Wound Healing Potential of Commiphora gileadensis Stems Essential Oil and Chloroform Extract

Abstract

Essential oils (EOs) prepared from the fresh and dried stems of Commiphora gileadensis were compared qualitatively and quantitatively. Although the components were closely similar, the amount of oil decreased from 2.23 to 1.77% upon drying. Both samples showed equal potencies in the antimicrobial testing. The chloroform extract (CE) of the fresh stems with reported antimicrobial activity was compared with the EO sample of the fresh stems for wound healing potential. For the wound healing assay, 11 mm-diameter full-thickness skin excision wounds were made on the backs of four groups of rats (n = 6). The negative control group I was treated with the cream base. Group II was treated with 2% Fucidin cream, which served as a reference, and groups III and IV were treated with 1% EO- and 3% CE-containing creams, respectively. Treatments were applied topically one time daily. The wound healing potential was evaluated by recording the wound contraction percentages, epithelialization period, and histopathological changes of wounds. The topical application of CE significantly promoted the healing of wounds in rats. The effectiveness was demonstrated through the speed of wound contraction and the shortening of the epithelialization period in an animal treated with CE cream when compared to the NC group. Histopathological studies of the CE cream-treated group also expressed the effectiveness of CE in improving the wound healing process. These findings suggested that CE cream can enhance the process of wound healing in rats.

1. Introduction

The resins obtained from members of the genus Commiphora are used for the management of microbial infections, wounds, tumors, obesity, pain, inflammation, arthritis, gastrointestinal diseases, and fractures [1]. Plants of Commiphora gileadensis (synonymous with Commiphora opobalsamum) have many applications in traditional medicine [2]. The bark of the plant is used to treat infected wounds [3], a fact supported by the broad-spectrum antimicrobial activity against both Gram-negative, Gram-positive bacteria, and fungi [4,5]. C. gileadensis can disturb the bacterial lectin-dependent adhesion process essential for the survival of Pseudomonas aeruginosa. The traditional claims of the ability of balsam obtained from the plant to control infections were also supported by their ability to interfere with bacterial lectin-dependent adhesion [6]. The leaves’ methanol extract expressed antiviral activity against two enveloped viruses: HSV-2 and RSV B [7].

We recently reported on the isolation of four ent-verticillane-type diterpenes, namely ent-Verticillol, (13S,14S)-ent-13,14-epoxyverticillol, (9S,10S)-ent-9,10-epoxyverticillol, (1S,3E,7E,11R)-(+)-verticilla-3,7,12(18)-triene, as well as gileadenol with novel diterpene skeleton from the fresh C. gileadensis stems’ CHCl₃ extract. Four triterepenes were also isolated, and the antimicrobial potential of the isolated compounds was demonstrated [8].

The main goal of the current study was the evaluation of the wound healing potential of the different C. gileadensis extracts and the essential oil and the correlation of this effect with the antimicrobial activity.

2. Materials and Methods

2.1. Plant Material

The plants of Commiphora gileadensis (L.) C.Chr were described earlier [8].

2.2. Chemicals

Hematoxylin, eosin, Masson trichrome, petrolatum, and sorbitan monolaurate were purchased from Merck, KGaA, Darmstadt, Germany. Mueller–Hinton agar, Sabouraud dextrose agar, M_H broth, S-D broth, M_H agar, and S-D agar were obtained from Scharlau, Barcelona, Spain. Tween 80 was obtained from Chem Sino, China. All solvents used were of analytical grades.

2.3. Preparation of the Oils

Samples of 300 g of the fresh stems and 200 g of shade dried stems (obtained from drying 300 gm of fresh stems) of C. gileadensis were used to prepare the essential oil by hydrodistillation for 5 h using Clevenger apparatus. Fresh stems were cut into pieces about 2 cm long and were utilized for oil isolation, while the intact stems were dried in in the shade under a controlled temperature for two weeks, then ground and used for oil preparation. The resulting oil layers were separated, and the condensates were extracted with ether. The ether extract was added to the separated oil and dehydrated using anhydrous sodium sulfate. The ether was then evaporated under the reduced pressure of 350 m bar leaving the essential oil (EO). Each experiment was repeated three times.

2.4. GC/MS Analysis

Diluted EO samples (5 ppm) in methanol (1 uL of 5 ppm concentration) were injected (1 uL) into GC/MS apparatus (Agilent Model 7890 MSD) fitted with capillary column (30 m × 0.25 mm i.d., 0.25 μm coating) HP-5MS using the Autosampler and applying the splitless mode. The starting temperature was set at 60 °C for 10 min and raised at the rate of 4 °C/min until it reached 220 °C where it was held for 5 min. The temperature was raised again at a rate of 10 °C/min to 290 °C and was kept isothermally for 5 min. The carrier gas was Helium (99.999% purity) with a flow rate of 1.0 mL/min. Quadrupole MS analysis conditions were set to an electronic impact ionization mode at 70 eV with a mass range of 30 to 600 m/z. The components of the EO were identified by comparing the obtained mass spectra with that stored in the library of the National Institute of Standards and Technology (NIST 2017) (Figure 1). The results were analyzed and controlled by MASSHUNTER software (Agilent MassHunter Workstation Software-Quantitative Analysis program version B.04.xx, Agilent Technologies, Inc., Santa Clara, CA USA).

2.5. GC Analysis

The GC chromatograms were recorded on GC Agilent 7890B, fitted with an HP-5 19091J-413 capillary column (30 m × 0.25 mm) and FID detector using the same conditions of GC/MS analysis. The relative retention index (RRI) to n-alkanes series was applied for the identification of peaks. Computerized peak areas were used for the quantitative determination of each compound.

2.6. Extraction

Fresh stems (5.8 kg) were macerated in CHCl₃ at room temperature (10 L × 5) to yield 117.5 g of the CHCl₃ soluble extract (CE) after the solvent was evaporated using a rotary vacuum under it to reduce pressure. The fresh stems were then similarly extracted with MeOH (10 L × 5) to yield 226.7 g of the MeOH soluble extract (ME). Extraction of the fresh leaves (2.3 kg) was performed in the same fashion and provided 32.2 g of the CHCl₃ soluble extract and 90.25 g of the MeOH soluble extract.

2.7. Antimicrobial Activity

2.7.1. Bacterial Strains

American Type Culture Collection (ATCC) strains and National Collection of Type Culture (NCTC) maintained in the Microbiology Laboratory at the College of Pharmacy/Prince Sattam University (Al-Kharj- Saudi Arabia), Bacillus subtilis and Staphylococcus aureus, Escherichia coli, and Klebsiella pneumonia, as well as the fungus Candida albicans, were used in the study. The strains were grown aerobically at 37 °C. The suspension of organisms equivalent to a 0.5 McFarland standard was utilized.

2.7.2. Antimicrobial Assay

The MIC of the tested materials was measured following the broth dilution method adopted by the Clinical and Laboratory Standards Institute guidelines [9]. Both EO and CE were prepared in 10 mg/mL, where serial dilutions were prepared. The testing was performed in the range of 50–3.125 µg/mL. From each tested organism, 10 µL of the culture was inoculated with the used concentrations. Sterility was assured by the incubation of Mueller–Hinton broth (MHB, Scharlau) alone. Negative control results were obtained by the incubation of MHB with different concentrations of DMSO. After the incubation period of 24 h at 37 °C, the lowest concentration that inhibited visible microbial growth was designated as the MIC [9,10].

2.8. Evaluation of Wound Healing Activity

2.8.1. Experimental Animals

In this study, 24 healthy male Wistar rats weighing 180–200 g were experimented on. Rats were bred and housed in the lab animal unit, College of Pharmacy; University of Prince Sattam bin Abdulaziz in ventilated cages (Rat IVC Blue Line, Techniplast, Buguggiate VA, Italy). The animals were maintained in controlled environmental conditions (25 ± 1 °C and 12 h/12 h light/dark cycle) with food and water ad libitum. The care and handling complied with the internationally accepted guidelines for use of animals 33. Furthermore, the animal experiments were approved by the Bioethical Research Committee (BERC) at Prince Sattam bin Abdulaziz University (ref No. BERC-008-04-21).

2.8.2. Preparation of Creams

The topical creams were prepared by melting petrolatum (21.16 g) in a water bath at 70 °C. Tween 80 (2 g) was dispersed in the oil phases, respectively, and 0.5 g of EO or 1.5 g of EC were added. Quantities of glycerol (4.67 g) were mixed together accordingly with the aqueous phase composed of water (17.17 mL) and sorbitan monolaurate (5 g). The oily phase was added to the aqueous phase slowly with continuous stirring at 500 rpm using a Kenwood kitchen mixer. After the addition of the oil phase was completed, further mixing for another 5 min was applied before the cream was allowed to set [11].

2.8.3. Experimental Design

Creams of essential oil (EO) and fresh stems (CE) of C. gileadensis were evaluated for their wound healing potential in rats using the excision wound model [12]. Twenty-four rats were anesthetized using ketamine hydrochloride (5 mg/kg i.p.) and xylazine (2 mg/kg i.p.). In the dorsal area of each rat, the skin was shaved by an electrical clipper and disinfected with 70% alcohol. A uniform wound of 11 mm in diameter was excised from the shaved region of each rat with the aid of sterile toothed forceps and sharp pointed scissors (Figure 2). Rats were randomly grouped into four groups, with 6 rats/group.

• Group 1: Negative control group (NC); treated with the plain cream base topically.
• Group 2: Reference group (REF); topically with 2% Fucidin cream.
• Group 3 and 4 were treated with either 1% EO or with 3% CE creams, respectively.

Different treatments were distributed topically over the wound area once a day starting from the day of wounding (day 0) until the complete healing of wounds was achieved. The wound area was assessed every 4 days by drawing its borders with the help of a transparent sheet. These wound drawings were retraced on a sheet of 1 mm² graph paper. The wound areas were obtained by counting the squares [13].

The percentage reduction of wound contraction was calculated based on the initial wound area [14].

$$\% \mathrm{Wound} \mathrm{Contraction}=\frac{\mathrm{wound} \mathrm{area} \mathrm{on} \mathrm{day} 0-\mathrm{wound} \mathrm{area} \mathrm{on} \mathrm{day} \mathrm{n} }{\mathrm{wound} \mathrm{area} \mathrm{on} \mathrm{day} 0}\times 100$$

where n = number of days (4th, 8th, 12th, 16th, and 20th day).

The epithelialization period was calculated as the period of days required for the scab to fall off and leave no raw wounds behind [15].

2.9. Histopathological Examination

At the end of the study, tissues from the wounded area were collected in 10% buffered formaldehyde and prepared in an automatic tissue processing machine (ASP300s, Leica Biosystems, Deer Park, IL, USA). After that, tissue samples were soaked in paraffin wax blocks, and sections of 5 µ thickness were prepared using a rotary microtome (SHUR/Cut 4500, TBS, Durham, NC, USA) [16]. Two sections of each block were stained either by hematoxylin and eosin (H&E) or Masson trichrome (MT) [17]. For hematoxylin and eosin, stain sections were dewaxed and rehydrated with descending grades of alcohol to water. The sections were stained in hematoxylin (HX082464, MERK, Darmstadl, Germany) for 10 min, washed with running tap water until the sections were ‘blue’ for 5–10 min, then stained in 1% eosin Y for 1–3 min and washed with running tap water for 1–3 min. Sections were then dehydrated with alcohol and mounted in DPX [18]. Masson trichrome techniques were used according to Hamad et al., (2016) [17].

2.10. Data Analysis

The data are presented as mean ± SEM. Analysis of the results was performed by SPSS version 19 to apply a one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison tests. Graphical representation was carried out using Microsoft Excel 2010. The differences between mean values were considered significant at p < 0.05.

3. Results and Discussion

3.1. Preparation of the Oil and GC-MS Study

The average yield of EO from the fresh and dried stems of C. gileadensis showed a 0.46% loss in the dried sample (

Table 1. Yield of essential oil from fresh and dried stem samples of C. gileadensis.

Condition	Weight (g)	Weight of Oil (g)	% w/w
Fresh	300.00	6.69	2.23
Dried	200.00	3.55	1.77

). Regarding the oil composition revealed by GC-MS analysis, little difference was found in the number of compounds (

Table 2. Components of fresh and dried stem samples essential oil of C. gileadensis.

Components	RT	RRI	Area %
			Fresh	Dry
β-Pinene (1)	9.7833	978	62.974	4.462
Eugenol	26.7184	1365	5.834	35.366
Caryophyllene	28.7625	1444	4.543	34.469
Humulene (2)	29.8557	1460	-	1.718
cis-Calamenene	32.0615	1533	12.29	0.687
Isoeugenol acetate	32.2167	1618	1.926	0.483
1S,3E,7E,11R)-(+)-verticilla-3,7,12(18)-triene (3)	63.1821	2040	11.374	20.638
Total			98.941	97.823

). However, great changes were observed in the percentage of the components. It was noticed that the lighter monoterpene components, such as β-Pinene (1) (Figure 1), dramatically declined from 62.974% in the oil obtained from the fresh plant samples to 4.462% in the oil derived from the dried samples. Components with higher molecular weights were expected to be less volatile, and consequently, their relative percentages increased in the oil obtained from the dried samples. For example, Eugenol percentage increased from 5.834% in the fresh oil samples to 35.366% in the essential oil samples prepared from the dried plants (Table 2). The diterpene hydrocarbon 1S,3E,7E,11R)-(+)-verticilla-3,7,12(18)-triene (3) was identified via direct comparison with the isolated compound [8], and its percentage increased to 20.638 in the essential oil of the dried samples.

3.2. Antimicrobial Activity

Two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), two Gram-negative bacteria (Escherichia coli and Klebsiella pneumonia), and the fungus (Candida albicans) were utilized to study the antimicrobial effect of the EO obtained from the fresh and dried stems. The activity of the CE and methanol extract (ME) of the fresh stems of C. gileadensis was previously reported [8]. The MIC was determined for all of the tested materials (

Table 3. MIC (mg/mL) of different essential oil and different extracts.

	Staph. aureus	B. subtilis	E. coli	k. pneumonia	C. albicans
Fresh Stem Oil	0.75	0.75	0.5	0.5	0.25
Dried stem Oil	0.75	0.75	0.5	0.5	0.25
Fresh stem CHCl3 Ext.	0.75	0.75	0.75	0.25	0.125
Fresh stem MeOH Ext.	75	25	75	50	25
Fresh Leaves CHCl3 Ext.	0.75	0.5	0.75	0.25	0.125
Fresh Leaves MeOH Ext.	75	25	75	25	75

). The two essential oil samples were active against all of the tested organisms with equal potencies, although the yield of the oil was less in the cases of dried samples, and the relative percentages of the components were different. This indicated that the effect is due to combined action of all the components [19,20]. The CE expressed stronger activity against k. pneumonia and C. albicans than the EO.

3.3. Wound Healing Activity

The wound healing properties of C. guidottii and C. myrrha have been reported, while C. gileadensis was mentioned in the early Islamic era to be used by the Prophet and his companions for the treatment of wounds [21,22]. As the wound healing effect can be correlated to the antimicrobial activity, both EO and CE of the fresh stem sample were selected for the wound healing study. Both products were formulated as creams [11] containing 1% EO and 3% CE.

In the experiment for the evaluation of the wound healing activity of EO and CE, all animals survived, and no complications related to the procedure were observed. The sizes of wounded areas, percentages of wound contraction, and the periods of epithelialization are represented in Figure 3, Figure 4, Figure 5 and Figure 6. On day 0, the wound areas in all groups were almost similar, and there was no significant difference between different groups. The topical application of EO and CE creams on the wounds of rats reduced their areas in comparison to the NC group. The group treated with Fucidin cream had a smaller wound size than that of the other tested creams (Figure 3).

In this study, the percentages of wound contraction were calculated on the 4, 8, 12, 16, and 20 post-wounding days, as shown in Figure 4. Wound contraction facilitates the re-epithelialization of the wounded skin and helps in restoring the function of skin as a physical barrier. The topical application of the reference Fucidin cream on the wounds resulted in a significant increase in the wound contraction rate when compared to the NC group. Significant effects were also observed for the groups treated with EO and CE creams compared to the NC group (Figure 4). Interestingly, the percentages of wound contraction were significantly higher in CE-treated rats, reaching 96.49 ± 1.71 and 100.0 ± 0.00% after 16 and 20 days, respectively (Figure 4). In EO-treated rats, 77.37 ± 2.43 and 92.63 ± 1.51% of wound contractions were recorded after 16 and 20 days of topical application, respectively.

The rates of epithelialization (in days) were also evaluated in wounded rats, with epithelialization being the proliferation and migration of epithelial cells across the wound. The time for compelling this process is an important parameter to evaluate the wound healing potential. The group treated with the cream base took a longer time (30.67 ± 2.36 days) to achieve epithelialization (Figure 5). The mean time taken for complete epithelialization in groups treated with EO and CE creams was reduced. Moreover, the mean healing time in CE-treated rats (17.50 ± 1.50 days) was comparable to that of the reference Fucidin cream-treated group (14.67 ± 1.86 days).

3.4. Histopathological Study

Histopathological examination using Mayer’s hematoxylin stain and Masson trichrome technique was used as a general parameter for the evaluation of wound healing potential. Masson trichrome develops a blue color with collagen, giving an indication about fibrosis of the examined tissues [23]. The histological examination of skin samples of the NC group showed multiple areas of tissue damage, degeneration, and a wide area of necrosis (Figure 6A). Furthermore, the skin of NC rats showed multiple areas of lost collagen fibers (Figure 7A). Skin samples of the REF group showed normal skin tissue samples (Figure 6B) with normal contents and distribution of collagen fibers (Figure 7B). Skin samples of the EO-treated rats showed moderate improvement (Figure 6C) with areas of lost collagen (Figure 7C). The skin of the CE-treated rats showed much improvement (Figure 6D) and an almost normal content of collagen fibers (Figure 7D).

Our recent phytochemical study on the CE of the fresh stems resulted in the isolation of diterpenes and triterpenes, all with significant antimicrobial activity [8]. EO lacks these components due to their higher molecular weight. This can explain the stronger effects of the CE in the antimicrobial testing and as a wound healing promoter compared with the EO.

4. Conclusions

The process of drying had a great impact on the yield and percentage of components of the EO prepared from the fresh and dried stems of C. gileadensis. Antimicrobial testing using Gram-positive and Gram-negative bacteria in addition to pathogenic fungus (Candida albicans) indicated that the EOs of the fresh and dry stems were equally active. The CE of the fresh stems was more active than the EO, while the ME was almost inactive.

The present study results suggest that CE and EO creams enhance wound healing in rats. The CE cream was more effective and comparable with that of Fucidin cream, as shown by the reduction in the percentage of wound contraction, reduction in the period of epithelization, and the improvement in the skin histopathological parameters. Further, the wound healing efficacy of CE seems to be correlated with its antimicrobial effect. Our findings suggest that CE cream is valuable for the treatment and management of wounds.