In pathology, a wound is a potentially challenging problem, and its early and late complications are a frequent cause of death. Many attempts have been made to reduce the burden of wounds, understand the physiology of wound healing and care by emphasising new treatments and developing technology to manage acute and hard-to-heal wounds.1,2
Wounds are a tissue injury that results in ‘loss of integrity of the epithelium with or without loss of underlying connective tissue’.3 Wounds can be a simple failure in the epithelial integrity of the skin or deeper, and can damage subcutaneous tissue or other structures such as tendons, muscles, vessels, nerves, parenchymal tissue and even bone.1
There are different causes and various classification criteria for wounds. Upon tissue damage, wound healing processes initiate and include four continuous, overlapping and carefully planned phases: rapid coagulation and haemostasis; inflammation; proliferation (mesenchymal cell differentiation, proliferation and migration); and proper angiogenesis and rapid re-epithelialisation of the wound site and wound reconstruction (with proper synthesis, cross-linking and collagen balance).1,4
Proper clinical management has a positive effect on the wound healing process and reduces potential complications. Disorders, mistakes or prolonged stages may lead to hard-to-heal wounds or delayed wound healing. Accelerating wound healing is considered a principle in treatment, the purpose of which, in addition to increasing the speed of recovery, is to heal hard-to-heal wounds in diseases such as diabetes.4
Medicinal plants have been part of human health care for thousands of years. According to the World Health Organization (WHO), more than 80% of the world's population is dependent on herbal medicine.5 Finding the proper medicinal plants to produce new, more effective, and powerful medicines is of interest to pharmaceutical researchers.
Dorema ammoniacum belongs to the Apiaceae (alternative Umbelliferae) family. Dorema ammoniacum is distributed in southwestern and central Asia. Gum ammoniacum consists of volatile oil, resin and gum. It contains: volatile oil 0.1–0.5%; resin 65–70%; gum circa 20%; along with free salicylic, valeric and butyric acids, but no umbelliferone is present.6 The major phenolic constituent of resin is ammoresinol. The volatile oil (circa 0.5%) contains various terpenoids with ferulene as the major component. It also contains linalool, linalyl acetate, and citronellyl acetate. The demonstration of the broad spectrum and antimicrobial activity of gum ammoniacum has supported its traditional use for chest infections.7,8
The essential oil obtained from different parts of Dorema ammoniacum has been screened in different studies. Phytochemical analysis showed hexadecanal (11.1%), α-cadinol (6.6%), sesquicineol-2-one (6.6%), ethyl linoleate (6.3%), ledol (5.1%), and γ-eudesmol (4.4%) to be the major constituents of the oil from stems and 2-pentadecanone (19.1%), β-eudesmol (17.2%), germacrene D (5.8%), α-eudesmol (5.8%) and spathulenol (5.0%) to be the major components in the essential oil of seeds.9
The major constituents of the essential oil are oxygenated monoterpenes (58.4%), sesquiterpene hydrocarbons (31.7%), (Z)-ocimenone (22.3%), (E)-ocimenone (18.1%), β-cyclocitral (9.9%) and ar-curcumene (6.4%).10 The essential oil in the leaves of Dorema ammoniacum contains sesquiterpene-rich oil (90.2%) which has shown the presence of high amounts of α-gurjunene (49.5%) and β-gurjunene (19.0%); δ-cadinene (16.24%), liguloxide (8.69%), δ-amorphene (8.43%), α–selinene (7.21%), β-selinene (6.62%) and α-himachalene (6.41%) were identified as the major components of the stem oil; β-bisabolene (15.1%), hexadecanal (13.2%) and (E)-nerolidol (11.3%) in roots were characterised as the major components.6,9
Iranian traditional medicine (ITM) is one of the most ancient medical systems in the world. In ITM, herbal extracts are used for treating a wide range of diseases. In ITM books, the subject of wounds and wound treatment fully describes the herbs and medicinal compounds, preparation methods and how to use them. Ammoniacum gum is used in ITM to heal various types of wounds. When the ammoniacum gum is applied to the wound area, it can remove extra discharge and corrupt tissues, induce growing healthy tissue and prevent scar formation on the injured site.11,12
There are a few studies on the therapeutic effects of Dorema ammoniacum gum, and more research needs to be carried out to confirm the therapeutic effects of this plant. To our knowledge, no research has been performed on ammoniacum gum on wound healing. However, in several studies, some of the pharmacological properties of this gum have been investigated.6,13,14,15
Some studies have reported an antibacterial effect for Dorema ammoniacum. The results showed that antibacterial properties are another reason for the wound healing effect of this plant.13,14 Also, antioxidant6 and anti-inflammatory15 effects of this plant have been reported in various studies.
The objective of the present study is to investigate the wound healing potential of the Dorema ammoniacum gum on a full-thickness skin wound model in Wistar rats and compare it to control groups.
The present study was designed to determine the effectiveness of the topical application of an ointment containing three different concentrations of ammoniacum gum ethanolic extract on accelerating wound healing, serum level of the growth factors, hydroxyproline content of repaired tissue, and histological parameters.
Method
Ethical approval
All of the ethical guidelines for use of laboratory animals were followed carefully. The experimental procedure has been approved by the Animal Ethical Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (approval number: IR.AJUMS.ABHC. REC.1397.039). This study is reported in accordance with the ARRIVE guidelines (Animals in Research: Reporting InVivo Experiments).16
Materials
Dorema ammoniacum gum was purchased from a local herb store in Tehran, Iran. The gum was identified by the Pharmacognosy Department of Jundishapur University of Medical Sciences, Ahvaz, Iran.
Preparation of topical products
The ammoniacum gum was pulverised by a grinder then macerated in a hydroalcoholic solution (80:20) for 24 hours at room temperature, and the ethanolic extract of ammoniacum gum was obtained. The resulting extract was filtered, and the solvent evaporated in a vacuum rotary. The extraction efficiency of ammoniacum gum was 69.75%. The concentrated extract was dried in a vacuum oven and stored at –20°C. Products containing 5%, 10% and 20% of this extract were prepared in Eucerin (Mahdaroo Co., Iran).
Animal experiment procedure
The 48 healthy male Wistar rats (200±20g) were selected from the animal laboratory of Jundishapur University of Medical Science, Ahvaz, Iran. The animals were housed individually in separated clean polypropylene cages to avoid them biting and scratching each other's wounds. They were kept under controlled conditions of temperature (22±2°C), humidity (60±5%) and a 12-hour light/darkness cycle. They also had free access to a standard commercial pellet diet and tap water. They were acclimatised for one week before the study. The animals received care for any health problems before and during the experimental study; the cages were cleaned and the bedding changed daily to prevent wound infection. No animals were subjected to any medicine or substance nor used in any investigation before the experiment. We selected a small sample size because the Dorema ammoniacum gum was being evaluated in vivo for the first time with regard to wound healing.
Wound creation
The animals were weighed and deeply anaesthetised by intraperitoneal (i.p.) administration of ketamine hydrochloride (10mg/100g). The animals' backs were shaved and disinfected with 70% ethanol; two circular full-thickness excision wounds were created on the dorsum region with a sterile histological punch (0.8cm diameter) comprising all the skin layers. After wounding, the rats were randomly divided (block randomisation using the AABB method) into six groups of eight animals and kept in separate cages. All cages were coded.
Treatment
Treatment started as follows:
- Group 1: test group treated with ammoniacum gum extract 5% (AGE 5%) applied locally on wounds
- Group 2: test group treated with ammoniacum gum extract 10% (AGE 10%) applied locally on wounds
- Group 3: test group treated with ammoniacum gum extract 20% (AGE 20%) applied locally on wounds
- Group 4: control group treated with phenytoin cream 1% applied locally on wounds
- Group 5: control group treated with Eucerin applied locally on wounds
- Group 6: control group without treatment.
During a 14-day treatment period, the animals received topical medicines once daily. In this way, after rinsing the wound with physiological serum, the wound was photographed, then 10g of the drug was gently applied to the wound with a spatula. To avoid wound infection, beds in cages were changed daily.
In this research, investigators who treated the animals, and the histologist who performed the histological examination on wound specimens, were blind to the composition of ointments.
Wound area measurement
In order to microscopically examine the surface of the wounds, imaging of the wounds was performed daily. Wound area photography was carried out between 9am and 1pm.
Under non-infectious conditions, after light anaesthesia, the animals were fixed in the standard position on a flat surface; a ruler was placed next to the wound and photographed using a digital camera embedded at a fixed distance from the wound. The situation was the same for all animals. On days 0, 5, 7, 10, 12 and 14 after wounding, wound surface measurements were performed. The wound surface was calculated by Digimizer software (MedCalc Software, Belgium). The percentage of wound healing was measured using the following formula:
( A 0 − A t ) / ( A 0 × 100 )
where A0 is the wound area on the first day and At is the wound area after time interval t.
Histopathological study
The animals were euthanised 7 and 14 days after wounding, and samples of the repaired tissue were taken from the wound area. All ethical principles were observed during the work with the animals tested. After separating the tissues, a sequence of processes was performed on these samples. These steps include fixing the tissues in formalin, dehydrating the tissues, moulding them in paraffin and preparing paraffin blocks. After preparing the paraffin blocks, cutting was performed with a rotary microtome, and slices of 5μm thickness were prepared. The slices were stained with haematoxylin and eosin (H&E) (for morphological observations) and Masson trichrome (to detect collagen fibres). Tissue samples were evaluated for the following histological criteria: re-epithelialisation, inflammation, neovascularisation, granulation tissue formation and collagen disorganisation.
Hydroxyproline assay
Tissue samples were weighed, then hydrolysed with a 6N hydrochloric acid solution (10mg tissue per 1ml acid), and incubated for 2 hours at 120°C to complete acid hydrolysis. The pH of the samples was then adjusted to 7.0.
Standard hydroxyproline solutions were prepared with concentrations of 1.0–4.0μg/ml. Standard solutions and samples were subjected to oxidation with chloramine T solution. Then Ehrlich reagent was added, and the coloured complex read at 60°C with a spectrophotometer (SPEKOL 2000; Analytik Jena AG, Germany). The absorbance values were measured at 550nm and compared to those of the standard curve to determine the concentration of hydroxyproline in the tissue samples.17,18
Growth factors assay
On days 7 and 14, under sterile conditions, blood samples were taken and centrifuged at 1000rpm (112×g) for 15 minutes and then stored at –80°C. The levels of TGFβ1, PDGF, EGF and VEGF in serum samples were measured by enzyme-linked immunosorbent assay (ELISA), based on the manufacturer's instructions (Bioassay Technology Laboratory, China).
Statistical analysis
Data were expressed as mean±SD (standard deviation). Statistically significant differences were determined using one-way analysis of variance (ANOVA) with the least significant difference (LSD) post hoc for multiple comparisons. For all tests, values of p<0.05 were considered statistically significant. Statistical analyses were performed using the SPSS16 statistical software (IBM SPSS Statistics, US).
Results
Wound area measurement
Fig 1 shows the gradual wound healing in experimental groups during the treatment. On day 5 after wounding, the rate of wound closure in the group receiving the ointment containing 5% ammoniacum gum extract (AGE 5%) was higher than the other groups. On days 7, 10 and 12 after wounding, the acceleration of contraction and wound closure in the group treated with an ointment containing 20% ammoniacum gum extract (AGE 20%) was higher than the other groups, so that by day 12, the wounds were completely healed.
For an accurate evaluation of the wound healing process on days 0, 5, 7, 10, 12 and 14 after wounding, wound surface measurement was performed using the Digimizer software. The results are presented in Fig 2.
On days 5 and 7 after wounding, the percentage of wound healing in the treated group with AGE 5% ointment significantly increased compared with all control groups (treated with phenytoin cream, treated with Eucerin and untreated groups) (p<0.001 and p<0.01, respectively). The increase in the wound healing rate in the AGE 10% ointment-treated group on day 5 compared to the Eucerin and untreated groups was statistically significant (p<0.01). Also, this significant increase in wound healing compared to all control groups was observed on day 7 (p<0.05). On days 5 and 7, the group treated with AGE 20% ointment showed a significant increase compared to the untreated group in terms of wound healing percentage (p<0.05).
On day 10 after wounding, the improvement rate in all treated groups (AGE 5%, AGE 10%, AGE 20%) showed a significant difference compared to the control groups (p<0.001). By day 12, all animals treated with these herbal medicines had fully recovered. On day 14, there were no significant differences in the rate of wound healing between the experimental groups. No infection occurred in any of the animals.
Histopathological study
Microscopic examination of the repaired tissue on day 7 revealed the proliferation of keratinocytes in the borders of the wound in all experimental groups. The granulation tissue formation in the treatment groups showed a significant difference compared to the control groups (p<0.05). The low accumulation of inflammatory cells at the wound site in the treatment groups indicates that the severity of the inflammation was lower in these groups compared to the control groups. In the treatment groups, the migration of keratinocytes beneath the scab began on day 7 and was complete by day 14.
On day 14 after wounding, a large percentage of the wound site was filled with granular tissue. This tissue On day 14 after wounding, a large percentage of the wound site was filled with granular tissue. This tissue had more fibroblasts and blood vessels in the treatment groups. The wound surface in the treatment groups was completely covered by keratinised epithelium, and keratin layers were observed on the surface of the healed wound in the treatment groups.
Tri-chromium staining indicated the formation of collagen fibres in all groups. The collagen disorganisation showed a significant decrease in all three AGE-treated groups compared to the untreated group (p<0.01).
Photomicrographs of the wound area in the treatment and control groups on different days are shown in Figs 3–6.
For quantitative evaluation of histological parameters, six microscopy slides per animal were examined for assessment of histological changes such as re-epithelialisation, collagen disorganisation, inflammation, neovascularisation and granulation. Each histological parameter was graded into four categories: normal (0), weak (1), moderate (2) and intense (3), and the averages were considered. The results of the assessment of histological parameters are shown in Figs 7 and 8.
Hydroxyproline assay
Collagen is one of the proteins that contain large amounts of the amino acid hydroxyproline. Collagen extracted from the skin contains 10–14g/100g of hydroxyproline. The hydroxyproline content is therefore an indicator of the amount of collagen formation in the tissue.19
On day 7 after wounding, the amount of hydroxyproline measured in the AGE 10%-treated group showed a significant difference compared to the phenytoin-treated group (p<0.05) and the untreated and Eucerin-treated groups (p<0.001). In the AGE 20%-treated group, the production of hydroxyproline showed a significant increase compared to the untreated and Eucerin-treated groups (p<0.001). The AGE 5%-treated group had a significant difference in terms of hydroxyproline content compared to the untreated and Eucerin-treated groups (p<0.05).
On day 14, the hydroxyproline content in the repaired tissue in the AGE 10%-treated group showed a significant increase compared to the phenytoin-treated, untreated and the Eucerin-treated groups (p<0.001).
In the AGE 20%-treated group, there was a significant difference in the content of hydroxyproline compared with the phenytoin-treated group (p<0.05) and the untreated and Eucerin-treated groups (p<0.001). In the AGE 5% group, the hydroxyproline amount showed a significant increase compared to the untreated and Eucerin-treated groups (p<0.05 and p<0.001, respectively). The results of measuring the hydroxyproline content in the healed tissues in the experimental groups is shown in Table 1.
Table 1. Comparison of hydroxyproline content of repaired tissue in the experimental groups (μg hydroxyproline per mg tissue sample)
| Experimental groups | Day 7 | Day 14 |
|---|---|---|
| AGE 5% | 920.10±11.25 *† | 1893.73±63.38 *††† |
| AGE 10% | 1355.77±169.72 ***##††† | 2263.20±85.79 *** ### ††† |
| AGE 20% | 1111.67±73.78 ***††† | 2027.14±109.3 ***#††† |
| Phenytoin | 973.20±33.81 * | 1775.78±32.58 |
| Eucerin | 686.20±18.41 | 1345.49±168.50 |
| Untreated | 706.07±15.02 | 1579.96±24.21 |
| Hydroxyproline content of normal skin | 2948.85 ± 18.48 | 2948.85±18.48 |
The results are expressed as mean±SEM (standard error of mean), n=8
#p˂0.05,
##p<0.01, p˂0.001 compared to the group treated with phenytoin
†p<0.05,
††p<0.01,
†††p<0.001 compared to the group treated with Eucerin
*p<0.05, p˂0.01,
***p<0.001 compared to the group without treatment
AGE 5%—group treated with 5% ammoniacum gum extract; AGE 10%—group treated with 10% ammoniacum gum extract; AGE 20%—group treated with 20% ammoniacum gum extract; phenytoin—group treated with 1% phenytoin cream; Eucerin—group treated with Eucerin; Untreated—untreated group
Growth factors assay
Serum levels of growth factors (EGF, PDGF, VEGF, TGF-β) in the blood of animals treated with AGE 5%, AGE 10% and AGE 20% ointments, compared with the control groups on days 7 and 14 after wounding, is shown in Fig 9.
Discussion
Four overlapping phases have been described in the classical model of wound healing and include homeostasis, inflammation, proliferation and re-epithelialisation.20 The haemostasis phase involves the accumulation of platelets and the activation of the blood coagulation cascade.
A large number of growth factors are released from the platelets and attract neutrophils, fibroblasts, endothelial cells and keratinocytes into the wound area by chemotaxis.21,22
The inflammatory phase involves changes in the activity of neutrophils, macrophages and mast cells,23 leading to the migration of neutrophils and macrophages into the tissue. Neutrophils are the first cells that arrive at the wound site and cleanse the wound of bacteria and dead tissue. About 48 hours after wounding, tissue macrophages arrive and produce cytokines and growth factors.21,22
The proliferation phase includes collagen, proteoglycans and fibronectin production to form the extracellular matrix (ECM), re-epithelialisation and neovascularisation. The fibroblasts that produce matrix and collagen are the dominant cells at this phase. The re-formation stage involves the synthesis, decomposition and reorganisation of collagen fibres through proteolytic enzymes produced by fibroblasts, neutrophils and macrophages.22
Due to the importance of wound healing in medicine, several studies have been conducted in this field to find an effective drug in accelerating wound healing and, at the same time, with low complications and low cost. Meanwhile, the effectiveness of medicinal plants has been proven extensively through experimental research.24 Herbal products have been used for centuries in wound care for their therapeutic effects, including anti-inflammatory, antioxidant and antimicrobial activities.25,26 Numerous in vitro and in vivo studies, and a few clinical trials, have been performed to confirm the effect of herbal products on wound healing.27
Dorema ammoniacum is one of the most important species of the genus Dorema. The presence of potent compounds such as polyphenols, sesquiterpenes, coumarins and flavonoids in oleo-gum resin extract or essential oil, which have a biological activity such as antioxidant, antimicrobial, and even anti-Alzheimer, confirms the high potential of this plant for medicinal use.28
Wound reduction is a good criterion for assessing the extent of wound healing. During the healing process, as a result of wound contraction and connective tissue formation, the wound area decreases. Fibroblasts have contractile properties, and they are able to stretch the epidermal layer and reduce the size of the wound.29
In the current research, macroscopic assessment has shown that topical application of these herbal ointments accelerates wound healing and increases the rate of skin wound contraction. The results of the semi-quantitative evaluation of histopathological parameters in animals treated with ammoniacum ointment indicate an increase in the formation of new epithelium and granular tissue formation, which ultimately increases the elongation of the epithelium and reduces the epithelial gap. These findings suggest that ammoniacum gum has a predominant effect on wound healing rate.
In an experimental study, the effectiveness of different concentrations of ethanolic extract of Boswellia serrata oleo-gum resin on wound healing was investigated. In this study, the healing effect of the cream containing different concentrations of Boswellia serrata oleo-gum resin extract (5, 10 and 15%) on the excision wound model in animals was compared with the standard treatment (povidone–iodine solution). The results showed that the cream containing 15% Boswellia serrata oleo-gum resin extract positively affected fibroblasts and collagen synthesis and increased the tensile strength of healed wounds, resulting in faster wound healing.30
Another study on the wound-healing activity of a 70% ethanolic extract of Shorea robusta resin in albino rats demonstrates that this extract accelerates wound contraction and increases the tensile strength of repaired tissue.31
In the current study, microscopic and statistical evaluations showed that during the study period, topical application of ammoniacum gum in the treated groups caused the various phases of the wound healing process that lead to wound closure to progress well; while in the untreated group, all events were delayed. There were no significant changes in the Eucerin group in terms of histological parameters, hydroxyproline content and growth factors compared to the untreated group; Eucerin was used to keep the wound moist but it was not enough to aid wound healing. In the positive control group (phenytoin), repair proceeded in the usual way. Phenytoin enhances granulation tissue formation, promoting collagen deposition and accelerating wound healing in rats compared to the group without treatment.
Recent studies on plant extracts have indicated that phytochemical components such as flavonoids32 and triterpenoids33 promote wound contraction and an increased rate of epithelialisation in the wound healing process.
Previous studies have determined that the ammoniacum gum is rich in flavonoids, sesquiterpene, coumarins and terpenoids.28,34,35
The wound-repair ability of ammoniacum gum is probably due to the presence of these phytochemical components in the plant; although, additional studies are necessary to isolate and determine the active compounds of the ammoniacum gum responsible for these pharmacological effects.
Collagen production and ECM remodelling are important in wound healing. Collagen has a large content of the amino acid hydroxyproline, which has been used as a biochemical marker for tissue collagen. In the proliferative phase, macrophages stimulate fibroblasts to produce collagen. Accumulation and organisation of collagen fibres and other cellular matrix proteins increase the strength of repaired tissue. Disruption in any of the repair processes can lead to a non-healing wound or the appearance of atrophic or hypertrophic scars.36,37
AGE improved the contraction of wounds, and this effect was related to the improvement of collagen synthesis as indicated by the higher hydroxyproline content in wounds treated with AGE (Table 1).
In this study, measurement of growth factors in the rat blood showed that topical application of ammoniacum gum increases serum levels of VEGF, EGF, VEGF and TGF-β. These results were similar to those obtained in the research conducted by Shahouzehi et al. on Pistacia atlantica resin.38 The results showed that Pistacia atlantica resin extract increases VEGF levels in the wound site, which results in the enhancement of inflammatory cells in the wound area, promotes migration and proliferation of endothelial cells and subsequently leads to wound contraction and tissue healing.38
It has been reported that TGF-β produced by macrophages promotes the differentiation of fibroblasts to myofibroblasts and increases collagen synthesis, resulting in ECM contraction and is followed by wound closure.39
TGF-β1 is a multifunctional cytokine that regulates various cellular functions in all phases of wound healing. It has been reported that TGF-β produced by macrophages and fibroblasts promotes the differentiation of fibroblasts to myofibroblasts and enhances granulation tissue formation and increases collagen synthesis, resulting in ECM contraction, and is followed by wound closure.39,40,41
PDGF is a potent chemoattractant for neutrophils, monocytes and fibroblasts. It also stimulates the synthesis of fibronectin, glycosaminoglycan and collagenase.42
Elevated serum levels of PDGF and TGF-β in AGE-treated groups increased fibroblast migration into the wound site and differentiation into myofibroblasts, respectively, and resulted in wound healing. The increased number of myofibroblasts can be seen on photomicroscopic images (Fig 4, not marked).
VEGF is the key factor in the wound healing process. It has been shown that VEGF increases vascular permeability as well as stimulating the secretion of growth factors and cytokines necessary for wound healing.43 VEGF enhances neovascularisation during the process of wound repair by stimulating the migration of endothelial cells through the ECM; it also mediates angiogenic activity during the proliferative phase.44,45
On the other hand, the increase in serum levels of VEGF on day 7 after treatment in the AGE-treated groups compared to the control groups indicates an increase in the formation of new blood vessels at the wound site, which increases blood flow to the damaged tissue and accelerates wound healing.
In 1990, Bakhtiarian et al. demonstrated that ammoniacum gum has dose-dependent anti-inflammatory and analgesic effects in animal models and the anti-inflammatory effect of this extract in high doses is comparable to indomethacin.15
According to an in vivo study, Dorema ammoniacum gum showed significant anti-inflammatory and analgesic activity in a dose-dependent manner in animal models. Dorema ammoniacum gum contains phenolic compounds which have a significant role in the biological activity of the gum. The highest analgesic and anti-inflammatory effects of the ammoniacum gum were found at a dose of 500mg/kg, which significantly reduced the pain in the acute and chronic phase and was comparable to indomethacin. Most of the peripheral pain is associated with the formation of inflammatory cytokines, interleukins and bradykinins. The results revealed that the analgesic effect of ammoniacum gum was probably achieved by inhibiting the proinflammatory cytokines, which lead to inhibition of pain peripherally.15
In an in vivo study, the effectiveness of the Dorema ammoniacum gum aqueous extract on acute inflammatory conditions was evaluated. The results of this study showed that topical application of the extract significantly suppresses oedema produced by carrageenan without producing erythema and oedema, which shows the efficacy of the extract in the inhibition of the synthesis, secretion and function of inflammatory mediators. The findings indicated that topical treatment with an aqueous extract of Dorema ammoniacum has anti-inflammatory properties similar to diclofenac as a reference drug.46
The results of a previous study demonstrated that the ethyl acetate extract of the roots showed antioxidant activity; additionally, the antibacterial evaluation of ethyl acetate and chloroform extracts of the roots represented potent antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Essential oils were also effective against Shigella dysenteriae.6,10
According to another study, the dichloromethane-methanol extract of ammoniacum gum (from Dorema ammoniacum) indicated broad-spectrum antimicrobial properties against seven Gram-positive bacteria (Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus faecalis), one Gram-negative bacterium (Bordetella Bronchiseptica), one yeast (Saccharomyces cerevisiae) and one fungus (Aspergillus niger). The minimum inhibitory concentration (MIC) of this extract was 40mg/ml.14
Previous studies on this gum showed that hexane, chloroform, ethyl acetate and methanolic extracts of the roots and shoots of Dorema ammoniacum have antioxidant effects.6 In the wound area, the amount of oxygen free radicals is high, and the presence of these free radicals causes oxidative stress and ultimately cytotoxicity and delay in wound healing, which results in a hard-to-heal wound. Therefore, the removal of these free radicals is an important factor in the wound healing process.47
The findings of a study demonstrated the methanolic extract had a high level of total phenolic and flavonoid contents, good inhibition of AChE and BChE enzymes and memory-enhancing effect, and exhibited high free radical scavenging potential. This extract probably activates the enteric nervous system signalling, increases the level of ACh, and strengthens neuronal activity that might be involved in memory processing.34
A dichloromethane extract of the gum resin from Dorema ammoniacum has shown acetylcholinesterase (AChE)-inhibitory activity. Four active compounds have been isolated and characterised in the extract (doremone A, an analogue of doremone A, dshamirone and ammoresinol). The results showed that dshamirone is the most active compound for AChE-inhibitory activity, whereas the other substances showed weak activity.48
Evaluation of the cytotoxicity of the essential oil of Dorema ammoniacum (extracted by hydrodistillation) showed that among four cell lines tested, cancer cells (MCF and SW480) were more sensitive to Dorema ammoniacum oil than the other two fibroblast cell lines (HFLP and HFSF). These results demonstrated that the essential oil of Dorema ammoniacum has low cytotoxicity.28
Overall, it seems that ammoniacum gum is probably involved in wound healing by reducing inflammation and cell proliferation due to its compounds with anti-inflammatory, antimicrobial, wound healing and regenerative effects.
Limitations
There have been no previous studies on the therapeutic effect of Dorema ammoniacum gum on wound healing, and this is the first study in this field to our knowledge. In future studies, topical formulations containing this gum can be designed and used in wound healing. One of the limitations of this study was the small number of animals in the groups. This was necessary in order to comply with the principles of medical ethics in working with laboratory animals.
Conclusion
In the present study, histological investigations showed a positive effect of ammoniacum gum on tissue repair and the regeneration of skin wounds, and this enhancement of tissue regeneration was associated with increased collagen fibres, granular tissue and fibroblast cell proliferation in the wound site.
The results showed that ammoniacum gum extract is effective in stimulating the proliferation of epidermal layer cells, which ultimately leads to accelerated wound contraction in the treated groups. Based on macroscopic findings, it can be concluded that topical application of AGE significantly increases the wound healing rate in rats and reduces wound healing time.
This study showed that 20% ethanolic extract of ammoniacum gum has significant effects on wound healing in the wound model in rats. Further research is recommended in the following areas: experimental research is needed to understand the mechanism of ammonia gum wound healing. A clinical trial should be performed on ammoniacum gum to determine the effectiveness of this gum in diabetic foot ulcers.
Reflective questions
- How does Dorema ammoniacum gum affect wound healing?
- Does an increased concentration of ammoniacum gum have an improved effect?
- How does topical use of ammoniacum gum compare with phenytoin in wound healing?