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Evaluation of wound healing and anti-inflammatory activity of the rhizomes of Rumex abyssinicus J. (Polygonaceae) in mice
© Mulisa et al. 2015
Received: 3 November 2014
Accepted: 25 September 2015
Published: 30 September 2015
Rumex abyssinicus Jacq (Polygonaceae) is widely used in Ethiopia for treatment of wound and other diseases. Although reports are available in the literature on some of the claimed activities, nothing has so far been reported about the wound healing activity of R. abyssinicus. Thus, this work was initiated to investigate the wound healing and anti-inflammatory activities of 80 % methanol extract of the rhizomes of R. abyssinicus in mice.
Following extraction of the rhizomes of the plant with 80 % methanol, the extract was formulated as ointment (5 % & 10 % w/w) with simple ointment base B.P. The ointment was then evaluated for wound healing activity using excision and incision wound models. Parameters, including wound contraction, epithelization time and hydroxyproline content were determined using the excision model, whereas tensile strength was measured from the incision model. In parallel, anti-inflammatory activity of the rhizome was evaluated with carrageenan induced hind paw edema model by dissolving the 80 % methanol extract in 1 % carboxyl methyl cellulose and administering orally in various doses (250, 500 and 750 mg/kg).
Wound treated with 5 % and 10 % (w/w) hydroalcoholic extract ointment exhibited significant wound healing activity in both models, as evidenced by increased wound contraction, shorter epithelization time, higher tissue breaking strength and increased hydroxyproline content. The hydroalcoholic extract also produced dose-related significant reduction (p < 0.05–0.001) of inflammation.
The results of this study demonstrated that the hydroalcoholic extract of the rhizomes of R. abyssinicus facilitated wound healing at least in part via its anti-inflammatory activity, supporting its traditional claim as a wound healing agent.
Wound may be described in different ways but the most common definition is “loss or breaking of cellular and anatomic or functional continuity of living tissues” [1, 2], and it can be broadly categorized as acute or chronic wound [3, 4]. The healing of wound is a complex process. At the time of injury, capillary will be damaged and blood clot is formed. This is followed by an early stage of inflammation. During the early phase of inflammation, cells like neutrophils and monocytes clean bacteria and necrotic tissue through phagocytosis as well as release of enzymes and toxic oxygen products. This is followed by migration of macrophage to wound area, which marks the transition from early to late phase of inflammation. The proliferative phase, which follows, is characterized by granulation and tissue proliferation formed mainly by fibroblast and the angiogenesis process. Finally, reformulation and improvement in the components of the collagen fiber that increases the tensile strength will occur during the remodeling stage . Thus, alterations in the inflammatory phase will impact the overall integrity of the healing wound.
Globally, wounds are a main cause of disability and lost productivity. Apart from the incidence, chronic wounds are known for causing different health problems. A study done in North European countries indicated that foot ulcers cause up to 85 % of amputations [5, 6]. In sub-Saharan African and South Asian countries, 1 % to 2 % of the population faces chronic wound at least once in their lifetime [7, 8]. More than 1.25 million people sustain burns and approximately 5 million suffer from non-healing wounds each year in the US . The cost of wound care is substantial. Chronic wounds cost European countries 1 % to 2 % of their annual health care budget and $1 billion/year in the US . This cost is unthinkable for most people in developing countries suffering from infected wounds . Indirect costs such as psychosocial damage and poor quality of life are also major concerns .
Majority of the population in both developing and some developed countries use traditional medicine for their primary health care [12, 13]. In developing countries, like Ethiopia, management of skin diseases and dermatological disorders such as cuts, wounds, and burns has relied on medicinal plants for a long period of time. Herbs are known to make the wounded area moist, which is very essential for healing [14, 15]. Nowadays, discovery is shifted towards wound healing promotion in order to reduce hospitalization cost and serious complications . Several lines of evidence have shown the benefits of a wide variety of plants in their wound healing capacity [2, 17]. Thus, plant products are considered to be the best and cost-effective substitutes for wound treatment.
Rumex abyssinicus Jacq (Family; Polygonaceae) is the most common traditional medicinal plant in the highlands of tropical Africa and distributed throughout North Africa and Ethiopia. In the Ethiopian traditional medicine, the rhizomes of R. abyssinicus (“Mekmeko” in Amharic) are used to treat malaria, gonorrhea, poisoning, hepatitis, constipation, sciatic neuralgia, hypertension, migraine, rheumatism, breast cancer, stomach distention, earache, liver diseases, hemorrhoids, typhus, rabies and wound [18–22].
Previous reports indicated that 80 % methanol extract of the rhizomes of R. abyssinicus possessed antimicrobial and in vitro anti-inflammatory activities , diuretic and analgesic , and antimalarial  activities. To date, however, no scientific report could be found in the literature concerning the wound healing activity of the plant, although there is an ample ethnobotanical claim for this property. This study was therefore conducted to provide scientific evidence for the folklore use of the plant in wound healing processes.
The fresh rhizomes of R. abyssinicus was collected from Menagesha forest, located 40 km west of Addis Ababa, Ethiopia. The plant was identified by a taxonomist and a voucher specimen (E001/10) was deposited at the National Herbarium, College of Natural and Computational Sciences, Addis Ababa University (AAU) for future reference.
Healthy, adult Swiss albino mice of either sex (25–30 g, and 6–8 weeks of age) purchased from the animal house of the Ethiopian Public Health Institute and adult Wistar rats of both sexes (200–300 g, aged 3–4 months) obtained from the animal house of the School of Pharmacy, AAU, were used for the study. The animals were housed in cages under standard conditions (25 ± 2 °C, 55 ± 5 % relative humidity, and 12 h light and dark cycles) and provided with pellet diet and water ad libitum. The study protocol was approved by the Ethics committee of the School of Pharmacy, AAU. Animal handling and care was carried out throughout the experiment following international laboratory animal use and care guidelines .
Rhizomes of R. abyssinicus were sliced to smaller pieces and dried for three weeks under shade. The dried rhizomes were then grinded to coarse powder. Five hundred gram of the powder was then macerated with 80 % methanol for three days in a conical flask with occasional stirring and shaking. The extract was then filtered (Whatman No. 1) and the residue was re-macerated twice to obtain maximum yield. The combined filtrate was then evaporated in a ventilated oven at 40 °C until dried. The resulting dry extract was weighed and provided a percentage yield of 16.2 % (w/w). The dried extract was stored in a refrigerator for the preparation of topical formulation (ointment).
Formula used for preparation of the ointment
White soft paraffin
Acute dermal toxicity
For dermal toxicity, a total of 10 (5 female and 5 male) rats were used. Animals showing normal skin texture were housed individually in a cage and acclimatized to the laboratory condition for five days prior to the test. Following acclimation, around 10 % of the body surface area fur was shaved 24 h before the study from the dorsal area of the trunk of the test animals. A limit test dose of 2000 mg/kg of the 10 % formulation was applied uniformly over the shaved area for 24 h. At the end of the exposure period, residual test substance was removed and the animals were observed for development of any adverse skin reactions daily for 14 days.
Grouping and dosing of animals
For excision model, four groups of mice, each containing six animals were used. The first group was treated with simple ointment, and served as a negative control. The second and third groups were treated with 5 % and 10 % of the extract ointment, respectively. The fourth group was treated with nitrofurazone (0.2 %) and served as a positive control. For incision model, five groups of mice, containing six mice per group were used. The animals of Group I-IV were treated in a similar fashion with excision wound model, but animals in Group V were not treated with any agents and served as untreated controls.
For assessment of anti-inflammatory activity, five groups of mice containing six animal per group were used. Group I was treated with 1 % carboxyl methyl cellulose (CMC) and served as a negative control. Groups II-IV were treated with 250 mg/kg, 500 mg/kg, and 750 mg/kg of the extract. Group V was treated with indomethacin (10 mg/kg). Dose levels were chosen based on acute oral toxicity results described elsewhere  and dose calculation was based on Deshmukh et al. , with slight modification. All administrations were performed orally, with a maximum volume of 10 ml/kg. The extract and the standard drug were dissolved in 1 % CMC to obtain an oral suspension.
Excision wound model
On wounding day, animals were anesthetized using subcutaneous injection of ketamine (1 ml/kg) and the back hair of the animals was depilated by shaving. About 300 mm2 circular area was then marked and the full thickness of the marked area was carefully excised by using sharp sterilized scissors. After 24 h of wound creation, the ointments were applied gently once daily, according to the respective grouping as described under grouping and dosing section, to cover the wounded area until complete healing was achieved. Wound contraction, epithelization period and hydroxyproline content were monitored. Wound contraction was measured as percent contraction every 2 days until complete wound closure was achieved .
Measurement of wound contraction
Epithelization time measurement
The period of epithelization was calculated as the number of days required for falling off of the dead tissue remnants without any residual raw wound .
Estimation of hydroxyproline content
Incision wound model
Where so is simple ointment, and lu is left untreated.
Where Co is the average inflammation (hind paw edema) of the control group at a given time; and Ct is the average inflammation of the plant extract or indomethacin treated mice at the same time.
Raw data obtained from both wound and mouse paw edema models are expressed as mean ± SEM. The data were analyzed using SPSS version 17.0 and differences among groups were compared with one-way ANOVA followed by Post Hoc Tukey test. The data were considered significant at p < 0.05.
Acute dermal toxicity
Effect on excision wounds
Effect of topical application of the ointment formulated from 80 % methanol extract of the rhizomes of Rumex abyssinicus on wound contraction of excision wound model in mice
Wound area (mm2) on post-wounding days
234.2 ± 11.8
202.0 ± 10.6
159.1 ± 13.0
110.0 ± 8.5
33.5 ± 5
20.5 ± 2.7
9.2 ± 3.0
5 % HEO
179.3 ± 23.3
150 ± 15.0
80.2 ± 21.7a2
27.3 ± 11.4a2
10 ± 6a2
2 ± 1.4a3
10 % HEO
82.4 ± 8.1 a3,c2
72.67 ± 9.2a3,c2
39.1 ± 8.1a3
11.0 ± 3.0a3
1.8 ± .5a3
130.7 ± 17.0a2
108.5 ± 17.4a3
54.5 ± 10.7a3
17.2 ± 4.8a3
4.8 ± 2a3
1.67 ± 1.3a3
Effect of topical application of the ointment formulated from 80 % methanol extract of the rhizomes of Rumex abyssinicus on epithelization period
Epithelization period (days)
17.3 ± 0.333
5 % HEO
14.5 ± 0.764a2
10 % HEO
13.3 ± 0.333a3
13.5 ± 0.671a3
Estimation of hydroxyproline content
Hydroxyproline content of excision wounds following topical application of the ointment formulated from 80 % methanol extract of the rhizomes of Rumex abyssinicus
Hydroxyproline (mg/100 g)
10.6 ± 0.92
5 % HEO
13.9 ± 0.89a1
10 % HEO
18.5 ± 0.70a3
21.6 ± 0.86a3
Effect of topical application of the ointment formulated from 80 % methanol extract of the rhizomes of Rumex abyssinicus on tensile strength of incision wound
Tensile strength (g) (mean ± SEM)
% Tensile strength
205.4 ± 19.5
244.67 ± 17.1
5 % HEO
333.33 ± 18.2a1
10 % HEO
384.50 ± 19.6a3
438.17 ± 19.1c2,a3
Anti-inflammatory activity of 80 % methanol extract of the rhizomes of Rumex abyssinicus on carrageenan-induced mice paw edema following oral administration
Mean change in the paw volume (ml)
0.50 ± 0.04
0.61 ± 0.02
0.7 ± 0.03
0.69 ± 0.04
0.65 ± 0.03
Extract (250 mg/kg)
0.50 ± 0.02
0.60 ± 0.03 (1.64 %)
0.58 ± 0.04 (17 %)
0.55 ± 0.02a1 (20.29 %)
0.51 ± 0.02a2 (21.54 %)
Extract (500 mg/kg)
0.50 ± 0.03
0.55 ± 0.02 (9.84 %)
0.47 ± 0.04a3 (32.86 %)
0.44 ± 0.02a3 (36.23 %)
0.30 ± 0.03a3c3 (53.85 %)
Extract (750 mg/kg)
0.51 ± 0.01
0.48 ± 0.04 (21.31 %)
0.43 ± 0.02a3,c1 (38.57 %)
0.40 ± 0.04a3,c2 (42.03 %)
0.25 ± 0.02a3c3 (61.54 %)
Indomethacin (10 mg/kg)
0.53 ± 0.03
0.48 ± 0.03 (24.6 %)
0.35 ± .02a3c3 (50 %)
0.24 ± 0.02a3c3 (65 %)
0.10 ± 0.02a3c3 (84.6 %)
Wound healing is a complex process that cannot be understood by using a single model or relying on an in vitro study. Better understanding of the healing process necessitates the use of two or more different in vivo models . Wound healing includes an acute inflammatory phase accompanied by collagen synthesis, which finally form a scar. Currently available drugs can’t affect all phases of wound healing, calling for the need for developing new drugs from different sources. Rapid healing requires fast wound contraction, shorter epithelization period and adequate gain of tensile strength. Biochemical markers, including tissue DNA, RNA, total protein, and hydroxyproline are indicative of better healing quality of drugs [37, 38]. Thus, in the present study, an attempt was made to evaluate the potential wound healing activity of R. abyssinicus, a medicinal plant used for wound healing in Ethiopian folklore medicine using the two most important wound models i.e., excision and incision wound models.
In excision wound model, the ointment formulated from the hydroalcoholic extract of the rhizomes of R. abyssinicus showed significant increase in percentage closure of excision wounds (Table 2 & Fig. 4). During the proliferative phase of wound healing, wound contraction enhances closure of the defect by pulling the edges of the wound towards the center . Contraction decreases healing time because it decreases the size of the wound and reduces the amount of extracellular matrix needed to repair the defect. Contraction also facilitates re-epithelization by shortening the distance migrating keratinocytes must travel . Moreover, the wound will close at fast rate, if the medication is more efficient . The effect of the rhizomes of R. abyssinicus on wound contraction points to the fact that the extract is endowed with pro-healing action as wound contraction accounts for 88 % of the healing process (the remaining healing is due to scar formation) . Although the mechanism by which the extract increased wound contraction remains to be seen, it is plausible to suggest that it might be attributed to either the plant’s anti-inflammatory effect (present study) or induction of macrophage cell proliferation .
In this study, the epithelization time was also found to be significantly shorter in animals treated with ointments containing the crude extract (Table 3). Epithelization involves the proliferation and migration of epithelial cells across the wound bed . Therefore, the shorter epithelization time in the hydroalcoholic extract might be due to facilitated proliferation of epithelial cells and/or increasing the viability of epithelial cells . A large body of evidence indicates that antimicrobial activity correlates with wound healing. Infection can seriously delay healing process by causing poor quality granulation tissue formation, reduced tensile strength of connective tissue, impaired epithelization and odor [14, 44]. Hence, increased rate of wound contraction and decrease in period of epithelization in the animals treated with the extract in excision wound model could also be attributed to the R. abyssinicus’s antimicrobial activity, as such activity is reported earlier for the plant . Wound re-epithelization is a hallmark of successful wound care . Moreover, better healing activity is distinguished with short epithelization period . Thus, shorter epithelializtion periods in animals treated with the extract reinforces the notion that R. abyssinicus has a potential application as a wound healing agent.
Hydroxyproline is an amino acid that is found in collagen and measuring its level indicates collagen turnover . The hydroxyproline content in mice treated with ointment containing the extract was significantly higher than controls, but comparable to the standard (Table 4). Higher hydroxyproline content in the extract and standard treated group might be related to enhancement of the proliferation and migration of fibroblasts and collagen deposition . Thus, it is conceivable to suggest that the observed decrease in epithelization and increase in wound contraction with application of the extract could be due to the plant’s potential to increase collagen synthesis through its effect on hydroxyproline content, as wound contraction begins almost concurrently with collagen synthesis .
Better efficacy of the crude extract in wound healing was further evidenced by the breaking strength in incision wounds (Table 5). Higher tensile strength is an indication of better wound healing  and it mainly depends on the increase in collagen concentration and stabilization of the fibers. Hence, based on the amount of hydroxyproline detected, it could be assumed that the crude extract enhanced the strength of the incision wound by increasing the collagen levels, which could stick the wound edges together at the repaired site .
Inflammation is important in wound healing. During acute wound it makes wound bed to be healed by removing necrotic tissue, debris, and bacterial contaminants as well as recruiting and activating fibroblasts. Under normal conditions, inflammation is a self-limiting process. Excessive inflammation, however, limits wound healing . The extract showed anti-inflammatory activity in carrageenan induced paw edema model in mice (Table 6). Carrageenan is widely used to induce hind paw edema for the discovery and evaluation of anti-inflammatory drugs . Carrageenan injection induces inflammation within short period of time. It produces three distinct phases: first (0–1.5 h), second (1.5–2.5 h) and third (2.5–5 h) phases of inflammation. The mediators of inflammation involved are histamine and serotonin, bradykinin, and prostaglandins for the three phases, respectively. The third phase is mostly used to assess anti-inflammatory effect of traditional medicinal plants, as this phase is sensitive to most clinically effective anti-inflammatory drugs . Neither of the treatments produced effect in the first hour, suggesting less responsiveness of phase one mediators to the tested agents (Table 5). Inhibition was, however, apparent following the 2nd hour, the only exception being 250 mg/kg of the extract. This observation could possibly indicate that phase 2 mediators’ response might vary with dose. On the other hand, all doses of the extract were effective from the 3rd hour onwards, regardless of the dose levels used, pointing to the fact that phase 3 mediators are the most likely targets for the extract. There appears also to be an in vitro and in vivo correlation, as in vitro anti-inflammatory activity for the plant has been described in previous reports . Moreover, this finding is concordant with earlier reports showing that plants having anti-inflammatory activity do also exhibit wound healing effect .
The 80 % methanol extract of the rhizomes of R. abyssinicus had been reported to possess secondary metabolites, including, among others, tannins, saponins, flavonoids, steroids and anthraquinones . Flavonoids and tannins have been shown to be important for wound healing due to their antioxidant, anti-inflammatory and antibacterial activities. In line with this notion, published reports indicate that hydroalcoholic extract of Rumex crispus , R. maderensis , R. acetosa, R. patientia , Polygonum convolvulus, Rheum undulatum, and R. acetosella  possess wound healing activity because of their phenolic (such as flavonoids and tannins) content. Active constituents such as chrysophanol, emodin and physcion were also previously isolated from the rhizomes of R. abyssinicus . Emodin [37, 56] and chrysophanol  isolated from other plant species are reported to have antimicrobial, anti-inflammatory and wound healing activities. In addition, majority of the Rumex species have also been reported to have antimicrobial action due to the presence of physcion and rumicin . Taken together, these findings collectively indicate that phytoconstituents individually or synergistically may initiate a host of mechanisms responsible for the wound healing activity of R. abyssinicus. Although histopathological studies are not performed that could have provided data with regeneration of the epithelium and disposal of the collagen, the measured parameters consistently showed that the plant is endowed with favorable wound healing activities. Preparations are underway to isolate the compound (s) responsible for the observed activities in subsequent studies.
The data generated from the present study indicated that the crude extract of the rhizomes R. abyssinicus increased wound contraction, breaking strength of the repaired tissue and hydroxylproline content. These findings highlight the potential wound healing activity of the plant and uphold the traditional claims of the plant for the treatment of wounds. The hydroalcoholic extract was also endowed with significant anti-inflammatory activities that explain, at least in part, its wound healing activity.
The authors would like to thank Addis Ababa University for the financial support extended to complete the research work.
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