Chromatographic analysis (LC-MS and GC-MS), antioxidant activity, antibacterial activity, total phenol, and total flavonoid determination of Cleome arabica L. growing in Jordan

ABSTRACT The pivotal Cleome arabica L. species belongs to the Cleome genus with fascinating secondary metabolites consisting of main classes of phenols, flavonoids, and glucosinolate derivatives. The current study was carried out on three crude extracts of Cleome arabica L. plant of Jordanian origin. In terms of the valuable resources for natural new drug development. The phytochemical contents, antioxidants, and antibacterial activities, in addition to the oil composition of this species were identified. Forty-six compounds representing 96.98% of the total composition were detected from the essential oil obtained by hydrodistillation (0.06%). The oil composition was dominated by nitrogen-sulfur-containing compounds (35.66%), esters (19.64%), and oxygenated sesquiterpenes (11.92%). Butanol extract had significantly the best scavenging activities of DPPH (P < .0001), ABTS (P < .0001), and hydroxyl radicals (P < .0001) while the Aq.MeOH extract exhibits the highest flavonoid content (173.5 ± 0.002 mg Quercetin/g dry extract) and had significantly the most ferrous chelating effect (P = .0011). Also, most of the tested extracts showed antibacterial activities against bacterial strains. Our findings exhibited that the Cleome arabica L. plant from Jordan origin might be a good candidate as potential biologically compounds that could be used as a source of natural antioxidants and antibacterial agents.


Introduction
Plants, in general, were used for many decades in the treatment and prevention of various dietary and pathogen-related diseases. Several studies showed that the biological activities including antioxidant, antimicrobial, anticancer, anti-inflammatory, and antidiabetic activities of these plants are related to the presence of phenols, flavonoids, alkaloids, terpenoids, carotenoids, vitamins, and tannins. [1] These compounds showed hydrogen donators capacity, free radical scavengers, and metal chelating potential. Also, they have been known as antimicrobial agents. [2,3] Cleome, known as a spider flower, is the largest genus of the Cleomaceae family comprising about 200 species spread around the world, especially in North Africa. [4,5] Cleome species have traditionally been known for their medicinal treatments against earache, skin diseases, rheumatism and headache. [6,7] Phytochemical screening studies carried out on Cleome species revealed the presence of different important secondary products including flavonoids, saponins, coumarins, terpenoids, phenolics and alkaloids. [7][8][9] The

Preparation of crude extracts
Freshly plant samples were air-dried at room temperature for a month. Then, it was ground to a powder and the fatty acids were removed using petroleum ether (40-60°C) in Soxhlet extraction. The remaining plant material was dried and then extracted with methanol. A rotary vacuum evaporator was used to concentrate the methanol extracts and dried them. Then, the dried extract was separated between CHCl 3 and H 2 O (1:1 v/v) solvent system. After that, the dried CHCl 3 fraction was separated between 10% aqueous methanol and hexane. n-butanol was used to extract the polar organic compounds. Thus, the obtained three extracts (10% aqueous methanol, n-butanol and water) were directly used for testing.

Essential oil extraction
The essential oil from fresh aerial parts of the Cleome arabica L. plant was extracted following the protocol described in the literature. [11,25] Briefly, small pieces of fresh aerial parts of the Cleome arabica L. plant (200 g) were hydrodistillated for 4 hours using a Clevenger-type apparatus. Then, anhydrous sodium sulfate was used for drying the oil. Then, the oil is directly stored in GC-grade n-hexane at 4°C for (GC-MS) analysis.

LC-MS analysis of phytochemicals
LC-MS analysis procedure was conducted according to Abu-Orabi et al. [26] Briefly, A Bruker Daltonik (Bremen, Germany) Impact II ESI-Q-TOF System equipped with Bruker Dalotonik Elute UHPLC system (Bremen, Germany) was used for screening flavonoids and phenolics compounds of interest in both positive (M + H) and negative (M-H) electrospray ionization modes. Briefly, Chromatographic separation was conducted using a 120, C18 reversed phase column [100 x 2.1 mm, 1.8 µm (120 A°)] at 30°C, autosampler temperature 8.0°C with total run time 20.0 min using gradient grade. The elution gradient consisted of mobile phase A (water/methanol (90:10%) with 5 mM ammonium formate and 0.1% formic acid)) and solvent B (methanol with 5 mM ammonium formate and 0.1% formic acid). MS/MS analysis was performed in negative ion mode with an ion spray voltage of −4,500 V.

Gas chromatographic analysis of the essential oils
Chemical extractions procedure was described by Al-Qudah et al. [27] Briefly, 1 μL extracted oil diluted in 10.0 μL GC grade n-hexane was analyzed by GC-MS (Model Varian Chrompack CP-3800 GC/MS, Saturn, Netherlands) system equipped with a DB-5 GC capillary column (5% diphenyl, 95% dimethyl polysiloxane, for quantitative analysis, Hewlett-Packard HP-8590 GC equipped with optima-5 column (5% diphenyl, 95% dimethyl polysiloxane) (30 m × 0.25 mm, 0.25 μm film thickness) and a splitsplitless injector (split ratio 1:50) was used. The relative percent concentrations of the detected compounds were calculated using relative peak areas. A hydrocarbon mixture of n-alkanes (C8-C20) was analyzed separately under the same chromatographic conditions. Essential oils with different chemical constituents were identified by comparing their mass spectra with those found in the database library (Wiley 275 library, New York, USA).

Phytochemical screening
The phytochemical screening of butanol, Aq. MeOH and water extracts from Cleome arabica L. were performed using a standard procedure. [28]

Total phenolic contents (TPC) and total flavonoid contents (TFC)
Follin-Ciocalteu method and aluminum chloride assay were conducted to quantify TPC and TFC, respectively. [27,29] For TPC analysis, 0.5 mL of the extract was mixed with 2.5 mL of 0.2 N Follin-Ciocalteu reagent and 2 mL of Na 2 CO 3 (75 g/L). The absorbance of the final solution was then recorded at 765 nm wavelength. The TPC content of the plant was represented as (mg GA/g dry extract). For TFC analysis, aliquots of (1 mL) extracts of (1 mg/mL) concentration were taken in different volumetric flasks (10 mL). Then, 4 mL of distilled water was added to each flask, followed by the addition of 0.3 mL of sodium nitrite (5% NaNO 2 , w/v). After 15 min, the absorbance was measured at 510 nm. The TFC content for different extracts of the plant was represented as (mg quercitin/g dry extract). Methanol was used as blank.

Antioxidants activity
Antioxidant activity of the Cleome arabica L. plant was investigated for butanol, Aq. MeOH and water crude extracts. DPPH, ABTS, FIC and Hydroxyl radical (HO•) scavenging assays were used to determine the antioxidant activities of the extracts according to the literature. [27,29] IC 50 value, the concentration of the substrate that causes 50% loss of the radical activity, was used for the interpretation of the results from all used methods. The IC 50 values were calculated using the linear regression method of the tested crudes. Measurements were performed in triplicates. Percentage of inhibition in all assays was calculated as: Scavenging effect (%) = (Ac -AS/Ac) ×100.
where Ac represents the absorbance of the blank and As represents the absorbance in the presence of the extract.

Antibacterial activity
The antibacterial activity of the Cleome arabica L. extracts was evaluated using both gram-positive and gram-negative bacterial strains (provided by ATCC). Gram-positive bacteria included Staphylococcus aureus (ATCC 29213), which was used as standard and Staphylococcus aurus (BAA-41), which was used as a resistant isolate. Whereas gram-negative bacteria included Escherichia coli (ATCC 25922), which was used as a standard and Escherichia coli (BAA-2452), which was used as a resistant strain. Broth micro-dilution method with some modifications was used to determine the Minimum inhibitory concentration (MIC) of the Cleome arabica L. extract against susceptible bacteria as described by Kuete et al. [30] Briefly, each extract was dissolved in 10% (DMSO) and diluted in MHB to reach a 100 mg/mL concentration. After that, 50 µl of inoculums (standardized to 1 × 10 5 CFU/ mL by adjusting the optical density to (0.08-0.13) at 600 nm) was added into a 96-well plate containing 50 µl of different concentrations. The bacterial culture (10% DMSO and broth) without the plant extract was considered as a positive control, while the culture containing the broth-only was considered as a negative control. The plates were incubated at 37°C for 24 h. For the minimum bactericidal concentration (MBC) determination, 10 µl from each well was added to 90 µl of phosphate buffer saline (PBS) and diluted eight times. Then 10 µl of each well was placed on MHA medium and incubated at 37°C for 24 h. The lowest concentration exhibited no growth was corresponded to MBC.

Statistical analysis
Data were analyzed using PC SAS (v. 9.2; SAS Institute, Cary, NC, USA). Mean and standard error were calculated for each antioxidant and fraction. One-way ANOVA (analysis of variance) was used to assess significant differences between different methods of the extraction at different concentrations at α = 0.05. A post-hoc Tukey-Honest test was used to estimate pairwise comparisons for significant results from the One-way ANOVA. Graphs of different parameters were made using Graph Pad Prism 7. Linear Pearson correlation for each of butanol, methanol, and water extracts was conducted between total phenolic content and antioxidant activity. R Gui version 4.0.3 was used to visualize the correlation matrix and pair-wise scatter plots using the chart correlation function.

LC-MS profiling
The results of the LC-MS/MS analysis of Cleome arabica L. are shown in (Table 1). LC-MS analysis of the three extracts of the plant revealed the presence of 30 compounds in different amounts. The most abundant detected compounds were isoorientin, hesperidin and nevirapine. The relative amount (%) of each compound in butanol, Aq. MeOH and water extracts were, respectively: 47.33, 31.29 and 11.08 for isoorientin, 18.40, 4.45 and 25.40 for hesperidin and 2.49, 11.90 and 18.36 for nevirapine. In comparison with other studies, four compounds including β-sitosterol, 11-α-acetylbrachy-carpone -22(23)-en, 17-α-hydroxycabraleactone and amblyone were detected in Cleome arabica L. from Tunisia. [19] The variation in the content of the same species possibly depends on the plant's origin (the site from where the plant sample was collected).

Chemical composition of essential Oil of Cleome arabica L. growing in Jordan
The chemical profile of the oil retention indices (RI) and the percentage area under the GC peak of the oil constituents are shown in (Table 2). A total of 46 compounds representing 96.98% of the total composition were investigated. The principal components of the plant oil were 2-Methyl butyl isothiocyanate (27.11%), ethyl dodecanoate (16.57%), isopropyl isothiocyanate (6.94%) and   2-Methyl-(3E)-octen-5-yne (6.48%). The detected oil constituents of the plant were classified as follows: monoterpene hydrocarbons (1.29%), oxygenated monoterpenes (4.87%), sesquiterpene hydrocarbons (4.79%), oxygenated sesquiterpenes (11.92%), nitrogen-sulfur containing compounds (35.66%), esters (19.64%), one carboxylic acid (2.75%) and other compounds (16.06%). Essential oils are mixture of natural products that give plants their smell and flavor. Several Cleome species from different origins have been analyzed by many researchers for their essential oils composition. For example, it was reported that oxygenated diterpenes and (Z)-Phytol were the major compounds of the oil isolated from Indian Cleome rutidosperma DC. [31] Essential oils analysis of Cleome amblyocarba Barratte & Murb, Cleome rupicola vicary and Cleome ramosissma Webb ex Parl. grown in Saudi Arabia exhibited their enrichment with a variety of compounds dominated by isothiocyanate and cubenol. [11,12] Also, it was reported that the essential oils of two Jordanian Cleome species namely, Cleome droserefolia and Cleome trinervia were rich with terpenoids and sulfur and nitrogen containing compounds. [12] The chemical composition variations of the essential oils present in different Cleome species could be attributed to the plant's origin, which represents several factors affecting the plant chemical composition, such as climatic change, genetic variability, seasonal variation and the nature of the soil. The presence of high amount of sulfur-nitrogen containing compounds in the Cleome arabica L. plant is supported by the presence of glucosinolates that can produce nitrile and isothiocyanate compounds via hydrolysis. [12] It is worth mentioning that this is the first study analyzing the essential oil composition of Cleome arabica L. plant in Jordan.

Phytochemical analysis of Cleome arabica L extracts
Plants contain a wide variety of phytochemical compounds including phenols, flavonoids and terpenoids. In this study, the phytochemical screening of the Aq. MeOH and butanol extracts from Cleome arabica L. plant revealed the presence of tannins, flavonoids, saponins and glycosides, whereas, the water extract from the same plant contained only saponins and glycosides. However, the phytochemical tests applied on the three crude extracts showed the absence of the alkaloids, anthraquinones and terpenoids (Table 3). In previous studies, several extracts including methanol, ethanol, acetone and butanol have been used for plant extraction [32,33] exhibiting differences in the content of phytochemical compounds and antioxidant activities among them. These differences could be related to the influence of plant materials by the solubility of solvent, degree of phenols polymerization and the interaction of phenol compounds with other plant constituents. [34] Our data of phytochemical analysis showed an agreement with the studies reported that Cleome arabica L. plant from different origins contain several types of phytochemical compounds . [9,19,35] In addition, our data showed that there are qualitative and quantitative differences in the phytochemical compounds detected in the three extracts (Aq. MeOH, butanol and water) from the same Cleome arabica L. plant. This can be attributed to the differences in the polarity of the extraction solvents, which could bring a wide variation in the levels of the extracted bioactive compounds. [36]

Total phenolic and total flavonoid contents
Total phenolic content (TPC) of the three extracts were identified using Folin-Ciocalteu method and calculated as (mg gallic acid (GA)/ g dry weight of extract). The total phenolic content was calculated using the following regression equation: Y = 2.00 × 10 −3 X -2.98 × 10 −2 , R 2 = 0.9698 (based on the calibration curve) where Y is the absorbance at 765 nm and X is the TPC in the extract. Table 3 shows the amount of TPC in the three extracts of Cleome arabica L. The results showed that butanol extract had the highest TPC (539.46 ± 12.00 × 10 −3 mg GA/g dry weight), then Aq. MeOH (321.8 ± 9.00 × 10 −3 mg GA/g dry weight), and the lowest TPC was determined in the water extract (189.46 ± 21.00 × 10 −3 mg GA/g dry weight). Total flavonoid content (TFC) of the three extracts was determined using the aluminum chloride assay and calculated as (mg Quercetin/g dry weight of extract). TFC was identified according to the following regression equation based on the calibration curve. Y = 3.00 × 10 −4 X + 3.92 × 10 −2 , R 2 = 0.9956, where Y is the absorbance at 510 nm and X is the TFC in the extract. The obtained results showed that Aq. MeOH extract had the highest content of flavonoid (173.5 ± 2.0 × 10 −3 mg quercetin/g dry weight), then butanol (146.9 ± 5.0 × 10 −4 mg quercetin/g dry weight), whereas water extract had the lowest content of flavonoid (4.66 ± 1.0 × 10 −3 mg quercetin/g dry weight) ( Table 4). Variation in the amount of phenolic and flavonoid compounds between extracts could be attributed to the differences in solubility and polarity of solvents used during extraction. [37]

Relationship between phenolic content and antioxidant activity of butanol, Aq.MeoH and water extracts
The relationship between the total phenolic content and the scavenging % of DPPH, ABTS and HO radicals and FIC effect showed that there was a strong positive linear correlation between them. The correlation coefficients (R) for DPPH, ABTS, HO and FIC were, respectively: 0.79, 0.65, 0.90 and 0.69 in butanol extract (Figure 3a (Figure 3c). Phenolic and flavonoid are major compounds that exhibit a wide spectrum of chemical and biological activities including radical scavenging properties. [38] Our results showed that there is a positive linear correlation between phenolic content of extracts and their antioxidant activities and these results agree with several studies that reported that there is a strong positive linear relationship between high content of phenolic compounds and their biological activities of plants. [9,31,39]

Antibacterial activity
The phenomenon of bacterial resistance to antibiotics leads to the emergence and spread out of diseases for which no treatment yet exists. So, the search for novel antibacterial agents from medicinal plants has become a highly relevant and important subject for the research. [40] In this study, the effectiveness of the three extracts (Aq. MeOH, butanol, and water) of Cleome arabica L. plant from Jordan against four bacterial strains was determined using microdillution method. The results showed that all the tested extracts had antibacterial activity against gram-positive bacteria (Staphylococcus aureus) at 25 and 50 mg/mL compared with the positive control (+ve) ( Tables 5, Tables S1 and S2). On the other hand, butanol and water extracts showed antibacterial activity at 50 mg/mL against gram-negative bacteria (Escherichia coli) ( (Tables 5,Tables 3S and 4S). The lower sensitivity of gram-negative than gram-positive bacteria toward the Cleome arabica L. extracts may be due to the cell wall impermeable to foreign substances (antibacterial agents) which is composed of structural lipopolysaccharides. Moreover, gramnegative bacteria exhibit multiple efflux pumps preventing the intracellular accumulation of antibacterial agents. [40] Thus, the antibacterial activity could be associated with levels of tannins present. Tannins present  in Cleome arabica L. extracts may react with the bacterial proteins forming a stable water-soluble compound resulting in killing the bacterial cells through a direct damage that occurs to their cell membranes. In general, the antibacterial activity of the Cleome arabica L. extracts is based on their contents of phenols, flavonoids, tannins, and glycosides. [40,41]

Conclusion
In this study, the analysis of chemical composition of essential oil isolated from Jordanian origin Cleome arabica L. plant lead to identify 46 compounds dominated by esters and sulfur-nitrogen containing compounds. The major ones were 2-Methyl butyl isothiocyanate, Ethyl dodecanoate, Isopropyl isothiocyanate and 2-Methyl-(3E)-octen-5-yne. Phytochemical screening of different extracts from aerial parts of Cleome arabica L. revealed their enrichment with tannins, flavonoids, saponins, and glycosides. All tested extracts showed the presence of phenolic and flavonoid compounds but with different quantities. In addition, all extracts showed radical scavenging potential and ferrous chelating effect with little differences in the scavenging percentage among them. Also, most of the tested extracts showed antibacterial activities for both gram-negative and gram-positive bacterial strains. To the best of our knowledge, this is the first study analyzing the essential oil and identified the phytochemical constituents and antioxidant and antibacterial activities of Cleome arabica L. grown in Jordan. However, more research has to be conducted to isolate the bioactive compounds of essential oil and extracts to determine their potential for therapeutic uses against different diseases and to synthesize natural antioxidants and antibacterial agents.