the present study, stress and toughness values were found to be significantly increased in HP groups compared to groups 3, 4, and 5. Similar studies exist regarding the beneficial effects of HP on the mechanical properties of the dermis. Süntar et al²⁷ also found increased tensile strength on wounded rat and mice tissues. The present study has concordant findings with the above literature.

Wound healing is a complex biological process requiring well-coordinated and balanced interactions of all constituents from the inflammatory to remodelling phase. Wound maturation, including the differentiation of fibroblasts to myofibroblasts, is of great importance for the ultimate healing of diabetic wounds.³¹

Normally, collagen synthesis and degradation reach an equilibrium on the remodelling phase. Adequate collagen synthesis is necessary for appropriate wound healing. However, excessive collagen synthesis due to a high involvement of dermal fibroblasts may cause collagen deposition on dermis and subcutaneous tissues. Hypertrophic scars and keloids may occur and result in disfigurement, contractures, pruritus, pain, and organ dysfunction.³²

The histological evaluation of the authors’ study revealed less fibrosis in the HP groups compared to the animals in group 4. However, Yadollah-Damavandi et al¹⁷ found the number of fibroblast cells increased in animals that were treated with HP compared to the control group. The healing process of a tissue is a very complex biological process involving numerous sequential biochemical steps. The transforming growth factor (TGF) superfamily is one of the most important proteins in wound healing. Isoforms of this cytokine were proposed to have different effects. Transforming growth fator beta 1 is shown to mediate fibrosis, while TGFβ3 may promote reduced scarring.³³ Further investigation is needed on this topic.

The mechanical properties of the skin are largely associated with collagen fibers. The structure and the composition of the collagen play a substantial role in the function of the healing structure. Type I collagen is a major component of the skin and has an important role in the healing and remodelling of skin structure.³⁴

Several factors have been accused of diminishing healing capacity of tissues belonging to animals with DM. The inflammatory phase has been shown to be prolonged in diabetic mice. Imbalance in synthesis and degradation of collagen is thought to be the reason behind the inferior biomechanical properties of diabetic skin. Dysregulated inflammation, proteolysis, and a higher ratio of type III/I collagen ratio may influence the healing capacity towards decreased strength and stiffness in diabetic wounds.³⁵ Connizzo et al³⁶ showed that diabetic tissue had lower type I collagen levels than healthy tissue.

Diabetic wounds are a difficult entity to deal with in regards to many factors such as decreased angiogenesis, impaired growth factor production, and altered inflammatory and immune responses. Among more than 100 factors influencing the abovementioned mechanisms, matrix metalloproteinases deserve attention due to an imbalance of accumulation and remodelling of extracellular matrix components. Lower maturation, rather than the formation of collagen content, is suggested to be considered as the factor for impaired healing of diabetic wounds.³⁵ Inadequate oxygenation due to local ischemia, especially seen in DM, leads to the formation of highly toxic reactive oxygen metabolites, which results in increased lipid peroxidation. Malondialdehyde is known to be the end product of lipid peroxidation. Therefore, high MDA levels might be suggested as an indicator for impaired endothelial cells, permeability of capillaries around keratinocytes, and collagen metabolism resulting in inappropriate wound healing.³⁷ These findings point to defective posttranscriptional protein synthesis leading to impaired baseline tissue integrity, which is associated with the predisposition of patients with diabetes to wounds.³²

In the present study, HP application is shown to reduce the MDA levels in comparison to the animals in group 4. Due to the findings, it might be assumed that the olive oil and the extract forms of HP decreased the oxidative stress in the diabetic tissue permitting better healing. 

The authors did not measure collagen synthesis parameters in this study. However, stress, strain, and toughness variables were examined as the final outcome of collagen synthesis for wound healing.^38,39 The increase in stress, strain, and toughness may be linked with the stimulation of collagen synthesis and collagen crosslinking.

In this study, Hypericum perforatum content was not analyzed due to financial reasons. The authors aim to further investigate the various ingredients of the extract concentrations in future studies to better understand the findings with the analyzed concentrations. 

The care and survival of diabetic rats were far more difficult compared to nondiabetic animals. Infection of diabetic tissue and diminished healing capacity are to be considered. These factors should be taken into consideration for the number of rats assigned to each group, especially for long duration studies.

Complementary parameters, including hydroxyproline, inflammatory, and some oxidative stress markers, would be beneficial to better analyze the mechanisms involved in wound healing.

As mentioned in the text, the authors could not find a similar investigation on diabetic rats evaluating biomechanical, biochemical, and histopathological parameters all together. Therefore, there was a dearth of related literature to more fully discuss herein.

]In this study, lower MDA levels and higher mechanical quality of HP-applied groups with lesser fibrosis delineated better tissue healing in diabetic rats. Further investigation is needed to assess the tissue collagen including hydroxyproline content, the enzymatic metabolism, and additional factors such as nitric oxide metabolism to widely discuss the effects of the plant on wound healing and its constituents. 

From the Faculty of Medicine, Medical Student, Mersin University, Mersin, Turkey; Faculty of Medicine, Department of Biophysics, Mersin University; Faculty of Pharmacy, Department of Biochemistry, Mersin University; Faculty of Medicine, Department of Pathology, Mersin University; Faculty of Medicine, Department of Endocrinology, Mersin University; Department of Biostatistics, Mersin University; and Faculty of Medicine, Department of Otorhinolaryngology, Mersin University

Address correspondence to:
Ülkü Çömelekoğlu, PhD
Mersin University 
Faculty of Medicine
Department of Biophysics 
Mersin, 33343,Turkey
ucomelek@yahoo.com

Disclosure: The authors received a grant and support from TUBITAK (Turkish Scientific and Technical Research Institute), and it was presented at the 10th Annual Middle East Update in Otolaryngology Confrence, Dubai, United Arab Emirates. The authors have no financial or other conflicts of interest to disclose.