Background
Wounds, which happen due to physical and chemical injuries or microbial infections, are inevitable events in life. The aim of wound healing is to bring back the structure and function of the injured tissues to the nearly pre-wound conditions. In other words, healing is an intricate body’s natural process to regenerate the integrity of damaged tissue [
1‐
3], thus the collaborative efforts of many different tissues and cell lineages are vital for healing. Several stages are involved in a wound healing process including inflammatory, proliferation and finally remodeling phases [
2,
4]. Research on drugs capable of managing each step would be of great interest in modern biomedical sciences [
1]. Interestingly, up to 80% of the world’s population use herbal medicines in the treatment of various skin disorders, and known drugs obtained from plant sources have proved to enhance the healing of different wounds [
1,
5]. In Iranian folklore, medicinal plants are used to heal skin wounds [
6]; however, the potential of many traditional herbal extracts in wound healing still remains unexplored.
Fumaria genus belongs to Fumariaceae family and comprises 60 species of herbaceous flowering plants with a worldwide distribution, particularly in Asia [
7].
Fumaria vaillantii Loisel. is one the seven species grown in different parts of Iran with the local name of “Shatareh” [
8]. Aerial parts of
F.vaillantii as an infusion have widely used by the traditional healers in the treatment of psoriasis, jaundice and fever [
8]. Interestingly, the ancient Iranians used
Fumaria spp. as a topical formulation, traditionally named “Zemad “or “Marham” to heal skin disorders [
9,
10]. Most notably, native people of Jandagh, located in the central part of Iran, empirically used
Fumaria vaillantii L. for the healing of skin wounds [
6]. A vast majority of pharmacological properties including antioxidant, anti-inflammatory, anti-fungal and anti-bacterial have also been reported for this plant, which was attributed to the presence of diverse secondary metabolites [
11‐
15]. Among a large number of compounds present in this species, alkaloids and flavonoids are two important classes [
16‐
18]. With inflammation as an important stage involved in wound healing,
Fumaria vaillantii possessing anti-inflammatory as well as antioxidant properties, due to the presence of metabolites, seems to be effective in the primary stages of wound repair [
11,
19]. To the best of our knowledge, only a single study has been performed on the wound healing potential of
F. indica extract [
19] . To further our knowledge, the present study was undertaken to assay the wound healing property of
F.vaillantii gel formulation in both an excision and an incision wound models in rats.
Recently, wound dressing has taken a large attraction due to creating a moist environment to support faster wound healing [
20]. Amongst the existing dressings, hydrogels are reported to be optimal for use in all steps of wound healing as a drug delivery system [
21]. In the present work, we aimed to formulate a hydrogel for topical administration of
F.vaillantii extract, which is suitable for dermatological application including wound healing. To the best of our knowledge, this is a first report on the therapeutic potential of hydrogel formulation composed of
F.vaillantti extract as a wound healing promoter in an in vivo model.
Methods
Plant material and extraction procedure
F.vaillantii plant was collected from North of Iran in August 2014. A voucher specimen (No. 6563 TEH) was deposited at the Herbarium of Faculty of Pharmacy at Tehran University of Medical Sciences and authenticated by Dr. Gholamreza Amin. Following separating the aerial parts of the plant, they were dried in the dark for 3 days. Total extract was prepared by thoroughly mixing 320 g of dried powder with ethanol: water (80:20) three times at room temperature for 72 h via maceration procedure. The extracts were evaporated to dryness and then kept at 4 °C.
Weighed quantities of different thickening agents including HPMC (Hydroxypropyl methylcellulose) 4000 cP, HPMC 15 cP and Carbomer 940, which were kind donations of Hakim Pharmaceutical Company, were separately added to distilled water and allowed to soak for 24 h. The hydroalcoholic extract of Fumaria vaillantii (10%) was solubilized in propylene glycol (Sepidaj, Iran). The latter solution was transferred to the aqueous dispersion of each thickening agents, individually at different concentrations. The mixtures were then stirred gradually to find the best formulation. Triethanolamine was added to neutralize carbomer solution.
Evaluation of the gel
pH measurement
In order to determine the pH of the gel, the glass electrode was completely dipped into a solution containing 10% of the hydrogel in deionized water.
Rheology of the gel
Viscosity was determined at 25 °C using a Brookfield digital viscometer-RVDV-III (Brookfield, Massachusetts, USA) and spindle no. 52 at different rpm.
Centrifugation test
Hydrogels were centrifuged at 6000 rpm for 30 min and then examined for phase separation.
Animals
In our study, male albino Wistar rats weighing 220–250 g were purchased from the National Animal Center (Pasteur Institute of Karaj) and maintained in a 12/12-h light–dark cycle, with food and water supplied ad libitum. Animals were treated in accordance with the guidelines approved by the animal ethics committee of Pasteur Institute of Iran (IR.PII.REC.1397.027, 2019-01-09). Animals were divided into three groups (n = 5). Group I: Received administration of 10% F.vaillantii hydrogel formulation. Group II: Received topical application of gel base. Group III: Served as the negative control. The animals were euthanized with intra-peritoneal injection of sodium pentobarbital at 60 mg/kg on the 10th and 21st days after wounding.
Wound healing activity assessment
Excision and incision models were used to investigate the wound healing activity of gel formulation of F.vaillantii total extract.
Excision wound model
Rats were anesthetized using ketamine + xylazine and the hair on the back was clipped with electric clippers. Cutaneous square wounds of 225 mm
2 width with 2 mm depth were inflicted on the depilated ethanol-sterilized dorsal thoracic region of rats with the help of sterilized surgical scissors under a semi-aseptic condition. The
F.vaillantii hydrogel formulation along with the gel base was topically applied once a day for 21 days. Wound photos were taken and the wound area was measured in mm
2 by putting a transparent sheet over it. Upon tracing the wound margin by a permanent marker, the wound area was recorded using graph paper and Image J software on 2nd, 4th, 6th, 8th, 10th, 12th, 14th 16th,18th and 21st post wound days to monitor the percentage of wound closure. The percentage of wound contraction was calculated using the formula [
22]: (Initial wound size – specific day wound size)/ Initial wound size × 100.
The healed wound along with the surrounding skin obtained on day 21 were excised and then dissected into two equal parts to be further examined by histopathological analysis and hydroxyproline level determination method.
Histopathological evaluation
The healing tissues obtained from all the three groups of animals in our excision wound model were processed for histopathological analysis. Following fixation of the tissue samples in 10% formalin, samples were dehydrated using graded alcohol series. Afterwards, skin samples were cleared in xylene and then embedded in paraffin wax. Serial sections of 5 μm were prepared and stained with hematoxylin and eosin (H&E) for routine histopathological evaluation. All slides were investigated in a blinded manner by a pathologist.
Hydroxyproline determination
Hydroxyproline contents were spectrophotometrically measured by Woessner’s method [
23]. In brief, after the weighed tissue samples were hydrolyzed in 6 N HCl for 18 h at 115 °C, the residue was evaporated to dryness and remixed with a known volume of water. Following incubation of one ml of the sample with 0.5 ml of 0.05 M chloramine T solution (Sigma-Aldrich, USA) for 20 min at room temperature, 1 ml of Erlich’s solution was added and further incubated for 15 min at 60 °C. Absorbance was measured at 550 nm using a spectrophotometer (Cecil Company, UK). Hydroxyproline level was calculated from a linear standard curve and presented as μg/100 mg of dry content.
Incision wound model
Rats were anesthetized with ketamine + xylazine and the dorsal fur of the animals was shaved whit an electric clipper to make the incision wound. Incision of 3 cm was made at least 2 cm lateral to the vertebral column and parallel to it by a sharp scalpel with sufficient care. Afterwards, the incision was closed with surgical sutures at intervals of 1 cm. The hydrogel containing 10% of
Fumaria vaillantii L
. total extract and the gel base were topically applied once a day, starting from the initial day for 10 days. Sutures were removed on the 8th day and breaking strength of the healed wound and normal skin were measured using uniaxial tensile test (Model Z 2.5, Zwick GmbH & Co, Ulm-Einsingen, Germany) [
24] on the 10th day.
Confirmation of quercetin in total extract by HPLC
Quercetin content was evaluated using Waters LC 600 model chromatograph (Waters, Massachussets, USA) coupled with UV detector. Separation was performed by isocratic elution at a flow rate of 1 ml/min on Acclaim™ 120 C18 analytical column (150 mm × 4.6 mm; 5 μm). Mobile phase was a mixture of 2% acetic acid in water and acetonitrile in a 2:1 ratio and the sample injection volume was 20 μl. Stock solution of total extract of F.vaillantii was prepared (10 mg/ 10 ml) in ethanol and then passed through a 0.45 μm membrane filter. A 370 nm wavelength was applied for analysis. Peak areas were integrated automatically by Waters Software.
Statistical analysis
The data are expressed as mean ± SEM of at least triplicate determinations, and comparisons were based on ANOVA followed by Tukey’s post test using a GraphPad Prism 6.0 Software. A value of p < 0.05 was considered as statistically significant.
Discussion
Breakdown in normal anatomic structure and function of the skin, termed skin wound, which could occur through several causes including physical injuries, may lead to the opening and disruption of the skin [
27]. Wound healing is a dynamic process in which dermal and epidermal tissues regenerate as closely as possible to the normal status. A sequence of events occurs to repair the damage following injury. These events have been classified into several stages including inflammatory, proliferative and remodeling [
28]. Cytokines are usually released in the inflammatory stage due to the phagocytosis of bacterial pathogens leading to the migration of the cells involved in the proliferative stage. A subsequent chain of events including angiogenesis, collagen deposition, granulation, tissue formation, epithelialization and wound contraction take place at the proliferative stage [
29]. In the final stage, collagen remodeling takes place along the tension lines [
30]. Non-toxic, novel and cost-benefit therapeutic agents that contribute to increased healing rate, hastened epithelialization, inhibition of bacterial infection and supporting tissue remodeling have gained a great deal of attention among researchers worldwide. Therefore, we herein attempted to make a hydrogel formulation containing 10%
F.vaillantii total extract and applied it on an animal model of skin wound to verify the hypothesis that this formulation could demonstrate a distinguished treatment in healing wounds by providing an enhanced tissue repair.
Our results revealed that the topical administration of our hydrogel formulation using HPMC 4000 cP (2.5%) on a rat excision wound model leads to a significant acceleration in wound healing after 6, 10 and 14 days, a finding confirmed by an increased wound contraction compared to the negative control group. This enhanced potential of wound healing may be due to anti-inflammatory, antimicrobial and astringent properties of the plant, which are well documented in the literatures [
31]. In this regard, our literature survey identified several phytochemicals including flavonoids, alkaloids, tannins and saponins present in the
F.vaillantii total extract [
11], which may be responsible for its wound contraction and enhanced rate of epithelialization 21 days post wound healing. This assumption is supported by our previous study indicating a considerable amount of flavonoids in the
F.vaillantii total extract [
32]. As flavonoids including quercetin are known to possess antioxidant and anti-inflammatory effects [
33,
34], and according to the HPLC results performed here, confirming the presence of 2.25% of quercetin in total extract of
F.vaillantii, the wound healing activity of
F.vaillantii extract may be attributed to this property in the inflammatory phase. Furthermore, the alkaloids present in
F.vaillantii are also responsible for its antimicrobial property [
7,
35], which in turn leads to a better wound healing in the inflammatory phase.
Hydrogels exhibit several advantages as they provide the required moist environment to the wound area and also act as a suitable carrier for topical administration of substrates. Moreover, they cause a slow release of substances over time. What this information brings out noticeably is that hydrogel formulation can be a suitable candidate to promote wound healing. Thus, we prepared a hydrogel formulation with 10% total extract of
F.vaillantii using 2.5% HPMC 4000 cP displaying an optimum consistency and spreadability. Consequently, the proper hydrogel spreading would assist in the uniform administration of the gel to the skin. Additionally, based on our results, our formulated herbal gel contributed to a faster wound healing compared to the negative control group. Surprisingly, collagen fiber thickness and hydroxylproline content appeared to be more or less similar but basically higher in the gel base than
F.vaillantii gel formulation -treated groups, which can necessarily be explained by the therapeutic effect of hydrogels alone. In addition, topical administration of
F.vaillantii hydrogel was found to significantly increase the number of vessels as well as hair follicles in the gel-treated compared to the negative control wounds. Similar observations have also been reported by Xiao-bo Wu et al. (2012) who concluded that angiogenesis in granulation tissues results in improvement of circulation needed for supplying oxygen and nutrients vital for the healing process [
36].
The establishment of an incision wound model needs to be further worked out in order to determine breaking strength, confirming the wound healing activity of 10% total extract hydrogel of
F.vaillantii. In our study, control rats exhibited a wound breaking strength (area up to f
max) of 164.5 ± 68.26 N/mm on the 10th post wound day, whereas gel base and the hydrogel extract-treated groups displayed no significant breaking strength (259.2 ± 53.09 and 240.8 ± 30.63 N /mm), respectively. These data are in agreement with the results of our excision model and highlight the role of the gel base in collagen production, which leads to stabilization of fiber formation and subsequent stable intra- and inter- molecular crosslinks [
37,
38].
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