Lepidium meyenii (Maca) Roots: UPLC-HRMS, Molecular Docking, and Molecular Dynamics

Lepidium meyenii or Maca is widely cultivated as a health care food supplement due to its nutritional and medicinal properties. Although there are a few in-depth studies evaluating Maca antihypertensive effects, the correlations between the chemical constituents and bioactivity of the plant have not been studied before. Thus, the roots were extracted using different solvents (aqueous, methanol, 50% methanol, and methylene chloride) and investigated for their antihypertensive and antioxidant activities through several in vitro assays. The methanolic extract exhibited the best renin and angiotensin converting enzyme (ACE) inhibitory activities with IC50 values of 24.79 ± 1.3 ng/mL and 22.02 ± 1.1 ng/mL, respectively, along with the highest antioxidant activity. In total, 120 metabolites from different classes, e.g., alkylamides, alkaloids, glucosinolates, organic acids, and hydantoin derivatives, were identified in the methanolic extract using ultrahigh-performance liquid chromatography/high-resolution mass spectrometry (UPLC/HRMS). Molecular docking simulations were used to investigate the potential binding modes and the intermolecular interactions of the identified compounds with ACE and renin active sites. Glucotropaeolin, β-carboline alkaloids, succinic acid, and 2,4-dihydroxy-3,5-cyclopentyl dienoic acid showed the highest affinity to target the ACE with high docking scores (S ranging from −35.32 to −22.51 kcal mol–1) compared to lisinopril (S = −36.64 kcal mol–1). Interestingly, macamides displayed the greatest binding affinity to the active site of renin with docking scores (S ranging from −22.47 to −28.25 kcal mol–1). Further, β-carbolines achieved docking scores comparable to that of the native ligand (S ranging from −13.50 to −20.06 kcal mol–1). Molecular dynamics simulations and MMPBSA were also carried out and confirmed the docking results. Additionally, the computational ADMET study predicted that the compounds attaining promising docking results had proper pharmacokinetics, drug-likeness characteristics, and safe toxicological profiles. Ultimately, our findings revealed that Maca roots could be considered a promising candidate as an antihypertensive drug.


INTRODUCTION
Maca (Lepidium meyenii Walp., Brassicaceae family), also known as Peruvian ginseng, is a well-known crop in Peru widely used as a health care food supplement and medicine. 1,2 The Food and Agriculture Organization recommended Maca as a safe edible food in 1992. 2 After that, it was considered one of the star products in the global health care market due to its various potential effects. The plant is well known for its nutrient compositions, such as minerals, proteins, starches, amino acids, sugars, and fatty acids, and commonly purchased in the form of capsules or powder worldwide as a nutritional supplement. 3 Traditionally, L. meyenii root is used as an active component in oils, coffee, or chocolate to relieve fatigue, strengthen the body, and improve fertility and sexual function. 4,5 Many pharmacological activities have been reported, such as aphrodisiac, antiproliferative, antioxidant, learning and memory enhancing, antidepressant, antirheumatic, UV radiation protection, hepatoprotective, antifatigue, immunomodulatory, neuroprotective, and fertility increasing. 6 Additionally, it could be used for chemical and physical stress responses, premenstrual discomfort and menopausal symptoms, prostatic hyperplasia, osteoporosis, and locomotion. 3,4 The main compounds found in L. meyenii are glucosinolates, isothiocyanates, (thio)hydantoins, macaenes, polysaccharides, fatty acids, alkaloids, macamides, and flavonols. 6 Hypertension (HTN) is a growing public health concern where one in three adults worldwide has HTN, which is exceedingly more than 10% of the worldwide population. 7 The prevalence of HTN has been shown to increase with age. Poor control of HTN is closely linked to the burden of cardiovascular diseases and high mortality rates. 8 Several classes of HTN medications have been developed; however, many of these drugs have been reported to have undesirable adverse effects. It is known that plant extracts are less toxic, contributing to their preference over synthetic drugs. Moreover, plants consumed in diet have been found to be useful in the management of HTN. 9 For this purpose, the investigation of plants used in traditional medicine for potential hypotensive effects is an important strategy for the development of new and safe drugs. Based on the strong relationship between oxidative stress and HTN, the use of a single agent with antioxidant and antihypertensive properties could provide an effective strategy for the prevention and treatment of HTN. 10 Interestingly, many plants and functional foods displayed high antioxidant effects and a great ability to scavenge free radicals in oxidative stress. 11 Liquid chromatography coupled with high-resolution mass spectrometry is a widely employed technique to identify chemical constituents in different plant extracts with high sensitivity and specificity. 12 It allows the separation of many kinds of substances in a high throughput manner. 13,14 The high-resolution mass analysis allowed accurate dereplication of monoisotopic masses of known natural products using commercial databases such as the Dictionary of Natural Products (DNP), LipidMaps, and KEGG. 13 As a computer-assisted approach, molecular docking plays an important role in predicting the binding modes and abilities of small molecules toward the targeted enzymes and receptors. In addition, it has exhibited the efficient screening ability of bioactive components with multiple targets with a high degree of accuracy. Implementation of molecular docking methods facilitates the discovery of novel therapeutic drugs that can treat a variety of chronic diseases. 15,16 Being a plant with many health effects, Maca has attracted a lot of attention as a potential antihypertensive agent. 17 For this purpose, the objective of our study was to evaluate the antihypertensive effects of different Maca extracts through in vitro inhibition of Renin and ACE enzymes and to assess their antioxidant effects using several assays, i.e., total antioxidant capacity (TAC), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS), ferricreducing antioxidant power (FRAP), and iron-reducing power. Furthermore, a comprehensive profiling of the active extract was performed through a sensitive and accurate UPLC/HRMS method to identify the bioactive compounds in the extract. The potential binding modes and intermolecular interactions of the identified compounds with ACE and Renin active sites were studied using molecular docking simulations to better investigate the mode of interaction underlying the inhibitory effects for the first time. In addition, molecular dynamics simulations and PBSA analysis were also carried out to further validate the binding of the most promising candidates as well as to examine the stability of the complexes formed. Finally, ADMET computational parameters were evaluated to predict the physicochemical properties and toxicity profiles of the tested compounds.

RESULTS AND DISCUSSION
2.1. In Vitro Antioxidant Assays. There is a strong indication that many threatening pathophysiological processes such as cardiovascular diseases are associated with the accumulation of free radicals in the body. As innate antioxidant defense mechanisms are inefficient, the dietary intake of antioxidants is vital to protect our cells from damage by free radicals. To evaluate the antioxidant potential of the four extracts, five in vitro complementary assays were used. Some methods utilize radical producers, e.g., DPPH and ABTS, and some use metal ions for oxidation, e.g., iron-reducing power assay. Our results showed that the methanolic extract exhibited the most potent activity, as depicted in Table 1, with an IC 50 value of 41.44 ± 2.44 μg/mL and EC 50 of 60.05 ± 2.38 μg/mL in the DPPH and iron-reducing power assays, respectively, as compared to ascorbic acid with IC 50 = 22.44 ± 1 μg/mL and EC 50 = 57.76 ± 4.75 μg/mL, respectively. In addition, the methanolic extract showed significantly higher total antioxidant capacity and FRAP and ABTS scavenging activities, 303.91 ± 6.5, 293.99 ± 2.5, and 1072.93 ± 7.77, respectively, calculated as μM ascorbic acid equivalent/g extract.
2.2. In Vitro Antihypertensive Assays. The Renin− angiotensin system is considered an important determinant of blood pressure and fluid homeostasis in the human body. Renin catalyzes the first and rate-limiting step that initiates the production of angiotensin-I, which is subsequently converted by the action of ACE to angiotensin-II, a potent vasoconstrictor that contributes to blood pressure elevation. Therefore, simultaneous inhibition of Renin and ACE activities would efficiently regulate the Renin−angiotensin system, providing a new alternative way to treat hypertension efficiently. 18,19 The results obtained from the inhibitory effects on Renin and ACE activities of Maca extracts (Table 2) showed that the methanolic extract exhibited the highest inhibitory effects compared to the other extracts (aqueous, 50% methanol, and methylene chloride). In our study, the IC 50 values of the methanolic extract for Renin and ACE inhibitions were 24.79 ± 1.3 ng/mL and 22.02 ± 1.1 ng/mL, respectively, indicating a good hypotensive effect when compared to Quinapril with IC 50 = 15.01 ± 0.8 ng/mL and 13.97 ± 0.7 ng/mL, respectively.
2.3. UPLC-HRMS Analysis of the Secondary Metabolites. The chemical constituents of the active methanolic extract were analyzed via UPLC-HRMS in both positive and negative ionization modes to highlight possible different classes of bioactive compounds that may be correlated to the hypotensive effects of the Maca extract. All compounds Imidazole alkaloids, β-carboline alkaloids, macaridines, and common amide alkaloids are the most characteristic alkaloids in Maca. 2 In total, 35 alkaloids (Table 3 and Figure 1) were tentatively identified in the Maca extract in the positive ionization mode.
2.3.1.1. Identified Imidazole Alkaloids. Many imidazole alkaloids such as lepidilines A−G were previously isolated and characterized from Maca roots. 20,21 They are tertiary and quaternary alkaloids derived from histidine with the 1,3dibenzylimidazolium nucleus. 20,21 Lepidiline alkaloids were reported to increase fertility and inhibit human ovarian, bladder, and pancreatic cancer cells. 22 Thirteen imidazole alkaloids were identified based on their MS/MS fragmentation and reported literature. 2,22 Lepidiline A (compound 49, Table 3 and Figure S1) showed a precursor ion [M − Cl] + at m/z 277.1700 and a product ion at m/z 185.1062 generated by splitting of the C 7 H 7 group from the parent ion.
Lepidiline C (compound 50, Table 3 and Figure S2) had a molecular ion at m/z 307.1805 and gave the same characteristic ion as lepidiline A (m/z 185.1062) besides a fragment ion at m/z 121.0645, indicating the formation of a methoxy benzyl ion (C 8 H 10 O) + .
For compound 51 (lepidiline B, Table 3 and Figure S3), the [M − Cl] + ion was at m/z 291.1855, and the base peak was at m/z 199.1232 (C 13 H 15 N 2 ) + , which resulted from the loss of C 7 H 7 .
Lepidiline D (compound 54, Table 3 and Figure S4) displayed a molecular ion at m/z 321.1932 and produced a characteristic product ion due to the loss of phenyl, methoxy, benzyl, and methoxybenzyl groups. Thus, fragment ions at m/z 246.2423, 199.1232, and 121.0654 were obvious in the MS 2 spectrum of lepidiline D ( Figure S4). Following the same dissociation pathways, the remaining imidazole alkaloids were assigned.
The mass spectrum of compound 43 was identified as 1pentyltetrahydro-β-5,6-carboline-3-carboxylic acid (Table 3 and Figure S9) based on its MS/MS data and previously reported literature. 2 It showed a [M + H] + ion at m/z 285.1596 and two fragments at m/z 239.1545 (C 16 H 20 N 2 ) + and m/z 121.0645 (C 7 H 9 N) + , which were generated from the multiple breakages of phenyl, pentyl, and carboxylic groups as demonstrated in Figure S9.
Two more β-carboline alkaloids (peak nos. 34 and 59, Table  3) were in accordance with the aforementioned fragmentation All data are presented as (mean ± SD). Different superscripts letters for a given value within a column are significantly different from each other (Duncan's significant difference multiple range post-hoc test, P < 0.05).   Table 3 and Figures S11−12) in the positive mode.
Compound 45 (Table 3 and Figure S11) was identified as 3benzyl-1,2-dihydro-4-methoxy-N-hydroxypyridine. It Figure S12). In addition, the cleavage of C 6 H 5 , C 9 H 6 NO 2 , C 7 H 7 , and C 7 H 7 NO 2 groups from the precursor ion was observed. 2 Furthermore, common amide alkaloids such as 2-phenylacetamide, N-methyl-3-hydroxy-benzeneacetamide, and Nbenzylformamide (Table 3) were deduced by comparing their fragmentation pattern together with the literature. The  4 They are benzylalkylamides, which exhibit many biological functions such as improving sexual potential and neuroprotective, antiinflammatory, anticancer, and antifatigue effects. 31−35 They are better ionized and detected in the positive ionization mode. 36 The molecular ions, fragment ions, and retention times for 19 macamides detected in the methanolic extract (Table 3 and Figure 1) were identical to the previously reported chemical profiling of Maca roots. 2,37,38 All macamides showed a high intensity of protonated molecular ions [M + H] + in their mass spectra, while the intensity of other fragment ions corresponding to an alkyl chain was relatively low in the spectra due to the relative instability of the long-chain aliphatic group in the mass spectrometer. 51 (Table 3 and Figures S13−14). Compound 108 exhibited a molecular ion at m/z 400.3185 and fragment ions at m/z 121.0651 and m/z 138.0906 characteristic of the N-(3-methoxybenzyl)-type macamides. 39,40 Compound 110 showed a molecular ion at m/z 370.3127 beside the product ion at m/z 108.0808 characteristic of the Nbenzyl-type macamides. 39 The remaining macamides (Table 3) were identified following the same dissociation pathways which include the formation of the distinct ions at m/z values of 108, 121, and 138 which corresponded to the formation of Nbenzyl, 3-methoxybenzyl, and N-3-methoxy benzyl ions, respectively. 21,27 2.3.3. Identification of Macaenes and Fatty Acids in Maca. Macaenes are characteristic polyunsaturated fatty acids found in Maca roots. 21 In the present study, six macaenes were observed mainly in the negative ionization mode (Figure 2). Among them, 55-oxo-6E,8E-octadecadienoic acid (compound 74, Table 3 Figure S15). The fragments ions at m/z 279.2331 and 152.9949 indicated the loss of CH 2 and C 10 H 18 groups, respectively, from the molecular ion ( Figure S15). The fragment at m/z 125.0964 was generated by the cleavage of a CO group from the fragment ion at m/z 152.9949, while the product ion at m/z 185.1174 was attributed to the loss of a C 9 H 16 group from the parent ion. 2 The MS behaviors of the other identified macacenes were in line with that of compound 74 (Table 3).
Twenty-six fatty acids, mainly polyunsaturated and hydroxylated, such as oleic acid, linolenic acid, and hydroxy-linoleic acid, were tentatively identified in Maca extract ( Table 3). The diagnostic fragment ions generated by loss of carbon dioxide and water molecules were predominant in their MS/MS spectra. 41 Product ions were generated by loss of C 6 H 6 , C 5 H 6 O 2 , and C 8 H 8 O 2 groups from the molecular ions. 42 2.3.4. Identification of Glucosinolates in Maca. Glucosinolates are common secondary metabolites present in Maca and are responsible for the pungent flavor of the roots. Glucosinolates are assumed to exhibit many biological effects such as cancer chemoprotective, antioxidant, and apoptosispromoting activities. 29 Furthermore, glucosinolates can modulate insulin resistance and improve metabolic disorders. 43 Glucotropaeolin, the most abundant glucosinolate in Maca, was observed in the negative mode at a retention time of 3.92 min (compound 26, Table 3 and Figure 2). The mass spectrum of glucotropaeolin ( Figure S16) showed a molecular [M − H] − ion at m/z 408.0435 and a glucosinolate specific fragment ion at m/z 259.0134 corresponding to the neutral loss of R-NCS from the parent ion. Moreover, another diagnostic peak at m/z 195.0338, corresponding to the formation of a D-thioglucose fragment (C 6 H 11 O 5 S) − was observed in the spectrum. 44 Two product ions at m/z 328.0877 (C 14 Table 3 and Figure S17). It produced a protonated molecular ion at m/z 261.1234. MS2 data showed fragments at m/z 231.0294, 184.0737, and 121.0644, indicating loss of OCH 3 , C 6 H 5 , and C 6 H 8 N 2 O 2 groups, respectively, from the molecular ion. 6,45 Meyeniin C (compound 47, Table 3), a thiohydantoin derivative, that was recently isolated from Maca roots 46 has been detected in both ionization modes. In the negative ion mode at a retention time of 9.57 min (Figure 2), meyeniin C revealed a parent ion with m/z 307.0724 ( Figure S18). The successive breakdown of the hexahydroimidazo[1,5-c]thiazole ring in MS 2 experiment was responsible for the fragmentation behavior of meyeniin C (Figures S18−19). 6 2.3.6. Identification of Organic Acids and Miscellaneous Compounds in Maca. Acids undergo better fragmentation and give higher abundant ions in the negative ionization mode. The fragmentation patterns of identified organic acids were consistent with the previously reported data. 2,47 In agreement with the previous literature, the loss of CH 2 , CO 2 , and H 2 O groups was considered as the representative fragmentation pathway in acids (Table 3). Based on the accurate mass measurements and MS/MS data, seven organic acids have been identified, such as succinic acid, hydroxy succinic acid, and 2,4-dihydroxy-3,5-cyclopentyl dienoic acid (Table 3).
Finally, 24 miscellaneous compounds were identified in Maca extract including amino acids, organic esters, sugars, and benzyl isocyanate (Table 3). Their identities were confirmed by comparing their retention times and fragmentation patterns with data given in the literature, 48 17 the correlations between Maca chemical constituents and enzyme inhibitions have not yet been studied. Therefore, in this study ACE and renin inhibitory properties of different extracts of Maca were evaluated followed by metabolite profiling of the active extract using UPLC/HRMS. Moreover, the possible mechanisms of action based on structure−activity relationships and molecular docking were investigated.
To examine the possible ligand-structure/enzyme-binding relationships and to confirm the experimental data, selected compounds from each class, viz., lepidilines, β-carbolines, macaridines, macamides, glucosinolates, etc., were docked into the active site of ACE and renin.
First, docking of the identified compounds into the threedimensional X-ray crystallographic structure of the angiotensin converting enzyme (PDB code: 1O86) in complex with lisinopril was accomplished using Molecular Operating Environment (MOE) 2010.10. The antihypertensive drug, lisinopril, forms hydrogen bonds with Tyr 520, Lys 511, His 353, and Tyr 523 of ACE through its carbonyl and carboxylate groups, in addition to ionic bonds with Glu384 and Zn701 via charged amino and carboxylate groups ( Figure S20). Interestingly, zinc is an essential catalytic component of ACE. Thus, interaction with the zinc-binding motif is crucial for ACE inhibition.
To confirm the accuracy of the used docking process, validation was achieved by redocking of the cocrystallized ligand into the active site of ACE. The validation results showed that the docking protocol is suitable for the anticipated docking study. This is confirmed by the perfect alignment between the coordinates of lisinopril and its self-docked pose with root-mean-square deviation (RMSD) of 0.811 Å and docking score (S) of −36.64 kcal mol −1 . Moreover, the selfdocked pose was able to attain the same binding interactions shown by the cocrystallized ligand with the key amino acids in the binding site of ACE.
The results of the docking study in the active site of ACE showed that out of all studied compounds, ten compounds showed promising results. Glucotropaeolin, succinic acid, 2,4dihydroxy-3,5-cyclopentyl dienoic acid exhibited high affinity to the target enzyme in descending order. Interestingly, the identified β-carboline alkaloids except for (1R,3S)-1-methyl-βcarboline-3-carbaldehyde (compound 59) showed high binding affinity to ACE active site. The affinity of the compounds to the target enzyme was judged by comparing the binding free energy (docking score) and the interactions displayed by the docked compounds with that of the cocrystallized ligand  ( Table 4). Upon docking in the active site of ACE, all the mentioned compounds showed ionic bond interactions with the zinc-binding motif (Figures 3 and 4). Additionally, glucotropaeolin with the highest docking score (S = −35.32 kcal mol −1 ) displayed multiple hydrogen bond interactions, with the amino acids Lys511, Tyr 520, Gln281, and Tyr523 achieving a binding mode like that of the native ligand lisinopril, which can be attributed to the presence of ionized sulfonic acid and hydroxyl groups (Figures 3A and 4A). Second, docking of the identified compounds into the threedimensional X-ray crystal structure of renin (PDB code: 2V0Z) complexed with aliskiren was performed. The cocrystallized ligand forms multiple hydrogen bonds with Arg74, Gly34, Asp32, Asp215, Ser76, and Tyr14 through its amino, hydroxyl, and methoxy groups, in addition to water-mediated hydrogen bonds with Thr295 and Gln128 ( Figure S21). The validation results revealed a perfect alignment between the coordinates of the inhibitor and its self-docked pose with root-mean-square deviation (RMSD) of 1.33 Å and docking score (S) of −20.33 kcal mol −1 .
The obtained results revealed that 15 compounds showed good binding interactions. Macamides attained very poor docking scores toward ACE. However, upon docking of the different classes of the identified compounds into the active site of renin, several macamides (Table 4)     6A. Interesingly the eight identified β-carbolines have achieved docking scores comparable to that of the native ligand (S ranging from −13.50 to −20.06 kcal mol −1 ). For example, 1pentyltetrahydro-β-5,6-carboline-3-carboxylic acid displayed a hydrogen bond interaction with Asp215 in addition to three hydrogen−π interactions with Ser76 and Tyr75, as shown in Figures 5B and 6B. It is worth mentioning that renin contains two catalytic residues, namely, Asp32 and Asp215, located at its active site. 50 In the present work, several mecamides and carbolines bound to Asp215, as shown in Table 4, suggesting that they can compete with the substrate for the active site.
Finally, the molecular docking outcomes suggest that the promising inhibitory activity of the methanolic extract against ACE might be attributed to glucotropaeolin, β-carbolines, succinic acid, and 2,4-dihydroxy-3,5-cyclopentyl dienoic acid. In the same vein, macamides and β-carbolines have the highest  affinity toward renin which could also justify the promising antihypertensive activity of the extract.

Molecular Dynamics and Binding Free Energy
Calculation. The stability of the ligands within the binding pockets of the enzyme was examined through the comparison of the RMSD of the ligand with the protein (Figure 7a−d). The RMSD of the ACE complexes with both 1-pentyltetrahydro-β-5,6-carboline-3-carboxylic acid and glucotropaeolin showed flucctuations of 0.1 Å. This is slightly higher than the fluctuations seen in the renin complexes with N-(3methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide and 1-pentyltetrahydro-β-5,6-carboline-3-carboxylic acid, which was found to be 0.05 Å. However, both values are small, suggesting that the ligand−protein complex is stable. Moreover, the trajectories resulting from the molecular dynamics simulations were visually examined to further study the stability of the ligands within the binding site.
Additionally, the number of hydrogen bonds formed between the enzymes and the ligands throughout the simuation were also examined ( Figure 8). The ACE glucotropaeolin was found to form an average of four hydrogen bonds throughout the simulation, while both the 1pentyltetrahydro-β-5,6-carboline-3-carboxylic acid complex with ACE and N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide complex with a renin enzyme were found to form an average of two hydrogen bonds throughout the simulation. Finally, the renin−1-pentyltetrahydro-β-5,6-carboline-3-carboxylic acid complex was found to form only an average of one hydrogen bond throughout the simulation. This is in agreement with the results observed in the docking analysis.
Besides, the solvent-acceptable surface area (SASA) ( Figure  9) was also alnalyzed for the four complexes, which could indicate changes in the teritiary structure of the protein. The results showed that the SASA of the four complexes showed small fluctuations throughout the simulations, suggesting that there was no change in the structure of the protein during the simulation. The estimation of the binding energies is shown in Table 5. The binding of 1-pentyltetrahydro-β-5,6-carboline-3carboxylic acid with ACE showed the most promising result, with the binding energy estimate calculated at −27.03 ± 5.8 kcal mol −1 , with a significant contribution by electrostatic and van der Waals interactions. The binding energy for the glucotropaelin−ACE complex was estimated to be −25.78 ± 9.6 kcal mol −1 , which is also promising with a significant contribution by van der Waals. On the other hand, the results obtained from the interaction energy analysis for the complexes with a renin enzyme showed less promising results, where the binding energy estimate for N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide with renin was −7.20 ± 5.6 kcal mol −1 , whereas the binding energy estimate for 1pentyltetrahydro-β-5,6-carboline-3-carboxylic acid with renin was found to be 17.5406 ± 7.04 kcal mol −1 . The poor results for the renin complexes may be attributed to the high free solvation energy.
Human gastrointestinal absorption (HIA), blood−brain barrier (BBB) penetration, substrate or nonsubstrate for glycoprotein (P-gp) permeability, and the interaction of molecules with cytochrome P450 isomers (CYP) were     predicted. Also, bioavailability scores were calculated based on the Lipinski rule of five 51 and other models. Moreover, the oral bioavailability radar chart of the studied compound indicated their good predicted oral bioavailability and promising pharmacokinetic properties ( Figure 10).

Toxicity Prediction.
The selected compounds were further passed through one more web server, Osiris Property Explorer (http://www.organic-chemistry.org/prog/peo/), to predict their potential toxicities. Prediction through this program depends on the functional group resemblance of the tested compounds with the widely in vitro and in vivo studied compounds comprised in its database. The output data are demonstrated as color coded: red, green, and yellow. The green color suggests low toxic potential; yellow proposes mild toxicity; and red displays a high probability of toxicity. 52,53 The outcomes revealed that all the examined compounds are expected to be safe, displaying no toxicity concerning mutagenicity, tumorigenicity, and irritant effects (Table S1).

CONCLUSION
In this study, we established an effective strategy based on the metabolite profiling and molecular docking for screening and determining compounds with ACE and renin inhibitory effects in Maca extract, whereas the docking simulations predicted the molecular interactions and revealed the binding modes of 17 compounds within the putative binding pockets of ACE and renin. The results of the molecular dynamics simulations further investigated the interactions formed between the most promising candidates and the ACE and renin enzymes. Moreover, the molecular dynamincs simulations also investigated the binding energies and stability of the complexes. The outcomes of the MD simulations suggested that the screened compounds have a higher potential to act as ACE inhibitors rather than renin inhibitors, due to the higher binding energy values estimated by the PBSA calculations. Proper drug likeness properties and safe toxicological profiles were attained by some selected compounds, which was illustrated by the computational ADMET study. Finally, our results specified Maca compounds as potential bioactive markers that could lay a foundation for the development of new hypotensive drugs derived from natural sources. For extraction, four different solvents (water, methanol, 50% methanol, and methylene chloride) were used to extract the chemical components from Maca roots. The powders (100 g each) were extracted separately using an Ultra Turrax T50 homogenizer (IKA, Janke & Kunkel, Germany). Solvents (methanol, 50% methanol, and methylene chloride) were removed with a rotary evaporator to obtain the extracts in the yield of 1.67%, 1.89%, and 0.95% (w/w), respectively. The water extract was dried in a freeze-drier to obtain an extract in the yield of 1.50% (w/w).
4.3. Antioxidant Assays. DPPH, ABTS radical scavenging, TAC, and the iron-reducing power assays were assessed as described in previous works. 54 Ascorbic acid was used as a standard.
In DPPH: The DPPH radical scavenging activity (%) was calculated as follows: IC 50 = [(Ac − As)/Ac] × 100, where Ac was the absorbance of control (DPPH solution without a test sample) and As was the absorbance of the sample [DPPH solution + sample (extract/standard)]. The IC50 is defined as the concentration of substrate that causes 50% reduction of the DPPH color.
In iron-reducing power, the EC 50 value (μg/mL) is the effective concentration giving an absorbance of 0.5 for reducing power and was obtained from linear regression analysis.
In FRAP, ABTS, and TAC, total antioxidant was expressed as ascorbic acid equivalent (AAE) (μmol/g of extract) after construction of the ascorbic acid calibration curve.
4.4. Renin and ACE Inhibition Assay. The renin inhibitory potentials of the samples and ACE were tested according to the method described in the previous literature. 55 The inhibitory activities of the extracts were represented as percentages of inhibition in a concentration range of 10−150 ng/mL. An antihypertensive agent, Quinapril, was used as a standard at a concentration of 1−20 ng/mL. All experiments were performed 3 times.
4.5. Statistical Analysis. Each experiment was done in triplicate (n = 3). Statistical analysis was performed using SAS software ver. Sixteen (SAS Institute Inc., Cary, NC, USA). One-way ANOVA was performed, and the resultant values at P ≤ 0.05 were considered significant. Different letters indicate the significance according to Duncan's multiple range test. EXCEL 2016 and SPSS software were used for data analysis.
4.6. UPLC/HRMS Analysis. The extract was resuspended in methanol, and 2 μL was injected and separated on a reversed phase (RP) C18 column, using a UPLC system. The mass spectra were obtained in positive and negative ionization modes using a heated electrospray ionization (HESI) source in combination with an Exactive, Orbitrap-type HRMS. 56 4.7. Data Mining and Processing. The RAW data were converted with ProteoWizard 3.0 into mzXML format and imported to MZmine 2.53 software. Mass ion peaks were isolated with a centroid mass detector threshold with the noise level set to 1.0 × 10 4 and an MS level of 1−2. The chromatogram builder was used with a minimum time span set to 0.001 min and the minimum height and m/z tolerance to 1 × 10 6 and 0.001 m/z or 5.0 ppm, respectively. Chromatogram deconvolution was performed using a baseline cutoff algorithm with minimum peak height of 1 × 10 6 , peak duration range of 0−0.1 min, and baseline level of 1× 10 4 . The separated peaks were then deisotoped using the function of isotopic peaks grouper (m/z tolerance: 0.001 m/z or 5.0 ppm, retention time tolerance: 0.05 absolute (min), maximum charge: 2, and representative isotope: most intense). The parameters for data filtering and gap filling were set to m/z tolerance 0.001 m/z or 5.0 ppm, retention time tolerance 0.05 absolute (min), and minimum standard intensity 1 × 10 4 . An adduct search was performed for Na + , K + , NH +4 , formate, and ACN + (RT tolerance: 0.05 absolute (min), m/z tolerance: 0.001 m/z or 5.0 ppm, max relative adduct peak height: 50%). The processed data were then subjected to formula prediction by selecting atoms C, H, N, O, and S and adjusting parameters with a heuristic element count with all suboptions to get the isotope pattern filter working with all features with isotope peaks. Finally, peak identification was used to search for unidentified metabolites using KEGG and LipidMaps online databases or Dictionary of natural products, 2015 (DNP, 15).
4.8. In Silico Studies. 4.8.1. Docking Study. All the molecular docking studies were achieved using Molecular Operating Environment (MOE, 2010.10) software. The X-ray crystal structure of the angiotensin converting enzyme (PDB code: 1O86), 57 in complex with lisinopril and renin cocrystallized with aliskiren as an inhibitor (PDB code: 2V0Z), 58 were imported from the protein data bank. Regarding ACE protein structure, it was prepared for docking by removal of all water molecules, whereas the cofactors, zinc, and two chloride ions were retained throughout the docking process. As for renin protein structure, the enzyme was prepared by removal of chain B and all ligands and water molecules that are not involved in the binding. The protein structures were prepared for docking study using the Protonate 3D protocol in MOE with default options. The structures of the compounds were constructed using ChemBioDraw Ultra 17.0, and their SMILES were copied to MOE. 3D Protonation of the compounds was carried out using the precise mode in "Protonate" tool, and the most prevalent ionized form was selected for subsequent steps. Energy minimization of the structures using the MMFF94x force field and a gradient of 0.05 was applied using the "Energy Minimize" tool. The partial charges were automatically calculated for each molecule. Conformational analysis was run using the default settings for a systematic search. The lowest energy conformer of each molecule was saved to another database to be docked into the binding site of the target enzymes. The Triangle Matcher placement method and London dG scoring function were used for the evaluation of the binding pattern and binding affinity of the ligands. The cocrystallized ligands were used to define the active site for docking. A docking protocol was first validated by redocking of the cocrystallized ligands in the active site of the enzymes with an energy score (S) = −36.64 kcal mol −1 and root-mean-square deviation (RMSD) of 0.811 Å for ACE and energy score (S) = −20.33 kcal mol −1 and root-mean-square deviation (RMSD) of 1.33 Å for renin. The validated docking protocol was then used to study the ligand−target interactions for the identified compounds in the active site of the target enzymes. This was performed to predict their binding modes and binding affinities to rationalize the promising antihypertensive activity.
4.8.2. Molecular Dynamics and Binding Free Energy Calculation. Molecular dynamics simulations were carried out to further validate the molecular docking results for the compounds showing the highest activity. The selected compounds were as follows: 1-pentyltetrahydro-β-5,6-carbo-line-3-carboxylic acid which showed promising results for both enzymes, ACE and renin, in addition to N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide and glucotropaeolin, which showed promising results for renin and ACE, respectively. The best docked pose for each enzyme−ligand complex was selected and used for the 30 ns molecular dynamics simulation using GROMACS (Groningen Machine for Chemical Simulations). 59 Both the topology and the coordinate file for the proteins were generated using the AMBER99SB force field, 60 while the ligand parametrization was carried out using the ACPYPE (AnteChamber Python Parser Interface) 61 and the automated topology builder (http://bio2byte.be/acpype/ ). 62 The system was solvated using TIP3P water molecules, and 15932 and 15941 water molecules were added to the renin-N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide and renin-1-pentyltetrahydro-β-5,6-carboline-3-carboxylic acid systems, respectively. Similarly, 25 663 and 25 665 water molecules were added to the ACE−1-pentyltetrahydro-β-5,6carboline-3-carboxylic acid and ACE-glucotropaeolin systems, respectively. Na + and Cl − ions were added to the system to achieve a final concentration of 0.15 mM as well as to neutralize the system. For the renin system, 57 Na + and 49 Cl − ions were added, while 93 Na + and 79 Cl − were added to the ACE system. The systems were then equilibrized through energy minimization, followed by two restrained molecular dynamics simulations. Molecular dynamics simulations were carried out for 30 ns, at a constant temperature of 300 K and 1 bar. The resultant trajectories were analyzed using XMGrace 63 and were visually inspected using VMD (Visual Molecular Dynamics). 64 In addition, the binding free energy of the previously mentioned complexes was calculated using molecular Mechanic/Poisson−Boltzmann Surface Area (MM-PBSA). GMX_pbsa 65 was used to calculate the binding free energy over the 30 ns of the MD simulation. 4.8.3. In Silico ADMET Study. 4.8.3.1. Pharmacokinetic and Drug-Likeness Properties. SMILES notations of the studied compounds were inserted directly into the Swiss ADME (http://www.swissadme.ch/) 66 web server followed by running the prediction of the pharmacokinetics and druglikeness characteristics of the studied compounds. 4.8.3.2. Toxicity Prediction. The computational tool, Osiris Property Explorer (http://www.organic-chemistry.org/prog/ peo/), was used for estimation of the potential toxicities, viz., mutagenicity, tumorigenicity, and irritant effect.