Exploring the Chemical Constituents, Antioxidant, Xanthine Oxidase and COX Inhibitory Activity of Commiphora gileadensis Commonly Grown Wild in Saudi Arabia

The use of the synthetic drugs has increased in the last few decades; however, these drugs exhibit various side effects. Scientists are therefore seeking alternatives from natural sources. Commiphora gileadensis has long been used to treat various disorders. It is commonly known as bisham or balm of Makkah. This plant contains various phytochemicals, including polyphenols and flavonoids, with biological potential. We found that steam-distilled essential oil of C. gileadensis exhibited higher antioxidant activity (IC50, 22.2 µg/mL) than ascorbic acid (IC50, 1.25 µg/mL). The major constituents (>2%) in the essential oil were β-myrcene, nonane, verticiol, β-phellandrene, β-cadinene, terpinen-4-ol, β-eudesmol, α-pinene, cis-β-copaene and verticillol, which might be responsible for the antioxidant and antimicrobial activity against Gram-positive bacteria. The extract of C. gileadensis exhibited inhibitory activity against cyclooxygenase (IC50, 450.1 µg/mL), xanthine oxidase (251.2 µg/mL) and protein denaturation (110.5 µg/mL) compared to standard treatments, making it a viable treatment from a natural plant source. LC-MS analysis revealed the presence of phenolic compounds such as caffeic acid phenyl ester, hesperetin, hesperidin, chrysin and transient amounts of catechin, gallic acid, rutin and caffeic acid. The chemical constituents of this plant can be explored further to investigate its wide variety of therapeutic potential.


Introduction
In the last few decades, the use of synthetic drugs has increased massively; however, the use of such products comes with undesired side effects. Scientists are therefore looking for alternatives from natural sources. Since time immemorial the traditional medicine practitioners have been using the mixture of herbs or mixture of plant extracts to treat various disorders [1]. Commiphora gileadensis is one such plant. It has been used for centuries to treat various disorders [2,3]. In Arab countries, C. gileadensis is traditionally known as balm of Makkah, basin and bisham [4]. It belongs to the Burseraceae family, in which there are around 190 species that belong to the genus Commiphora distributed in regions such as southern Arabia (Yemen and Oman); in northeastern African countries such as Somalia, Ethiopia, Sudan; and in subcontinental countries such as India and Pakistan [5][6][7]. Its resin is widely used in perfumes and, due to its medicinal properties, it is used in medicinal products to treat various disorders including arthritis, obesity, pain, and gastrointestinal and parasitic infections [7,8]. It is found in the Sarawat mountains in the west of Saudi Arabia. One study used an aqueous extract of C. gileadensis as an analgesic, diuretic and

Total Phenolics and Flavonoids
The total phenolics and flavonoids in 70% ethanolic extracts of C. gileadensis were 18.90 mg GAE/g extract and 59.41 mg QE/g extract, respectively (Table 3). Table 3. Total phenolics and flavonoids in ethanolic extracts of C. gileadensis.
The percentage of these compounds was low compared to the major compounds identified in monoterpenes. Dudai et al. [16] obtained similar findings regarding a high percentage of monoterpenes compared to sesquiterpenes.
However, there are other reports that contrast with the present study. The chemical components of the essential oil of the aerial parts and flower of C. gileadensis, collected in Makkah, Saudi Arabia, were dominated by sesquiterpenes and the absence of monoterpenes, with the exception of terpinene-4-ol (8.5% and 9.8%) [12]. A similar observation was observed in the essential oil of the stem and bark of C. gileadensis collected from Ein-gedi Gardens [17]. In contrast to our investigation, which was carried out on the aerial parts of C. gileadensis collected from Saudi Arabia, this variation in the chemical constituents may be due to time of collection, difference in the sample processing, or diversity among specimens of the same plant species.

LC-MS/MS Analysis
The targeted analysis of ethanolic extract of C. gileadensis revealed the presence of CAPE, hesperetin, hesperidin and chrysin, as well as small amounts of catechin, gallic acid, rutin and apigenin (Table 4). It also contained transient amounts of caffeic acid and myricetin. These phenolic compounds are involved in biological activity such as DPPH free radical scavenging, xanthine oxidase inhibition and inhibition of protein denaturation. The ethanolic extract displays moderate antibacterial activity against S. aureus. The extract did not exhibit appreciable antibacterial activity against E. coli (Figure 1) due to the presence of lipophilic compounds which cannot cross the Gram-negative bacterial cell wall.
Makkah, Saudi Arabia, were dominated by sesquiterpenes and the absence of monoterpenes, with the exception of terpinene-4-ol (8.5% and 9.8%) [12]. A similar observation was observed in the essential oil of the stem and bark of C. gileadensis collected from Eingedi Gardens [17]. In contrast to our investigation, which was carried out on the aerial parts of C. gileadensis collected from Saudi Arabia, this variation in the chemical constituents may be due to time of collection, difference in the sample processing, or diversity among specimens of the same plant species.

LC-MS/MS analysis
The targeted analysis of ethanolic extract of C. gileadensis revealed the presence of CAPE, hesperetin, hesperidin and chrysin, as well as small amounts of catechin, gallic acid, rutin and apigenin (Table 4). It also contained transient amounts of caffeic acid and myricetin.
These phenolic compounds are involved in biological activity such as DPPH free radical scavenging, xanthine oxidase inhibition and inhibition of protein denaturation. The ethanolic extract displays moderate antibacterial activity against S. aureus. The extract did not exhibit appreciable antibacterial activity against E. coli (Figure 1) due to the presence of lipophilic compounds which cannot cross the Gram-negative bacterial cell wall.

Antioxidant Activity
Antioxidants protect cells from the adverse effect of foreign molecules, drugs, carcinogenic substances and free radicals [18]. Antioxidants can reduce free radicals by scavenging it. Free radicals are generated in various metabolic processes and are associated with a number of stress-related diseases including cancer, diabetes, dementia and necrosis of the myocardial cells due to interaction with DNA, which causes causing mutation [18][19][20]. Extract of C. gileadensis showed antioxidant activity due to the presence of various chemical constituents such as polyphenols, flavonoids, catalase and oxidase. Therefore, plant-based products or mixtures that include polyphenols and flavonoids possess antioxidants that can eliminate free radicals and protect human health and wellbeing [21]. In the present study, antioxidant activity was analyzed using DPPH. Free radical scavenging activity was also evaluated using DPPH, and it was found that the ethanolic extract and essential oil showed potent antioxidant activity with IC 50 values of 56.5 ± 0.4 and 22.2 ± 0.5, respectively, compared to ascorbic acid (1.25 ± 0.05 µg/mL) which is a known antioxidant molecule. The presence of compounds such as α-pinene, β-pinene, γ-terpinene and terpinen-4-ol may have contributed to the antioxidant activity observed [22]. Ethanolic acid was also evaluated for inhibition using a β-carotene bleaching assay. The ethanolic extract showed appreciable activity with an IC 50 value of 75.8 ± 7.7 µg/mL compared to rutin (4.50 ± 0.35 µg/mL). This might be due to the presence of gallic acid, caffeic acid, rutin, hesperidin and other compounds present in ethanolic extract and the aerial parts of the tree. The phenolic compounds and flavonoids (gallic acid, caffeic acid and rutin) present in the ethanolic extract inhibit the denaturation of protein. The results are presented in the Table 5. The aqueous extracts of C. gileadensis leaf and twig have been shown to be successful in treating alloxan-induced diabetes in hypercholesterolemic male rats, where they restored all biochemical parameters to normal [23].

COX-1 Inhibitory Activity
Evaluation of COX activity is the basic strategy for developing NSAIDs to treat inflammation-related disorders. To test the COX-1 inhibitory effect of the ethanolic extract, a COX-1 inhibitory screening assay was performed using commercial assay kits (ab204698, Abcam, Tokyo, Japan) containing SC560 as a standard. The assay was performed as per the instruction provided with the kit. Different concentrations of ethanolic extract in 5% DMSO were used, with 5% DMSO used as a control. The preliminary screening results indicated that the ethanolic extract of C. gileadensis displayed COX-1 inhibitory activity at a concentration of 450 µg/mL. Under comparable conditions, the IC 50 of the standard compound SC560 was 5 ng/mL. It is possible that the CAPE, caffeic acid, gallic acid and other phenolic compounds are responsible for COX-1 inhibitory activity. More investigations would be required to evaluate COX-inhibitory activity on the fractionated ethanolic extract. Furthermore, NSAIDs' primary mode of action prior to Vane's discovery [24] of their inhibitory activity against cyclooxygenase is the suppression of protein denaturation [25]. Ben-Yehoshua et al. [26] have reviewed the COX-2 inhibitory activity of chloroform extract of C. gileadensis.

Xanthine Oxidase Activity
One of main characteristics of the ischemic injury is the overproduction of superoxide anion and the conversion of xanthine dehydrogenase to xanthine oxidase, which produces superoxide anions when xanthine is converted to uric acid [27]. Some natural products containing polyphenols have shown a dose-dependent inhibitory effect [28]. The flavonoids and phenolic acids present in plant products are potent inhibitors of enzymes such as cyclooxygenase, xanthine oxidase and lipo-oxygenase [29]. These enzymes control inflammation, hyperuricemia and gout. The chemical constituents gallic acid, rutin, caffeic acid and CAPE play important roles in the inhibition of xanthine oxidase. Inhibitors of xanthine oxidase and uricosuric agents are used in the treatment of diseases such as gouty arthritis and inflammatory diseases. At present, drugs such as allopurinol are used in the treatment of gout, but these synthetic drugs have various side effects [30]. Drugs with greater therapeutic values and lesser side effects are required. As the ethanolic extract of C. gileadensis contains flavonoids and phenolic compounds like rutin, apigenin, catechin, hesperetin, hesperidin, CAPE and caffeic acid, the xanthine oxidase activity of its ethanolic extract was investigated.
A literature survey on xanthine oxidase activity revealed no publications on the inhibitory activity of C. gileadensis in the treatment of xanthine oxidase and gout. The oil extracted from the aerial part of the plant exhibited significant xanthine oxidase activity. Inhibitory activity data is expressed as inhibitory concentration (µg/mL, Table 5). Wang et al. [31] reported that the caffeic acid, its derivatives and other flavonoids can inhibit xanthine oxidase.

Collection of Plant Material and Sample Preparation
The sample was collected in spring 2019 from the Badr area in Medina district, Al-Hijaz, Saudi Arabia. The aerial parts of the plant were washed, dried and ground for sample processing (Figure 2).

Extraction of Essential Oil
The essential oil of C. gileadensis was extracted from 100 g of the prepared sample in 500 mL of water by hydro-distillation for four hours in a Clevenger-type apparatus. After extraction, the essential oil was dried over anhydrous sodium sulfate and stored in lightresistant, inert glass tubes. The essential oil was kept at 2-8 ℃ until further investigation.

Total Flavonoid Content
Using the technique outlined by Al-Jaber et al. [32], the total flavonoid content in the ethanolic extract was determined calorimetrically using the reported technique without any modification. The flavonoid concentration was expressed as mg quercetin equiva- The ground aerial parts (0.25 kg) were soaked in 50% ethanol (2.5 L) with occasional shaking in two different containers for two days. The content was warmed briefly (60 • C) for 5 h and then filtered under a vacuum using a sintered glass funnel G2. The filtrate was evaporated collectively using a Buchi R-100 Rotary Evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland). The samples were then stored in liquid nitrogen for analysis.

Extraction of Essential Oil
The essential oil of C. gileadensis was extracted from 100 g of the prepared sample in 500 mL of water by hydro-distillation for four hours in a Clevenger-type apparatus. After extraction, the essential oil was dried over anhydrous sodium sulfate and stored in lightresistant, inert glass tubes. The essential oil was kept at 2-8 • C until further investigation.

Total Flavonoid Content
Using the technique outlined by Al-Jaber et al. [32], the total flavonoid content in the ethanolic extract was determined calorimetrically using the reported technique without any modification. The flavonoid concentration was expressed as mg quercetin equivalent/g of dry extract (mg QE/g).

Total Phenolic Content
The Folin-Ciocalteu method was used for measuring the total phenol content in the extract. Briefly, a mixture of 2.5 mL Folin-Ciocalteu reagent (2 N diluted tenfold) and 2 mL of Na 2 CO 3 solution were mixed with 0.5 mL of the extract. The resultant mixture was allowed to stand at room temperature for 15 min. The absorbance of the solution was determined at 765 nm using methanol as the blank solution. Then, total phenol content was given as mg gallic acid equivalent/g of dry extract (mg GAE/g). Each measurement was carried out three times [32].

GC-MS Analysis
The procedure outlined by Halub et al. and Naik et al. [15,33] using a Shimadzu QP2020 GC-MS (Shimadzu Corporation, Kyoto, Japan) supplied with a split-split less injector, was utilized to analyze samples and separate the volatile components using a DB5-MS fused silica column. Besides the published data, every chemical component's mass spectrum was compared to the corresponding reported spectra for GC-MS, with reference to the ADAMS-2007 and NIST 2017 mass spectrometry libraries. To ascertain the identified compound, a comparison was made between reported values and relative retention indices (RRI) in reference to n-alkanes (C 8 -C 30 ).

LC-MS/MS Analysis of the Extract
Analysis of flavonoids and phenolics compounds was performed using a SciEx UPLC (Exion-UPLC, USA) equipped with the LC-ESI-PDA-MS/MS-4500-QTRAP system (AB Sciex Instrument, Framingham, MA, USA), utilizing Analyst 1.7 software for data analysis. The chromatographic separation was conducted at 30 ± 1 • C using a Phenomenex column (3.0 × 50 mm, 5 µm). The elution gradient consisted of a mobile phase A (5 mM ammonium format in water: methanol (95:5; v/v)) and methanol (1 mM formic acid). The gradient program with the following proportions of solvent B was applied (%B, min): 5-90% B (0.00-8.00 min), 90-90% (8.00-12.00 min), 90-5% (12.01-15.00 min). The solvent flow rate was 0.35 mL/min and the injection volume was 5 µL. MS/MS analysis was performed in positive and negative ion mode. Nitrogen gas at a pressure of 60 psi was used as the nebulizing and drying gas. The mass spectra were obtained over an m/z range of 100-900 amu. According to Tulini et al. [33], measuring antioxidant activity involves establishing the amount of antioxidant needed to decrease the initial DPPH concentration to 50% (IC 50 ). A DPPH stock solution (0.002 percent w/v) in ethanol was prepared. As a serial dilution process, various ethanoic concentrations of the extract or essential oil (4-500 µg/mL) were prepared. DPPH solution (150 µL) was mixed in an ELISA plate with 150 µL of the sample at various concentrations. ELISA plates were incubated in the dark for 30 min until the color developed. Using a multi-mode ELISA reader (Synergy HTX, Biotek, CA, USA) the developed color was measured at 517 nm. DPPH radical scavenging activity was determined and the IC 50 was calculated using SigmaPlot ver. 11 [34]. % Free radical Scavenging activity = [Abs.control − Abs.sample] Abs.control × 100 (1)

β-Carotene Bleaching (BCB) Assay
A solution of β-carotene/linolenic acid was prepared by dissolving 10 mg of β-carotene as a stock solution into 100 mL of chloroform. In another flask, linolenic acid (25 mg) and Tween 20 (215 mg) were mixed and 3 mL of β-carotene solution was then added. The chloroform was evaporated by flushing with nitrogen gas. Next 75 ml of distilled water was added to the β-carotene/linolenic acid solution. Immediately after preparation, the absorbance of this solution was recorded at 470 and 700 nm. A standard antioxidant (rutin) solution was prepared by dissolving 3 mg of rutin in 10 mL methanol (300 µg/mL) as stock, and then various concentrations of rutin were prepared (0.6-19 µg/mL). Different strength solutions of extract (15-500 µg/mL) were prepared in methanol in an ELISA plate; 275 µL of β-carotene/linolenic acid solution was added to all the cells, each of which contained 25 µL of extract, methanol and rutin standard. All the solutions (control and test) were incubated (50 • C) for 1 h. The absorbance was taken at 470 nm and 700 nm at zero time, then after 1 h and 2 h. The control sample contained the equivalent amount of methanol. The degradation rate and antioxidant activity were calculated using the formula: Degradation rate of β-carotene = Ln (A initial/A Sample)/60 (2) Antioxidant activity (100%) = Degradation rate of control − degradation rate of sample degradation rate of sample × 100 (3)

COX-1 Inhibitory Activity
COX-1 inhibitory activity was measured using a COX-1 kit (ab204698, Abcam, Japan) as per the instructions provided by the supplier [35]. The samples were mixed with a cofactor, COX-1 enzyme and arachidonic acid. The enzymatic reaction was stopped by adding a 0.2 mL aliquot of 1 N HCl. The positive control for the reaction was SC-560.

Xanthine Oxidase Inhibitory Activity
Xanthine oxidase (XO) inhibitory activity was measured by observing the formation of uric acid in the xanthine oxidase system, as described in [36]. The assay kit consisted of 0.6 mL phosphate buffer (100 mM; pH 7.4), 0.1 mL sample, 0.1 mL XO (0.2 U/mL) and 0.2 mL xanthine (1 mM; dissolved in 0.1 N NaOH). For the analysis, the reaction was initialized by the addition of enzymes with or without inhibitors. Changes in the absorbance of the reaction mixture at 290 nm for 15 min were determined by comparing it with the absorbance of the reagent blank. The enzymatic reaction was stopped by adding a 0.2 mL aliquot of 1 N HCl. The positive control for the reaction was allopurinol.

Inhibition of Protein Denaturation
Inhibition of protein denaturation was estimated using the reported procedure [37] with minor modifications. Various solutions for the assay procedure were prepared: test solution, test control, product control and standard solution. All the required solutions were prepared using a pH 6.3 buffer. The samples were incubated for 20 min at 37 • C, then the temperature was raised to 50 • C and they were incubated for a further 15 min. After cooling, the absorbance was determined at 416 nm with a Synergy HTC multimode reader (BioTek, Winooski, VT, USA). The percent inhibition of protein denaturation was calculated using the given formula.

Antimicrobial Activity
Gram-positive (S. aureus ATCC 6538) and Gram-negative (E. coli ATCC 8739) bacteria were used to assess the ability of the extract and moxifloxacin (standard drug) to inhibit bacterial growth [38]. Sterilized nutrient agar was placed in a 9 cm sterilized petri disc. The plates were inoculated with bacterial culture using sterilized cotton swaps. Next, 6 mm holes were created with sterilized 6 mm surgical punches. The wells were filled with 40 µL of extract solution (250 µg/mL), essential oil and moxifloxacin standard solution. A 5% DMSO solution was used as a control. The plates were incubated at 37 • C for 24 h. Each experiment was performed in duplicate. Zones of inhibition were measured and are reported in Figure 1 and Table 6. Table 6. Antibacterial activity of the essential oil and ethanolic extract against E. coli and Staphylococcus aureus.

Sample Name
Inhibition Zone (mm)

Statistical Analysis
The results obtained in the present study are expressed as mean ± standard deviation (SD). For the statistical analysis of the experimental data, Graph-Pad Prism 5 (Graph-Pad Software, San Diego, CA, USA) was used.

Conclusions
It can be concluded that due to the presence of various chemical constituents such as gallic acid, rutin, apigenin, caryophyllene, α-pinene, β-pinene, γ-terpinene and terpinen-4ol, the C. gileadensis extract exhibited significant biological activity. The various chemical constituents present in the plant extract make it a viable natural resource that can be used in various pharmacological formulations. The plant extract showed significant biological activity, which contributes to its success as a traditional medicine used by traditional practitioners to treat various ailments including inflammation, pain and gouty arthritis. The chemical components can be explored further for their various biological and pharmacological benefits.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28052321/s1, Figure S1: GC-MS analysis of ethanolic extract of aerial parts of Commiphora gileadensis; Figure S2: GC-MS analysis of Essential oil collected from aerial parts of Commiphora gileadensis.