key: cord-0997481-6xkztzba
authors: Kaur, Bhupinder; Maity, Himadri Sekhar; Rakshit, Madhulekha; Srivastav, Prem Prakash; Nag, Ahindra
title: Cryo-Ground Mango Kernel Powder: Characterization, LC-MS/MS Profiling, Purification of Antioxidant-Rich Gallic Acid, and Molecular Docking Study of Its Major Polyphenols as Potential Inhibitors against SARS-CoV-2 M(pro)
date: 2021-10-10
journal: ACS Food Sci Technol
DOI: 10.1021/acsfoodscitech.1c00179
sha: 1cfa8bd040a6a0d3590572f48feaa3ce3488854d
doc_id: 997481
cord_uid: 6xkztzba

[Image: see text] Mango processing waste (MPW) is an inexpensive and rich source of valuable substances. Hence, the mango kernel powder (MKP) from four cultivars (Chausa, Neelum, Barahmasi, and Dashehari) was characterized for the selection of the best cultivar. The MKP of the best cultivar (Dashehari) was analyzed for the profiling of polyphenols using LC-MS/MS in both modes of ionization (positive and negative) and indicated the presence of 50 compounds with specific retention times. After identification, gallic acid (GA), an important industrial compound, was targeted and purified followed by its confirmation using NMR (600 MHz) and HRMS. The antioxidant activity (IC(50): 1.96 μg/mL) of extracted GA proposes its use as a natural antioxidant in novel food formulations. Additionally, SARS-CoV-2 main protease (M(pro)) was selected for molecular docking based virtual screening of seven major polyphenols (MKP), and the results were compared with hydroxychloroquine. The docking scores of targeted polyphenols revealed that three compounds (epicatechin, mangiferin, and quercetin) exhibited appreciable proteolytic activity against M(pro). In this way, it is a favorable approach toward environmental safety on the standpoint of green chemistry owing to the use of food processing waste and elimination of the waste dumping/composting problems.

The agricultural industries are responsible for generating millions of tons of byproducts at all stages of the supply chain, which are inexpensive and excellent sources of bioactive compounds. 1−4 If not processed, they can cause serious environmental issues such as pollution, combustion, vegetation damage, and greenhouse gas emission. 5 Moreover, byproducts can be utilized to and produce valuable products from their metabolites. The food processing waste can be utilized after valorization of valuable constituents, which in turn contributes to environmental safety. 6, 7 The extraction of the beneficial compounds from these wastes can be a cost-effective approach, as an alternative to the expensive compounds. 8 However, the potential application of byproducts in food supplementation depends strongly on their various properties.

Mango (Mangifera indica) is recognized as a "superfruit" due to the presence of many promising functional ingredients and has more than 1500 varieties (India). In 2017−18, India was the largest producer (21. 253 MT) of mangoes accounting for almost 40% of the total worldwide production of mango. 9 Industrial processing of mango juice, pickles, puree, and other products generates redundant byproducts representing 40−50% of the fruit weight. Mango seed is a single flat oblong that can be fibrous or hairy on the surface, depending on the cultivar. The kernel inside the seed represents 45−75% of the seed and about 20% of the whole fruit. 10 Though not utilized on a commercial basis due to the need for further processing, mango seeds are said to contain functional and health beneficial properties which can be utilized after profiling of their cultivars. 7, 11 In contrast to the pulp, the mango kernel contains more polyphenols. Goḿez-Caravaca et al. 12 observed that mango pulp had the lowest concentration of bound phenolic compounds with 2.51 mg/100 g d.w. as compared to that of peel and seed (33.69 and 41.71 mg/100 g d.w.). Moreover, the concentrations of phenolic compounds in the mango pulp was 4.6 times lower than that of mango kernels. 13 Furthermore, mango kernel has higher bioactive compounds than other commercially grown fruits like avocado, jackfruit, and tamarind kernels. 14 Although several studies 11,15−18 suggest the antioxidant, anticancer, antiproliferative, antimicrobial, and cytotoxic activities of mango polyphenols including gallic acid (GA), but the complete knowledge of molecular basis for their activity remains unknown. Though, there are two results of GA metabolism in the body; the first is conversion to nonpharmacological active compounds, and the second one is transformation into compounds which exhibit pharmacological activity after metabolism. 19, 20 Being a histone acetyltransferase inhibitor, GA could counteract amyloidinduced neurotoxicity by selectively suppressing NF-κB activation in the animal model of Alzheimer's disease. 21 In addition, García-Rivera et al. 22 showed that GA could regulate IκK/IκB/NF-κB, MAPK, and MEK1/p90RSK/MSK signaling pathways in MDA-MB231 breast cancer cells. It is also responsible for the inhibition of p300/CBP mediated p65 acetylation and subsequent IκB/NF-κB signal activation, which could reduce the expression of inflammatory mediators in A549 lung cancer cells. 23 Recently, the novel coronavirus disease (COVID-19) has been spreading around the world causing the deaths of 222,000,588 people as of September 7, 2021 (https://www. worldometers.info/coronavirus/). The researchers are urgently looking for potential inhibitors to the main protease (Mpro) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to tackle the pandemic. 24 Even though vaccination ensures the buildup of immunity, reoccurrence of coronavirus disease has been seen. Bioactive components from natural sources might enhance immunity and reduce the chance of the occurrence of this disease. 25 Thus, this calls for the need for predicting plausible drug candidates from natural sources to inhibit COVID-19 virus, and this could be explored using molecular docking. 26 Molecular docking is a technique for computer-aided drug design by docking a ligand with the receptor's active site. It describes the actual site of compound interaction and also reveals the names of amino acids involved in the interaction. 27 The docking method yields a scoring function that correctly ranks drug candidates and also can propose structural hypotheses of drug target binding sites. 28 Grinding of agricultural produce is an old technique which involves frictional forces. The intensive use of energy causes heat generation when larger particles are fractured into smaller sizes. The increase in temperature is unfavorable for the aroma, flavor, phenolics, and overall quality of the final product. 7, 29 According to Reblova, 30 the easily oxidizable phenolic acids (i.e., gallic, gentisic, protocatechuic, and caffeic) showed a decrease in antioxidant activity with increasing temperature (in comparison with activity at 90°C) at a slower rate than the less oxidizable ones (i.e., syringic, ferulic, and sinapic acids, and especially vanillic acid). On the other hand, cryogenic grinding maintains the low-temperature environment owing to the use of liquid nitrogen and results in retention of valuable constituents. 31 The cryogenic liquid absorbs the heat which is being generated due to frictional forces during grinding and helps to preserve the wholesomeness of the final product.

There are few studies on the phenolic composition of mango kernel powders obtained by regular grinding techniques. 32−36 However, a detailed literature review has shown that cryoground MKP has not yet been characterized. Hence, this study was proposed to elucidate the chemical, morphological, and phytochemical profiling of cryo-ground MKP along with isolation of antioxidant-rich gallic acid. In addition, major polyphenols derived from MKP were scrutinized for their potential interactions with SARS-CoV-2 M pro . In this way, the industries can earn profit by cutting the transportation and dumping cost of waste. This would not only bring in extra revenue for the producer but also reduce the amount of accumulated waste to be disposed of. 2.2. Preparation of Mango Kernel Powder. The mango seeds were cracked using a nut shelling unit to obtain kernels and subjected to tray dryer (50 ± 3°C) for 48 h. The grinding operation was carried out in cryogenic ball mill along with liquid nitrogen as cryogenic liquid (bp −196.5°C). Our previous study 7 parameters for cryogenic grinding were used to acquire cryo-ground MKP. The obtained samples were sieved using a sieve shaker (Retsch GmbH, Haan, Germany) by passing through a set of standard sieves (710, 500, 300, 250, 212, 180, and 150 μm). The MKP (150 μm) samples were packed in airproof containers and stored in a refrigerator (4 ± 2°C) for further analysis. . MKP (1 g) mixed with ethanol (100 mL) was subjected to homogenization (handheld minihomogenizer: MT-13K) for 10 min and the obtained suspension was kept in a water bath (45°C) for 60−120 min; afterward it was centrifuged at 8000 g for 20 min 3 followed by filtration through filter paper (Whatman No. 4). After filtration, the clear extract was examined for total phenolics, total flavonoids, and antioxidant activity. 38 2.3.3. Microstructure and Mineral Analysis of Mango Kernel Powder. The samples were made conductive by gold coating before their introduction to the SEM characterization cell. The Merlin SEM-EDX (scanning electron microscopy with energy-dispersive X-ray spectroscopy), based on the GEMINI II column (Carl Zeiss, Merlin FE-SEM, Oberkochen, Germany), examined the shape, geometry, and mineral profile. The instrument was coupled with ZeissSmartSEM and INCA PentaFET × 3 (Oxford Instrument, High Wycombe, UK) analysis software. The study was carried out in three iterations. For the MS/MS analysis, argon gas with collision energy of 30 eV was employed to fragment bioactive compounds. The analysis was carried out in three iterations. 2.5. Purification of Gallic Acid Using Column Chromatography. The ethanolic MSKE was at first dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness. The crude residue was purified by flash column chromatography on silica gel (60−120 mesh). The mobile phase of ethyl acetate:hexane (EtOAc:Hx) was optimized with different proportions (0−100% (v/v)) for the fractionation. The effluent from the outlet of the column was collected in test tubes and analyzed using TLC plate (Merck 60F 254 ). The TLC plates were continuously monitored with a UV detector and mixed according to their purity. The fractions containing GA were mixed together and evaporated to dryness for rechromatography on silica gel (230−400 mesh). The purified GA was then quantified and further analyzed by NMR (600 MHz) and HRMS for structural confirmation.

2.6. Confirmation of Purified Gallic Acid by NMR and HRMS. The NMR spectroscopic data was obtained using a Bruker Avance DPX200 (600 MHz) instrument along with trimethylsilane (TMS) as internal reference. 1 H NMR and 13 C NMR spectra were recorded via broadband decoupled mode at 600 and 150 MHz, respectively. The solvent used was DMSO-d 6 with proton and carbon shifts as δ 2.50 and 39.52 ppm, respectively. The compound concentration used for analysis was 20 mg/mL.

The high resolution mass spectrometer (HRMS) of Agilent 1260 Infinity II coupled with AdvanceBIO 6545XT LC/Q-TOF was used to confirm the mass of purified gallic acid.

2.7. DPPH Scavenging Activity of Purified Gallic Acid. The antioxidant activity of isolated GA was determined according to Rafiee et al. 40 and compared with rutin and quercetin with slight modification.

2.8. Molecular Docking Studies of Major Polyphenols of MSK against SARS CoV-2 M pro . The RCSB Protein Data Bank (http:// www.rcsb.org) (PDB ID: 6Y2F) was referred for obtaining the crystal structure of the SARS CoV-2 main protease, 41 and the ligands were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov) database. The docking of the main protease with seven polyphenols of mango was conducted by using AutoDock Vina software. The binding energies of M pro and selected compounds were evaluated using the Results are expressed as mean ± SD of triplicates. SD followed by different letter superscripts within each column is significantly different (P ≤ 0.05). 

3.1. Characterization of Mango Kernel Powder. 3.1.1. Proximate Analysis of Mango Kernel Powder. MKP has an impressive amount of essential nutrients, minerals, phenolics, and flavonoids required for human well-being. The moisture content of any powdered material is a critical parameter for its life span.

The moisture content of all the varieties ranged from 8.13 ± 0.010% to 9.54 ± 0.012% (Table 1) which is bit higher than other powdered food products. So, they were properly stored in airtight containers. Chausa (2.20 ± 0.035%) and Neelum (2.13 ± 0.025%) had high ash content with an insignificant difference, which gives an indication of the minerals present in it. They play a very important role in biochemical reactions as catalyst (metalloenzyme), which helps to maintain the body's metabolism. The main attraction of mango waste is mango kernel oil (MKO), and Chausa (8.83 ± 0.207%) had the highest fat content among all the varieties, which can be utilized in chocolates as a cocoa butter substitute. 2 3.1.2. Phytochemical Profile of Mango Kernel Extract. Mango kernel is a rich source of polyphenols, viz., phenolic acids, lignans, stilbenes, and flavonoids. The data for yield, TPC, TFC, and antioxidant activity (IC 50 ) in the MSK of four cultivars is expressed in Table 1 . The yield of total polyphenols from ethanolic MSKE was observed as (7.58 ± 0.111)−(10.33 ± 0.092) g/100 g d.w., which is higher than that of mango peel and seed dehydrated by various drying treatments and Egyptian mango cultivars. 4, 34 The probable reason for higher values could be environmental factors and cryogenic grinding that helps to preserve the beneficial heat-sensitive compounds. The samples contained much higher total flavonoids than total phenolics, ranging from 73.3 ± 0.006 to 169.4 ± 0.024 mg QE/g, the highest levels being present in Dashehari and Barahmasi (169.4 ± 0.024 and 169.3 ± 0.112 mg QE, respectively).

The findings of Ajila et al. 42 explored that bound phenolics in mango peel (ripen) dietary fiber from both Raspuri (29.52 ± 1.73 mg GAE/g) and Badami (28.10 ± 0.10 mg GAE/g) cultivar were comparable to the data of present study. Among all the cultivars, the lowest TPC value of Chausa (25.8 ± 0.193 mg GAE/g) was closer to the highest values reported by Tunchaiyaphum et al. 43 The papaya seed [(8.55 ± 0.49)− (22.59 ± 0.06 mg) GAE/g] and MKP from Egyptian cultivar named Zebda (23.90 ± 0.33 mg GAE/g) showed lower TPC values. 35, 44 The IC 50 value was calculated from a linear regression equation to range between 21.40 ± 0.143 and 34.95 ± 0.131 μg/ mL (R 2 = 0.97). The IC 50 is inversely proportional to the antioxidant activity. The Barahmasi cultivar exhibited the highest antioxidant activity (IC 50 : 21.40 ± 0.143 μg/mL). The antioxidant activities of MSKE were higher than those of standard BHT and tocopherol, respectively. 36, 45 Comparing our previous results of mango peel powder 7 with our present results, the MKP explored that both the total flavonoid and total phenolic contents are higher in the seed as compared to the peel, while there was not much difference observed in IC 50 values for all the cultivars.

3.1.3. Microstructure of Mango Kernel Powder. SEM with a wide range of resolution was used to visualize the internal geometry and surface morphology of MKP. The typical The microstructure of mango kernel changed during grinding operation, which could have a significant impact on physicochemical, functional, and rheological properties. During the transformation of an ordered to a disordered structure, there is breakage of bonds. 46 For all the cultivars, smooth, oval, and irregularly shaped particles can be clearly seen at all magnifications. The micrographs of cryogenic fracture surfaces of Dashehari at 2000× and 4000× showed the lumpy structure. This might be due to its high moisture content. At 10,000×, the particles of all cultivars can be seen packed closely, which indicates their poor flowability. The combination of high mechanical impact force and attrition produced small-sized particles, some of which contributes to amorphous domain. It can be said that cryogenic grinding was effective at altering the actual shape and structure of the MSK. The biological origin, varietal difference, maturity index, and physiology are the possible reasons for variation in size and shape of all the samples.

Powder. The mineral profile indicated the amount of inorganic matter present in the sample. The SEM-EDX was used, and particular areas were focused to obtain the spectrum (Figure 2) . Potassium, calcium, manganese, and chlorine were observed as highlighted peaks in almost all the cultivars. Among all the cultivars, only Chausa was revealed to have sodium which helps to maintain the acid−base balance. It also had the highest calcium (21.49 ± 0.02%) which is a component of bones and teeth; additionally, it plays an important role in muscle contraction and blood clot-related mechanisms.

Although Barahmasi (MKP) does not contain many minerals, it has significant amounts of phosphorus (8.83 ± 0.06%), potassium (63.51 ± 0.017%), and iron (13.87 ± 0.05%), which aid in formation of healthy teeth and bones, maintain fluid balance, and help in functioning of the central nervous system (CNS), respectively (SI Table 1 ). Among all observed minerals, potassium was the dominant element in mango peel and pulp. 7, 47 These finding are in line with the study undertaken for Barahmasi (MKP) that also showed potassium (63.51 ± 0.017%) as the highlighted element. This Na−K interaction helps in nerve transmission and muscle contraction/relaxation. The ratio of potassium to sodium content is very much important, especially considered for patients with high blood pressure. The LC-MS analysis was carried out in both ionization modes to obtain protonated/deprotonated molecules (SI Figure 1(a, b,  c, d, e, f) ). However, the negative mode was more favorable for the detection of phenolic compounds due to their acidic behavior. The retention time, precursor ion, diagnostic fragments, molecular formula, molecular weight, and tentative identification of compounds is summarized in Table 2 . The retention time and fragmentation data of available standards were used to confirm a compound. Despite the lack of authentic standards, very reliable and tentative identification of 50 compounds was proposed by comparing the obtained fragmentation patterns with previously reported daughter ions in the literature.

During the initial run time of 1−5 min, organic acids, viz., citric acid, cinnamic acid, malic acid, gallic acid, and quinic acid (1, 2, 4, 5, and 9) were explored. MS/MS spectra of compounds 2, 4, and 5 showed fragment ions at m/z 103, 115, and 125, respectively. Fruits are a rich source of citric acid (1) and hydroxycinnamic acids (3, 10, 21, and 39); moreover, many researchers have reported their potential as strong antioxidant agents. 48 Compound 3 was tentatively assigned as a p-coumaric acid which presented fragments at m/z 241 and 119 due to the loss of CO 2 from the carboxylic group. Caffeic acid (10) and chlorogenic acid (21) yielded minor ions at m/z 135 and m/z 191, which correspond to elimination of CO 2 and deprotonated quinic acid, respectively. Compound 39 was designated as ferulic acid, 49 which produced fragment ions due to cleavage of the methyl group (m/z 178) and sequential loss of CO 2 (m/z 135). Compound 4 was identified in the negative mode at m/z 133 with fragment ion at m/z 115 corresponding to the malic acid fragmentation pattern.

Compounds 5 and 9 were assigned as gallic acid (SI Figure 2 ) and quinic acid with their base peak at m/z 125 due to loss of CO 2 . The MS data of these compounds were also matched with commercial standards.

Mangiferin (11) was identified in both negative and positive ion modes with major ions at m/z 423/421 along with daughter ions at m/z 331 (90 Da) and 301 (120 Da) indicating the loss of C-glycoside xanthone. (+)-Catechin (13), a flavan-3-ol, produced the product ions at m/z 285 and 151 due to loss of sugar moiety and CH 3 . 38 The precursor ion at m/z 289 produced the product ions at m/z 245, 202, and 161. Such a fragmentation pattern confirmed the presence of another flavan-3-ol compound 26 (Epicatechin). Caffeoyl glucose (23) showed a pseudomolecular ion at m/z 343 and characteristic fragment ion at m/z 179 (Caffeic acid). In positive ionization mode, compounds 31 and 32 showed a pseudomolecular ion at m/z 517 with base peak at m/z 191 corresponding to quinic acid in structure. These compounds were identified as 1,3-dicaffeoylquinic acid (31) and 4,5-dicaffeoylquinic acid (32), based on the obtained MS data and the literature. 50 The deprotonated ion peak at m/z 353 for compound 38 (RT 21.74) showed product ions at m/z 191, 173, and 109 and was characterized as 1aaffeoylquinic acid. 51 Quercetin was assigned to compound 37 (RT 21.63 min) with the precursor ion at m/z 301; additional confirmation was done by comparison of their mass fragments with standard compound and previously published data. Two compounds at 11.08 and 15.01 min exhibited the same precursor ion at m/z 463 and common fragment at m/z 300 confirming the elimination of the hexoside moiety. Additionally, compound 18 (ellagic acid-O-hexoside) gave minor ions at m/z 283 and 257 corresponding to the loss of H 2 O and 2CO. In contrast, compounds 22 and 24 fragmented at m/z 301, suggesting the presence of Quercetin derivatives and tentatively acknowledged as qutin and quercetin-O-hexoside, respectively. 52 Compound 25 (coumaryl-hexoside) showed minor ions at m/z 163 (pcoumaric acid) and 119 due to removal of CO 2 from carboxylic group. 53 Two flavone derivatives 33 and 35 were tentatively characterized as luteolin-7-O-glucuronide and luteolin-4′-Orutinoside with a common fragment obtained at m/z 285 and 255, corresponding to the loss of sugar moiety and CHO, respectively. Moreover, the methoxy-substituted flavones comprising nevadensin and cirsimaritin, which were not the focus of the present work, were confirmed by matching the fragments with Ola et al. 54 Compound 14 had a molecular weight of 302 and showed fragment ions at m/z 283, 172, and 145 due to the loss of H 2 O, the presence of the shikimate moiety, and the loss of a neutral alkyl moiety, respectively. The structure was unambiguously identified as ellagic acid and increased the probability of identifying its derivatives. Dimethyl-O-ellagic acid (27) and methy-O-ellagic acid (30) showed characteristic fragment ions at m/z 314 and 299, respectively, due to the elimination of CH 3 . 49 The pseudomolecular ion at m/z 285 gave product ions at m/z 227 owing to the loss of 2CHO and assigned as kaempferol (19) . Moreover, another compound (29: kaempferol-3-O-rutinoside) explored a daughter ion at m/z 285 (kaempferol) and 227 corresponding to the loss of Ohexoside and 2CHO. The fragment of compound 20 at m/z 273 resulted from the elimination of CH 3 OH; such results confirm the presence of methyl brevifolincarboxylate, and compound 45 was confirmed as oleanolic acid by comparison of RT and fragmentation patterns with those reported by Kumar et al. 49 Nine compounds (6, 12, 15, 16, 17, 34, 36, 41 , and 43) displayed fragment ions at m/z 169 (Table 2 ) as a consequence of losing gallate. According to their fragmentation, the tentatively identified compounds were β-glucogallin (6), galloylhexose (12), ethyl gallate (15), trigalloylglucose (16), methyl gallate (17), digalloylglucose (34), digalloyl-hexoside (36) , tetragalloylglucose (41) , and dihydroxybenzoicacetatedigallate III (43) . Moreover, compounds 15 and 17 showed base peak at m/z 125 and produced the product ion at m/z 169 (gallic acid) due to loss of C 2 H 5 and CH 3 , respectively. Three compounds (50, 42, and 49) exhibited pseudomolecular ions at m/z 938/786/634 with common daughter ions at m/z 301 (loss of HHDP-glu). These fragmentation patterns were identical to trigalloyl-HHDP-glucose, digalloyl-HHDP-glucose, and galloyl-HHDP-glucose, as reported by Yang et al. 55 Compound 48 (m/ z 939) gave fragments at m/z 787 (152 Da: loss of galloyl unit) and 769 (170 Da: loss of gallic acid), which is the typical fragmentation pattern of pentagalloylglucose. 56 3.3. Purification of Gallic Acid by Column Chromatography. Initially, the column was run with hexane in order to remove the fatty part to avoid the isolation complexity of polar compounds. The MKE was separated into nine fractions (F 1−9 ) according to the polarity. All the fractions were monitored against standard GA for their purity using TLC. The fractions resulted into mixture (impure) of compounds as it was observed that chromatogram (TLC) showed more than one band under UV lamp (SI Figure 3(a, b, c) ). F 6−9 were discarded because they showed no traces of gallic acid. However, F 2−4 contained GA along with other compounds, so they were combined for rechromatography (silica gel: 230−400 mesh) to afford the pure GA. As a result, the active fraction of GA was obtained from the solvent system of EtOAc:Hx (2:3). The pure solvent fractions were collected in a preweighed container and quantified GA as 1.28 g/100 g of MSK powder. When TLC of the final fraction was seen under UV light, there was no spot other than at Rf = 0.40, which was the same as that of standard GA. The obtained GA was off-white in color with mp of 262°C.

3.4. Confirmation of Purified Gallic Acid by NMR and HRMS. The structural confirmation of GA was guided by NMR and mass spectroscopy analysis (Table 3) . Owing to the high polarity of GA, DMSO-d 6 was used as the NMR solvent for 1 H NMR and 13 C NMR spectroscopy. Data are presented as follows: chemical shift δ (ppm), integration, multiplicity (ssinglet and bs-broad singlet), and coupling constant in J (Hz). The detailed NMR spectrum depicted the structure as gallic acid (SI Figure 4(a, b) ). The 1 H NMR and 13 C NMR data of purified GA was in line with those reported in the literature for GA. 57, 58 It was observed that proton peaks appeared at 9.18 ppm (bs, 2H) and 8.84 ppm (bs, 1H), indicating the presence of three phenolic hydroxyl groups, whereas the proton signal at 12.24 ppm (bs, 1H) revealed the confirmation of a carboxyl group. In particular, the sharp proton signal at 6.91 ppm attributed to a singlet suggests the existence of two aromatic protons. The mass of the purified GA was determined by HRMS (Figure 3 3.5. Antioxidant Activity of Purified Gallic Acid. The DPPH radical-scavenging activity of purified GA was compared with standard GA, quercetin, and rutin, and the IC 50 values were found to be 1.96, 1.03, 18.61, and 13.63 μg/mL, respectively (SI Figure 5 ). These results are in line with reported data, 59,60 which justifies the superiority of GA as a strong radical scavenger. Brand-Williams et al. 61 reported the highest antiradical activity of GA with a stoichiometry of 4 reduced DPPH molecules per molecule of GA.

Furthermore, the superiority of GA as an antioxidant compared to other polyphenols, such as pyrogallol, methyl gallate, α-tocopherol, and chlorogenic acid, is supported by Rafiee et al., 16 Asnaashari et al., 62 and Cheel et al. 63 The probable reason for variation of antioxidant activity among different polyphenols might be the increased stability of the phenoxy radicals, which depends on the number of electron donating hydroxy and methoxy groups. 62 Hence, the study undertaken explored that gallic acid, with three hydroxyl and one carboxylic acid groups, was observed to be the most active as compared to quercetin and rutin.

3.6. Molecular Docking Studies of Major Polyphenols of MSK against SARS CoV-2 M pro . The previous literature has reported an antimalarial drug as a good inhibitor toward the active site of M pro . 64 Among the recommended drugs, hydroxychloroquine has been used in the present study as a MET 49 , and CYS 44, exhibiting a docking score of −6.07 kcal/ mol. Seven major polyphenols of MKP underwent molecular docking, and the study revealed that some of the mango polyphenols such as mangiferin, epicatechin, and quercetin have shown effective docking scores of −6.49, −7.23, and −6.52 kcal/ mol, respectively (SI Table 2 ). The docking scores of these three polyphenols are comparable with hydroxychloroquine. Mangiferin complex with M pro of SARS-CoV-2 was stabilized by MET seen that all three polyphenols were capable of binding to the catalytic dyad of HIS 41 and CYS145 of M pro . Interestingly, the molecular docking investigation explored that, among seven major polyphenols, three showed good binding affinities. Furthermore, the in vitro and in vivo investigation of these proposed compounds needs to be done for validation.

The study undertaken revealed that the mango kernel is a rich source of natural antioxidants, which have a tendency to replace artificial ones. All four cultivars studied during this investigation had significant nutritional value including minerals, and the morphology expressed the presence of irregularly shaped particles. On the basis of characterization, Dashehari was considered superior to all other cultivars and was selected for the identification of bioactive compounds by LC-MS/MS, which tentatively screened 50 bioactive compounds based on their MS fragmentation patterns. The separation and purification process of antioxidant-rich gallic acid using column chromatography has been successfully developed in this study. The antioxidant activity of GA proposes its use as a natural antioxidant. Molecular docking studies revealed that three polyphenolic compounds, viz., mangiferin, epicatechin, and quercetin, exhibited effective docking scores along with good binding affinities.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsfoodscitech.1c00179. Table 1 . Mineral composition of cryo-ground MSKP from different cultivars (n = 4). Table 2 

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Profiling of phenolic compounds using UPLC-Q-TOF-MS/MS and nephroprotective activity of Indian green leafy vegetable Merremia emarginata

Characterization of phenolic constituents and evaluation of antioxidant properties of leaves and stems of Eriocephalus africanus

Rapid qualitative and quantitative analysis of bioactive compounds from Phyllanthus amarus using LC/MS/MS techniques

MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits. Food Chem

HPLC/DAD/MS characterisation and analysis of flavonoids and cynnamoil derivatives in four Nigerian green-leafy vegetables

Analysis of hydrolyzable tannins and other phenolic compounds in emblic leafflower (Phyllanthus emblica L.) fruits by high performance liquid chromatography-electrospray ionization mass spectrometry

A 1 H NMR investigation of the interaction between phenolic acids found in mango (Manguifera indica cv Ataulfo) and papaya (Carica papaya cv Maradol) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals

Isolation and characterization of gallic acid and methyl gallate from the seed coats of Givotia rottleriformis Griff. and their anti-proliferative effect on human epidermoid carcinoma A431 cells

Antioxidant activity of Phyllanthus niruri extract, rutin and quercetin

Comparison of radicalscavenging activities for selected phenolic acids

Use of a Free Radical Method to Evaluate Antioxidant Activity

Antioxidant activity of gallic acid and methyl gallate in triacylglycerols of Kilka fish oil and its oil-in-water emulsion

Free radical scavenging activity and secondary metabolites from in vitro cultures of Sanicula graveolens

Effects of antimalarial drugs on neuroinflammation-potential use for treatment of COVID-19-related neurologic complications