Unravelling the Phytochemical Composition and Antioxidant Potential of Different Parts of Rumex vesicarius L.: A RP-HPLC-MS-MS/MS, Chemometrics, and Molecular Docking-Based Comparative Study

Rumex vesicarius L. Polygonaceae is a wildly grown plant in Egypt, North Africa, and Asia with wide traditional uses. Several studies reported its biological activities and richness in phytochemicals. This research addresses a comprehensive metabolic profiling of the flowers, leaves, stems, and roots via RP-HPLC-QTOF-MS and MS/MS with chemometrics. A total of 60 metabolites were observed and grouped into phenolic acids, flavonoids, phenols, terpenes, amino acids, fatty acids, organic acids, and sugars. Principal component analysis and hierarchal cluster analysis showed the segregation of different parts. Moreover, the antioxidant capacity was determined via several methods and agreed with the previous results. Additionally, an in silico approach of molecular docking of the predominant bioactive metabolites was employed against two antioxidant targets, NADPH oxidase and human peroxiredoxin 5 enzyme (PDB ID: 2CDU and 1HD2) receptors, alongside ADME predictions. The molecular modelling revealed that most of the approached molecules were specifically binding with the tested enzymes, achieving high binding affinities. The results confirmed that R. vesicarius stems and roots are rich sources of bioactive antioxidant components. To our knowledge, this is the first comprehensive metabolic profiling of R. vesicarius giving a prospect of its relevance in the development of new naturally based antioxidants.


Introduction
Numerous researchers have focused on exploring new vegetable sources of natural antioxidants for preserving health in response to the increasing need for a healthy lifestyle.Currently, medicinal plants have the potential to be a viable substitute for commercial pharmaceuticals in medicine [1][2][3].Additionally, natural plant-based substances have better pharmacological activity, are less expensive and more effective, and have fewer adverse effects [4,5].Furthermore, investigating the novel functions that the chemical compositions of medicinal plants could have in enhancing or improving the qualities of products derived from plants is still necessary in the research of these compositions [6].Phenolic compounds are one of the main classes of secondary metabolites made by plants.They act as antioxidants and exert powerful protection on food from oxidative damage [7].In recent years, there has been a surge in interest in natural antioxidants, particularly phenolic compounds from plants.

. Phenolic Compounds Phenolic Acids and Phenols
One hydroxybenzoic acid was annotated with m/z 153.019, expressing decarboxylation, and hence was characterized as dihydroxybenzoic acid [35].As for hydroxycinnamic acids, they were distinctive markers in differentiating the studied parts, where three isomers of caffeoyl quinic acid were noticed only in the flowers and were characterized by the appearance of quinic acid at m/z 191.05, followed by its dehydrated ion at m/z 173.05, as well as the presence of the caffeic acid ion at m/z 179.03 followed by its dehydrated ion at m/z 161.02, and decarboxylated ion at m/z 135.04 [22].In this line, Figure 3A demonstrates the fragmentation pattern of caffeoylquinic acid III.Furthermore, eight hydroxycinnamic acids were noticed only in the stems for the first time in the genus Rumex.They occurred as esters of coumaric acid and/or ferulic acid moieties with sucrose.They portrayed the loss of coumaroyl moieties at m/z 146.03, coumaric acid at m/z 163.04, feruloyl moieties at m/z 175.04, and/or ferulic acid at m/z 193.05.Their occurrence and fragmentation are in agreement with several reports of Polygonaceae [31][32][33][34].In this context, helonioside B was noticed with m/z 735.21 expressing the neutral loss feruloyl and ferulic acid moieties as 6 ′ -acetyl-3,6-diferuloylsucrose.Hydropiperoside (m/z 779.22) also portrayed the neutral loss of two coumaroyl moieties followed by dihexosyl moieties loss as β-d-(1,3,6tri-p-coumaryl)-fructofuranosyl-α-d-glucopyranoside [22,32].Two isomers of vanicoside B (m/z 955.27) were observed, showing the sequential loss of dicoumaroyl moieties followed by the neutral loss of ferulic acid and a coumaroyl hexosyl moiety as tricoumaroyl monoferuloyl sucrose, Figure 3B [34].Similarly, two isomers of lapathoside A (m/z 985.28) expressed the sequential loss of coumaroyl, feruloyl, and hexosyl moieties as 1,6 ′ -diferuloyl-3,6-dip-coumaroyl sucrose.Furthermore, lapathoside B isomers I-II (m/z 1015.30)expressed a similar pattern of fragmentation to that of lapathoside A, with a loss of feruloyl moiety instead of a coumaroyl one, as 1,6,6 ′ -triferuloyl-3-p-coumaroyl-sucrose. Figure 3C illustrates the fragmentation pattern of lapathoside A I.
Moreover, 6-gingerol and 8-gingerol were annotated in all parts of R. vesicarius except for the stem in the case of 8-gingerol.It is worth noting that their description is the first in Rumex, and their fragmentation pattern is in agreement with a previous report with the loss of the neutral alkyl moiety CH 3 (CH 2 ) 4 CHO among other fragments [36].

Flavonoids
With regards to flavonoids, they were a distinctive marker in differentiating flowers and stems from other parts in general; they were most abundant in flowers in qualitative and quantitative aspects (Figure 1A,E).From this perspective, they were classified into flavones (14 derivatives), flavonols (12), and a hydroxyflavonoid (Table 1).

Flavones
Flavones were derivatives of apigenin and luteolin.In this context, peak 37 with m/z 269.04 and molecular formula C 15 H 10 O 5 indicated the presence of apigenin innate fragments of m/z 225.05 accounting for apigenin -CO 2 and 151.00 and 117.03 accounting for the characteristic fragments of retro-Diels-Alder (RDA) reactions of flavonoids for the ions [ 1,3 A] − and [ 1,3 B] − , respectively [22,41].Moreover, five constitutional isomers were noticed for apigenin hexoside with m/z 431.10 and molecular formula C 21 H 20 O 10 .In fact, the employment of the state-of-the-art technique of RP-HPLC coupled with high-resolution QTOF-MS and tandem MS/MS enabled the differentiation between such constitutional isomers, where two of them occurred as apigenin-C-hexoside I-II expressing the fragmentation patten of C-glycosides with the neutral loss of n(CHOH, 30 Da) moieties [21,22], whereas three isomers were characterized as apigenin-O-hexoside expressing a hexosyl moiety loss (162 Da) and apigenin aglycone (m/z 269.04) [21,25].It is worth mentioning that apigenin-C-hexoside was reported in Rumex as vitexin and isovitexin, while apigenin-Ohexoside was reported as apigenin-7-O-glucoside [30].Also, four isomers with m/z 563.14 expressing the fragmentation pattern of C-glycosides with the appearance of the ions of m/z 117.03 for the [ 1,3 B] − characteristic apigenin ion were annotated as apigenin-Cpentoside-C-hexoside I-IV [35] and were described for the first time in Rumex, where it was observed in Polygonaceae as isoschaftoside [30].Figure 4A demonstrates the pattern of fragmentation of apigenin-C-pentoside-C-hexoside II.Concerning luteolin derivatives, luteolin aglycone itself was detected with the characterized ions of retro-Diels-Alder (RDA) fragmentation of [ 1,3 A] − and [ 1,3 B] − of m/z 151.00 and 133.03, respectively [22,41]; see Table 1.Additionally, three constitutional isomers of luteolin hexoside were noticed where two isomers exhibited C-glycosylation fragmentation patterns, while the former one portrayed an O-glycosylation fragmentation pattern with the appearance of luteolin aglycone at m/z 285.04 [25,35].It is noteworthy that orientin, isoorientin, and luteolin-7-O-glucoside were reported in Rumex [30].

Flavonols
Twelve flavonol derivatives were detected, mainly in the flowers of R. vesicarius, with the presence of galangin (m/z 269.04), kaempferol (m/z 285.04), and isorhamnetin (m/z 315.05) as aglycones (Table 1).Regarding quercetin derivatives, they occurred mainly as glycosides showing the neutral loss of conjugated moieties followed by the appearance of quercetin aglycone at m/z 301.03 with feature fragments of quercetin, viz., quercetin-CH 2 O (m/z 271.02), quercetin-H 2 O-CO (m/z 255.02), [ 1,2 A] − (m/z 179), and [ 1,3 A] − (m/z 151) [22,41].In this regard, peak 20 was annotated as quercetin-O-hexoside with a hexosyl moiety loss (162 Da) alongside quercetin fragmentation.It bears noting that it was described in R. aquaticus as quercetin-3-O-β-D-galactopyranoside [28].Peaks 24 and 27 exhibited the same fragmentation pattern as peak 20 with an additional loss of an acetyl moiety (42 Da) and hence were characterized as quercetin-O-acetylhexoside I and II (Figure 4B) and described in Polygonaceae as quercetin-3-O-(6 ′′ -acetyl-glucoside).Likewise, two isomers of quercetin-O-diacetylpentoside indicated the neutral loss of two acetyl and one pentosyl moieties followed by quercetin fragmentation (Table 1).Peak 23 showed a quercetin-O-hexoside fragmentation pattern with a prior loss of a hydroxymethylglutaryl moiety (m/z 144 Da) and hence was characterized as quercetin-O-hydroxymethylglutaryl hexoside, which was observed in R. tunetanus [14].Furthermore, three peaks were observed with a fragmentation pattern similar to that of the aforementioned compound with an additional loss of an acetyl moiety (42 Da) (Table 1), and they were characterized as quercetin-O-hydroxymethylglutaryl acetylhexoside I-III as new proposed structures (Figure 4C).
A hydroxyflavonoid was detected in the roots with m/z (395.22) and molecular formula C 25 H 30 O 4 and was characterized as kazinol A (Table 1) [30].

Fatty Acids
Ten fatty acid derivatives were noticed and were classified into short-chain fatty acids as hydroxydecanoic acid and hexanoic acid anhydride, where the former was observed only in the leaves while the latter was noticed only in the roots (Table 1) [30].
As for long-chain fatty acids, three isomers of hydroxylinolenic acid, two isomers of hydroxylinoleic acid, and three isomers of hydroxyoleic acid were characterized in different parts of R. vesicarius [24,40,42].
2.1.5.Amino Acids, Organic Acids, and Sugars Leucine/isoleucine and quinic acids were noticed in the flowers only, and azelaic acid was present in both the flowers and leaves (Table 1) in agreement with previous reports [22,35].
Four sugar derivatives were found only in the roots, namely a disaccharide, two isomers of disaccharide glycerol, and gluco-hepatonic acid hexoside [14,30].

Comparison of the R. vesicarius Different Parts by Multivariate Data Analysis
Multivariate data analysis was employed, considering the relative abundance of the characterized metabolite peaks, for discrimination between the different parts in the conducted study by principal components analysis (PCA) and hierarchical clustering analysis (HCA) [22,43].In this sense, the first three components PCA demonstrated made up 93.57% of the total variance in the score plot.Both the roots and the stems were in one quadrant, as were the flowers and leaves, yet the flowers and stems were closely related to each other (Figure 5A).The biplot of variables and observations showed several metabolites that contributed to the parts' segregation from each other, where some markers were distinguished and aided in parts segregation as galangin, kazinol A, 6-gingerol, vanicoside B I-II, lapathoside B I-II apigenin C hexoside I-II, disaccharide glycerol II, hydroxyoleic acid III, and hydroxylinoleic acid I (Figure 5B).
In the same manner, HCA revealed two main clusters where the leaves were distant from the other parts, as they were clustered in one main cluster while the other cluster was for the remaining parts.Moreover, stems and roots were near each other, as they were located in a subcluster (Figure 5C).In this line, PCA and HCA served in the chemotyping of different R. vesicarius parts for marker distinction and/or to facilitate bioactive metabolites standardization of each part.

Antioxidant Evaluation of Different Parts of Rumex vesicarius Extracts
Oxidative stress is a state that occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to neutralize them with antioxidants.ROS are highly reactive molecules that can damage lipids, proteins, and DNA if left unchecked [44].
Oxidative stress has been implicated in the pathogenesis of many diseases, including cancer, cardiovascular disease, neurodegenerative disorders, diabetes, and aging [45].Overall, maintaining a healthy balance between ROS production and antioxidant defenses is critical for preserving cellular function and preventing oxidative damage [46].Many natural plants have been investigated in cell culture and animal studies, as well as in some human clinical trials, for their potential to prevent or treat conditions linked to oxidative stress.The research is still emerging, but the evidence suggests natural antioxidants may be a useful complement to conventional therapies [47].
Antioxidant activities are evaluated by measuring DPPH, ABTS, H 2 O 2 , FRAP, and TAC scavenging activities in leaf, flower, root, and stem extracts compared to ascorbic acid standard and to each other, as shown in Table S1 and Figure 6, and the determination of IC 50 depends on the percentage of inhibition of activity, as shown in Tables S2-S6 and Figures S1-S5.
The results showed that the stems are the most active part, as they exhibited the highest antioxidant activity with IC 50 14.11± 0.52, 12.28 ± 0.47, 14.99 ± 0.11, 21.37 ± 4.01, and 10.16 ± 0.77 for DPPH, ABTS, H 2 O 2 , FRAP, and TAC scavenging activities, respectively, close to standard values, followed by flowers extract, which showed similar antioxidant values that were close to those of stems.
Our results agreed with previous works which suggest that R. vesicarius is rich in polyphenolic compounds, such as flavonoids, phenolic acids, and tannins [15].These phytochemicals are known to have potent antioxidant and free radical scavenging abilities [48].Several studies have shown that extracts of R. vesicarius exhibit strong free radical scavenging activity and reducing power and metal chelating ability in cell-free assays.The antioxidant capacity of the plant has been attributed to its high content of phenolic and flavonoid compounds [20].
R. vesicarius extracts have been found to enhance the activities of endogenous antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, in experimental models [49].In a previous study, R. vesicarius extracts demonstrated the ability to protect against oxidative stress-induced damage in various organs, including the liver, kidneys, and brain [50].The antioxidant properties of the plant may contribute to its observed protective effects against conditions like diabetes, inflammation, and heavy metal toxicity [45].Based on the available evidence, R.vesicarius shows promise as a natural source of antioxidants that could be useful in the prevention or management of diseases linked to oxidative stress.However, more clinical research is needed to fully understand the therapeutic potential of this plant and its active constituents.In summary, the robust antioxidant potential of R. vesicarius is primarily attributed to its rich phytochemical profile, particularly its high content of polyphenolic compounds.Further investigation is warranted to explore the possible therapeutic applications of this natural antioxidant source.

Antioxidant Evaluation of Different Parts of Rumex vesicarius Extracts
Oxidative stress is a state that occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to neutralize them with antioxidants.ROS are highly reactive molecules that can damage lipids, proteins, and DNA if left unchecked [44].
Oxidative stress has been implicated in the pathogenesis of many diseases, including  R. vesicarius extracts have been found to enhance the activities of endogenous antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, in experimental models [49].In a previous study, R. vesicarius extracts demonstrated

Docking against Antioxidant Molecular Targets
The virtual molecular study of the predominant polyphenol components, identified from various parts of R. vesicarius methanolic extracts, were carried out within the binding sites of NADPH oxidase (NO) and human peroxiredoxin 5 enzyme (PRDX5).The results are recorded in Table 2 and Figure 7 and Figure S6 and revealed that most of the examined compounds showed inhibitive activity against both tested enzymes, with varieties in the scores that showed similarity to FAD and benzoic acid co-crystallized ligands.the ability to protect against oxidative stress-induced damage in various organs, including the liver, kidneys, and brain [50].The antioxidant properties of the plant may contribute to its observed protective effects against conditions like diabetes, inflammation, and heavy metal toxicity [45].Based on the available evidence, R.vesicarius shows promise as a natural source of antioxidants that could be useful in the prevention or management of diseases linked to oxidative stress.However, more clinical research is needed to fully understand the therapeutic potential of this plant and its active constituents.In summary, the robust antioxidant potential of R. vesicarius is primarily attributed to its rich phytochemical profile, particularly its high content of polyphenolic compounds.Further investigation is warranted to explore the possible therapeutic applications of this natural antioxidant source.

Docking against Antioxidant Molecular Targets
The virtual molecular study of the predominant polyphenol components, identified from various parts of R. vesicarius methanolic extracts, were carried out within the binding sites of NADPH oxidase (NO) and human peroxiredoxin 5 enzyme (PRDX5).The results are recorded in Table 2 and Figures 7 and S6 and revealed that most of the examined compounds showed inhibitive activity against both tested enzymes, with varieties in the scores that showed similarity to FAD and benzoic acid co-crystallized ligands.

Interactions with the NADPH Receptor
NADPH oxidase (NOX) is one of the crucial oxidative bursts and consists of seven members implicated in various vital human physiological processes that are medicated to release reactive oxygen species (ROS).ROS have two distinct responsibilities that change based on the situation.Numerous illnesses and dysfunctions, including immunodeficiency and lung and cardiovascular disorders have been linked to ROS-mediated stress.On the other hand, the NADPH oxidase (NOX)-mediated release of ROS results in the removal of invasive germs in neutrophils and macrophages and hence acts as an inflammatory mediator [51][52][53].Kazinol A and quercetin-3-O-6 ′′ -(3-hydroxyl-3-methylglutaryl)-D-glucopyranoside, followed by hydropiperoside, exhibited the best fit position within the enzyme active site, more than the co-crystalline ligand, displaying binding affinities (BA) of −7.7, −7.6, and −7.5 Kcal/mol, respectively (Table 2).The formation of many linkages was explained by this fitting within the active site of the NADPH-oxidase enzyme.Hydropiperoside established four conventional-H bonds with Arg246, Asn34, Glu250, and Met33 and two C-H bonds with Asn34 and Pro117, together with three different pi-bonds including pi-cation and pi-sigma bonds with Lys116 and pi-sulphur bonds with Met33 together with the formation of multiple Van interactions (Figures 7A and S6
Regarding the in silico results for both enzymes, they supported the obtained in vitro results, wherein hydropiperoside, vanicoside B, lapathoside A and lapathoside B existed only in the stems and, for instance, kazinol A existed only in the roots when compared to other organs and it appears that the highest potent antioxidant activity is dependent Plants 2024, 13, 1815 20 of 28 on the subclass hydroxy-cinnamic acids present in the stems organ, as revealed in all the investigated assays.
formed three hydrophobic alkyl bonds with Ile119, Leu116, and Lys49 and one conventional H-bond with Thr147 (Figures 8D and S6(11B)).All abovementioned compounds showed several Van der Waals interactions in Figures 8 and S6B.
Regarding the in silico results for both enzymes, they supported the obtained in vitro results, wherein hydropiperoside, vanicoside B, lapathoside A and lapathoside B existed only in the stems and, for instance, kazinol A existed only in the roots when compared to other organs and it appears that the highest potent antioxidant activity is dependent on the subclass hydroxy-cinnamic acids present in the stems organ, as revealed in all the investigated assays.

ADME Predictions of the Selected Compounds
ADME parameters were estimated to the major compounds identified in the four different organs of R. vesicarius methanolic crude extract which make them a promising option for drug development.Interestingly, one phytoconstituent was found to achieve Lipinski's rule of five with a good to poor bioavailability score of most phytoconstituents.It is also important to note that the majority of the compounds are shown to be soluble in water in the results that is a crucial property for absorption and distribution within the body.The possibility for developing a transdermal medicine delivery method must be evaluated by measuring the skin's permeability, or the rate at which a molecule penetrates the stratum corneum.It was discovered that the crude extracts identified phytochemical ingredients, all had good skin penetrability.Finally, the majority of the detected phyto-compounds were discovered to be P-gp inhibitors or substrates.P-gp is the primary component of ATP-binding cassette transporters, also known as ABC transporters, which are employed to safeguard the central nervous system (CNS) against exogenous agents and are a primary technique for identifying active efflux across biological membranes.It was discovered that the majority of the phytoconstituents were not CYP3A4 inhibitors, which is the isoenzyme in charge of 60% of xenobiotic metabolism, which includes carcinogens, drugs, eicosanoids, and steroids.
ADME tool results showed that all compounds have poor bioavailability (0.11-0.17) except compounds 5, 6, and 12 with a high score (0.55), and only compound 6 fulfilled all drug-likeness rules without any violations.These three compounds had moderate synthetic accessibility (4.34-5.12),which showed an explicit synthetic route, meanwhile, other compounds (1-4, and 7-11) showed a moderate to difficult synthetic accessibility range from 5.04-8.12.In addition, all compounds were varied from being poor to being highly soluble in water, especially compound 1 was observed as a high soluble in water, on the other hand compounds 2, 4, 5, 9, 10, and 12 were observed with good solubility and only compound 3 was showed as a moderately soluble in water.
Sweilam et al., 2023 with their co-workers mentioned that the absorption and diffusion of the compounds through the blood-brain barrier were predicted by using the BOILED-Egg tool [52].It is worth mentioning that compound 6 was the only one that has high GIT absorption properties, on the other hand, all compounds (1-12) predicted poor diffusion through the BBB, and all have high permeability values for skin permeable parameters.Compound 1 is thought to enter the skin at a log Kp value of greater than 2.5 cm/h, which had the best skin penetrability, followed by compound 2 then compound 10.Seven compounds (1-3, 7-8, and 10-11) showed binding affinity to the P-gp substrate, whereas only compound 6 showed inhibition activity against CYP3A4 and CYP2C9 human isoforms.Skin permeation (log Kp), distribution, and ADME properties were analysed by the Swiss-ADME software and recorded in Table 3 and Figure 9.The final conclusions +revealed that one of the twelve compounds fulfilled the oral drug ability of Lipinski's rule of five, with five slightly meeting the criteria of this rule.No MR: molar refractivity, "Å 2 " polar surface area, "TPSA" Topological polar surface area, and Gentiobiosylglycerol (1), Helonioside B (2), Hydropiperoside (3), Isoorientin (4), Isovitexin (5), Kazinol A (6), and Lapathoside A (7), Lapathoside B (8), Orientin (9), Quercetin-3-O-6 ′′ -(3-hydroxyl-3-methylglutaryl)-D-glucopyranoside (10), Vanicoside B (11), Vitexin (12).SwissADME web tool [55].

Preparation of Different Parts Extracts
The dried powdered flowers, stems, leaves, and roots (200, 500, 100, and 10 g, respectively) were defatted with n-hexane followed by maceration in 80% methanol, (20 mL solvent for each gram of powder under ultrasonication for one hour per day twice) till exhaustion.Extracts were collected, filtered, pooled, and evaporated under vacuum (at 40 • C) until free from solvent to give dark residues (10.23, 24.23, 4.32, and 1.1 g) for the flowers, stems, leaves, and roots, respectively.All extracts were saved for further examination at −20 • C [23].
The system was connected to a 6530-quadruple time of flight (Q-TOF) LC/MS (Agilent Technologies) that had a dual ESI interface, as described in references [22,25].Regarding the operating conditions, they were governed by MassHunter Workstation software B.06.00 (Agilent technologies).Briefly, the drying gas temperature was 325 • C (10 L/min flow; 20 psig nebulizer pressure).The temperature of the sheath gas was 400 • C, and it flowed at a rate of 12 L/min.The capillary voltage was set at 4000 V, the nozzle voltage at 500 V, the fragmentor voltage at 130 V, the skimmer voltage at 45 V, and the octupole radiofrequency voltage at 750 V.The mass to charge (m/z) ranged from 70 to 1500 Da.
The data processing and characterization of metabolites were conducted using Mass-Hunter Qualitative Processing B.06.00 (Agilent Technologies) as stated in references [21,24,25] taking into consideration observed m/z, RT, associated fragments for m/z and generated molecular formulae.

Evaluation of the Antioxidant Activity Using DPPH Scavenging
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was produced in a fresh methanol solution and kept in the dark at 10 • C. the test compound was prepared.A 40 µL aliquot of methanol was added to 3 mL of DPPH solution.Using a UV-visible spectrophotometer, absorbance values were promptly recorded (Milton Roy, Spectronic 1201).The absorbance for (control) and the reference compound ascorbic acid were also detected.All the determinations were performed in three times and averaged [56,57].The percentage inhibition (PI) of the DPPH radical was calculated using the Formula (1): where A C = Absorbance of the control and A T = absorbance of the sample + DPPH.The 50% inhibitory concentration (IC 50 ), (the concentration needed to achieve 50% DPPH radical scavenging activity) was calculated from dose response curve graphic plots.

Evaluation of the Antioxidant Activity Using ABTS Radical Scavenging
ABTS scavenging capacity is a decolorization assay which is determined according to the procedure described by others [57,58].Measurements were taken at room temperature.The extracts were diluted at a ratio of 1:10 with (80%) methanol.
The absorbance at 734 nm was measured every minute for 13 min following initial mixing.Ascorbic acid and methanol were used as the standard antioxidant and the negative control, respectively.Experiments were performed three times with three replicates for each sample.The percent free radical scavenging activity was calculated according to Formula (2): where A n is the final absorbance values of the negative control, and A s is the final absorbance values of the sample.The 50% inhibitory concentration (IC 50 ) was determined as showed in DPPH method.

Evaluation of the Antioxidant Activity Using H 2 O 2 Scavenging Assay
Antioxidant activity using H 2 O 2 acavenging assay was performed as previously described [59].The H 2 O 2 radical scavenging percentage of the extracts was calculated using the following Formula (3): The 50% inhibitory concentration (IC 50 ) was determined from graphic plots of the dose response.

Evaluation of the Antioxidant Activity Using FRAP Scavenging
The reduction of ferric to ferrous ions by the extract is an indication of the potential antioxidant property.The reducing power of the extract was evaluated as previously described [60,61].This method is based on the reduction of ferricyanide relative to extracts.Samples (1 mg/mL) in 1 mL of methanol were mixed with 2.5 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 mL of potassium ferricyanide [K 3 Fe (CN) 6 ] (1%, w/v).After 20 minutes of incubation at 50 • C, the reaction mixture was acidified with 2.5 mL of trichloroacetic acid (10%, w/v).The reaction mixture was centrifuged at 1000× g for 10 minutes.The supernatant solution (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of freshly prepared ferric chloride (0.1%, w/v).The absorbance of the resulting solution was measured at 700 nm versus a blank using the previously mentioned spectrophotometer.The reducing capability percentage (%) was calculated as follows (4): where, A 0 : absorbance of the control solution.A s : sample absorbance.The 50% inhibitory concentration (IC 50 ) was calculated using the DPPH method.

Determination of the Total Antioxidant Capacity (TAC)
The TAC was investigated by means of the method previously reported [62].Ascorbic acid and methanol were used as the standard antioxidant and the negative control, respectively.Experiments were performed three times with three replicates for each sample.The total antioxidant activity percent was calculated according to the following Formula (5): % Activity = [(A n − A s ) × 100]/An (5) where A n is the final absorbance values of negative control, and A s is the final absorbance values of sample.The 50% inhibitory concentration (IC 50 ), was estimated from graphic plots of the dose response.

Statistical Analysis
Statistical analysis was done employing one-way analysis of variance (ANOVA) where the results are expressed as the mean ± standard deviation of the mean (SD) followed by Tukey as a post-hoc test.Values with p < 0.05 are considered significantly different using Graphpad Prism software 5.04 (San Diego, CA, USA), the conditional formatting was performed by Microsoft Excel 365 (Redmond, WA, USA), and the bubble plot was created by Minitab 17 (Minitab, Inc., State College, PA, USA) [21].

Conclusions
The conducted study portrays a comprehensive metabolic profiling of different parts of R. vesicarius, revealing 60 metabolites.The stems were most abundant in hydroxycinnamic acids, whereas the flowers were most abundant in flavonoids.PCA and HCA enabled the discrimination of the studied parts.The stems showed antioxidant potential similar to ascorbic acid, the used standard.Overall, high concentrations of the polyhydroxy derivatives identified in the different organs of R. vesicarius ascertain its antioxidant characteristic properties.Further in vitro and in silico studies are needed to confirm this activity.Herein, future prospective in vivo pharmacological analysis is necessary to support the confirmation of these properties of the identified molecules as antioxidant agents.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants13131815/s1,Table S1.IC 50 for polar fractions of leaves, flowers, roots, stems extracts and ascorbic acid as std against DPPH, ABTS, H 2 O 2 , FRAP, and TAC scavenging activities, Results are shown as means ± S.D. (measured in triplicate; n = 3).Means in the same column that do not share a letter are significantly different (p < 0.05) using ANOVA followed by Tukey as a post-hoc test.Table S2.DPPH scavenging % activity (IC 50 value) of four plant parts extract at different concentration.Table S3.ABTS scavenging % activity (IC 50 value) of four plant parts extract at different concentration.Table S4.H 2 O 2 scavenging % activity (IC 50 value) of four plant parts extract at different concentration.Table S5.FRAP scavenging % activity (IC 50 value) of four plant parts extract at different concentration.Table S6.TAC scavenging % activity (IC 50 value) of four plant parts extract at different concentration.

Plants 2024 , 29 Figure 1 .
Figure 1.Base peak chromatograms (BPCs) of (A) flowers, (B) leaves, (C) stems, and (D) roots of R. vesicarius and bubble plot of the observed masses m/z vs. the retention time concerning metabolite classes and peak areas for the (E) flowers, (F) leaves, (G) stems, and (H) roots.

Figure 1 .
Figure 1.Base peak chromatograms (BPCs) of (A) flowers, (B) leaves, (C) stems, and (D) roots of R. vesicarius and bubble plot of the observed masses m/z vs. the retention time concerning metabolite classes and peak areas for the (E) flowers, (F) leaves, (G) stems, and (H) roots.

Figure 2 .
Figure 2. Structures of the major bioactive metabolites in the different parts of R. vesicarius L.Figure 2. Structures of the major bioactive metabolites in the different parts of R. vesicarius L.

Figure 2 .
Figure 2. Structures of the major bioactive metabolites in the different parts of R. vesicarius L.Figure 2. Structures of the major bioactive metabolites in the different parts of R. vesicarius L.

Plants 2024 ,
13, x FOR PEER REVIEW 14 of 29 of different R. vesicarius parts for marker distinction and/or to facilitate bioactive metabolites standardization of each part.

Figure 7 .
Figure 7. Two-dimensional (2D) molecular interactions of the most dominant chemical compounds: (A) compound 3; (B) compound 6; and (C) compound 10 identified in different R. vesicarius organs with the active site of the NADPH oxidase (NO) receptor (PDB ID:2CDU), (dimensions X:19.1672,Y: 17.5909, Z:29.7368), (root mean square deviation) RMSD < 2. 2.4.1.Interactions with the NADPH Receptor NADPH oxidase (NOX) is one of the crucial oxidative bursts and consists of seven members implicated in various vital human physiological processes that are medicated to release reactive oxygen species (ROS).ROS have two distinct responsibilities that change based on the situation.Numerous illnesses and dysfunctions, including
Figure S1: Evaluation of Antioxidant Activity using DPPH scavenging.
Figure S2: Evaluation of Antioxidant Activity using ABTS radical Scavenging.

Figure S3 :
Evaluation of Antioxidant Activity using H 2 O 2 Scavenging Assay.

Table 1 .
Metabolites characterized in the flowers, leaves, stems, and roots of R. vesicarius L.

Table 2 .
Docking analysis of the predominant compounds identified in different R. vesicarius organs against antioxidant enzymes (binding energies, interaction type derived from the best conformations of each compound into the macromolecule).

Table 3 .
In silico ADME-physicochemical predictions of the predominant compounds 1-12 identified in different R. vesicarius organs.