Investigation and Biological Assessment of Rumex vesicarius L. Extract: Characterization of the Chemical Components and Antioxidant, Antimicrobial, Cytotoxic, and Anti-Dengue Vector Activity

The objective of this study was to assess the biological potency and chemical composition of Rumex vesicarius aboveground parts using GC–MS. In this approach, 44 components were investigated, comprising 99.99% of the total volatile compounds. The major components were classified as fatty acids and lipids (51.36%), oxygenated hydrocarbons (33.59%), amines (7.35%), carbohydrates (6.06%), steroids (1.21%), and alkaloids (0.42%). The major components were interpreted as 1,3-dihydroxypropan-2-yl oleate (oxygenated hydrocarbons, 18.96%), ethyl 2-hydroxycyclohexane-1-carboxylate (ester of fatty acid, 17.56%), and 2-propyltetrahydro-2H-pyran-3-ol (oxygenated hydrocarbons, 11.18%). The DPPH antioxidant activity of the extracted components of R. vesicarius verified that the shoot extract was the most potent with IC50 = 28.89 mg/L, with the percentages of radical scavenging activity at 74.28% ± 3.51%. The extracted plant, on the other hand, showed substantial antibacterial activity against the diverse bacterial species, namely, Salmonella typhi (23.46 ± 1.69), Bacillus cereus (22.91 ± 0.96), E. coli (21.07 ± 0.80), and Staphylococcus aureus (17.83 ± 0.67). In addition, the extracted plant was in vitro assessed as a considerable anticancer agent on HepG2 cells, in which MTT, cell proliferation cycle, and DNA fragmentation assessments were applied on culture and treated cells. The larvicidal efficacy of the extracted plant was also evaluated against Aedes aegypti, the dengue disease vector. As a result, we may infer that R. vesicarius extract increased cytocompatibility and cell migratory capabilities, and that it may be effective in mosquito control without causing harm.


Introduction
Wild plants are considered pharmacologically as a source of bioactive molecules. Early people treated their ailments using plants; hence, the history of medicinal plants is as long as the history of mankind [1,2]. About 80% of the world's population, primarily in Africa and other underdeveloped countries, still depends mainly on traditional medicine for disease treatment. African medicinal plants have a vast range of biological qualities that need to be identified, recorded, and researched. Over 1300 medicinal plants are used in European countries, out of which 90% are wild [3]. These confidential pharmaceutical drugs accessible from natural sources have fewer unfavorable side-effects than synthetic sources [4].
Egypt's flora includes around 2080-2094 species of seed plants and vascular cryptogams [5,6]. The family Polygonaceae is a temperate-zone cosmopolite. Around the globe, there are 46 genera and 1100 species. This family is represented in Egypt by 28 species under eight genera [5,7]. The plants of genus Rumex comprise almost 150 annual, biennial, and perennial species broadly distributed in temperate climates worldwide [7,8]. Rumex is a common household herb, which is beneficial in the treatment of constipation. It has cleaning effects and can be used to treat skin disorders [9,10]. Rumex spp. are reported to have hepatoprotective, phytotoxic, antioxidant, and antimicrobial properties [11][12][13][14][15].
The properties of natural substances from plants such as phenolics, flavonoids, and terpenoids have been broadly described. Rumex vesicarius L. (Hammeidda) is an annual semi-succulent pale green leafy herb with 15-30 cm height [7,16]. This herb, which is endemic to northern Africa and Asia [17], grows yearly throughout the wet seasons of autumn and spring. Many studies have shown that it is regarded to be a nutritious supplement plant due to its high levels of β-carotenes [18], vitamins (especially vitamin C), lipids, proteins, and organic acids. It also includes minerals such as potassium, sodium, calcium, magnesium, iron, manganese, and copper [16,19,20]. Recent studies have concentrated on the characterization of active chemical components, as well as biological uses of extracted plants. The whole plant is medicinally significant and heals a variety of ailments such as tumors, hepatic illnesses, poor digestion, constipation, calculi, heart problems, aches, spleen diseases, and hiccough [21,22].
Aedes aegypti is a mosquito that can spread dengue fever, chikungunya, Zika fever, Mayaro, and yellow fever viruses, as well as other disease agents. The continuous application of synthetic insecticides causes the development of resistance in vector species, biological magnification of toxic substances through the food chain, and adverse effects on environmental quality and nontarget organisms including human health [23,24].
Previous phytochemical investigation revealed the presence of phenolics, flavonoids anthraquinones, sesquiterpenes, and minerals [15,25,26], However, the anti-dengue vector activity of R. vesicarius extract was not previously studied. Despite its significance, there have been little studies on the plant. Therefore, the current study sought to analyze the chemical ingredients of the Egyptian ecospecies of Rumex vesicarius methanol extract using GC-MS spectroscopy analysis in order to investigate the biochemical components responsible for the biological effects. The work was continually extended in order to investigate the antioxidant properties of the extracted plant using the DPPH free-radical scavenging test, while the antibacterial and cytotoxic properties of the extracted plant were examined in vitro against several bacterial species and cancer cell lines. Furthermore, the larvicidal activity against Aedes aegypti was assessed.

Antioxidant Activity-DPPH Assay
The antioxidant activity was evaluated for the methanol extract of R. vesicarius the DPPH colorimetric assay. Ascorbic acid was used as the reference standard for a parison of the obtained results. The investigated extract showed potent antioxidant ities as compared to the results of ascorbic acid (IC50 = 12.48 mg/L). Subsequently, t sults, as presented in Table 2, verified that the shoot extract had the most potent an dant scavenging activity with IC50 = 28.89 mg/L. The prevalence of fatty acids and derivatives (51.36%), as well as oxygenated hydrocarbons (33.59%), was the major controlling the mechanism of the reactions involved in the evaluation of the antio aptitude of the investigated extracts. The present results of Rumex vesicarius are in ment with the results of Nishina et al. [28], Demirezer et al. [29], Al-Ismail et al. [30] [31], and Li and Liu [32].

Antioxidant Activity-DPPH Assay
The antioxidant activity was evaluated for the methanol extract of R. vesicarius using the DPPH colorimetric assay. Ascorbic acid was used as the reference standard for a comparison of the obtained results. The investigated extract showed potent antioxidant activities as compared to the results of ascorbic acid (IC 50 = 12.48 mg/L). Subsequently, the results, as presented in Table 2, verified that the shoot extract had the most potent antioxidant scavenging activity with IC 50 = 28.89 mg/L. The prevalence of fatty acids and their derivatives (51.36%), as well as oxygenated hydrocarbons (33.59%), was the major factor controlling the mechanism of the reactions involved in the evaluation of the antioxidant aptitude of the investigated extracts. The present results of Rumex vesicarius are in agreement with the results of Nishina et al. [28], Demirezer et al. [29], Al-Ismail et al. [30], Özen [31], and Li and Liu [32]. Fatty acids and lipids isolated from Chenopodium ambrosioides and Euphorbia lathyrus displayed potent antioxidant activities for scavenging free radicals in solution [33,34]. The major compounds 2-propyltetrahydro-2H-pyran-3-ol, ethyl 2-hydroxycyclohexane-1-carboxylate, and 1,3-dihydroxypropan-2-yl oleate (11.18%, 17.56%, and 18.96%, respectively) act as substantial antioxidant agents. Many factors influence the mechanism of antioxidant activity; in general, the antioxidant capacity of bioactive compounds is determined by the ability of reactive oxygen species, such as phenolics, fatty acids, terpenes, oxygenated hydrocarbons, or carbohydrates, to scavenge or stabilize free radicals [35][36][37]. In this study, the shoot extract outperformed the other wild plant extracts in terms of antioxidant activity. Furthermore, antioxidants mainly come from plants in the form of phenolic compounds such as flavonoids, phenolic acids, ascorbic acid, and carotenoids. According to our previous study, this plant contains nonvolatile compounds such as flavonoids and phenolics [15,22].
The chemical constituents of plant extracts also tend to form a complex with DPPH solution and accordingly stabilize or scavenge the free radicals in solution [38][39][40]. The antioxidant activity was attributed to the number of free hydroxyl groups [41][42][43].

Antibacterial Activity
The antibacterial potential of the extracted R. vesicarius was assessed using the agar disc diffusion assay [43]. In this course, four Gram-negative and four Gram-positive bacterial strains were selected to estimate the antimicrobial effects of the investigated extracts. Accordingly, the extracted sample and standard antibiotics were prepared in a concentration of 10 mg/L ( Table 3). The results indicated that the extract revealed potent antibacterial activities toward diverse bacterial species. The S. Typhimurium was the mainly influenced bacterial strain, while P. aeruginosa and S. xylosus were the main resistant species ( Table 3). The tested organisms could be ordered according to their sensitivity as follows: S. Typhimurium < B. cereus < E. coli < S. aureus < K. pneumoniae < S. haemolyticus < S. xylosus < P. aeruginosa. The standard antibiotics, ampicillin, azithromycin, cephradin, and tetracycline, showed variable activities. P. aeruginosa was entirely resistant to ampicillin, cephradin, and tetracycline, while S. Typhimurium was resistant to ampicillin, azithromycin, cephradin, and penicillin (Table 3). Moreover, the plant extract revealed potent antibacterial activity toward Escherichia coli, Salmonella Typhimurium, and Bacillus cereus species (21 ± 0.80, 23.46 ± 1.69, and 22.91 ± 0.96 mm), but had less potency toward P. aeruginosa and S. xylosus compared to the antibiotics. Table 3. Antibacterial activity of the methanol extract from the aerial parts of R. vesicarius and some selected reference antibiotics at a concentration of 10 mg/mL. The methanol extract of R. vesicarius in the present study showed about threefold greater activity toward S. aureus, K. pneumoniae, and E. coil than the Indian variety [44,45]. Several experiments on different plant parts of different species of Rumex confirmed their potent antibacterial activity toward both Gram-positive and Gram-negative bacteria [15,[46][47][48]. These differences in the antimicrobial potential could be attributed to environmental conditions such as climate and soil type, which can influence the extract components [49].

Microbes
The methanol extract of R. vesicarius appeared to exhibit potent antibacterial activity regardless of whether species were Gram-negative or Gram-positive species. Hence, the assorted results achieved for the same extract were a function of its numerous components. Thus, the composition of the extract was evaluated to determine the effectiveness of specific compounds against bacterial species. In particular, by studying the role of the main chemical components of the extracted R. vesicarius as antibacterial agents, we found that terpenes, oxygenated hydrocarbons, and carbohydrates presented higher antibacterial potential [50][51][52]. Moreover, fatty acids and lipids in the essential oils of some extracted plants display improved antimicrobial characteristics [53][54][55]. This plant contains nonvolatile chemicals such as flavonoids and phenolics, according to our study [15]. These chemicals showed antibacterial efficacy against bacteria with multiple resistances [45,47].

Cytotoxicity and Cell Migration Analysis
Recently, studies have been conducted for the development of novel treatment protocols using herbs in the creation of novel treatment approaches for numerous cancer varieties [56]. Moreover, the utility of herbal extracts with characteristic therapeutic properties is beneficial in pharmaceutical chemistry to achieve increased efficiency and biological potency [57]. Therefore, the utility of plant extracts is predicted to result in reduced sideeffects compared to chemotherapeutic drugs [58]. Herein, R. vesicarius extract was assessed for antitumor activity using the MTT assay. This assay fits cell numbers to a growth curve. The MTT experiments were performed in the dark. Hepatocellular carcinoma (HepG2) cells were chosen to assess the antitumor activity of R. vesicarius extract. The IC 50 (half-maximal inhibitory concentration) values were obtained through plotting the cell viability against drug concentration (µM). Thus, the cytotoxic potential is inversely proportional to the used concentrations and IC 50 values. A control sample of MTT solution was seeded in one well. Serial dilutions of five extract concentrations were used (31.3, 62.5, 125, 500, and 1000 µg/mL).
The half-maximal inhibitory concentrations (IC 50 , µg/mL) and the absorbance reads for each sample concentration are shown in Table 4. The results revealed that the shoot extract possessed weaker cytotoxic activity with an IC 50 value at 352.4 µg/mL. The mode of action of cytotoxicity is dependent on the phytochemical composition, concentration, and nature of cancer cell lines [59], in addition to the surface morphology, size, and aggregation of the extract particles. Furthermore, compounds such as polyphenols and flavonoids may be responsible for the anti-inflammatory and cytotoxic properties of plant extracts [15,60]. According to our investigation [15], R. vesicarius is composed of nonvolatile compounds including flavonoids and phenolics.
Recently, cytotoxicity, apoptosis, proliferation, cell-cycle progression, genetic damage, and apoptotic cell death were reported in HepG2 cancer cells [60][61][62]. Herein, the improved cell migration properties of R. vesicarius extract were also estimated using a cell motility assay [63][64][65]. The films revealed perfect wound healing activity in HepG2 cell lines. The cell lines of HepG2 migrated to the center of the scratch, covering the main part of the scratch after staining. The cell migration exemplified by microscopic images at definite intervals of time is presented in Figure 3. The plant extracted displayed no migration at 0 h or after 26 h of incubation. In contrast, after staining, the images showed a better healing rate at the varying doses of the extracted plant with a much faster healing rate upon increasing the concentration of the investigated extract. Consequently, the maximum healing rate was noted when using the plant extract at the IC 50 dose after staining. The migration of cells from the edges to the center of the scratch was achieved after diffusion of the extract into the generated scratch, thus closing the scratch with a higher rate of migration. The cell migration activity for wound healing is attributed to the effect of several components of the plant extract, e.g., minerals, vitamins, activated enzymes, polyphenolics, and polysaccharides [66]. The impact of these components improves the healing of wounds through increased growth factors, angiogenesis, and reduced aggregation of platelets, while serving as exceptional scavengers for free radicals t the wound site [67]. The induced cell growth results from the impact of phytochemicals, e.g., reactive oxygen species, accompanied by respectable antioxidant potency and improved wound closure activity. Accordingly, on the basis of the substantial therapeutic value of the extracted R. vesicarius, it can be considered as a proficient biocompatible material for wound dressing; future in vivo studies and clinical analysis are required to confirm its practical applicability.

DNA Fragmentation
DNA fragmentation was used as an indicator of apoptosis for the investigation of apoptotic cells. Oxygenated hydrocarbons display strong antioxidant effects and, accordingly, can protect the cellular DNA from oxidative damage in many degenerative diseases associated with oxidative stress [68]. Thus, the occurrence of DNA fragmentation, to estimate the proapoptotic effect of R. vesicarius, was discovered in all of the intended groups using the DNA gel electrophoresis procedure. The densitometry analysis of the treatmentdependent DNA laddering effect identified a noteworthy increase in the percentage of DNA fragmentation (MDR1: 40.23%; CD44: 70.53%) as compared to the untreated control groups (Figure 4). According to the results, the methanol extract of R. vesicarius could change the cell morphology, thereby inhibiting cell growth in a time-and dose-dependent manner, leading to DNA degradation.

The EC50 Value of R. vesicarius Extract
The dose-response relationship is plotted in Figure 5. The dose-response curve in Figure 5a was normalized along the X-axis by its EC50 value (Figure 5b). The IC50 of the R. vesicarius extract was estimated by plotting the sample absorbance against the log of doses

DNA Fragmentation
DNA fragmentation was used as an indicator of apoptosis for the investigation of apoptotic cells. Oxygenated hydrocarbons display strong antioxidant effects and, accordingly, can protect the cellular DNA from oxidative damage in many degenerative diseases associated with oxidative stress [68]. Thus, the occurrence of DNA fragmentation, to estimate the proapoptotic effect of R. vesicarius, was discovered in all of the intended groups using the DNA gel electrophoresis procedure. The densitometry analysis of the treatmentdependent DNA laddering effect identified a noteworthy increase in the percentage of DNA fragmentation (MDR1: 40.23%; CD44: 70.53%) as compared to the untreated control groups (Figure 4). According to the results, the methanol extract of R. vesicarius could change the cell morphology, thereby inhibiting cell growth in a time-and dose-dependent manner, leading to DNA degradation.  DNA Fragmentation DNA fragmentation was used as an indicator of apoptosis for the investigation of apoptotic cells. Oxygenated hydrocarbons display strong antioxidant effects and, accordingly, can protect the cellular DNA from oxidative damage in many degenerative diseases associated with oxidative stress [68]. Thus, the occurrence of DNA fragmentation, to estimate the proapoptotic effect of R. vesicarius, was discovered in all of the intended groups using the DNA gel electrophoresis procedure. The densitometry analysis of the treatmentdependent DNA laddering effect identified a noteworthy increase in the percentage of DNA fragmentation (MDR1: 40.23%; CD44: 70.53%) as compared to the untreated control groups (Figure 4). According to the results, the methanol extract of R. vesicarius could change the cell morphology, thereby inhibiting cell growth in a time-and dose-dependent manner, leading to DNA degradation.

The EC50 Value of R. vesicarius Extract
The dose-response relationship is plotted in Figure 5. The dose-response curve in Figure 5a was normalized along the X-axis by its EC50 value (Figure 5b). The IC50 of the R. vesicarius extract was estimated by plotting the sample absorbance against the log of doses The dose-response relationship is plotted in Figure 5. The dose-response curve in Figure 5a was normalized along the X-axis by its EC 50 value (Figure 5b). The IC 50 of the R. vesicarius extract was estimated by plotting the sample absorbance against the log of doses at different concentrations of the serial dilution ( Figure 5). The response increased upon increasing the extract concentration, with the vertical point of the curve revealing the EC 50 value [69,70]. The data analysis showed that a dose of 500 µg/mL had a cytotoxic influence on HepG2 cell lines.
at different concentrations of the serial dilution ( Figure 5). The response increased upon increasing the extract concentration, with the vertical point of the curve revealing the EC50 value [69,70]. The data analysis showed that a dose of 500 µg/mL had a cytotoxic influence on HepG2 cell lines.

Larvicidal Bioassay
Data given in Table 5 indicate the larvicidal activity of the methanolic extract of R. vesicarius against the third-instar larvae of Ae. aegypti at 24 and 48 h post treatment. The results revealed a significant increase in larval mortality with all concentrations of the methanolic extract of R. vesicarius. The maximum larval mortality was recorded at concentrations of 1200 mg/L after 24 and 48 h (28.9% and 42.6%, respectively), while the lowest mortality was 7.7% after 24 h when the concentration was 250 mg/L and 1.1% after 48 h when the concentration was 125 mg/L. The LC50 (the concentration of extract that causes 50% mortality) of the extract was 19.99 and 14.97 mg/L, while the LC90 (the concentration of extract that causes 90% mortality) was 36.12 and 27.43 mg/L after 24 and 48 h, respectively. The toxicity of plants depends on their various bioactive compounds, including phenolics, terpenoids, flavonoids, and alkaloids, either as single or joint compounds [71]. Moreover, desert plants such as R. vesicarius are easily available and a rich source of bioactive compounds, which function as larvicides, pupicides, or mosquito repellents [15,72].
These results are consistent with those obtained by Nasir et al. [71], who used essential oils from some medicinal plants against Ae. albopictus, where ginger was revealed to be most effective with the lowest LC50 values after 8 and 16 h, followed by peppermint, basil, eucalyptus, and neem. Ullah et al. [72] [73] used a petroleum ether extract from leaves of Lantana camara against larvae of An. multicolor, recording the highest larval mortality (100.0%) at 140 mg/L. Shehata [74] found that a petroleum ether extract from leaves of Prunus domestica and Rhamnus cathartica was more effective against C. pipiens (LC50: 33.3 and 63.4 mg/L, respectively) than chloroform (LC50: 70.8 and 192.1 mg/L) and methanolic (LC50: 132.7 and 273.5 mg/L, respectively) extracts. Dey et al. [75] found that the aqueous extract of Piper longum showed the highest larval mortality after 24 h. of treatment against Ae. aegypti, An. Stephensi, and C. quinquefasciatus. Farag et al. [76] observed that the peel powder of Punica granatum, extracted with petroleum ether, had potential toxicological effects against third-instar larvae of C. pipiens with an LC50 value of 95.66 mg/L.

Larvicidal Bioassay
Data given in Table 5 indicate the larvicidal activity of the methanolic extract of R. vesicarius against the third-instar larvae of Ae. aegypti at 24 and 48 h post treatment. The results revealed a significant increase in larval mortality with all concentrations of the methanolic extract of R. vesicarius. The maximum larval mortality was recorded at concentrations of 1200 mg/L after 24 and 48 h (28.9% and 42.6%, respectively), while the lowest mortality was 7.7% after 24 h when the concentration was 250 mg/L and 1.1% after 48 h when the concentration was 125 mg/L. The LC 50 (the concentration of extract that causes 50% mortality) of the extract was 19.99 and 14.97 mg/L, while the LC 90 (the concentration of extract that causes 90% mortality) was 36.12 and 27.43 mg/L after 24 and 48 h, respectively. The toxicity of plants depends on their various bioactive compounds, including phenolics, terpenoids, flavonoids, and alkaloids, either as single or joint compounds [71]. Moreover, desert plants such as R. vesicarius are easily available and a rich source of bioactive compounds, which function as larvicides, pupicides, or mosquito repellents [15,72]. Mortality is expressed as the mean ± SE (standard error) of three replicates. Different superscript letters within each treatment (column) express significant variation at a probability level of 0.05 (Duncan's test). * p < 0.05.
These results are consistent with those obtained by Nasir et al. [71], who used essential oils from some medicinal plants against Ae. albopictus, where ginger was revealed to be most effective with the lowest LC 50 values after 8 and 16 h, followed by peppermint, basil, eucalyptus, and neem. Ullah et al. [72] recorded LC 50 and LC 90 values of 50.27 and 203.99, and 17.77 and 206.49 mg/L for Cassia fistula and Nicotiana tabacum extracts against larvae of C. quinquefasciatus. Hassanain et al. [73] used a petroleum ether extract from leaves of Lantana camara against larvae of An. multicolor, recording the highest larval mortality (100.0%) at 140 mg/L. Shehata [74] found that a petroleum ether extract from leaves of Prunus domestica and Rhamnus cathartica was more effective against C. pipiens (LC 50 : 33.3 and 63.4 mg/L, respectively) than chloroform (LC 50 : 70.8 and 192.1 mg/L) and methanolic (LC 50 : 132.7 and 273.5 mg/L, respectively) extracts. Dey et al. [75] found that the aqueous extract of Piper longum showed the highest larval mortality after 24 h. of treatment against Ae. aegypti, An. Stephensi, and C. quinquefasciatus. Farag et al. [76] observed that the peel powder of Punica granatum, extracted with petroleum ether, had potential toxicological effects against third-instar larvae of C. pipiens with an LC 50 value of 95.66 mg/L.

Plant Material and Extraction Process
The aerial parts of Rumex vesicarius were acquired from Wadi Hagoul, north of the Eastern Desert, Egypt (29 • 54 47.46 N 32 • 12 28.10 E) during the flowering season (April 2021) from different populations. The plant was identified according to Tackholm [5] and Boulos [7]. A voucher specimen (Mans. 0161822009) was prepared and deposited in the Herbarium of Botany Department, Faculty of Science, Mansoura University, Egypt.
The collected samples were cleaned and naturally dried. Then, 10 g of dried plant materials were placed in a 250 mL conical flask containing 150 mL of methanol. The mixture was then transferred to a water bath shaker (Memmert WB14, Schwabach, Germany), with continuous shaking for 2 h at room temperature. Whatman filter papers (No. 1, 125 mm, Cat No. 1001 125, Germany) were used for the filtration step of the mixture. The final concentrations of the extracted plant issues were investigated, and the extracts were stored at 4 • C [77].

Gas Chromatography-Mass Spectrometry Analysis (GC-MS)
The chemical composition of the extracted R. vesicarius plant was characterized by implementing the plant extract on a Trace GC-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness) [78]. The temperature of the column oven was firstly held at 50 • C, raised subsequently by a rate of 5 • C per minute to reach 250 • C, and held for 2 min, before raising the temperature to the final temperature (300 • C) by 30 • C per minute and holding for 2 min. The injector and MS transfer line temperature were kept at 270 and 260 • C, respectively. Helium (He) was used as the inert carrier gas at a constant flow rate of 1 mL/min. The solvent was released after 4 min, and diluted samples of 1 µL were injected directly using an Autosampler AS1300 coupled with GC in split mode. EI mass spectroscopy was collected at 70 eV ionization voltage over a range of 50-500 for m/z in packed scan mode. The temperature of the ion source was fixed at 200 • C. The chemical components of the individual extracted plant materials were interpreted through a comparison of their mass spectral data with those in the WILEY 09 and NIST 14 mass spectrometry databases. The GC-MS analysis indicated five conceivable components for each significant peak. In the case of dissimilar proposed components, the factors of probability and the fragmentation patterns of the main structure were used to determine the selected structure.

Antioxidant DPPH Assay
A stock solution of extracted R. vesicarius was diluted in methanol to the desirable concentrations (5,10,20,30,40, and 50 mg/L). DPPH solution (1 mL, 0.135 mM) was added to each prepared concentration of sample solution. Catechol was used as a standard with the same concentrations as the tested samples. The samples were kept at room temperature in the dark for 30 min, and the absorbance was measured at λ = 517 nm using a UV/Vis spectrophotometer (Spekol 11 spectrophotometer, analytic Jena AG, Jena, Germany). The percentages of antioxidant scavenging activities were calculated using the following equation, in which the DPPH solution in methanol was used as a control: The procedure was applied following previous reports [79,80] with insignificant modifications. The inhibitory concentrations (IC 50 , mg·L −1 ) were calculated by applying the plotted exponential curve [81], which specified the relationship of the sample concentration with the percentage of remaining DPPH • radicals. Microbial testing: The antimicrobial activity of the extracted plant issues was estimated using an agar well diffusion assay [82] with inocula containing 10 6 bacterial cells/mL to spread on nutrient agar plates. The sterilized filter paper discs (Whatman No.1, 6 mm in diameter) were immersed overnight in the extracted issues of the plant until saturation, and another set of filter paper discs were immersed in methanol as a control. The discs were placed on the surface of agar plates seeded with definite bacterial microorganisms. The plates were incubated at 37 • C for 18-24 h, and the inhibition zone diameters (mm) were measured [83].
The MTT assay was used for evaluation of the extracted R. vesicarius [84,85]. HepG2 cell lines were seeded at a concentration of 3 × 10 3 cells/well suspended in 100 µL of complete medium in 96-well plates, and the experiments were implemented in duplicates. Subsequently, the plates were incubated for 24 h in 5% CO 2 at 37 • C until settling and adherence. The plant extract was prepared in serial dilutions (31.3, 62.5, 125, 500, and 1000 µg/mL) and applied to cell lines for 48 h. The medium was removed by aspiration, and MTT (0.5 mg/mL), dissolved in the culture medium, was added onto cells and incubated at 37 • C and 5% CO 2 for 4 h. SDS (100 µL) was added to each well, and the cell growth was measured at λ max. = 570 nm (BioTek, Elx800, Winooski, VT, USA), expressing the results as a percentage of the control. The IC 50 values were estimated using Origin 8.0 ® software (Origin Lab Corporation, Northampton, MA, USA). The IC 50 values were estimated using the equation IC 50 = (0.57−b)/a. The inhibition percentage was estimated using the following equation (Equation (2)): % Inhibition = Absorbance control -Absorbance control Optical density control × 100.
The relative cell viability percentage was estimated using the following equation: % Cell viability = Absorbance samples -Absorbance blank Optical density control -Absorbance blank × 100.

Cell Motility Assay
Cells were seeded in a six-well plate. The monolayer cells were scratched using a 10 µL pipette tip to create a wound, and cells were washed twice by PBS and then treated using the IC 5 , IC 10 , IC 25 , and IC 50 of the R. vesicarius extract. The wounds were detected using phase-contrast microscopy on an inverted microscope. Images were taken regularly at 0 h, after staining, and after 26 h [86].

Conventional PCR
The conventional PCR technique was used for determination of the mRNA expression of CD44 and MDR1. The cells were treated with R. vesicarius extract for 24 h and collected to extract cellular RNA; then, total RNA was reverse-transcribed to cDNA (Qiagen, Germantown, MD, USA). The reaction included 1 µL of cDNA in a total volume of 20 µL Larvae of Ae. aegypti were obtained from the Center for Disease Vector in Jizan, and they were reared for six generations at the Center of Environmental Research, Faculty of Science, Jazan University under controlled conditions (temperature: 27 ± 2 • C; RH: 70-80%; 12 h/12 h light-dark regime). Adult mosquitoes were kept in 30 × 30 × 30 cm wooden cages and were provided daily with cotton pieces soaked in 10.0% sucrose solution for a period of 3 days after emergence. After this period, females were allowed to take a blood meal from a pigeon host, which is necessary for laying eggs (anautogeny). A plastic cup (15 × 15 cm) containing dechlorinated tap water was placed in the cage for laying eggs. The resulting egg rafts were picked up from the plastic dish and transferred into plastic pans (25 × 30 × 15 cm) containing 3 L of tap water and left for 24 h. The hatching larvae were provided daily with a piece of bread as their diet. This diet was found to be the most preferable food for larval development and female fecundity [87].

Larvicidal Activity of Tested Plant Extracts
For larvicidal activity, the tested material of methanol extracts was dissolved in 0.1 mL of methanol, while the tested material of acetone and chloroform extracts were dissolved in two drops of Tween-80 as emulsifier to facilitate the dissolving of tested material in water. Different ranges of concentrations of each extract were prepared in order to detect mortalities. All experiments with the tested materials were performed in 100 mL of dechlorinated tap water contained in 200 mL plastic cups. Then, third-instar larvae (10 larvae) were placed immediately into plastic cups containing different concentrations of extracts. Three replicates were usually used for each tested concentration. All plastic cups were incubated under controlled conditions of the mosquito colony, and mortality was subsequently recorded. Control larvae received 0.1 mL of methanol or two drops of Tween-80 in 100 mL of water. Mortality was recorded daily, and dead larvae and pupae were removed until adult emergence [87]. Larval mortality was indicated by a failure to respond to mechanical stimulation. The larval mortality percentage was estimated by using the following equation [88]: Larval mortality = Number of dead larvae Number of treated larvae × 100.

Statistical Analysis
The antioxidant, antibacterial, and larvicidal activity studies were repeated three times with three replications, and the results were submitted to a one-way ANOVA to determine the significance of the differences between samples using the Costat software program (CoHort Software, Monterey, CA, USA).

Conclusions
In summary, 44 components were identified from the methanol extract of R. vesicarius shoots by GC-MS analysis. The majority of the characterized components were fatty acids and their derivatives, representing 51.36% of the total composition of the methanol extract. In addition, the major components were interpreted as 2-propyltetrahydro-2H-pyran-3-ol, ethyl 2-hydroxycyclohexane-1-carboxylate, and 1,3-dihydroxypropan-2-yl oleate (11.18%, 17.56%, and 18.96% of the composition). The R. vesicarius shoots displayed various biological properties such as antioxidant, antibacterial, and cytotoxic activities, as well as larvicidal activity against Aedes aegypti, the dengue disease vector, at low concentrations relative to the results of the respective standards. Precisely, the shoot extract exhibited the most potent antioxidant activity, indicating its higher potency to trap the free radicals in the DPPH solution. The extracted shoot of R. vesicarius revealed the most potent antibacterial activity against Gram-negative bacterial species, in which the extract showed broad-spectrum activity against Escherichia coli, Salmonella Typhimurium, and Bacillus cereus. The current study investigated the ability of the plant extract to improve the proliferation and viability of hepatocellular carcinoma cells in a wound closure in vitro assay. The remarkable percentages of terpenes, fatty acids, and their esters characterized from the R. vesicarius extract, as well as the considerable biological results, support the opportunity for future studies on this plant for drug discovery from a natural source. Furthermore, R. vesicarius extract appears to have great potential for controlling disease vector insects. Funding: The authors extend their appreciation to The Researchers Supporting Project number (ISP20-1), Jazan University, Saudi Arabia.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.