Bioactive Components Identification and a Pilot Health Properties Study of Tribulus terrestris Linn: A Native Saudi Arabian Plant

Received: 01-Apr-2024, Manuscript No. JDAR-24-135973; Editor assigned: 03-Apr-2024, Pre QC No. JDAR-24-135973 (PQ); Reviewed: 17-Apr-2024, QC No. JDAR-24-135973; Revised: 22-Apr-2024, Manuscript No. JDAR-24-135973 (R); Published: 29-Apr-2024, DOI: 10.4303/JDAR/236291

Introduction

Materials and Methods

Chemicals

These chemicals were obtained from Sigma-Aldrich Co., (USA); DPPH, ABTS, Folin- Ciocalteau reagent, Gallic acid, Quercetin, BHT, Trolox, Ferrozine, and phenolics standards Compounds. The others’ reagents and detergents were also informed in each analytical process according to the instrument used.

Plant collection

In May 2022, we collect Tribulus terrestris L. plant from the Al- Leith area of Saudi Arabia. Dr. Abdallah A. ALFeel from the Department of Arid Land Agriculture, King Abdulaziz University did the laboratory identification and recognized the studied taxonomy of the plant. A testimonial instance (TT52022) was deposited at Umm Al-Qura University’s Chemistry Department, Faculty of Applied lores, Al-Leith University College. We apply the plant material to drying the sun system before grinding it into a fine by using mortal and thinking in an electrical grinder for preparation the powder for the extraction process. The experiments steps in detail in this study are demonstrated in Figure 1.

Figure 1: Experimental design

Plant extraction

We use Hexane: Ethyl acetate (2:1, v/v) and methanol (150 mL) to obtain 2 separate extracts. the grind Tribulus terrestris L. leaves (15 g per solvent) were used in each extract process. We used an orbital shaker (Stuart, England) for vortex the extraction solutions for 12 hours at room temperature at 160 rpm. We use Whatman No. 1 filter paper to filter the obtain extracts separately.

We wash the plant residues and 2 additional times using 150 mL of each fresh solvent for each one separately. We use rotary evaporator (Heidolph Unimax 2010, Germany) to concentrate the obtained filtrates separately. The yielding extracts were Hexane: Ethyl Acetate Extract (HEAE) and Methanol Extract (MeE) [18,19].

Phytochemical analysis

Estimation of Total Phenolics (TP): To determine the TP concentrations in HEAE and MeE, we combined 1 mL of the extract with 10 mL of distilled water and 1 mL of Folin reagent. We waited for 5 minutes, then we added 10 mL of 8% Na₂CO₃ to the mixture. We then added 8 mL of distilled water and vortexed the mixture for 15 seconds before putting them in dark incubation for 90 minutes at 25°C ± 2°C [19]. We determined the optical density of color, the measuring components spectrophotometrically at 750 nm as compared to a blank sample. We expressed the values of TP as mg Gallic Acid Equivalents (GAE)/g, D.W. We conducted 3 assessments for each sample [19].

Estimation of Total Flavonoids (TF): To determine the TF concentrations in HEAE and MeE, we combined 1 mL of the extract with 4 mL of distilled water and 0.3 mL of NaNO₂ (5%). After waiting for 5 min, we added 0.3 mL of 10% AlCl₃ to the mixture. After waiting for an extra 6 minutes, we added 2 mL of NaOH (1M) and 2.4 mL distilled water, then vortexed the mixture for 15 seconds before allowing it to stand for 10 minutes at 25°C ± 2°C [18]. We determined the absorbance of the yield mixture at 510 nm compared to a blank sample. We calculate the amount of TF as mg Quercetin Equivalents (QE)/g, D.W. We apply 3 measurements for each sample [18].

HPLC phenolic profile of MeE: We vortexed 10 mg of MeE in 2 mL methanol for 15 min., then filtrated it using A 0.2 μm Millipore membrane filter. We injected the filtrate (5 μL) into an HPLC (Agilent Technologies 1260 series, Germany) utilizing its auto-sampling injector. We isolated phenolic compounds at a constant temperature of 40°C ± 2°C and 0.9 mL/min flow rate using a 4.6 mm x 250 mm Eclipse C-18 column with a particle size of 5 μm. A mixture of water (A) and acetonitrile with 0.05% trifluoroacetic acid (B) was used as the mobile phase, with a linear gradient of 0 min (82% A), 0 min-5 min (80% A), 5 min-8 min (60% A), 8 min-12 min (60% A), 12 min- 15 min (82% A), and 15 min-20 min (82% A). The DAD detector was checked at 280 nm [24]. The concentration of each specific phenolic component (mg/g of dry weight) is calculated by comparing its relative peak area with the reference standards such as Gallic acid, Chlorogenic acid, Catechin, Methyl gallate, Coffeic acid, Syringic acid, Pyro catechol, Rutin, and Ellagic acid from Sigma-Aldrich (Steinheim, Germany) [24,25].

Fatty acid and hydrocarbon analysis of HEAE

Fatty acid analysis by GC-FID: The former technique of Zahran and Tawfeuk was utilized to produce the Fatty Acid Methyl Esters (FAMEs) of HEAE. Hexane (1.0 mL) was added to 15 mg of the extract, and then 0.4 M sodium methoxide (1.0 mL) was added [26]. After 30 sec vortex, the mixture was allowed to settle for 15 minutes. The FAMEs were separated for gas chromatography (GC-FID) analysis from the upper phase. FAMEs were examined using a Perkin Elmer Auto System XL fitted with a ZBWax (capillary column, 60 mm x 0.32 mm) and a flame ionization detector (FID). We used helium as a carrier gas at one milliliter per minute flow rate. The oven was set to start at 50°C and increase in temperature by 4°C per minute. The injector and detector were adjusted to a temperature of 250°C [26].

Hydrocarbons analysis by GC/MS: The HEAE lipoidal matter was prepared by saponification, which involved refluxing 50 mg of the extract with 10% alcoholic potassium hydroxide for 5 hours in a water bath at 100°C ± 5°C. After evaporating the solvent, the precipitate was stirred with 50 mL of water. By using a separating funnel, the mixture was fractionated with ether (4 mL ÃÃÂ??? 80 mL). The pooled ethereal fractions were cleaned from any alkalinity using distilled water, then dried up over Na₂SO₄ anhydrous. The solvent was concentrated using rotary, and the hydrocarbons (unsaponifiable substances) were analyzed using GC/MS spectroscopy [27]. The GC/MS comprises an Agilent 8890 gas chromatography equipped with an Agilent 5977B mass spectrometer and HP-5MS capillary column (30 m (L), 0.25 mm (ID), and 0.25 mm thickness). The temperature of the oven started at 50°C, then scheduled to increase by 5°C per minute until 280°C, and then held at that temperature for 25 minutes. We used helium as a carrier gas at one milliliter per minute flow rate. Using a split ratio of 1:50, hydrocarbons (1 μL) was injected automatically in the GC at 230°C. Mass spectra was scanned using the Electron Impact (EI) mode at 70 eV with scan range of m/z=39 amu- 500 amu. The split peaks were detected by relating them to data from the NIST (National Institute of Standards and Technology) collection [27].

Antioxidant activity

DPPH• radical scavenging assay: After preparing 0.1 mM of DPPH• (2,2- diphenyl-1-picryl hydrazyl) in methyl alcohol, 1 mL of DPPH solution was added to 1 mL of HEAE and MeE at strengths of 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL. After 15 sec vortex, the mixture was kept standing at 25°C ± 2°C in a dark area for 30 min. The readings were shown at 515 nm compared to a control sample [3]. All mixture components except for the extract were used in the control sample. The positive control was Butyl hydroxytoluene (BHT). We calculated the capacity to scavenge the DPPH• radical using this formula: Scavenging impact (%) of

Where Ac: The control reading; As: The sample reading. The findings were expressed as IC₅₀ (the sample’s concentration in mg/mL that has a 50% DPPH• radical scavenging effect) [3].

ABTS•+ antioxidant assay: Using the 2, 2’-azinobis- 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) test, which was detailed by previous investigation [22]. The scavenging activity of HEAE and MeE was determined. Stalks of ABTS (7.4 mM) and potassium persulphate (2.6 mM) were freshly prepared using distilled water. The 2 stock solutions were then combined in identical parts to create the functioning solution. They were left to react at 25°C ± 2°C in a dark area for a duration of 12 hours to 16 hours. Then we mixed the functioning solution (1 mL) with methanol (60 mL) to make the ABTS solution, with a reading of 1.1 ± 0.02 at 734 nm by spectrophotometer. 150 μL of HEAE/MeE at concentrations of 250 μg/mL, 500 μg/mL, 750 μg/mL, and 1000 μg/mL was mixed with the ABTS solution (2850 μL), then kept standing for 2 hours at 25°C ± 2°C in the dark. The readings were shown at 734 nm compared to a control sample. All mixture components except for the extract were used in the control sample. The positive control was Trolox. We calculated the capacity to scavenge the ABTS radical using this formula:

Where Ac: The control reading; As: The sample reading. The findings were expressed as IC₅₀ (the sample’s concentration in mg/mL that has a 50% ABTS radical scavenging effect) [22].

Ferrous ion (Fe²⁺) chelating assay: Three milliliters of HEAE/MeE at various concentrations (0.75 mg/mL, 1 mg/ mL, 1.25 mg/mL, 1.5 mg/mL) were mixed with 60 μl of 2 mM FeSO₄. To initiate the reaction, 100 μl of 5 mM ferrozine was added, the mixture mixed, and maintained standing for 10 min at 25°C ± 2°C. Using spectrophotometry, the mixture readings were calculated at 562 nm compared to a control sample [28]. All mixture components except for the extract were used in the control sample. The positive control was EDTA. We calculated the capacity to inhibit ferrozine-Fe²⁺ complex formation using this formula:

Where Ac: The control reading; As: The sample reading. The findings were expressed as IC₅₀ (the sample’s concentration in mg/mL that inhibits 50% of ferrozine-Fe²⁺ complex formation) [28].

Cytotoxicity activity

Three human carcinoma cells: HepG-2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), and HeLa (cervical carcinoma) were bought from the American Type Culture Collection (ATCC, Rockville, MD) [23].

Cell line propagation: The cells were propagated on Dulbecco’s Modified Eagle’s Medium (DMEM) involving gentamycin (50 μg/mL) and inactivated fetal bovine serum (10%). The cells were persistent by sub-culturing up to 3 times weekly and kept at 37°C in an environment with 5% CO2 [23].

MTT cytotoxicity assay: In Corning® 96-well tissue culture plates, the tested cell lines (5 x 104 cells/well) were suspended in the medium, and the plates were incubated for 24 hours. After aspirating the media, new medium with varying doses (0.25 μg/mL-500 μg/mL) of dissolved HEAE and MeE in DMSO was applied to the cells. Each plate included 6 well controls, each of which was run with media or DMSO. The positive control was vinblastine sulfate (PubChem CID 5388983) as a standard chemotherapy cytotoxic agent against cancer cell as vinblastine sulfate blook cell division. The MTT test was used to count the live cells after a 24-hour incubation period, in accordance with the procedure outlined by previous investigation [23]. In brief, we removed the media from the plates and added 100 μl of fresh, phenol red-free medium. We added 10 microliters of 12 mM MTT (in PBS) to all the wells, including the control wells, and incubated the plates for 4 hours at 37°C ± 2°C with 5% CO₂. After that, we removed 85 μl of medium from each well and added 50 μl of DMSO. We mixed the solution using a pipette and incubated the plates at 37°C for 10 minutes. We estimated the number of viable cells by measuring the absorbance readings of the wells at 590 nm with a microplate reader [23].

We calculated the viability (%) using this formula:

where ODc is the control absorbance reading and ODs is the sample absorbance reading. The results were expressed in terms of IC₅₀, which is the concentration (in μg/mL) of the extract that prevents 50% of contact cancer cells from growing [23].

Antimicrobial activity

In this study, the following germs were tested: Staphylococcus aureus (ATCC-47077) and Bacillus cereus (ATCC-12228) an example of Gramâ?ÂÃÂ?positive bacteria, Escherichia coli (ATCC-25955) and Proteus vulgaris (ATCC-13315) an example of Gram-negative bacteria, filamentous fungus Aspergillus niger (ATCC- 16888), and yeast specie of Candida albicans (ATCC-10231). HEAE and MeE’s antimicrobial activity was evaluated using a modified agar well diffusion technique [29]. In summary, 10 milliliters of fresh medium were cultured with 100 microliters of the test bacteria or fungus till 108 cells/mL of bacteria and 105 cells/mL of fungus are reached. Then we smeared 100 microliters of the bacterial culture onto nutrient agar plates to test their susceptibility using the well diffusion method. We hit holes with a diameter of 6 millimeters in the agarose gel and added 100 μl of the extract at doses of 2.5 mg/mL, 5 mg/mL, and 10 mg/mL to the well. The Petri dishes were incubated for 24 hours-48 hours at 37°C for bacteria and yeast and for 48 hours at 28°C for filamentous mold fungi. We measured the inhibition zones in millimeters (mm) as a scale to determine the efficiency of the antimicrobial effect. Since DMSO exhibited no inhibitory zone against any of the examined microbes, it was utilized as a negative control and to dissolve the tested extracts. Additionally, positive controls were performed using conventional medications such as gentamycin, an example of an antibacterial drug, and ketoconazole, an example of an antifungal agent [29].

Statistical analysis

Every test was run in triplicate. The means ± Standard Deviation (SD) of the data is presented. Using the SPSS, software program (version No. 20), and one-way ANOVA, the values were expressed as the mean ± SD at the 0.05 significance level.

Results

Phytochemical analysis

In this work, we extracted bioactive components from Tribulus terrestris L. leaves using 2 distinct solvents: Hexane/ethyl acetate (2:1, v/v) and methanol. The polar solvent, methanol, had a maximum extraction yield of 11.63%, while hexane/ethyl acetate yielded 1.77%. The Methanol Extract (MeE) of Tribulus terrestris Linn possessed the greatest quantities of total phenolics (7.12 mg GAE/g ± 0.09 mg GAE/g, d.w) and total flavonoids (4.62 mg QE/g, d.w ± 0.08 mg QE/g, d.w), compared to 0.31 ± 0.003 (mg GAE/g, d.w) of total phenolics and 0.1 ± 0.002 (mg QE/g, d.w) of total flavonoids in the Hexane/ Ethyl Acetate Extract (HEAE) as presented in Figure 2.

Figure 2: Total phenolics (TP, mg GAE/g, d.w) and total flavonoids (TF, mg QE/g, d.w) of Hexane/Ethyl Acetate Extract (HEAE) and Methanol Extract (MeE) of Tribulus terrestris Linn. Data are expressed as Mean ± SD (n=3). Different letters for the same parameter are significantly different at p<0.05

The individual phenolic and flavonoid components of the MeE were evaluated by HPLC-DAD. A total of 90 phenolic compounds were found in the MeE (Table 1). The main ones include the flavonoids hesperetin (2321.29 μg/g, d.w), quercetin (1770.84 μg/g, d.w), and daidzein (686.23 μg/g, d.w), and the phenolic acids chlorogenic acid (323.50 μg/g, d.w), cinnamic acid (230.94 μg/g, d.w), and gallic acid (127.72 μg/g, d.w), Figure S1 shows HPLC chromatogram of Tribulus terrestris L. methanol extract, signals at 280 nm (Figure 3).

Table 1: HPLC-DAD phenolic profile of Tribulus terrestris Linn Methanol Extract (MeE)

The fatty acid composition and hydrocarbons composition of HEAE was detected and presented in Table 2. Six fatty acids in total were detected using GC-FID, and the main fatty acids found in HEAE were palmitic acid (C16:0), linoleic acid (C18:2n6c), and oleic acid (C18:1n9c) in quantities of 19.25%, 28.52%, and 35.82%, respectively. Five hydrocarbons were detected in HEAE using GCMS analysis, and the most abundant of which is squalene (39.5%), phytol (28.56%), and tetracosanol (19.99%), Figure 4 shows GC/MS chromatogram of Tribulus terrestris L. hexane/ethyl acetate extract hydrocarbons.

Table 2: Fatty acid composition (%) and hydrocarbons composition (%) of Tribulus terrestris Linn Hexane/Ethyl Acetate Extract (HEAE)

Figure 3: The chemical structures of the main analytes were identified using the HPLC-DAD technique

Figure 4: GC/MS chromatogram of Tribulus terrestris hexane/ethyl acetate extract hydrocarbons

Antioxidant activity

Using 3 different assays (DPPH•, ABTS•+, and Fe⁺² chelation), we assessed the Tribulus terrestris L. extract’s in vitro antioxidant activity. The findings were reported as IC₅₀ values. Since the IC₅₀ value and antioxidant activity are inversely correlated, analyte antioxidant activity increases with decreasing IC₅₀ value. The MeE exhibited the greatest antioxidant capacity in all 3 experiments, with IC₅₀ DPPH 0.138 mg/mL, IC₅₀ ABTS 0.287 mg/mL, and IC₅₀ Fe⁺² chelation 1.293 mg/mL. In comparison, the HEAE had IC₅₀ DPPH 0.174 mg/mL, IC₅₀ ABTS 0.721 mg/mL, and IC₅₀ Fe⁺² chelation 1.525 mg/mL. Figure 5 show individual (DPPH•, ABTS•+, and Fe⁺² chelation) scavenging activity (%) of Hexane/Ethyl Acetate (H/E) and Methanol (Me) extracts of Tribulus terrestris L. and Methanol (Me) extracts of Tribulus terrestris L.

Figure 5: A) Antioxidant activities (IC₅₀, mg/mL), DPPH scavenging, ABTS scavenging, and Fe⁺² chelation effects, of Hexane/Ethyl Acetate Extract (HEAE) and Methanol Extract (MeE) of Tribulus terrestris Linn. Data are expressed as Mean ± SD (n=3). Different letters for the same parameter are significantly different at p<0.05; B) DPPH• scavenging activity (%) of Hexane/Ethyl Acetate (H/E) and Methanol (Me) extracts of Tribulus terrestris Linn. Data are expressed as Mean ± SD (n=3). Different letters for each extract (MeE and HEAE) individually are significantly different at p<0.05; C) ABTS•+ scavenging activity (%) of Hexane/Ethyl Acetate (H/E) and Methanol (Me) extracts of Tribulus terrestris Linn. Data are expressed as Mean ± SD (n=3). Different letters for each extract (MeE and HEAE) individually are significantly different at p<0.05; D) Fe⁺² chelation activity (%) of Hexane/Ethyl Acetate (H/E) and Methanol (Me) extracts of Tribulus terrestris Linn. Data are expressed as Mean ± SD (n=3). Different letters for each extract (MeE and HEAE) individually are significantly different at p<0.05

Cytotoxicity activity

Using MTT assay, we assessed the Tribulus terrestris L. extract’s in vitro cytotoxicity efficiency against HepG- 2, MCF-7, and HeLa cancer cells as compared with Vinblastine sulfate as a standard. The findings were reported as IC₅₀ values. Since the IC₅₀ value and cytotoxic effect are inversely correlated, analyte cytotoxic effect increases with decreasing IC₅₀ value. The HEAE exhibited the most potent cytotoxicity against the HepG-2, MCF-7, and HeLa cell lines, with IC₅₀ values of 46.71 ± 1.43, 61.38 ± 1.97, and 75.41 ± 2.31 μg/mL, respectively. In comparison, the MeE showed IC₅₀ values of 103.79 ± 3.08, 107.57 ± 3.29, and 157.52 μg/mL ± 5.83 μg/mL for the same cell lines (Figure 6). As shown by the lowest IC₅₀ values, the HepG-2 cell line is the most sensitive to Tribulus terrestris L. extracts followed by MCF-7, then HeLa.

Figure 6: Cytotoxic effect (IC₅₀, µg/ml) of Hexane/Ethyl Acetate (H/E) and Methanol (Me) extracts of Tribulus terrestris Linn against HepG-2, MCF-7, and HeLa cancer cell lines as compared to Vinblastine sulfate standard. Data are expressed as Mean ± SD (n=3). Different letters for each extract MeE and HEAE, Vinblastine sulfate individually are significantly different at p<0.05

Antimicrobial activity

We assessed the in vitro antimicrobial effectiveness of Tribulus terrestris L. HEAE and MeE versus several bacteria, including S. aureus, B. cereus, E. coli, and P. vulgaris, as well as the fungus A. niger and yeast C. albicans, by employing the agar well diffusion method. The results were expressed in terms of the inhibition zone (mm). In general, HEAE and MeE demonstrated modest bactericidal activities depending on concentration but had no influence on the growth of the fungus A. niger (Table 3). The maximum growth inhibition against S. aureus, E. coli, P. vulgaris, and C. albicans was demonstrated by the HEAE at 10 mg/mL, with inhibition zones of 7.5 mm ± 0.3 mm, 8.5 mm ± 0.4 mm, 12.8 mm ± 0.3 mm, and 6.5 mm ± 0.1 mm, respectively. P. vulgaris, a gram-negative bacterium, is the most sensitive microbe to Tribulus terrestris L. HEAE and MeE.

Table 3: Antimicrobial effect of Hexane/Ethyl Acetate Extract (HEAE) and Methanol Extract (MeE) of Tribulus terrestris Linn

Discussion

Several herbs, wild and edible, have many different medical properties depending on their multiple range of bioactive components and benefits phytochemical. Medicinal plants are used global in traditional prescriptions worldwide in healing or preventing several human illnesses. Furthermore, the components, effects, and quantity of bioactive constituents that are separated and identified from medicinal plants are extremely dependent on the extraction and the solvents composition. Phenolic compounds and their glycosides, for example, are extract using polar solvents, whereas steroids and fatty acids are extracted using nonpolar solvents [29,30]. In this study, the polar solvent, methanol, was found to have a maximum extraction yield of 11.63%, which is higher than that of Tribulus terrestris L.’s aqueous (10%) and methanolic (9.3%) extracts [31]. Numerous investigations have reported the effects of many solvents on the quantity, effect, and composition of bioactive recovered from several medicinal plants [19, 30]. The maximum extraction yield of highly polar solvents, such as methanol, reveals that the crude extracts contain a high quantity of phenolic substances [30, 32].

The findings of current work indicate that the Methanol Extract (MeE) of Tribulus terrestris L. contained higher amounts of total phenolics and total flavonoids than the Hexane/Ethyl Acetate Extract (HEAE). The current findings agree with previous results investigation, who also reported that the methanol and ethanol extracts of Tribulus terrestris L. includes the highest levels of total phenolics and total flavonoids compared to the hexane extract [33]. The adequate quantities of total phenolics and total flavonoids of Tribulus terrestris L. collected from the Saudi Arabian Al-Leith region in current study are higher than that of Tribulus terrestris L. collected from the Saudi Arabian Najran region [7] and are comparable with that of Tribulus terrestris L. collected from various other countries [14,22,31,33,34].

In current investigation, methanol is the most proper solvent for phenolic and flavonoid extraction from Tribulus terrestris L. Given that the extract of phenolic compounds varies based on their structural characteristics, the HPLC analysis output indicated the presence of flavonoids hesperetin, quercetin, and daidzein, as well as phenolic acids chlorogenic acid, cinnamic acid, and gallic acid as the essential source phenolics in the MeE. These findings are comparable with recent studies that identified several flavonoids, phenolic compounds, and flavonoid glycosides in Tribulus terrestris L. from several geographical areas [14,22,35,36]. The geographic location, method of extraction, plant part utilized, and solvent utilized have a substantial impact on the concentrations of phenolics and flavonoids [14,22,30,32].

In the current study, we determine that hexane/ethyl acetate is the most suitable solvent for extracting fatty acids and hydrocarbons from Tribulus terrestris L. The key fatty acids identified in HEAE were palmitic acid (C16:0), linoleic acid (C18:2n6c), and oleic acid (C18:1n9c). The current results agree with those of previous finding, who demonstrated” that “the key fatty “acids detected in Tribulus terrestris L. collected from various areas in Turkey were linoleic acid, oleic acid, and palmitic acid [33]. In contrast to the previous finding who identified the principal fatty acids found in Tribulus terrestris L. as oleic, pentadecanoic acid, 9,12-octadecadienoic acid, 6,9,12,15-docosatetraenoic acid, and palmitic acid [37]. In the present experiment results, GC-MS analysis characters a group of beneficial contents such as squalene, phytol, and tetracosanol as the main hydrocarbons in the unsaponifiable fraction of HEAE of Tribulus terrestris L. Otherwise, previous investigations using GC-MS analysis have also revealed that Tribulus terrestris L. contains phytol and squalene, along with other hydrocarbons [7,38]. The variations seen in the fatty acid contents and hydrocarbon composition of Tribulus terrestris L. between this study and recent publications might potentially be referred to several factors such as the geographical climates, the extraction technique, method, utilized solvents, and the plant part. Research has shown that the chemical composition is closely related to the plant’s place of origin and, consequently, to the climate, that diversity give the studied plant different benefits, antibacterial, anti-cancer, and anti-oxidant [15,29,39].

The endogenous human antioxidants system is powerful and unfortunate for the current inactive lifestyle and Western diet. It is crucial to admit that human body health well-being needs more exogenous antioxidants from diet such as vegetables, fruits, and herbs. Antioxidants have a powerful capacity to protect and prevent cellular oxidative stress, decline DNA mutation, and keep human Healthline on wealthy activities, particularly from disorders connected to oxidative stress [39]. Therefore, assessing Tribulus terrestris L. extracts’ antioxidant capacities is considered to understand how effective the plant is in preventing and treating oxidative stress-related issues. The MeE showed the highest antioxidant capacity when tested using 3 different assays (DPPH•, ABTS•+, and Fe⁺² chelation) in comparison to HEAE. This can be attributed to the significantly higher quantities of phenolic and flavonoid compounds present in the MeE as reported in the current results. These findings are agreed upon with the previous study of [33]. They revealed that the methanol and ethanol extracts of Tribulus terrestris L., which contain high amounts of polyphenolics, showed stronger DPPH antioxidant properties in comparison to the hexane extract. There is genuine data demonstrating the significant relationship between plant extracts’ phenolic and flavonoid amounts and their antioxidant efficiency [23,30,32]. Numerous studies have indicated that different extracts of Tribulus terrestris L. showed antioxidant capacities using many tests such as DPPH, reducing power, ABTS, and FRAP [14,22,33,35,36]. This study found that MeE and HEAE exhibited their antioxidant activity by 2 different mechanisms: Metal chelating, as shown by the Fe⁺² chelation test, and electron or hydrogen atom transfer, as shown by the DPPH and ABTS analysis. The antioxidant influence of the extract is derived from the synergistic impact of the phytoconstituents, which is dependent on their concentration as well as their structure and interaction [23,32]. Because of their redox characteristics, phenolic compounds display antioxidant activity via a variety of potential pathways, including the decomposition of peroxides, neutralization and absorption of free radicals, transition of metal chelating activity, and/or hunting of singlet and triplet oxygen [39]. These findings suggest that Tribulus terrestris L. extracts, with their potent antioxidant activity, might be adequate choice for developing natural, safe, and healthful antioxidants for use in food preservers as heathy additive and pharmaceutical applications such as anti-inflammatory and anti-cancer [36,39,40].

Cancer is one of the most painful deadly causes of mortality worldwide. One of the cancer treatments is chemotherapy such as vinblastine sulfate, which has painful side effects on the healthy cell, tissue, and organ of the patient’s body [40,41]. In spite of several trails for select treatment methods without side effects, there is still a need for more and selectivity problems with chemotherapy medications, despite enormous attempts to develop effective ones. Therefore, several searches for new innovative medicines for cancer cells but not to healthy cells are concerning many researchers. Now, researchers are currently searching for new, safe, and valuable cytotoxicity agents derived from natural medicinal herbs [40]. As a result, testing Tribulus terrestris L. extracts for cytotoxicity activities are important to determine how effective the plant is in inhibiting cancer proliferation. The HEAE of Tribulus terrestris L. in the current study exhibited the strongest cytotoxic effect against HepG-2, MCF-7, and HeLa cell lines, compared to MeE. The present findings are comparable with the previous results that reported the cytotoxicity effects of Tribulus terrestris L. different extracts against different cancer cells including HepG2 cells, MCF-7 cells, cell lines of A2780 (ovary), HT29 (colon), and MCF7, cell lines of NCIADR/ RES (ovary), OVCAR-03 (ovary), U251 (glioma), 786-0 (kidney), PC-3 (prostate), NCI-H460 (lung), HT-29 (colon), and MCF7, and cell lines of SK-OV-3 (ovary), NCI-H522 (lung), HeLa, and MCF-7 [11,40,41,43]. The effects of HEAE in current study on MCF-7 and HeLa with IC₅₀ values of 61.38 μg/mL, and 75.41 μg/mL, respectively, are comparatively higher than those of methanol and 70% aqueous methanol extracts, with IC₅₀ values of 74.1 μg/ mL and 176.4 μg/mL and 221.2 μg/mL and 343.1 μg/mL, respectively [44]. The HEAE that exhibited the highest cytotoxic impact in current investigation was also the one with the lowest quantities of total phenolics, total flavonoids, and antioxidant effects. Therefore, the current outcomes reflect those bioactive substances other than phenolics, hydrocarbons of phytol and squalene or saponins, impact Tribulus terrestris L. cytotoxicity findings. In similar results previous report found an cytotoxicity effect of Tribulus terrestris L. saponin-enriched extract, attributing the action to the presence of gitogenin, protodioscin, and diosgenin saponins in the extract [43]. Previous researchers report that phytocomponents other than phenolics, such as stigmasterol and sitosterol, may be affected and result in the cytotoxicity action of Tribulus terrestris L. extract [11]. In addition, previous study reported that one of the phytocomponents affected the biological activity of Tribulus terrestris L. extract [44]. Tribulus terrestris L. extracts have antiinflammatory, antioxidant, and cytotoxicity impacts, may be due to the saponins of protodioscin and physcion [44]. Furthermore, many investigators surveyed the cytotoxicity activities of phytol and squalene hydrocarbons that agree with current results [45,46].

The current study also investigates the effect of Tribulus terrestris L. extracts as anti-bacterial. Therefore, bacteria resistance and infection are widely elevated due to many factors such as increased antibiotics used, climate change, sedentary lifestyle, and diet, which poses a severe threat to global public health. Therefore, continuing search for new innovative medicines as antibacterial is concern medical society. Herbs may yield a new, powerful source of antimicrobial activity, and a demand focus to phytochemicals and separate the biologically active components are commonly used in ancient and recent herbal medicine [29,47]. So, current work concerns reveal a pilot survey to study the effectiveness of the plant in preventing infectious disorders of Tribulus terrestris L. extracts through tested for antibacterial activity of several individuals’ microbes. In the current study, HEAE and MeE of Tribulus terrestris L. demonstrated modest bactericidal activities depending on concentration but had no influence on the growth of the fungus A. niger. The HEAE exhibited the maximum growth inhibition against S. aureus, E. coli, P. vulgaris, and C. albicans at 10 mg/mL, with inhibition zones of 7.5 ± 0.3, 8.5 ± 0.4, 12.8 ± 0.3, and 6.5 ± 0.1 mm, respectively. According to previous report that the methanol extracts of Tribulus terrestris L. fruits and leaves showed effective antibacterial and antifungal activity against the tested microbes [34]. Their impact against S. aureus was higher than the current HEAE and MeE results, with diminish zones of 17 mm ± 1.1 mm and 18 mm ± 0.86 mm, respectively. With inhibition zones of 19.23 mm and 14.04 mm and 19.88 mm and 14.60 mm, respectively, other study also reported that methanol and aqueous extracts of Tribulus terrestris L. leaves presented significant antibacterial effect against S. aureus and E. coli [31]. These impacts were superior to the current HEAE and MeE results. The antibacterial influences of Tribulus terrestris L. HEAE in this study may be because of bioactive other than phenolics, such as fatty acids or hydrocarbons like phytol and squalene, the main contents were identified in the HEAE. These findings disagree with previous studies, they attributed the antibacterial effects of Tribulus terrestris L. to the presence of phenolics and flavonoids only [14,34]. Several investigations have been conducted on the antibacterial qualities of phytol and squalene, which provide a confirmation to our theory regarding their implications in the antibacterial activity of HEAE [46,48]. Furthermore, there are many of data that points to the possibility concern study plant bioactives have the capacity to interrupt the construction of the bacterial cytomembrane, elevates its permeability, fluidity, membrane protein delocalization, and other antibacterial activity-related activities [49]. According to the current findings, Tribulus terrestris L. leaf extracts may be acceptable natural medication to replace or reduce the usage of antibiotics in bacterial illnesses.

In summary, Tribulus terrestris L. is considered as a member of Zygophyllaceae. The ancient plant is used in many traditional medicinal cultures such as Saudi Arabia, Chinese, Indian, and European traditions for many health benefits. Different studied extracts of T. terrestris reflect a content of antioxidants phytochemical components with anti-inflammatory activities, cytotoxicity, and antimicrobial that agree with current results [40-49]. Tribulus terrestris L. consider as great promise as a source of antioxidants with applications in both food and medicine. Its bioactive constituents make it an exciting candidate herb for further research.

Conclusion

The author concluded that the effect of primerary data Tribulus terrestris L. from the Al-Leith area of Saudi Arabia as the antioxidant, cytotoxicity, antimicrobial due to phytochemicals components. The author confirm presence of bioactive phenolic of Tribulus terrestris L. such as hesperetin, quercetin, daidzein, chlorogenic acid, and cinnamic acid, among the ninety phenolics in the Methanol Extract (MeE). In addition, the study reports the presence of hydrocarbons such as phytol and squalene, the fatty acids linoleic acid, and oleic acid in the Hexane/ Ethyl Acetate Extract (HEAE). We also assessed MeE antioxidant activity, as demonstrated by DPPH, ABTS, and Fe⁺² chelation and reported a significant antioxidant activity. The author concluded that HEAE had the strongest cytotoxicity activity against cancer cells HepG-2, MCF- 7, and HeLa. In addition, the HEAE had the strongest antibacterial effectiveness versus S. aureus, B. cereus, E. coli, P. vulgaris, and C. albicans. These findings suggest that Tribulus terrestris L. extracts as an excellent option for establishing natural, safe, and effective alternatives for study and use in food and pharmaceutical applications because of their strong antioxidant, cytotoxicity, and antibacterial impacts, that need further exploration in progress in author next investigation steps.

Acknowledgement

The author is thankful to Prof. Abdallah A. AL-Feel, King Abdulaziz University for authenticating the studied plant.

Conflict Of Interest

The author declares that she has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

1. A.G. Atanasov, S.B. Zotchev, V.M. Dirsch, C.T. Supuran, Natural products in drug discovery: Advances and opportunities, Nat Rev Drug Discov, 20(2021):200-216.

   [Crossref] [Google Scholar]

2. N. Barbhan, I. Miladi, S.I. Ali, E. Poupon, A.A. Mohamed, et al. Chemical constituents of Nitraria retusa grown in Egypt, Chem Nat Compd, 53(2017):994-996.

   [Google Scholar]

3. A.A. Mohamed, S.I. Ali, F.K. El-Baz, W.M. El-Senousy, New insights into antioxidant and antiviral activities of two wild medicinal plants: Achillea fragrantissima and Nitraria retusa, Int J Pharma Bio Sci, 6(2015):P708-P722.

   [Google Scholar]

4. S. Noori, A.R. Kiasat, M. Kolahi, R. Mirzajani, S.M.S. Nejad, Determination of secondary metabolites including curcumin in Rheum ribes L. and surveying of its antioxidant and cytotoxicity activity, J Saudi Chem Soc, 26(2022):101479.

   [Crossref] [Google Scholar]

5. S.A. Hassan, A.T. Al-Thobaiti, Morphological nutlet characteristics of some Lamiaceae taxa in Saudi Arabia and their taxonomic significance, Pak J Bot, 47(2015):1969-1977.

   [Google Scholar]

6. R. Ullah, A.S. Alqahtani, O.M.A. Noman, A.M. Alqahtani, S. Ibenmoussa, et al. A review on ethno-medicinal plants used in traditional medicine in the Kingdom of Saudi Arabia, Saudi J Biol Sci, 27(2020):2706-2718.

   [Crossref] [Google Scholar]

7. A.M. Alshabi, S.A. Alkahtani, I.A. Shaikh, M.A.A. Orabi, B.A. Abdel-Wahab, et al. Tribulus terrestris cytotoxicity against breast cancer MCF-7 and lung cancer A549 cell lines is mediated via activation of apoptosis, caspase-3, DNA degradation, and suppressing Bcl-2 activity, Separations, 9(2022):383.

   [Crossref] [Google Scholar]

8. A.K. Al-Asmari, A.M. Al-Elaiwi, M.T. Athar, M. Tariq, A. Al Eid, et al. A review of hepatoprotective plants used in Saudi traditional medicine, Evid Based Complement Alternat Med, (2014):890842.

   [Crossref] [Google Scholar]

9. A.V. Sirotkin, A. Kolesarova, Puncture vine (Tribulus terrestris L.) in control of health and reproduction, Physiol Res, 70(2021):S657.

   [Crossref] [Google Scholar]

10. S.I. Ali, G.A.E. El-Emary, A.A. Mohamed, Effects of gamma irradiation on FT-IR fingerprint, phenolic contents and antioxidant activity of Foeniculum vulgare and Carum carvi seeds, Res J Pharm Technol, 11(2018):3323-3329.

    [Crossref] [Google Scholar]

11. A. Khalid, A.S. Algarni, H.E. Homeida, S. Sultana, S.A. Javed, et al. Phytochemical, cytotoxic, and antimicrobial evaluation of Tribulus terrestris L., Typha domingensis pers., and Ricinus communis L.: Scientific evidences for folkloric uses, Evid Based Complement Alternat Med, (2022):6519712.

    [Crossref] [Google Scholar]

12. R. Stefanescu, A. Tero-Vescan, A. Negroiu, E. Aurica, C.E. Vari, A comprehensive review of the phytochemical, pharmacological, and toxicological properties of Tribulus terrestris L, Biomolecules, 10(2020):752.

    [Crossref] [Google Scholar]

13. R. Ranjithkumar, Q. Alhadidi, Z.A. Shah, M. Ramanathan, Tribulusterine containing Tribulus terrestris extract exhibited neuroprotection through attenuating stress kinases mediated inflammatory mechanism: In vitro and in vivo studies, Neurochem Res, 44(2019):1228-1242.

    [Crossref] [Google Scholar]

14. C. Tian, Y. Chang, Z. Zhang, H. Wang, S. Xiao, et al. Extraction technology, component analysis, antioxidant, antibacterial, analgesic and anti-inflammatory activities of flavonoids fraction from Tribulus terrestris L. leaves, Heliyon, 5(2019):e02234.

    [Crossref] [Google Scholar]

15. D. Dinchev, B. Janda, L. Evstatieva, W. Oleszek, M.R. Aslani, et al. Distribution of steroidal saponins in Tribulus terrestris from different geographical regions, Phytochemistry, 69(2008):176-186.

    [Crossref] [Google Scholar]

16. L.P. Kang, K.L. Wu, H.S. Yu, X. Pang, J. Liu, et al. Steroidal saponins from Tribulus terrestris, Phytochemistry, 107(2014):182-189.

    [Crossref] [Google Scholar]

17. Z.F. Wang, B.B. Wang, Y. Zhao, F.X. Wang, Y. Sun, et al. Furostanol and spirostanol saponins from Tribulus terrestris, Molecules, 21(2016):429.

    [Crossref] [Google Scholar]

18. S.I. Ali, A.A. Gaafar, S.A. Metwally, I.E. Habba, The reactive influences of pre-sowing He-Ne laser seed irradiation and drought stress on growth, fatty acids, phenolic ingredients, and antioxidant properties of Celosia argentea, Sci Hortic, 261(2020):108989.

    [Crossref] [Google Scholar]

19. A.A. Mohamed, S.I. Ali, M.Y. Sameeh, T.M.A. El-Razik, Effect of solvents extraction on HPLC profile of phenolic compounds, antioxidant and anticoagulant properties of Origanum vulgare, Res J Pharm Technol, 9(2016):2009-2016.

    [Crossref] [Google Scholar]

20. J.D. Hayes, A.T. Dinkova-Kostova, K.D. Tew, Oxidative stress in cancer, Cancer Cell, 38(2020):167-197.

    [Google Scholar]

21. S. Arfin, N.K. Jha, S.K. Jha, K.K. Kesari, J. Ruokolainen, et al. Oxidative stress in cancer cell metabolism, Antioxidants, 10(2021):642.

    [Crossref] [Google Scholar]

22. S.I. Ali, A.A. Gaafar, A.A. Abdallah, S.M. El-Daly, M. El-Bana, et al. Mitigation of alpha-cypermethrin-induced hepatotoxicity in rats by Tribulus terrestris rich in antioxidant compounds, Jordan J Biol Sci, 11(2018).

    [Google Scholar]

23. A.A. Gaafar, S.I. Ali, O. Kutkat, A.M. Kandeil, S.M. El-Hallouty, Bioactive ingredients and anti-influenza (H5N1), cytotoxicity, and antioxidant properties of Urtica urens L, Jordan J Biol Sci, 13(2020).

    [Google Scholar]

24. P. Goupy, M. Hugues, P. Boivin, M.J. Amiot, Antioxidant composition and activity of barley (Hordeum vulgare) and malt extracts and of isolated phenolic compounds, J Sci Food Agric, 79(1999):1625-1634.

    [Crossref] [Google Scholar]

25. A.W.A. Abdel-Aziz, N.M. Elwan, R.S. Shaaban, N.S. Osman, M.A.M Mohamed, High-performance liquid chromatography-fingerprint analyses, in vitro cytotoxicity, antimicrobial and antioxidant activities of the extracts of Ceiba speciosa growing in Egypt, Egypt J Chem, 64(2021):2-3.

    [Crossref] [Google Scholar]

26. H.A. Zahran, H.Z. Tawfeuk, Physicochemical properties of new peanut (Arachis hypogaea L.) varieties, OCL 26(2019):19.

    [Crossref] [Google Scholar]

27. W.S.A. Mettwally, H.A. Zahran, A.E. Khayyal, M.M.E Ahmed, R.M. Allam, et al. Calotropis procera (Aiton) seeds fixed oil: Physicochemical analysis, GC-MS profiling and evaluation of its in-vivo anti-inflammatory and in-vitro antiparasitic activities, Arab J Chem, 15(2022):104085.

    [Crossref] [Google Scholar]

28. K.X. Zhu, C.X. Lian, X.N. Guo, W. Peng, H.M. Zhou, Antioxidant activities and total phenolic contents of various extracts from defatted wheat germ, Food Chem, 126(2011):1122-1126.

    [Crossref] [Google Scholar]

29. A.A. Mohamed, S.I. Ali, F.K. EL-Baz, A.K. Hegazy, M.A. Kord, Chemical composition of essential oil and in vitro antioxidant and antimicrobial activities of crude extracts of Commiphora myrrha resin, Ind Crops Prod, 57(2014):10-16.

    [Crossref] [Google Scholar]

30. A.I. Dirar, D.H.M. Alsaadi, M. Wada, M.A. Mohamed, T. Watanabe, et al. Effects of extraction solvents on total phenolic and flavonoid contents and biological activities of extracts from Sudanese medicinal plants, South Afr J Bot, 120(2019):261-267.

    [Crossref] [Google Scholar]

31. S. Abubakar, B. Akanbi, V. Etim, O. Segun, J. Ogbu, Comparative study of phytochemical and synergistic anti-bacterial activity of Tribulus terrestris (L.) and Pandiaka heudelotii (Moq.) Hien on some clinical bacterial isolates, Pharm Biol Eval, 3(2016):83-91.

    [Google Scholar]

32. K. Muniyandi, E. George, S. Sathyanarayanan, B.P. George, H. Abrahamse, et al. Phenolics, tannins, flavonoids and anthocyanins contents influenced antioxidant and cytotoxicity activities of Rubus fruits from Western Ghats, India, Food Sci Hum Wellness, 8(2019):73-81.

    [Crossref] [Google Scholar]

33. N. Comlekcioglu, R. Cirak, Comparison of fatty acid composition and antioxidant contents of Tribulus terrestris L. collected from different localities, Turk J Agric Food Sci Technol, 9(2021):1448-1454.

    [Crossref] [Google Scholar]

34. R. Naz, H. Ayub, S. Nawaz, Z.U. Islam, T. Yasmin, et al. Antimicrobial activity, toxicity and anti-inflammatory potential of methanolic extracts of four ethnomedicinal plant species from Punjab, Pakistan, BMC Complement Altern Med, 17(2017):302.

    [Crossref] [Google Scholar]

35. H.M. Hammoda, N.M. Ghazy, F.M. Harraz, M.M. Radwan, M.A. ElSohly, et al. Chemical constituents from Tribulus terrestris and screening of their antioxidant activity, Phytochemistry, 92(2013):153-159.

    [Crossref] [Google Scholar]

36. F. Mubarik, A.A. Khalil, M.N. Akhtar, A. Khalid, S. Basharat, et al. Assessing the antioxidant characteristics and polyphenol content of Tribulus terrestris microwave-assisted fruit extract, Phytopharm Res J, 1(2022):1-10.

    [Google Scholar]

37. A. Javaid, F. Anjum, N. Akhtar, Molecular characterization of Pyricularia oryzae and its management by stem extract of Tribulus terrestris, Int J Agric Biol, 21(2019):1256-1262.

    [Crossref] [Google Scholar]

38. W.A. Aldaddou, A.S.M. Aljohani, I.A. Ahmed, N.A. Al-Wabel, I.M. El-Ashmawy, Ameliorative effect of methanolic extract of Tribulus terrestris L. on nicotine and lead-induced degeneration of sperm quality in male rats, J Ethnopharmacol, 295(2022):115337.

    [Crossref] [Google Scholar]

39. I.G. Munteanu, C. Apetrei, Analytical methods used in determining antioxidant activity: A review, Int J Mol Sci, 22(2021):3380.

    [Crossref] [Google Scholar]

40. A. Lichota, K. Gwozdzinski, Anticancer activity of natural compounds from plant and marine environment, Int J Mol Sci, 19(2018):3533.

    [Crossref] [Google Scholar]

41. H.J. Kim, J.C. Kim, J.S. Min, M. Kim, J.A. Kim, et al. Aqueous extract of Tribulus terrestris Linn induces cell growth arrest and apoptosis by down-regulating NF-κB signaling in liver cancer cells, J Ethnopharmacol, 136(2011):197-203.

    [Crossref] [Google Scholar]

42. A. Patel, A. Soni, N.J. Siddiqi, P. Sharma, An insight into the cytotoxicity mechanism of Tribulus terrestris extracts on human breast cancer cells, Biotech, 9(2019):58.

    [Crossref] [Google Scholar]

43. C.C.M. Figueiredo, A.C. Gomes, F.O. Granero, J.L.B. Junior, L.P. Silva, Antiglycation and antitumoral activity of Tribulus terrestris dry extract, Avicenna J Phytomedicine, 11(2021):224.

    [Google Scholar]

44. M.W. Abbas, M. Hussain, S. Akhtar, T. Ismail, M. Qamar, et al. Bioactive compounds, antioxidant, anti-inflammatory, anti-cancer, and toxicity assessment of Tribulus terrestris-in vitro and in vivo studies, Antioxidants, 11(2022):1160.

    [Crossref] [Google Scholar]

45. M.V.O.B. de Alencar, M.T. Islam, R.M.T. de Lima, M.F.C.J. Paz, A.C. Dos Reis, et al. Phytol as an anticarcinogenic and antitumoral agent: An in vivo study in swiss mice with DMBA-ÃÃÂ??ÂÃÂ?Induced breast cancer, IUBMB Life, 71(2019):200-212.

    [Crossref] [Google Scholar]

46. J.M. Lou-Bonafonte, R. Martinez-Beamonte, T. Sanclemente, J.C. Surra, L.V. Herrera-Marcos, et al. Current insights into the biological action of squalene, Mol Nutr Food Res, 62(2018):1800136.

    [Crossref] [Google Scholar]

47. A.A. Mohamed, S.I. Ali, H.F. Kabiel, A.K. Hegazy, M.A. Kord, et al. Assessment of antioxidant and antimicrobial activities of essential oil and extracts of Boswellia carteri resin, Int J Pharmacogn Phytochem Res, 7(2015):502-509.

    [Google Scholar]

48. M. Saha, P.K. Bandyopadhyay, In vivo and in vitro antimicrobial activity of phytol, a diterpene molecule, isolated and characterized from Adhatoda vasica Nees. (Acanthaceae), to control severe bacterial disease of ornamental fish, Carassius auratus, caused by Bacillus licheniformis PKBMS16, Microb Pathog, 141(2020):103977.

    [Crossref] [Google Scholar]

49. F.J. AÃÃÂ??ÂÃÂ?lvarez-Marti­nez, E. Barrajon-Catalan, Antibacterial plant compounds, extracts and essential oils: An updated review on their effects and putative mechanisms of action, Phytomedicine, 90(2021):153626.

    [Crossref] [Google Scholar]

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