Healing and topical anti-inflammatory activities of the total aqueous bark extract of Combretum glutinosum Perr. (Combretaceae)

Combretum glutinosum Perrot. Ex DCs (Combretaceae) is a plant used in Senegalese traditional medicine. Its trunk barks are used to treat burns and wounds. The objective of this study was to evaluate the healing effect and topical antiinflammatory activity of the aqueous bark extract of C. glutinosum in second degree burn and croton oil induced ear edema in mice . Phytochemically, the aqueous bark extract of C. glutinosum contains tannins and flavonoids, but not alkaloids. Experimental second degree burn was induced in dorsal flanks of the rats. Topical antinflammatory activity was performed in ear edema of mice induced with croton oil. The daily application of aqueous bark extract ointment of C. glutinosum 3 and 10% in petroleum jelly (CGPJ-3%, CGPJ-10%) induced concentration-dependent healing effect with complete tissue repair. In fact, the daily application of CGPJ-3% was associated with total cicatrization after 28 days of treatment (score 0). CGPJ-10% induced a shift in the healing curve to the left with almost complete tissue repair in 20 days (score 1). CGPJ-10% significantly prevented ear edema in mice. The percentage increase in ear edema was 42.78±4.94 vs 70.59±±18 in control group (p<0.05). C. glutinosum trunk bark induced cicatrization, probably involving tannins and flavonoids, which justifies its use in traditional medicine to treat burns and wounds.


INTRODUCTION
Healing is a complex process, involving a series of biochemical and cellular reactions that promote inflammatory process, repair and remodeling of damaged tissues (Sadaf et al., 2006). These reactions are triggered in response to an injury and restore the function and integrity of damaged tissues (Kumar et al., 2007). Inflammatory response is regarded as the first among many mechanisms involved in the healing process (Eming et al., 2007). However, when it is maintained, especially in chronic wounds, such as diabetic wounds, it becomes harmful (Rosique et al., 2015). The introduction of anti-inflammatory agents or healing products with antiinflammatory activity in wound management could be a beneficial strategy to improve wound healing (Dissemond et al., 2002). In traditional medicine, plants are widely used to facilitate wound healing with a high degree of therapeutic success (Muthu et al., 2006;Mesfin et al., 2009;Esimone et al., 2008). This is the case of the bark of Combretum glutinosum (Combretaceae) Perrot. Ex DCs which is used as a poultice to treat injuries and other wounds (Sereme et al., 2011).
In Senegal, C. glutinosum is the most widespread species of the genus Combretum (Kerharo and Adam, 1974). It is called "Ratte" in Wolof (Senegal). It belongs to an essentially tropical family, the Combretaceae. It is found in savannahs and forests, on sandy or clay soils and on lateritic soils as a shrub or small tree. It is also widespread in many African countries: Gambia, Mali, Guinea, Sierra Leone, Ivory Coast, Ghana, Benin, Nigeria, Niger, Cameroon, Central African Republic and Sudan (Dione, 1996).
The purpose of this study is to evaluate the topical healing and anti-inflammatory activity of C. glutinosum trunk bark on experimental burn model in rats and inflammatory croton oil edema in mice.

Plant material
The plant material is made of C. glutinosum bark harvested in the eastern region of Senegal (Tambacounda). The plant was identified at the Pharmacognosy and Botany Laboratory of the Faculty of Medicine, Pharmacy and Odontology, Cheikh Anta DIOP University (CADU), Dakar; where the voucher specimen (DPB-16-10) was deposited.
The barks were cut and dried at 25°C for 3 weeks. A brown powder was obtained after spraying.

Animal material
Wistar Kyoto strain rats weighing between 100 and 400 g and albino mice were used. The rats were obtained from the Pharmacology Laboratory, Faculty of Medicine, Pharmacy and Odontology, CADU. Meanwhile, the mice were obtained from the Pasteur Institute, Dakar and weighed between 27 and 38 g. The animals were housed in a cage under conditions of 25±2°C temperature, 12 h light cycle and provided with food and water ad libitum.
Experimental procedure Extraction C. glutinosum bark powder (225 g) was decocted in 3 L of boiling water for 30 m. After cooling, the decoction was filtered (filtrate 1) and the marc was placed in 2 L of water twice and the mixture was filtered (filtrate 2 and 3).
The combined filtrates were then concentrated using a rotary evaporator and then dried at a temperature of 40°C. The weight of the total dried aqueous extract of C. glutinosum was 29.38 g (13.06% (w/w)) dry bark powder. The total aqueous extract obtained was used to prepare the ointments for pharmacological tests (Chang et al., 2012;Mayba and Gooderham, 2017).

Phytochemical characterizations
Phytochemical characterization of flavonoids, tannins, alkaloids and sterols/triterpenes was performed on tubes and confirmed by thin layer chromatography (TLC).
Aluminum chloride was used to determine flavonoids, ferric chloride for evidence of tannins, Dragendorf's reagent for alkaloids identification and acetic anhydride combined with chloroform and sulfuric acid for sterols/ triterpenes characterization (Bassène, 2012) (Table 1).
Sodium benzoate was used as a reference preservative at the rate of 1.5 g per 1000 g of ointment. The sodium benzoate and extract powder were triturated in a mortar with a pestle. Petroleum jelly was gradually added while slightly triturating until the mixture homogenized. Two ointments (in 3 and 10% petroleum jelly) were obtained according to the proportions ( Table 2). The ointments were packaged in sealed jars and stored at room temperature away from the sun (Zdoryk et al., 2014).  Table 3. Scores of the evolution of experimental burns.

Score
Evaluation of the healing process 0 Healing is complete, tissue repair is complete 1 Tissue healing is almost completed 2 There are still vestiges of the crust, the lesion size decreases (skin reconstruction) 3 All dead tissue (scabs) are removed, ulceration, oozing 4 Necrosed skin is partially removed, ulceration, oozing 5 Necrosed skin completely covers the burned area
Sulfadiazine is an antibacterial sulfonamide, used topically in preclinical and clinical studies to demonstrate the healing activity of a new product (Adhya et al., 2015;Nasiri et al., 2015). The rats were then anesthetized with a 3% chloral solution by intra-peritoneal injection (1 mL/100 g). The dorsal flanks of the rats were shaved and cleaned. Experimental burns have been induced using a 3-cm diameter metal cylinder and heated for 5 min. The cylinder was applied for 20 s by slight pressing on the surface of the shaved skin of the rats to cause second degree burns (Hoşnuter et al., 2004).

Evaluation of the healing activity of the aqueous bark extract of C. glutinosum
Healing activity was evaluated according to the Kamoshida's method, which assigns scores ranging from 1 to 5 depending on the importance of the burn (Table 3). Evaluation of the scores has been done daily during 28 days.

Experimental protocol study of topical anti-inflammatory activity
Topical anti-inflammatory activity was evaluated in croton oilinduced ear edema in the experimental mice model (Tonelli et al., 1965;Tubaro et al., 1986).
The mice were divided into 3 lots of 3: -Lot 1: control -Lot 2: treated group with 10% C. glutinosum ointment -Lot 3: treated group with 10% indomethacin ointment In control group, the mice were treated locally with 10 μl of 1% croton oil in ethanolic solution applied to the right inner ear.
The ointments were applied on the left inner ear of mice. On the right inner ear, 10 μl of 1% croton oil in ethanolic solution and the ointments were also applied.
The treated animals were anesthetized with ethyl ether using a funnel 6 h after treatment. The ears were immediately cut along the cartilage and weighed immediately with a precision balance. The mice were then sacrificed.
Topical anti-inflammatory activity was evaluated using the following formulae: -RE: right ear, -LE: left ear.

Statistical analyses
The results were expressed as mean scores±standard error of mean (SEM). Statistical significance was determined by analysis of the variance followed by the Bonferroni's test (graphPadPrism 5).

Phytochemical characterization
The results of phytochemical characterization of the aqueous bark extract of C. glutinosum revealed the presence of flavonoids, tannins and sterols/triterpenes. Alkaloids were not detected (Figure 1, Table 4).

Healing activity
Evolution of experimental second-degree burn scores in rats without treatment: Seven days after the induction of experimental burn, the burned site was still covered with necrotic skin, corresponding to score 5. Three weeks after, in the absence of treatment, the burned site still showed an oozing ulcer (score 4).
Four weeks after, the experimental burn consisted of an open, oozing wound (score 2.75), corresponding to the absence of healing (Figure 2).

Evolution of experimental second-degree burn scores in rats after daily application of petroleum jelly:
The daily application of petroleum jelly on a second-degree burn did not show significant difference in scores compared to the untreated group after 2 weeks. The burn score of 4 corresponded to an oozing ulceration. The score changes from 2.75 to 2.25, respectively, after 21 and 28 days (Figure 2).

Evolution of experimental second-degree burn scores in rats after daily application of Sulfadiazine:
The experimental burn score in the sulfadiazine group after 2 weeks of treatment was not significantly different from the untreated group or after petroleum jelly application. Three weeks after, the burn score was 3.5 corresponding to an oozing ulceration. Tissue repair was incomplete after 4 weeks, confirming a second-degree burn. Meanwhile, sulfadiazine, which was indicated in first-degree burn

Group
Aqueous bark extract of C. glutinosum Figure  Flavonoids + Figure  showed no healing effect (Figure 2).

Evolution of experimental second-degree burn scores after treatment with an ointment of aqueous bark extract of C. glutinosum at 3 and 10% in petroleum jelly (CGPJ-3%, CGPJ-10%):
Daily application of CGPJ-3% was associated with burn healing. In fact, the score significantly decreased after 14 days of treatment (score 2.75). Tissue repair was complete after 28 days (score 0) (Figure 3). The cicatrization kinetic of C. glutinosum ointment is dependent on concentration. The daily application of CGPJ-10% shifts the healing curve to the left. Tissue repair was almost complete after 20 days (score 1) (Figure 4). Figure 5 shows the healing progress of the burns in rats treated with CGPJ-10% ointment.

Topical anti-inflammatory activity
Croton oil in ethanol induces inflammatory edema at percentage increase of 70.59±5.18. Application of the CGPJ-10% ointment significantly prevented ear edema in mice. The percentage increase in edema is 42.78±4.94 (p<0.05 versus control). The same profile is observed with the indomethacin ointment at 10%, used as a reference substance (Table 5).

DISCUSSION
In African traditional medicine, the barks of C. glutinosum are used in the treatment of burns. The objective of this   study was to highlight the healing and anti-inflammatory activities of the trunk bark of C. glutinosum, on experimental second-degree burn model in Wistar rats and inflammatory croton oil edema in mice.
In the present study, the induction of second-degree burn in rats was not associated with complete healing after 28 days of observation. Similar results were observed in the petroleum jelly treatment group, confirming that Carreiro and Fourtanier's (1983) observations according to which the vehicles usually employed to prepare ointments, such as petroleum jelly, did not have any specific effect in the healing process.  The aqueous extract of C. glutinosum trunk bark has a concentration-dependent healing activity in experimental second-degree burn model in wistar rats. Previous studies had already described the healing activity of plants belonging to the Combretaceae family. In fact, fractions from hydroalcoholic extract of stem barks of Terminalia arjuna showed healing effects, resulting in increased tensile strength and re-epithelialization in respective experimental models of incision and excision wounds (Rane and Mengi, 2003;Chaudhari and Mengi, 2006). Also, the aqueous leaf extract of Combretum micronatum (Combretaceae), induces healing by stimulating the viability of human keratinocytes and dermal fibroblasts (Kisseih et al., 2015). The healing effect of the bark of C. glutinosum may involve fibroblasts and proliferation stimulations during tissue reconstruction.
Phytochemically, the aqueous bark extract of C. glutinosum contains flavonoids, tannins and sterols/ triterpenes but not alkaloids.
Previous studies revealed the presence of tannins, flavonoids and triterpenes in the barks of C. glutinosum (Sereme et al., 2011;Yahaya et al., 2012;Niass et al., 2015;Sall et al., 2017). The phytochemical results of this study corroborates previous studies which described the presence of sterols/triterpenes, tannins and flavonoids.
The healing activity of different phytochemical components has been shown in several studies. In fact, epicatechin gallate, a natural polyphenolic compound, significantly improves scar healing and quality in an incision wound model. These effects may be in part, caused by an acceleration of angiogenic response and upregulation of nitric oxide synthase and cyclooxygenase enzymes (Kapoor et al., 2004). The total tannins present in the aqueous powder extract of Entada phaseoloides showed a healing action in excision wound model of rat. This effect could not only be favored by improvement in fibroblasts proliferation and migration, but also by an antibacterial activity attributed to these tannins (Su et al., 2017).
A probable action of C. glutinosum bark on the proliferation and/or migration of fibroblasts could be related to the abundant presence of tannins in the aqueous extract.
A pentacyclic triterpenoid molecule isolated from leaves of Lantana camara (Verbanaceae), induces healing action in incision and excision wound models in experimental rats. This effect involves faster epithelialization and a significant increase in breaking strength (Pravez and Patel, 2014). Studies have attributed the healing activity to triterpenic structured molecules. In fact, Asian acids, madecoside and asiaticoside, extracted from Centella asiatica, promote healing in experimental model of excision and incision wounds in healthy and diabetic guinea pigs (Shukla et al., 1999;Lee et al., 2012). Deoxyelephantopine, a sesquiterpene lactone isolated from leaves of Elephantopus scaber (Asteraceae) causes healing in excision and incision wounds in rat models (Singh et al., 2005;Lee et al., 2012).
In addition, ointment of C. glutinosum aqueous bark extract in petroleum jelly (CGPJ-10%), which showed a better healing effect, prevented croton oil-induced ear edema in mice. Olajide et al. (2003) reported the antiinflammatory activity of the methanolic leaf extract of C. micranthum (Combretaceae) in experimental carrageenan edema rat model. Recent studies have highlighted the anti-inflammatory activity of triterpene molecules (betulinic acid, cabralone), isolated from leaves of C. glutinosum (Sene et al., 2018).
The beneficial effect of the use of anti-inflammatories in wound and burn management has been widely demonstrated. Their use in the first phase of wound healing process promotes the reduction or control of inflammation. They also help accelerate healing and improve scar appearance (Rosique et al., 2015).
The healing activity of C. glutinosum trunk bark could probably be linked to the presence of tannins and sterols/ triterpenes, through a mechanism which essentially involves stimulating fibroblasts proliferation, accelerating reepithelialization and early control of inflammatory processes.