Extract of Berula angustifolia (L.) Mertens Enhances Wound Healing in Streptozotocin-induced Diabetic Rats

Introduction. Diabetes-impaired wound healing and other tissue abnormalities are considered to be a major concern. Objective. The aim of this study is to assess the wound healing activity of the methanolic extracts of Berula angustifolia leaves. Materials and Methods. Seven-week-old male Wistar rats with diabetes induced by streptozotocin injection were randomized into 5 groups of 6 rats based on allocated treatment. Wounds were created by an excision-based or incision-based wound model. For wound healing activity, the extracts were applied topically in the form of ointment and compared with the control groups. The healing of the wound was assessed based on excision, incision, hydroxyproline estimation, biomechanical, and biochemical studies. Results. The healing rate of the extract-treated groups was significantly different compared with the control group (P < .05). Hydroxyproline contents increased significantly in the extract-treated groups (P < .05). There were significant differences in the extract-treated versus nonextract-treated groups, particularly in terms of cellular infiltration, acute hemorrhage, congestion, edema, collagen production and density, reepithelialization, and neovascularization. Conclusions. The methanolic extract of B angustifolia enhances wound healing activity significantly in both studied wound models. From this animal study, enhanced wound contraction, decreased epithelialization time, increased hydroxyproline content, improved mechanical indices, histological characteristics, and biochemical studies suggest the extract of B angustifolia leaves may have therapeutic benefits in diabetes-impaired wound healing.

Diabetes-impaired wound healing and other tissue abnormalities are considered to be a major concern for clinicians.¹ The biochemical mechanisms involved in the healing process are mainly associated with disorders in collagen production that consequently delay reepithelialization in wounds and compromise migration and proliferation of keratinocytes and fibroblasts.² It has been reported that the treatments developed for this complex clinical problem are not effective.³

The prevalence of diabetes has become a major clinical problem and a serious issue for public health; impaired wound healing in patients with diabetes is a complication.⁴ Lack of cellular and molecular signals required for the normal wound repair process, including angiogenesis, granulation tissue formation, epithelialization, and remodeling, are encountered in these patients, which contribute to poor wound healing. The normal healing process in healthy individuals occurs at an optimal rate; however, this is usually delayed or even completely compromised in patients with diabetes.⁴

There is an increasing interest in the potential of traditional and complementary medicines in wound care that has led to studies investigating a range of plant extracts and other products as traditional wound healing agents.⁵ These agents usually influence 1 or more phases of the healing process, and they are involved in disinfection and provide a moist area to encourage the establishment of a suitable environment for wound healing.⁵

Plant extracts with wound healing properties have the potential for antioxidant, chelation, and antimicrobial activities and may act by 1 or more of these mechanisms.⁶ Natural antioxidants have been reported to play a major role in blocking the oxidative stress induced by free radicals.⁷ Therefore, it is important to explore other sources of safe antioxidants or natural agents. Recently, researchers have shown interest in edible and medicinal plants for their phenolic contents and related total antioxidant activities.^8,9

Wild edible plants with antioxidant activities are important constituents of traditional diets in Iranian culture; some of which have not been screened for wound healing potential. This information is necessary to validate the safe use of traditional plants and may be used to establish databases and evaluate other natural remedies with potential wound healing attributes.

Fructus lycii has been used as a remedy since ancient times in many countries for its diuretic, antipyretic, tonic, aphrodisiac, hypnotic, and hepatoprotective effects.⁹ The genus Lycium (Solanaceae) is represented by various species, including Berula angustifolia stock, an edible plant in Iran (locally called Khashk) with antioxidant properties.¹⁰

Results of a literature search show no systematic approach has been made to study the wound healing activity of B angustifolia leaf extract. In this study, the wound healing activity of the methanolic extract of B angustifolia leaves was investigated as an ointment form in 3 concentrations. Assessment of the healing process was based on excision, incision, hydroxyproline estimation, biomechanical, and biochemical studies.

This study protocol was reviewed and approved by Urmia University Ethical Committee (West Azerbaijan, Iran). All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 85–23, revised 1985).

Animal grouping

Sixty healthy male Wistar rats were used in the present study; 30 rats were used for the excisional wound model and the other 30 for the incisional wound model. The animals were divided into 5 groups of 6 animals for each model. The animals were housed in standard environmental conditions of temperature (22°C ± 3°C), humidity (60% ± 5%), and a 12-hour light/dark cycle. The animals were maintained on a standard pellet diet and tap water and were left in separate cages for 4 days at room condition for acclimation before wounding. All rats were closely observed for infection, and if they showed signs of infection, they were separated, excluded from the study, and replaced.

For histological assessments, the animals in the excisional wound model were assessed at 3 time points of 7, 14, and 21 days post wounding. For determination of hydroxyproline levels and biochemical analyses, the rats of the excisional wound model were sampled on day 21; the incisional wound model group was used for biomechanical testing. Rats were euthanized for assessments at the corresponding time points by overdose of the anesthetic agents.

Induction of diabetes

For insulin-deficient diabetes, rats were fasted overnight before receiving a single intraperitoneal injection (50 mg/kg in 0.9% sterile saline) of streptozotocin (STZ). Hyperglycemia (≥ 15 mmol/L) was confirmed at 2 and 21 days after induction by measurement of tail-vein blood glucose concentration (Ames Glucostix; Myles, Elkhart, IN). Rats underwent the procedures 3 days postinduction of diabetes.

Plant material and extract preparation

Plant samples were collected from the Western provinces of Iran in April and May 2015. The values for moisture and ash were 78.4 ± 2.3 and 20.8 ± 0.7, respectively. Specimens from the plant material were deposited and authenticated at the Department of Botany Sciences, the Hamadan Research Agricultural and Natural Rescores Center, Hamadan, Iran. The plant leaves were used in the study and the plant material crisp was powdered in an electric blender. For the methanolic extraction, 150 g of the fine powder was extracted with 600 mL of 80% methanol at 37°C for 3 hours. The sample then was centrifuged at 4500 rpm for 15 minutes and supernatants were used. The filtrate was placed in an oven to dry at 40°C. The obtained clear residue was used for the study. Moisture and ash contents were determined using standard methods.¹¹

Phenolic contents, flavonoid contents, and antioxidant activity of methanolic extract of B angustifolia

The radical scavenging capacity of the extract (2,2-diphenyl-1-picryhydrazyl; DPPH) was estimated using the Brand-Williams method.¹² The ferric reducing ability of plasma (FRAP) assay was performed based on a method described by Benzie and Strain with a slight modification.¹³ The total phenolic content (TPC) of the extract was determined using the Folin-Ciocalteu reagent based on a procedure described by others.¹⁴ The total phenolic content was expressed as gallic acid equivalents in mg/g of chloroform extract. The Trolox-equivalent antioxidant capacity (TEAC) assay was based on the ability of the extract to scavenge the stable 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals.¹⁵ The sample was mixed with 3.0 mL of ABTS solution and the absorbance was then measured at 734 nm. For the lipid peroxidation inhibition (LPI) assay, the extract (40 mL) was mixed with 4.1 mL of linoleic acid in absolute ethanol (2.51%), 8 mL of phosphate buffered (pH = 7.0; 0.05 M), and 3.9 mL of distilled water. The mixture was then placed in an oven at 40°C. Then, 70% ethanol (9.7 mL) and 30% ammonium thiocyanate (0.1 mL) were added to the mixture (0.1 mL). Three minutes after the addition of 0.1 mL of ferrous chloride (0.02 M in 3.5% hydrochloric acid) to the reaction mixture, the absorbance of red color was measured at 500 nm.¹⁶ The total flavonoid (TF) content of the extract was measured using a previously described method.¹⁷ The ferric reducing power of the extract was measured based on a previous method.¹⁸

Formulation of the ointment

The base formulation, consisting of Eucerin (30%; Beiersdorf AG, Hamburg, Germany) and Vaseline (70%; Unilever, Rotterdam, Netherlands) in about 1:2 proportions, were prepared based on previous studies.^19,20 The topical application form was prepared by mixing either 1 g, 2 g, or 4 g of powder extract of the plant material into the base formulation.

Excisional wound model and planimetric studies

For the excisional wound healing model, 30 healthy male Wistar rats, weighing about 160 g to 180 g and aged 7 weeks, were randomized into 5 groups of 6 rats: the control surgery group (control) included the creation of wounds but no treatment; the base formulation group (PO) had the creation of wounds and application of base formulation ointment; treatment group 1 (T1) had 1 g of plant extract ointment; treatment group 2 (T2) had 2 g of plant extract ointment; and treatment group 3 (T3) had 4 g of plant extract ointment.

After induction of anesthesia with xylazine hydrochloride (HCl) 2% (5 mg/kg/IP; Alfasan International, Woerden, Holland) and ketamine HCl 10% (60 mg/kg/IP; Alfasan International), all 30 animals were fixed in a ventral posture on an operating table. Following shaving and aseptic preparation, a circular wound measuring 12 mm in diameter, for secondary intention wound closure, was created by surgical incision with a scalpel until the dorsal muscular fascia was exposed. A predetermined, full-thickness area on the anterodorsal side of each rat measuring about 115 mm² was removed. All the test formulations were applied for 10 days starting from the day of wounding. Wound healing property was evaluated by wound contraction percentage and wound closure time. Photographs were captured immediately after wounding and on days 0, 7, 14, and 21 postoperatively by a digital camera (Cyber-Shot DSC-W350; Sony Corporation, Tokyo, Japan) while a ruler was placed near the wounds as a scale (Figure 1). Wound area was analyzed by Measuring Tool of Adobe Acrobat 9 Pro Extended software (Adobe Systems Inc, San Jose, CA) and wound contraction percentage (WCP) was calculated using the following formula:

Where A₀ is the original wound area and A_t is the wound area at the time of imaging.²¹

Determination of hydroxyproline levels

On postop day 21, a piece of skin from the healed wounded area was collected and analyzed for hydroxyproline content. As a major part of collagen, hydroxyproline has an essential role in collagen stability, which gives support and strength to the extracellular tissue. The hydroxyproline contents were estimated using a previously described method.²² Briefly, tissues were dried in a hot air oven at 60°C to 70°C to constant weight and were hydrolyzed in 6N HCl at 130°C for 4 hours in sealed tubes. The hydrolysate was neutralized to pH 7.0 and was subjected to chloramine-T oxidation for 20 minutes. The reaction was terminated by addition of 0.4 M perchloric acid and color was developed with the help of Ehrlich reagent at 60ºC and measured at 557 nm using ultraviolet-visible spectrophotometer (CamSpec M330; Cambridge, UK).

Incision wound model and biomechanical testing

Thirty healthy male Wistar rats, weighing about 160 g to 180 g and aged 7 weeks, were randomized into 5 groups of 6 (see previously). All animals of the 5 groups were anesthetized, as mentioned above, and a paravertebral long incision of 4 cm was made through the skin and cutaneous muscle at a distance about 1.5 cm from the middle on the right side of the depilated back. After the incision was made, the 2 ends of the wound were sutured at 0.5-cm intervals with 3-0 nylon. Treatments were applied in the same manner as corresponding excisional wound model groups (ie, control, PO, T1, T2, T3). Ointments were applied once daily for 9 days. On day 9, sutures were removed and a 7-cm long strip of skin with the same widths of wound diameter, in the manner that the wound was located at the middle of the strip, was removed by a double-blade scalpel. The skin was then wrapped in Ringer’s-soaked gauze, aluminum foils, and plastic bags and kept in a freezer at -20°C until mechanical testing. The TA.XTPlus Texture Analyzer mechanical test device (Stable Micro Systems, Surrey, UK) was used for the assessment. The samples were fitted with the appropriate clamps, with a 4-cm distance between clamps at the start of testing. The strips were loaded with 0 kg to 30 kg load cell with a strain rate of 1 cm per minute and the load elongation curves were obtained. Yield strength (yield point; kg), ultimate strength (kg), maximum stored energy (kg/cm), and stiffness (kg/cm) were measured from the load elongation curves.

Histological preparation and quantitative morphometric studies

Tissue samples were taken from excision wound model animals on postoperative days 7, 14, and 21 from the periphery of the wound along with normal skin and fixed in 10% buffered formalin, dehydrated and embedded in paraffin wax, sectioned at 5 µm, and stained with Masson’s trichrome. Photomicrographs were obtained under light microscope to assess the predominant stage of wound healing. Three parallel sections were obtained from each specimen. Cellular infiltration including the number of mononuclear cells, polymorphonuclear cells, and fibroblastic aggregation were quantitatively evaluated. Acute hemorrhage, congestion, vascularization, epithelialization, collagen production, and density also were evaluated qualitatively. Morphological findings were scored using image analyzing software (Image-Pro Express, version 6.0.0.319; Media Cybernetics, Silver Springs, MD). The histological parameters were classified according to the intensity of occurrence in 5 levels (- absence; + discrete; ++ moderate; +++ intense; and ++++ very intense).²²

Biochemical analyses

The frozen samples at -80°C were homogenized in phosphate-buffered saline and centrifuged at 5°C. The supernatant was used for analysis of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione S-transferase (GST) and Carbonyl proteins. The GST and SOD analyses were performed based on previously published methods.^23,24 Carbonyl proteins were performed based on a protocol previously described.²⁵ The biochemical data were quantified based on the Bradford method and normalized in relation to total protein levels in the supernatant.²⁶

Statistical analysis

For the differences among groups in the excisional model, hydroxyproline level tests were evaluated by Kruskal–Wallis variance analysis. When the P value from the Kruskal-Wallis test was statistically significant, multiple comparison tests were used to know the differences. Student’s t test was used for evaluation of other test results. Comparison among days was assessed by the Mann-Whitney U test. The Bonferroni correction was applied for all possible multiple comparisons. Statistical analysis was performed with SPSS Version 11.5 (SPSS Inc, Chicago, IL). A P value was set at .05.

Phenolic contents, flavonoid contents, and antioxidant activity

The methanolic extract of B angustifolia demonstrated total phenolic content (53.29 ± 0.64 mg of gallic acid equivalents/g of dry weight), TF content (29.16 ± 0.31 mg catechin equivalents/g of dry weight), antioxidant activity using the FRAP assay (426.0 ± 3.39 µmol Fe (II)/g of dry weight), antioxidant activity using the ABTS assay (711.0 ± 9.58 µmol Trolox equivalent/g of dry weight), DPPH radical scavenging activity (87.49% ± 2.08%), reducing power (714.0 ± 6.84 µmol Trolox equivalents/g of dry weight), and inhibition against lipid peroxidation (79.45% ± 2.03%).

Reduction in wound area

Wound contraction percentage in different groups during the course of study is shown in Table 1. The healing rate of T1-T3 groups was significantly different compared with the control group (P < .05). However, time had a significant effect on wound contraction of all wounds (P = .034).

Hydroxyproline content of the wounds

Proline is hydroxylated to form hydroxyproline after protein synthesis. Hydroxyproline contents in the control, PO, T1, T2, and T3 groups were found to be 42.36 ± 2.37 mg/g^-1, 45.52 ± 2.23 mg/g^-1, 65.38 ± 3.71 mg/g^-1, 66.58 ± 3.13 mg/g^-1, and 65.79 ± 2.68 mg/g^-1, respectively. Hydroxyproline contents were increased significantly in T1-T3 groups, which implies more collagen deposition compared with the control and PO groups (P = .001).

Biomechanical findings

The biomechanical indices, maximum stored energy, stiffness, ultimate strength, and yield strength obtained for T1-T3 groups were significantly higher than those obtained for the control and PO groups (P = .003; Figure 2).

Histological and morphometric findings

There were significant differences in comparisons of the T1-T3 groups and control and PO groups, particularly in terms of cellular infiltration, acute hemorrhage, congestion, edema, collagen production and density, reepithelialization, and neovascularization. During the study period, scores for reepithelialization and neovascularization were significantly higher in animals of the T1-T3 groups than the control and PO groups (P = .001). Polymorphonuclear and mononuclear cell count, fibroblast cell proliferation, and mean rank of the qualitative study of acute hemorrhage, edema, and collagen production scores in the T1-T3 groups were significantly higher than those of the control and PO groups (P = .001; Table 2; Figure 3, eFigures 4, 5, and 6).

Biochemical findings

The MDA values were significantly reduced in the T1-T3 groups in comparison with the PO and control groups on day 14 (P = .0032). On day 21, the MDA levels in the T1, T2, and T3 groups significantly decreased compared with the PO and control groups (P = .001; eFigure 7). The SOD levels were significantly higher in the T1-T3 groups compared with the PO and control groups on day 7 (P = .001). The SOD levels in the T1, T2, and T3 groups significantly decreased compared with the PO and control groups on day 14 (P = .0034; eFigure 8). There were no significant differences in GST levels among groups during the study period (P > .05; eFigure 9). Carbonyl proteins were significantly lower in the T1-T3 animals compared with the PO and control groups during the study period (P = .001; eFigure 10).

Wound healing in patients with diabetes is impaired and delayed due to high blood glucose levels, which hampers cell proliferation and decreases collagen production, resulting in decreased chemotaxis and phagocytosis.²⁷ Elevated blood glucose levels, a reduction in the levels of growth factors, and the inhibition of fibroblast proliferation all have been suggested to contribute to the observed impairment in wound healing.²⁷ Streptozotocin-induced diabetes in animals is one of the most extensively studied models of diabetes.² In this study, therefore, rats with STZ-induced diabetes were used as the model to study diabetic wound healing.

Collagen, the major component that strengthens and supports extracellular tissue, is composed of the amino acid hydroxyproline, which has been used as a biochemical marker for tissue collagen.²⁸ In the excisional wound model for the T1-T3 animals, there was a significant decrease in wound area. This indicated improved collagen maturation by increased cross linking. The balance between synthesis and breakdown shows collagen deposition is important in wound healing and wound strength development.²⁹

Hydroxyproline is a major component of the collagen that permits the sharp twisting of the collagen helix. It helps to provide stability to the triple-helical structure of collagen by forming hydrogen bonds. Hydroxyproline is found in few proteins other than collagen. For this reason, hydroxyproline content has been used as an indicator to determine collagen content.³⁰ Increased hydroxyproline content in the T1-T3 groups indicates increased collagen content since hydroxyproline is the direct estimate of collagen synthesis.²

Mechanical testing is sensitive to changes that occur during the progression of wound healing and can be used as a tool to measure the quality of healing. Mechanical property data provide a clinically relevant and functional assessment of wound healing quality. Histological analyses highlight cellular and connective tissue adaptation at the ultrastructural level in the repair process.³¹ When compared with other experimental groups, the T1-T3 animals showed a statistically significant difference in biomechanical parameters.

In the present study, histopathological examination and scoring revealed a significant difference by means of wound healing scores in the T1-T3 groups compared with the control and PO groups. The extract decreased the maturation time of granulation tissue and wound contraction, which means it enhanced reepithelialization with a significant effect on inflammatory infiltration and the number of fibroblasts in time-dependent activity.

Antioxidants have been reported³² to play a significant role in improving the wound healing process and protecting the tissues from oxidative damage. Wound healing mechanisms may contribute to stimulating the production of antioxidants in the wound site and to providing a favorable environment for tissue healing.³³

Increased oxidative damage may directly interfere with skin tissue repair.³³ In the present study, the T1-T3 animals showed a significant increase in SOD levels compared with the control and PO groups, demonstrating the antioxidant effect of the extract in all concentrations. It could be deduced that the antioxidant effect of the extract provided an important anti-inflammatory response that could be associated with stimulation of antioxidant enzymes, particularly SOD, which remained at high levels in the T1-T3 animals. Furthermore, lower levels of MDA and carbonylated proteins were observed in the T1-T3 animals, which are important markers of tissue stress.

Although the present study showed the promising effect of the extract of B angustifolia on wound healing in rats, data regarding the molecular mechanisms leading to its action remain to be investigated in depth. The authors did not provide molecular evidence for the action of the extract, which may be considered a limitation of this study.

The present study demonstrated methanolic extract of B angustifolia leaves had properties that render it capable of promoting accelerated wound healing activity compared with the controls. On the basis of the results obtained in the present study, it is possible to conclude that B angustifolia  extract ointment has significant wound healing activity in rats with diabetes. Dose-response studies should be conducted on the extract of B angustifolia leaves to determine maximal efficacy on diabetic wound healing.

The authors would like to thank the staff of the animal house of the Faculty of Veterinary Medicine of the Urmia University, Urmia, Iran, for their technical expertise and care of animals.

Affiliations: Department of Surgery and Diagnostic Imaging, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran; Department of Clinical Sciences, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Iran; and Department of Anatomical Sciences, Faculty of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran

Correspondence: Negin Sanaei, DVM, Department of Surgery and Diagnostic Imaging, Faculty of Veterinary Medicine, Urmia University, Urmia, Nazloo Road, Urmia, 57153 1177, Iran;
sanaei.negin@yahoo.com

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