Wound healing is a normal biological process of repair, following injury to the skin and other soft tissues, which is achieved through overlapping phases such as haemostasis, inflammation, proliferation and maturation.1 A fibrous scar containing large amounts of collagen is an end product of this process. Well-vascularised granulation tissue that is made within the wound area is responsible for synthesis of collagen, and provides strength and integrity to the dermis.2,3 Many important factors such as malnutrition, ageing, medication, radiation, and some diseases including diabetes, hypertension and obesity are associated with delayed wound healing.1,4
Wounds in patients with diabetes can be hard-to-heal, taking months despite essential and appropriate care.4,5 However, the exact pathogenesis of poor wound healing in patients with diabetes is not completely clear. The decrease of collagen content due to reduced biosynthesis and/or accelerated degradation of newly synthesised collagen and also the oxidative and inflammatory changes are thought to be the main causes.2,5
Wound care can be traced back to early civilisations, when use of herbal remedies were the basis of many treatments.6,7,8,9,10 Some plants have a high content of tannins, flavonoids, saponins, naphthaquinone, alkaloids, and triterpenes, which can increase the quality and the rate of wound healing.7,8,9,10
Iran has a rich flora widely distributed throughout the country, particularly in the west. In Iranian traditional medicine, herbal medicines have been the basis of treatments and cures many physiological conditions and diseases. Pimpinella anisum (anise), also known as ‘Badian Roomi’, from the umbelliferae family is one of the most important herbal medicines and is widely consumed in the west of Iran.
Anise is a grassy plant with white flowers and small green to yellow seeds, which grows annually in Iran, India, Turkey, Egypt and many other warm regions of the world.11 Chemical studies have demonstrated that the anise-fruits contain eugenol, methyl chavicol (estragole), trans-anethole, pseudoisoeugenol, coumarins (umbelliferone, scopoletin), anisaldehyde, caffeic acid derivatives (chlorogenic acid), minerals, proteins, flavonoids, polyenes, fatty oil and polyacetylenes as its major compounds.12
It was reported that Pimpinella anisum has several pharmacological effects such as analgesic, antiviral, antibacterial,13 anti-constipation14 and muscle relaxant properties.15
Furthermore, scientific evidence supports the hepatoprotective effects of different extracts, such as n-Hexane and hydroalcoholic extracts, and the essential oil of anise fruit, as well as the antioxidant and anti-inflammatory properties of phenolic compounds against cellular and oxidative damage caused by carbon tetrachloride (CCl4) in liver tissue.16 In oxidative degeneration caused by CCl4, n-hexane extract of anise seed decreased the lipid peroxidation and prevented cellular glutathione (GSH) consumption.16 Its anti-inflammatory effects are achieved by its inhibiting NF-kappaB, a protein complex regulating inflammatory process and myeloperoxidase (MPO), the enzyme which sets off the inflammatory process by oxidative burst.17,18 More importantly, its anti-diabetic activity was exemplified by reducing glucose and lipid levels alongside the attenuation of protein and lipid peroxidation.19
Its anti-ulcer activity was also reported by ameliorating necrosis, and inflammatory and dysplastic changes in gastric ulcers induced by ethanol.20 Trans-anethole, the major constituents of anise oil (93.9%) showed wound healing activity by enhancing fibroblast deposition and collagen fibre alignment and finally accelerating wound closure.13,21 Reduced wound index by Pimpinella anisum was also reported in the gastric mucosa of indomethacin-treated rats.20
Concurrent with these findings, it seems that the Pimpinella anisum genus can cope with the oxidative and inflammatory changes involved in diabetic wounds, stimulate fibroblast deposition and enhance wound closure by its compounds.
The present study was conducted to assess the dermal wound healing potential of Pimpinella anisum after topical application of its methanolic extract on experimentally-induced cutaneous wounds in a streptozotocin (STZ)-diabetic rat model.
Methods
Plant material and extract preparation
Anise seeds were obtained from the local market. The plants were identified by Dr. Mondani, Department of Agronomy and Plant Breeding, Razi University, Kermanshah, Iran. A voucher specimen with number ‘0012-Anise’ has been deposited in the herbarium of Veterinary Medicine Faculty, Razi University.
The plant materials were first cleaned and then dried at room temperature. The dried plants were chopped into small pieces (5mm) using a blade grinder (Matheo, model MFP71) and 300g were soaked in methanol for 72 hours, following which they were filtered using standard Whatman filter paper. This procedure was repeated twice to ensure maximal extraction from the anise. After extraction, the solvent was filtered and then evaporated by a rotary evaporator (Rotavapor, Buchi, UK). The achieved extract was then stored at –20°C until used in the experiment.
Animals
Male Sprague-Dawley rats weighing 180–200g were used. The rats were kept in 12 standard cages (70×40×25cm) under standard environmental conditions (23±1°C, with 55±5% humidity and a 12-hour light/dark cycle), and maintained with free access to water and standard pelleted food in the animal house of Veterinary Medicine Faculty, Razi University, Kermanshah, Iran.
Induction of diabetes by streptozotocin in rats
Diabetes was induced by a single intraperitoneal injection of 60mg/kg of STZ (Sigma, US) in all rats. The rats were fasted for 12 hours before and 12 hours after injection of STZ. Blood glucose levels above 250mg/dl, as well as levels of polyuria and polydipsia after three days post-injection were the main criteria for selection of diabetic rats in this study. The blood glucose in the diabetic rats are summarised in Table 1.
Table 1. Blood glucose above (mg/dl) in the diabetic rats in different groups (SD—standard deviation)
| Animals | Control | Basal cream | Tetracycline | Pimpinella anisum (10%) |
|---|---|---|---|---|
| Case no. 1 | 408 | 348 | 304 | 420 |
| Case no. 2 | 428 | 447 | 479 | 489 |
| Case no. 3 | 448 | 509 | 405 | 361 |
| Case no. 4 | 502 | 369 | 396 | 504 |
| Case no. 5 | 394 | 316 | 397 | 449 |
| Case no. 6 | 459 | 504 | 465 | 387 |
| Case no. 7 | 349 | 392 | 494 | 490 |
| Case no. 8 | 479 | 431 | 255 | 519 |
| Case no. 9 | 338 | 349 | 395 | 339 |
| Case no. 10 | 412 | 470 | 304 | 318 |
| Case no. 11 | 461 | 336 | 465 | 496 |
| Case no. 12 | 398 | 424 | 444 | 392 |
| Case no. 13 | 426 | 491 | 450 | 337 |
| Case no. 14 | 293 | 441 | 332 | 382 |
| Case no. 15 | 382 | 459 | 341 | 491 |
| Mean±SD | 411.80±56.05 | 419.07±63.70 | 395.07±73.30 | 424.93±69.93 |
Ethical approval
This study was performed under the approval of the state committee on animal ethics, Razi University, Kermanshah, Iran. Additionally, recommendations of European Council Directive (86/609/EC) of 24 November 1986, regarding the protection of animals used for experimental purposes were considered. This article does not contain any studies with human participants performed by any of the authors.
Wound creation
The rats were weighed prior to the surgical procedure. Anaesthesia was performed by injection of 1mg/kg xylazine hydrochloride (xylazine 2%; Alfasan, The Netherlands) and 60mg/kg ketamine HCl (ketamine 5%; Trittau, Germany) intramuscularly. The backs of the animals were shaved and then prepared for aseptic surgery. Under sterile conditions, a square shape, full thickness incision (2×2cm) was made in the skin and the incised piece was removed. The wound was left undressed and no local or systemic antimicrobial drugs were administrated.
Study design
The rats were randomly allocated into four main groups (n=15) as follows:
- control
- basal cream (placebo group)
- tetracycline
- Pimpinella anisum 10%
Once in there main groups, the rats were then further divided into three subgroups, representing days seven, 14 and 21 post-injury (n=5).
The groups were defined wound dressings were not used in the wound area of rats in the control group. In the tetracycline and basal cream groups, the injured area was covered with 1ml tetracycline (3%) and basal cream (Eucerin, Beiersdorf, Germany) daily, for 14 days post-injury. In the treatment group, the wound area was covered with 1ml Pimpinella anisum 10% (Pimpinella anisum powder, 10g was suspended in 90g Eucerin) for 14 days post-injury.
From each group, five animals were euthanised at days seven, 14 and 21 post-injury by intravenous injection of pentobarbital (50mg/kg). Tissue samples from all animals were collected and used for histopathological examination.
Rate of wound closure
Wounds were serially photographed using a digital camera on days seven, 14, and 21 post-injury. The wound area was measured using Image J software (Java 1.6.0.20; National Institute of Health, US). The following formula was used to calculate the rate of wound closure:22% wound closure =wound size day 0−wound size day (n)wound size day 0×100
n=numbers of days (seven, 14 and 21).
Histopathologic evaluation
For histopathological studies and biochemical analyses, full-thickness skin samples from the wound area including dermis, epidermis and subcutaneous were carefully dissected. These tissues were divided into two parts longitudinally; one part used for histopathological examinations and the other was washed rapidly with cold phosphate buffer solution (PBS) and was kept in a frozen condition (–80˚C) for measurement of the hydroxyproline antioxidant parameters and dry matter content.
For histopathological studies, the tissue samples were fixed in 10% neutral-buffered formalin, processed routinely, embedded in paraffin, sectioned at 5μm thickness, stained with haematoxylin and eosin (H&E) and evaluated microscopically. Histological examinations were done by two pathologists with a procedure reported by Oryan et al.9
Finally, photographs were taken with a digital camera (Dino capture; version 1.2.7) and transferred to computer software (Photoshop CS-4; Adobe, US) for digital analysis. For histopathologic analysis, five photomicrographs were selected from five microscopic fields of each tissue sample. The parameters that were assessed in histopathological sections consisted of fibrin deposition, haemorrhage, cornification of the epithelium, polymorphonuclear cell and mononuclear cell infiltration, re-epithelialisation, revascularisation, fibroblast and macrophage content, necrosis, presence of fibrocytes and collagen maturation and organisation.
Total cellularity (magnification ×200) and the number of fibroblasts, fibrocytes, macrophages, lymphocytes, neutrophils, and blood vessels (magnification ×800) of the wound area were counted and their mean and standard deviations (SD) were calculated.
Percentage dry matter content
The following equation was used to calculate the percentage of dry matter content of the collected skin:23% dry matter contect =dry weightwet weight×100
Measurement of hydroxyproline
Hydroxyproline content was measured according to the method of Oryan et al. and reported as mg/100mg of tissue.23 The data were then multiplied by 7.46 to convert them from hydroxyproline content to collagen content (according to Neuman and Logan, hydroxyproline can be converted to its equivalent of collagen through multiplication by the factor 7.46).24
Measurement of malondialdehyde, total antioxidant capacity and glutathione peroxidase
Frozen tissue from each rat (about 20mg) was homogenised in lysate buffer containing 500mmol Tris/HCl, pH 7.6, 10mM CaCl2 and 200mM NaCl and 1% Triton X-100, plus a protease inhibitor cocktail (catalogue number P2714, Sigma-Aldrich, UK). After 15 minutes of lysis process at 4°C, the samples were centrifuged at 14000×g for five minutes at 4°C. Finally, the supernatant was extracted to measure the protein level by Bradford method.25 Malondialdehyde (MDA) as an end product of oxidative damage to polyunsaturated fatty acids was measured in supernatant by the reaction of thiobarbituric acid with byproduct of lipid peroxidation.26 It was expressed as pmol/mg-1 of protein. Total antioxidant capacity of the supernatant was evaluated on the basis of the tissue's antioxidant power to reduce ferric ion (Fe3+) found in TPTZ (tripridyl-s-triazine) to the ferrous ion (Fe2+).27 It was expressed as nmol/mg protein. The glutathione peroxidase activity was measured using colorimetric method by glutathione peroxidase assay kit (Cayman, Ann Arbor, US) and expressed as U/mg protein.
Statistical analysis
Descriptive statistics including the median, mean, standard error (SE), minimum and maximum were calculated for all variables. For comparison of different parameters, the one-way analysis of variation (ANOVA) followed by Tukey post-hoc test were used. The data were analysed by SPSS software, version 22.0 (SPSS Inc., US) and p<0.05 was accepted as statistically significant. The plots were designed by GraphPad Prism version 6.0 (GraphPad Software, US).
Results
General observation
A total of 60 rats were used in the study, with each group containing 15 rats. At day seven post-injury, the wounds exhibited the formation of a scar covering thick granulation tissue in all rats. However, treatment with Pimpinella anisum produced more scar tissue than other groups when examined macroscopically. No re-epithelialisation was evident in the control, basal cream and tetracycline groups, while rats in the Pimpinella anisum group indicated minimal re-epithelialisation. At this stage, less cellularity, perivascular oedema and fibrin deposition and more collagen fibres were observed in Pimpinella anisum- treated wounds compared with the other groups.
At day 14 post-injury, a more organised pattern in the collagen fibres and better tissue alignment were seen in the Pimpinella anisum-treated group compared with the other groups. In all groups, although the epidermis was disorganised and thick, particularly when compared with the adjacent normal skin, but its size was decreased and its alignment was better in the Pimpinella anisum-treated lesions in comparison with the basal cream and control lesions. At this stage, evidence of pus accumulation, fibrin deposition, polymorphonuclear cells infiltration or oedema were not observed in the lesions of animals in all groups.
At day 21 post-injury, the wounds in groups were totally closed in the Pimpinella anisum-treated lesions. The size of scar tissue was also smaller and the collagen fibre alignment was better than in those of the basal cream and control group lesions. Additionally, the number of lymphocytes and macrophages was decreased in the Pimpinella anisum and tetracycline treated groups, and considerably greater tissue maturation and large capillary-sized blood vessels were observed compared with the other groups.
Quantitative analysis
At days seven and 14 post-injury, the diabetic rats in the control group displayed a significant delay in wound healing compared with those treated with Pimpinella anisum (p<0.05). At day 21 post-injury, the closure of the diabetic wounds treated with Pimpinella anisum (94.07±1.21%) was significantly faster than those of the control (77.19±2.97%), basal cream (79.77±4.06%) and tetracycline (83.24±2.53%) groups (p<0.05). The wounds were almost closed or greatly improved (between 98–100%) in two rats in the Pimpinella anisum group, while the wounds were not closed in other groups (Table 2, Fig 1a–l).
Table 2. Mean±standard deviation of wound closure (%) in groups on different days post-injury
| Days | Control | Basal cream | Tetracycline | Pimpinella anisum (10%) |
|---|---|---|---|---|
| Day 7 | 32.29±5.71* | 34.42±5.93* | 37.83±6.65*† | 43.34±3.65† |
| Day 14 | 58.63±3.63* | 62.51±5.07*‡ | 65.22±3.81†‡ | 70.63±2.59† |
| Day 21 | 77.19±2.97* | 79.77±4.06* | 83.24±2.53* | 94.07±1.21† |
Means within a column with *, † or ‡ denote significant differences. p<0.05 was accepted as statistically significant
The data obtained from the histopathologic evaluations are summarised in Table 3. At day seven post-injury, total cellularity was significantly decreased in the Pimpinella anisum-treated group in comparison with the control, basal cream and tetracycline groups (p<0.01; Fig 2a–d). The wounds treated with Pimpinella anisum had a higher number of blood vessels, fibroblasts, fibrocytes and a larger diameter of blood vessels compared with the other groups. The number of macrophages and lymphocytes in the Pimpinella anisum-treated group was significantly reduced compared with the other groups, but this difference was only statistically significant for the control group (p=0.003 and p=0.01, respectively).
Table 3. Histopathologic and histomorphometric analysis
| Day 7 | Control | Basal cream | Tetracycline | Pimpinella anisum (10%) |
|---|---|---|---|---|
| Total cell | 553.30±9.43* | 504.60±17.55* | 464.70±16.95* | 361.45±14.66† |
| Vascular number | 11.50±0.30* | 11.80±0.44* | 12.80±1.08* | 17.72±0.99† |
| Diameter of vessel (μm) | 7.81±1.37* | 8.49±1.80* | 11.40±1.47* | 16.87±1.69† |
| Fibroblasts and fibrocytes | 14.00±0.84* | 15.10±0.72* | 18.10±0.78*† | 22.40±1.66† |
| Fibrocytes | 3.80±1.61* | 4.30±1.41* | 5.81±1.66* | 5.90±2.72* |
| Fibroblasts | 10.20±1.01* | 10.80±0.61* | 12.20±0.90*† | 16.40±1.33† |
| Ratio | 0.43±0.07* | 0.41±0.05* | 0.55±0.12* | 0.37±0.03* |
| Lymphocytes | 17.00±0.89* | 15.70±0.96*† | 15.10±1.02*† | 12.00±0.80† |
| Macrophages | 18.20±1.31* | 16.10±0.94*† | 17.70±0.94*† | 13.30±0.95† |
| Neutrophils | 2.10±0.37* | 1.20±0.38*† | 0.40±0.22† | 0.70±0.33† |
| Day 14 | ||||
| Total cell | 420.88±21.24* | 373.40±25.32*‡ | 294.50±22.58†‡ | 260.50±15.66† |
| Vascular nnumber | 10.11±0.75* | 9.80±0.32* | 8.50±0.50*† | 6.20±0.51† |
| Diameter of vessel (μm) | 13.11±2.17* | 15.61±3.00*‡ | 19.29±2.87†‡ | 24.05±2.35† |
| Fibroblasts and fibrocytes | 20.40±0.65* | 19.30±0.89* | 25.20±1.20* | 32.60±1.74† |
| Fibrocytes | 7.66±1.80* | 7.00±1.41* | 10.70±1.25* | 16.40±4.24† |
| Fibroblasts | 13.10±0.48* | 12.30±0.57* | 14.50±1.19* | 16.20±0.87* |
| Ratio | 0.56±0.05* | 0.57±0.03* | 0.78±0.06* | 1.03±0.09* |
| Lymphocytes | 14.00±0.85* | 14.20±0.66* | 10.40±1.02*† | 7.70±1.16† |
| Macrophages | 13.60±0.79* | 11.40±1.18*b | 9.40±1.09*† | 8.70±0.47† |
| Neutrophils | 1.00±0.33* | 0.80±0.41* | 0.20±0.13* | 0.00* |
| Day 21 | ||||
| Total cell | 286.90±18.95* | 264.60±21.63* | 234.20±32.02* | 142.30±8.51† |
| Vascular number | 7.10±0.64* | 6.40±0.37* | 4.50±0.50*† | 2.40±0.33† |
| Diameter of vessel (μm) | 15.46±1.58* | 17.33±2.34* | 22.61±5.00† | 31.95±3.72‡ |
| Fibroblasts and fibrocytes | 28.70±1.37* | 29.70±2.27*† | 28.10±0.78* | 34.70±0.90† |
| Fibrocytes | 14.60±3.27* | 13.00±2.78* | 16.50±2.67* | 27.00±2.30† |
| Fibroblasts | 14.10±1.55*c | 16.70±1.73* | 11.60±0.58†‡ | 7.70±0.49† |
| Ratio | 1.21±0.19*† | 0.86±0.12* | 1.46±0.11† | 3.65±0.28‡ |
| Lymphocytes | 9.10±0.43* | 8.70±0.49* | 4.60±0.79† | 2.50±0.50† |
| Macrophages | 8.50±0.67* | 9.40±0.88* | 6.80±0.72*† | 3.70±0.36† |
| Neutrophils | 0.50±0.26* | 0.00* | 0.00* | 0.00* |
Five fields in each of five histopathologic sections were analysed for each group. Means within a column with different superscript letters (*, †, ‡) denote significant differences. p<0.05 was accepted as statistically significant
At day 14 post-injury, total cellularity in the Pimpinella anisum-treated group was significantly reduced compared with the control group (p<0.001), but the differences were not significant when compared with the basal cream and tetracycline groups (p>0.05; Fig 2e–h). At this stage, the wounds treated with Pimpinella anisum revealed a significantly lower number of blood vessels in comparison with the control lesions (p=0.016), but this reduction was not significant when compared with the basal cream and tetracycline groups (p>0.05). The treated lesions also had a higher number of fibrocytes and the larger diameter of blood vessels compared with the other group (p<0.05). The number of macrophages and lymphocytes had significantly decreased in the Pimpinella anisum-treated group when compared with the control (p=0.004 and p=0.006, respectively).
At day 21 post-injury, the wounds treated with Pimpinella anisum showed a significantly lower number of total cells as compared with the other groups (p<0.05; Fig 2i–l). The number of blood vessels was significantly lower in Pimpinella anisum group compared with the control and basal cream groups at this stage (p<0.001). The treated lesions had a larger diameter of blood vessels in comparison with the other groups (p<0.05). Additionally, the number of fibroblasts in the Pimpinella anisum-treated lesions was significantly lower than those in the basal cream and control groups (p<0.001), while the number of fibrocytes was significantly higher in the Pimpinella anisum group when compared with the other groups (p<0.001). The rats in the Pimpinella anisum group had a significantly lower number of lymphocytes and macrophages when compared with the control and basal cream lesions (p<0.05; Fig 2m–p).
Dry matter content
As is shown in Table 4, the dry matter content in the Pimpinella anisum-treated wounds was significantly increased compared with the control, basal cream and tetracycline lesions at days seven, 14 and 21 post-injury (p<0.001). However, the lesions in the tetracycline group had a higher dry matter content compared to the control and basal cream groups, but these differences were not statistically significant (p>0.05).
Table 4. Mean±standard deviation dry matter content, hydroxyproline and collagen in groups on different days post-injury
| Days | Control | Basal cream | Tetracycline | Pimpinella anisum (10%) |
|---|---|---|---|---|
| % dry weight | ||||
| Day 7 | 17.57±0.81* | 17.60±0.68* | 18.52±0.88* | 23.18±1.02† |
| Day 14 | 19.91±0.76* | 20.31±0.68* | 21.65±0.79* | 27.01±0.99† |
| Day 21 | 25.04±0.76* | 25.65±0.69* | 28.25±0.87* | 33.20±0.88† |
| Hydroxyproline (g/100g tissue) | ||||
| Day 7 | 3.52±0.14* | 3.58±0.09* | 3.87±0.13* | 4.75±0.18† |
| Day 14 | 4.24±0.14* | 4.14±0.24* | 4.53±0.13* | 5.37±0.18† |
| Day 21 | 4.63±0.16* | 4.70±0.15* | 5.14±0.33* | 6.36±0.27† |
| Collagen (g/100g tissue) | ||||
| Day 7 | 26.30±1.04* | 26.77±0.68* | 28.93±0.99* | 35.47±1.36† |
| Day 14 | 31.67±1.08* | 30.92±1.07* | 33.86±2.01* | 40.10±1.41† |
| Day 21 | 34.59±1.21* | 35.09±1.17* | 38.35±3.02* | 47.49±2.29† |
Means within a column with different superscript letters (*,†) denote significant differences. p<0.05 was accepted as statistically significant.
Hydroxyproline measurement
As is shown in Table 4, the level of hydroxyproline was significantly increased in the Pimpinella anisum-treated group when compared with the control, basal cream and tetracycline groups at days seven, 14 and 21 post-injury (p<0.001).
Oxidant/antioxidant measurement
As shown in Fig 3a, there was no significant difference in the total antioxidant capacity level in the wound area between different groups at days seven and 14 post-injury. However, at day 21 post-injury, it was significantly increased in the Pimpinella anisum-treated group compared with the other groups (p<0.01).
Pimpinella anisum decreased The MDA level was decreased in the Pimpinella anisum-treated wounds when compared with the control group at days seven, 14 and 21 post-injury. However, these differences were only statistically significant at day 21 post-injury (p<0.001) (Fig 3b). The activity of glutathione peroxidase was higher in the rats treated with Pimpinella anisum extract compared with the other groups at days 14 and 21 post-injury. In a comparison between different days, better curative effects of Pimpinella anisum was observed at day 21 post-injury where the level of glutathione peroxidase in the Pimpinella anisum-treated group was significantly higher than in the other groups (p<0.001) (Fig 3c).
Discussion
Cutaneous wounds are diverse, multifactorial, complex and persistent in patients with diabetes.28 Several mechanisms can cause cutaneous wounds, such as poor blood flow and oxygen release due to increased blood sugar. This condition is believed to be caused by impaired blood flow and oxygen release from increased blood sugar, decreased collagen and fibronectin synthesis from protein malnutrition, impaired local immune and cell defences, and decreased anabolic activity with decreased insulin and growth hormone.7
The study of wound healing medicines for patients with diabetes and the quest for better, more effective remedies is possibly one of the main challenges for investigators. The enormous costs of modern medicines lead the researchers to seek alternative strategies for the better management of wounds in their patients with diabetes. Medicinal plants are common remedies used by people in many countries.
Due to its reported hypoglycaemic, anti-inflammatory and antioxidant effects of Pimpinella anisum,16,17,18,19,29,30 and also its anti-ulcer activity and healing properties,20,21 we hypothesised that this plant could be used as a compound to accelerate wound healing process in diabetic conditions.
The results of this study indicate that topical application of Pimpinella anisum improved the quality of wound contraction, re-epithelialisation and scar formation in diabetic rats in the short-term. Over a longer time-period, topical application of this compound appeared to decrease total cellularity, improve maturation of fibroblast, increase fibroblast differentiation rate in lesion and ultimately reduce scar tissue size.
A main result of this experiment was the clear difference in wound contraction rate and re-epithelialisation rate between the Pimpinella anisum group and the other groups. Wound contraction is the procedure of mobilising normal skin around the wound to cover the denuded area and contains complex and coordinated interactions of cells, extracellular matrix (ECM) and cytokines.31 The higher rate of wound contraction and decrease in healing time in the wounds treated with Pimpinella anisum may be due to the plant's anti-inflammatory effects and its effect on the maturation and organisation of the granulation tissue. In this regard, recent studies have established that phytochemical constituents such as flavonoids can stimulate wound contraction and increase the rate of re-epithelialisation.32 Furthermore, the high level of trans-anethole composition with proven wound healing activity may represent the restorative effect of this compound.20,21
In diabetic wounds, high intracellular reactive oxygen species (ROS) generation comes from hyperglycaemia via activation of protein kinase C (PKC) signalling which suppresses the antioxidant system, similar to glutathione peroxidase.33 In this current study, imbalance of redox potential via diabetes was observed by a significantly lower total antioxidant capacity level and glutathione peroxidase activity, and a higher MDA concentration in wound area.
Interestingly, oxidative stress appeared to be ameliorated by the Pimpinella anisum through significant reversing of the previously mentioned oxidant/antioxidant parameters, particularly in the later phases of the study. It has been reported that ROS intervenes in proliferation of fibroblasts and keratinocytes in the later stages of wound healing.34 Therefore, the findings of our study confirm the restorative effect of Pimpinella anisum with an antioxidant mechanism. Similarly, its antioxidant properties against CCl4 and gentamycin-induced oxidative stress have been previously reported.16,35
Based on histopathological findings, granulation tissue formation appeared to be accelerated by applying Pimpinella anisum. The ability to accelerate wound healing was very prominent, especially when these observations were compared with other groups. Moreover, the increase in weight of dry granulation tissue in the Pimpinella anisum-treated rats suggested a higher content of protein at the wound site and ability to remodel the wound.
Generally, fibroblasts, collagen and new small blood vessels are the most important components of granulation tissue formed in the final part of the proliferative phase.32 Our results indicate that there is a positive correlation between the fibroblast count, number of blood vessels and collagen content.
In granulation tissue formation, the adherent cells, defined as fibroblasts, have a capacity to synthesise a new, impermanent ECM by excreting collagen and fibronectin. In the granulation tissue, collagen is the principal extracellular protein that is produced immediately after injury in the wound area. Furthermore, collagen plays an important role in providing strength and integrity to a tissue matrix, as well as in haemostasis.9 Better collagen maturity and alignment in this present study in wounds treated with Pimpinella anisum showed its novel healing effect.
Proliferation, maturation and migration of the epithelial cells and fibroblasts are dependent on an adequate oxygen supply. Therefore, vasculature in the wound area could supply nutrients and oxygen to the cells, enhance fibroblast maturation and support the production of considerably higher amounts of collagen.36 In addition, better healing in the wound area may also result from a better blood flow due to enhanced smooth muscle relaxation, the muscle relaxant properties of Pimpinella anisum have been previously reported.15
The presence of a large number of blood vessels in the Pimpinella anisum-treated wounds at the earlier stages of wound healing, indicate that the Pimpinella anisum is an angiogenic agent that enabled proper circulation of red blood cells for wound healing. In addition, a higher content of hydroxyproline, a specific marker of collagen, in the Pimpinella anisum- treated wounds indicate that Pimpinella anisum can accelerate production of collagen by fibroblasts and also their release into the ECM.
The wounds treated with Pimpinella anisum also showed a decreased total cellularity together with smaller macrophage and lymphocyte counts in days seven, 14 and 21 post-injury, while the presence of higher levels of macrophages and lymphocytes in the control group, even at the end of experiment, indicate the presence of chronic inflammation in these lesions.
The inflammatory phase is characterised by inflammation and haemostasis, followed by angiogenesis, collagen deposition and re-epithelialisation in the proliferative phase.8,9 Inflammatory cells, particularly macrophages, play an important role in facilitating fibroplasia during the inflammation phase. Several cytokines, pro-inflammatory mediators and growth factors are released by the macrophages that accelerate mesenchymal cell migration, proliferation, maturation and matrix production.9
The lower levels of inflammation in the Pimpinella anisum-treated group can be due to the presence of some phytoconstituents, including antioxidants and flavonoids such as isovitexin and epigenin, in this plant species which inhibited the activities of macrophages and production of chemical mediators by inhibiting NF-kappaB as mentioned before.16,17 Accordingly, in our study, Pimpinella anisum appeared to decrease inflammation and subsequently promote tissue organisation.
Limitations
The higher rate of the wound healing in rats, small population size, and the anatomical and physiological variations between rats and humans should be considered as limiting factors.
Conclusion
This present study established topical application of Pimpinella anisum can stimulate wound healing activity in diabetic rats. It was also observed that in the group treated with Pimpinella anisum, wound size, inflammation and cellularity were significantly reduced, and the re-epithelialisation rate, collagen content, fibroblastic response were enhanced. More scar tissue in the early stages of wound healing was also produced. In the later stages, Pimpinella anisum significantly decreased oxidative stress, scar tissue formation, and improved tissue maturity and remodelling. The wound-healing activity of Pimpinella anisum may be due to the individual or the additive effects of phytoconstituents present in the plant.
In this study, the effect of the phytoconstituents of Pimpinella anisum weres not investigated. More phytochemical studies are needed to characterise and identify the specific active compounds of this plant that are responsible for wound healing activity.
Reflective questions
- What is the role of plant extracts in the wound healing?
- How can use the medicinal plants in medicinal researches related to the wound healing?
- Is the Pimpinella anisum methanolic extract useful in the treatment of wound?