Antidiabetic, Antimicrobial, and Molecular Profiling of Selected Medicinal Plants

Natural products have been the center of attraction ever since they were discovered. Among them, plant-based natural products were popular as analgesics, anti-inflammatory, antidiabetic, and cosmetics and possess widespread biotechnological applications. The use of plant products as cosmetics and therapeutics is deep-rooted in Nepalese society. Although there are few ethnobotanical studies conducted, extensive research of these valuable medicinal plants has not been a priority due to the limitation of technology and infrastructure. Here, we selected 4 traditionally used medicinal plants to examine their bioactive properties and their enzyme inhibition potential. α-Glucosidase and α-amylase inhibitory activities were investigated using an in vitro model followed up by antioxidant and antimicrobial activities. The present study shows that ethyl acetate fraction of Melastoma melabathrium (IC50 9.1 ± 0.3 µg/mL) and water fraction Acacia catechu (IC50 9.0 ± 0.6 µg/mL) exhibit strong α-glucosidase inhibition. Likewise, the highest α-amylase inhibition was shown by crude extracts of Ficus religiosa (IC50 29.2 ± 1.2 µg/mL) and ethyl acetate fractions of Shorea robusta (IC50 69.3 ± 1.1 µg/mL), and the highest radical scavenging activity was shown by F. religiosa with an IC50 67.4 ± 0.6 µg/mL. Furthermore, to identify the metabolites within the fractions, we employed high-resolution mass spectrometry (LC-HRMS) and annotated 17 known metabolites which justify our assumption on activity. Of 4 medicinal plants examined, ethyl acetate fraction of S. robusta, ethyl acetate fraction of M. melabathrium, and water or ethyl acetate fraction of A. catechu extracts illustrated the best activities. With our study, we set up a foundation that provides authentic evidence to the community for use of these traditional plants. The annotated metabolites in this study support earlier experimental evidence towards the inhibition of enzymes. Further study is necessary to explore the clinical efficacy of these secondary molecules, which might be alternatives for the treatment of diabetes and pathogens.

methods might lead to the subsequent treatment of multiple diseases [7]. Diabetes and pathogens are postulated to have a high risk of emergence in Southeast Asia (SEA) [8,9]. Because of diabetes, 1.2 million people died in SEA in 2019 [8]. e death rate of people dying with infectious diseases is very high [9]. Medicinal plants possess immune-modulatory and antioxidant properties, leading to antibacterial activities.
ey are known to have versatile immune-modulatory activities by stimulating both nonspecific and specific immunity [10]. Phytochemicals such as vitamins (A, C, E, and K), carotenoids, terpenoids, flavonoids, polyphenols, alkaloids, tannins, saponins, pigments, enzymes, and minerals have hypercholesterolemic, anti-inflammatory, analgesic, antidiabetic, antimicrobial, antiplaque, antihypertensive, and anthelmintic activities [11][12][13][14]. e desorption/ionization methods for mass spectrometry had been technologically advanced for chemical identification over the past decades [15]; however, the characterization of plant extract using mass spectrometry is relatively difficult due to the lack of reliable extraction procedures [13,14,16]. Quadruple time-of-flight liquid chromatography-mass spectroscopy (Q-TOF LC/MS) technology provides an outstanding separation of major and trivial constituents of natural product extracts along with increasing selectivity and avoiding false-positive findings [17,18]. is high level of performance allows the elemental composition of each m/z value to be accurately calculated using the software. e high mass resolution of up to 20,000 makes it useful for exact mass measurement as well as for molecular formula generation of any unknown molecule, parent ion, and fragment ion in the plant extracts [19].
is method is rapidly becoming an effective analytical tool to annotate compounds within a short time frame and with high sensitivity. In this study, we prioritized four medicinal plants in Nepal. Along with bioactivity studies, we also characterized/annotated molecules via LC-HRMS. Further purification and characterization of therapeutically novel molecules could be the subject of future work.  117-39-5) were purchased from Sigma-Aldrich (Germany). Gallic acid and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Molychem (India) and Himedia (India), respectively. Dimethyl sulphoxide, sodium dihydrogen orthophosphate, and other chemicals were purchased from Fisher Scientific (India).  Table 1. e plants obtained were airdried in the shade at room temperature and ground into a fine powder.

Preparation of Crude Extracts and Fractionation.
e plant materials were shade-dried and ground into a fine powder. Extraction was done by the cold percolation method. Briefly, the powder of different plants was soaked in methanol for 24 hours at room temperature for three successive days. After 24 hours, the dissolved extracts were filtered through the Whatman-1 filter paper and collected and then concentrated at reduced pressure at 50°C using a rotary evaporator to obtain crude extracts. 50 g of the crude extract was then suspended in distilled water and successively partitioned with three different solvents based on their polarity, i.e., n-hexane, followed by dichloromethane and ethyl acetate.

Total Phenolic and Flavonoid Contents.
e TPC of the extracts was determined using the Folin-Ciocalteu reagent, as described previously [62,63] with slight modification. 20 μL of each extract (0.5 mg/mL) was separately mixed with Folin-Ciocalteau's reagent (100 μL, 1 : 10 v/v diluted with distilled water) and aqueous sodium carbonate (Na 2 CO 3 , 80 μL, 1 M) solution, and then the mixture was allowed to stand for 15 minutes at room temperature. e reaction mixture's absorbance was measured at 765 nm using a microplate reader (Synergy LX, BioTek, Instruments, Inc., USA). Gallic acid (GA) was used as a standard, and the total polyphenolic concentration in the extracts was expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g) of the extract. e assay was performed in triplicate.
e TFC of the extracts was determined using the colorimetric method described previously [64,65] with slight modification. Briefly, 20 μL of each extract (0.5 mg/ mL) was separately mixed with 60 μL ethanol and 5 μL aluminium trichloride (AlCl 3 , 10%). Subsequently, 5 μL of 1 M potassium acetate and 110 μL distilled water were added to each well, and the reaction mixture was allowed to stand for 30 minutes. e absorbance was then measured at 415 nm with a microplate reader against blank. Quercetin was used as standard, and the total flavonoid concentration in the extracts was expressed as milligrams of quercetin equivalent per gram of dry weight (mg QE/g) of extract. For each extract, triplicates of measurements were performed.
Quercetin was used as the standard reference compound for this assay. e degree of color change from purple (DPPH radical) to yellow (diphenyl picrylhydrazine) obtained after reduction at different concentrations was measured. e capability to scavenge the DPPH radical was calculated by using the following equation: where Ao is the absorbance of DPPH with 30% DMSO and A t is the absorbance of DPPH with a test or reference sample.

In Vitro α-Glucosidase and α-Amylase Inhibition
Assays. e crude extracts for α-glucosidase enzyme inhibitory activity were determined by the method described by Fouotsa et al. with slight modification [67]. e α-glucosidase (0.2 units) was premixed with 500 μg/mL of extract in 100 mM phosphate buffer saline (pH 6.8).
en, 0.7 mM pNPG in the PBS (phosphate buffer saline) buffer was added as a substrate. is reaction mixture was pre-incubated in a 96-well microplate at 37°C for 15 min. e α-glucosidase activity was determined by measuring the p-nitrophenol release from the hydrolysis of pNPG at 405 nm in a microplate with Gene 5 software. Acarbose was used as the standard compound for this assay. e % α-glucosidase inhibitory activity was calculated as given in where A o is the absorbance of enzyme-substrate reaction with 30% DMSO and A t is the absorbance of enzymesubstrate with plant extract. α-Amylase inhibition assay was carried out as described in our previous study [68]. Briefly, 20 µL of plant extracts and 80 µL of porcine pancreas α-amylase (1.5 U/mL) in phosphate buffer of pH 7.0 were loaded in microplate wells, and initial absorbance was taken. e plate was incubated at 37°C for 15 minutes. en, 100 µL of a substrate (0.5 mM), CNPG3 (in the same buffer), was added to start the reaction with incubation for 15 minutes, and change in absorbance was monitored at 405 nm [68]. e DMSO was taken as control. All the experiments were performed in triplicate in a final volume of 200 μL by using a microplate reader. e % α-amylase inhibitory activity was calculated using equation (2), mentioned earlier.

Antimicrobial Assay of Plant Extracts.
Antimicrobial assay of extracts was performed by the agar well diffusion method in Mueller Hinton Agar (MHA) plates [69]. e test organisms were inoculated in Muller Hinton Broth (MHB) and incubated at 37°C to adjust the turbidity to 0.5 McFarland standards giving a final inoculum of 1.5 × 10 8 CFU/mL. e MHA plates were lawn-cultured with the above maintained microbial inoculum. Plant extracts of 50 mg/mL concentration were prepared in 50% DMSO. Six wells of 6 mm were bored in the cultured lawn It is used in looseness of the bowels, the runs, hemorrhoids, leucorrhoea, wounds and cut lightening urinary issues, leucorrhea, urinary tract, and toothache [46,47]; antiviral activity, cytotoxicity activity [48], wound healing activity [49], antiulcer activity, antivenom activity [50], antidiabetic activity, antioxidant activity, antihyperlipidemic activity [47].

Determination of MIC and MBC.
e broth microdilution method was used to determine the MIC and MBC, according to Clinical and Laboratory Standards Institute (CLSI) [70]. Twofold serial dilutions of extracts were prepared directly in sterile 96-well microdilution plates with flat bottom wells containing MHB to obtain various concentrations. e bacterial inoculum was added at a final concentration of 10 6 CFU/mL by diluting 1 : 100 the 0.5 McFarland turbidity culture in MHB. Finally, five μL of bacteria was added to each well except for negative control.
Neomycin is a standard drug that was used as a positive control. e plate was covered with a sterile lid and incubated for 24 h at 37°C. Resazurin (0.003%) was added to each well of the microtiter plate and was incubated at 37°C for 3-4 hrs. e wells containing the bacterial growth turned into pink color, whereas the well without bacterial growth remained blue.
e MIC was considered as the lowest concentration of the extract that completely inhibits bacterial growth. e MBC was determined by streaking the content of wells onto NA plates with incubation of over 18 hours at 37°C [70]. Samples were analyzed on an Agilent 6520, Accurate-Mass Q-TOF Mass Spectrometer equipped with a G1311A quaternary pump, G1329A autosampler, and G1315D diode array detector (DAD). e solvent system consists of acetonitrile (ACN) and 5mM acetate buffer and water at the flow rate of 0.5 ml/min. e initial condition started from 5% ACN for 0.1 min to 30% ACN for 6 min, 80% ACN for 20 min, and back to its initial conditions. During the whole process, column temperature was maintained at 30°C. After passing through the DAD flow cell, the column elute was directed to Q-TOF HRMS fitted with an electrospray interface (ESI). e MS analysis was carried out using an ESI-positive ionization mode with mass ranges from 100-3000 Da. Raw data obtained from the LC/HRMS system were converted to mzML format using the Pro-teoWizard tool MSConvertGUI [71]. MZmine 2 was used for peak detection, peak alignment, and identification (target compound annotation) using centroid data [71][72][73].

Data Analysis.
e results were processed by using Gen5 Microplate Data Collection and Analysis Software and then by MS Excel. e IC 50 (inhibition of enzymatic hydrolysis of the substrate pNPG and CNPG3 by 50%) value was calculated using the Graphpad Prism software version 8. Values were expressed as a mean ± standard error of the mean of triplicate. e LC-HRMS metabolic was processed in an untargeted manner with MZmine 2 and used Mes-treNova 12.0 tool to annotate the molecules. e metabolites were searched using the Pubchem database, and Dictionary of Natural Products 2.

Total Phenolic and Flavonoid Contents.
e TPC and TFC were expressed as the GAE/g, and QE/g of extract using a calibration curve of gallic acid and quercetin, respectively. e TPC of plant extracts follows the order Likewise, the TFC of plant extracts follows the order S. robusta > M. malabathricum > F. religiosa > A. catechu, which are shown in Table 1S.

Free Radical Scavenging Activity.
In this study, the antioxidant activities of crude extracts of plants are shown in Table 2S. DPPH radical scavenging results were reported as IC 50 and compared with the IC 50 value of quercetin (6.3 ± 1.0 µg/mL) as a standard. e radical scavenging ability of plant extracts follows the order F. religiosa (67.4 ± 0.6 µg/mL) > M. malabathricum (74.9 ± 5.6 µg/mL) > A. catechu (84.9 ± 1.9 µg/mL) > S. robusta (111.4 ± 1.1 µg/ mL). e antioxidant ability is shown by providing hydrogen or electrons from the phytochemicals within extracts to free radicals.

α-Glucosidase Inhibitory Activity.
e results of α-glucosidase inhibitory activity of the extracts are given in Table 2. Among the tested fraction, ethyl acetate and water fraction showed the most potent activity with an IC 50 value ranging within 9-114.9 µg/mL against the α-glucosidase enzyme as compared to acarbose (IC 50  e MIC of different fractions against ATCC reference strains was found between 0.3 mg/mL and 25 mg/mL shown in Table 4. M. malabathricum and S. robusta extract showed inhibition at lower concentrations, but it requires more concentration to kill the bacterial growth, as evidence by MIC and MBC values. MIC of water fraction of A. catechu and ethyl acetate fraction of F. religiosa against K. pneumonia ATCC 700603 is shown in Figure 2S. Likewise, MBC of ethyl acetate fraction of A. catechu and M. malabathricum against S. sonnei ATCC 25931 is shown in Figure 3S.

Molecular Annotation.
e raw data of LC-HRMS were processed using MZmine and MestreNova 12.0 tool was used for further analysis. e total ion chromatogram (TIC) of the fractions subjected for LC-HRMS analysis is shown in Figure 4S. ese results were verified with available public compound databases. Compounds were annotated from the fractions of all 4 plants. e annotated molecules from our spectra are catechin ( Figure 5S

Discussion
Plants synthesize a wide range of secondary metabolites which showed broad-spectrum enzyme inhibitory potential. Nepal is a Himalayan nation with lopsidedly rich flora and fauna located at a transition zone between the flora of the Western Himalaya and the Eastern Himalaya [74].  [77,78]. Since natural products have contributed 48.64% as a source of new drugs for diabetes [79], we aim to annotate secondary metabolites from Nepalese plants covered by multiple pharmacological applications based on previous experiments.
Compounds like quercetin, ferulic acid, anthocyanins, catechin, and resveratrol, which are plant-based phenolics and flavonoids, are responsible for regulating glycemia through insulin secretion, increased glucose uptake, lipid peroxidation inhibition, and inhibition of enzymes like α-glucosidase, and α-amylase [80,81]. Due to the vast health benefits such as antioxidant, antimicrobial, antiulcer, antidiabetic, hepatoprotective, and anticarcinogenic activities of phenolic and flavonoid compounds, thus they are considered as a major class of phytochemicals [82,83].
Previously, evidence of antidiabetic activity of M. malabathricum leaves extract has been shown by in vivo experiments on diabetes-induced rats [47,84] to support our results. A. catechu (L.f.) Willd. leaf extracts showed an IC 50 of 0.4977 mg/mL against α-glucosidase [85]. Similarly, kaempferol, quercetin, naringenin, and baicalein were the flavonoids isolated from F. racemosa stem bark showing a significant decrease in blood glucose levels in diabetes-induced albino Wistar rats [86]. Additionally, gossypetin, herbacetin, kaempferol, leucoperalgonidin,  -     Evidence-Based Complementary and Alternative Medicine leucodelphinidin, and sorbifolin from Ficus species were shown as potential digestive enzymes inhibitors through in silico analysis [87]. Phenolic and flavonoid compounds play a major role in free radical scavenging activity via chain termination of free radicals that depend on hydroxyl groups' number and position by donating hydrogen atoms [88]. e structural features like many hydroxyl groups and their configuration, the ketonic functional group at C-4, and a double bond at C2-C3 on flavonoids enhanced the antioxidant ability [89,90]. Due to these structural features, extracts with higher phenolic compounds are already proved to have a higher antidiabetic ability via the formation of hydrogen bonds and hydrophobic interactions with enzymes and reduce their activity [91,92]. ere are high correlations between antioxidant capacity and total phenols and total flavonoids, so the presence of a high concentration of phenolic compounds also has high antioxidant activity [75]. ere is no correlation between high TPC and TFC value with high antioxidant activity in our study. Bioactive compounds present in plant extracts showed antidiabetic activity by inhibiting glucose absorption in the intestinal tract (inhibiting the different steps of carbohydrates digestion) or antioxidant ability [93].
Catechin, epicatechin, and taxifolin from A. catechu [26,96]; ursolic acid, 2α-hydroxyursolic acid, asiatic acid, β-sitosterol 3-O-β-D-glucopyranoside, quercetin, ellagic acid, and kaempferol from M. malabathricum [55]; and amarogentin, amaroswerin, gentianine, mangiferin, oleanolic acid, swechirin, swertiamarin, sweroside, swertanoone, ursolic acid, etc. from S. chirayita [102,103] were identified with antibacterial ability. e mechanism behind the antimicrobial activity of phenolic compounds and flavonoids is the inhibition of nucleic acid synthesis, cytoplasmic membrane function, energy metabolism, attachment and biofilm formation, and porin on the cell membrane, and membrane permeability alteration leading to cell destruction as well as attenuation of pathogenicity [104]. In our study, most of the extracts showed higher antibacterial activity against gram-positive bacteria as compared to gram-negative bacteria; it might be due to differences in the cell wall structure as the peptidoglycan layer in gram-positive bacteria is an ineffective permeability barrier as compared to gram-negative bacteria containing outer membrane and a unique periplasmic space rich in lipopolysaccharide molecules, which act as a barrier for the penetration of numerous antimicrobial compounds [105]. e most time-consuming steps are metabolite identification. Because of the time-consuming and the availability of plant materials, we performed LC/HRMS for metabolite profiling based on in vitro assay rather than performing highperformance liquid chromatography (HPLC), LC-HRMS/ MS, and nuclear magnetic resonance (NMR). Likewise, after performing LC-HRMS we proceeded with our raw data with MZmine and overlayed our ion chromatogram.  [106][107][108][109]. e fragmentation pattern of catechin/epicatechin is shown in Figure 20S. It is predicted to be gallocatechin/ epigallocatechin and procyanidin: in our spectra, we observed [M + H] + peak m/z 307.08 (C 15 H 14 O 7 ; t R 5.22); fragment peak at m/z 289 and 139 for gallocatechin/epigallocatechin. e fragmentation pattern of gallocatechin/ epigallocatechin is shown in Figure 21S. Likewise, the molecular ion peak at m/z 579. 15 Figure 22S. e absence of myricetin and the presence of proanthocyanidin in the leaves of S. robusta were very significant [111,112]. Base peak with m/z 303.051 (C 15 H 10 O 7 ; t R 8.04) was expected to be quercetin. e fragmentation pattern of the quercetin is shown in Figure 23S. Similarly, base peak with m/z 481.09 (C 21 H 20 O 20 ; t R 6.82) and the fragment ion at m/ z = 319.05 and 287.15 is interpreted as gossypin comparing our spectra with Giorio et al. and Petsalo et al. [113,114]. e base peak at m/z 329.08 (molecular formula C 14 H 16 O 9; t R 5.11) and quasi molecular ion peak [M + Na] + at m/z 351.06 are expected to be bergenin isolated from S. robusta with reference from the data collected from our spectra and Mukherjee et al. [60]. e other flavonoid is expected to be  [116]. Likewise, the base peak with m/z 435.09 (C 20 H 18 O 11 ; t R 7.83) and fragments peak at m/z 303.05, 287.05, 183.14, and 153.02 are predicted to be avicularin (quercetin 3-α -L-arabinofuranoside) as spectral figures match with the literature of Santos et al. [117]. Moreover, in our spectra, the base peak with m/z 419.09 (C 20 H 18 O 10 ; t R 8.22) and fragments peak at m/z 287.09 ([(M + H) − 132]) are annotated as kaempferol 3-O-α-L-arabinopyranoside taking reference of Vuković et al. [118].
M. melabathrium may contain isoquercetin since in our spectra at base peak m/z 465.10 (t R 7.58) and fragments ions at m/z 303.04 (quercetin) and 289.06 (kaempferol) exactly match with the spectra data of Liu et al. [119]. Base peak at m/z 617.11 (t R 7.16) and fragments ions at m/z 303.05 corresponding to [aglycone + H] + and at m/z 315.07 corresponding to [sugar + gallic acid + H] + are assumed to be quercetin 3-O-(6"-O-galloyl)-β-glucopyranoside taking reference of Şöhretoglu et al. [120]. e other metabolite was reported as kaempferol with base peak m/z 287.05 (C 15 H 10 O 6 ; t R 8.69) and fragments ion at m/z 259.12, 165.05, 153.0, 127.03, and 121.09 respectively. e spectra are similar to March et al. [121]. e fragmentation pattern of kaempferol is shown in Figure 24S. Similarly, from MZmine total ion chromatogram (TIC) we can say that the M. melabathrium and S. robusta may contain quercetin at base peak m/z 303 as we can observe the peak of quercetin coinciding in both plants.
In hexane fraction of F. religiosa, we did defatting by removing the first hexane fraction. But the chromatogram was also seen in our mass spectrometry with fairly fewer amounts.
en, we annotated dorsteniol secondary metabolites in F. religiosa. Literature search shows that Ficus species contain dorsteniol. Our spectra data shows base peak at m/z 263.09 (C 14 H 14 O 5 ; t R 8.14) and [M + Na] + peak at m/z 285.07 as in Alqasoumi et al. [122]. So, the predicted compound is dorsteniol.
In our study, we have annotated 17 secondary metabolites from different plant extract. To support our annotation, more spectroscopic data are required. Crude extracts showed potent activity due to the synergistic effect of compounds. Besides that, ethyl acetate and water fraction showed potent activity; this might be due to phenolic and flavonoid compounds present in those solvents. In some extracts, hexane and DCM showed good inhibition due to terpenes, steroids, and diterpenes present in extracts.

Conclusions
Due to the rising prevalence of diabetes and antimicrobial resistance, the quest for novel drugs from natural sources persists. e scientific study of different traditional medicinal plants used by diverse groups of people is necessary. Traditional medicinal plants that local peoples have used were screened for antioxidant, antimicrobial, and antidiabetic assay. Among 4 selected medicinal plants from Nepal, most extracts and their fractions can show antioxidant, antimicrobial, and antidiabetic activity, supporting these plants' traditional use. Further works can be carried out on isolation, identification, in silico, and kinetics of potential inhibitors from an active fraction of plant extracts used as a drug candidate for diabetes.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.
Disclosure is paper has been extracted from an MSc thesis by Babita Aryal and others carried out in Tribhuvan University, Kirtipur, Kathmandu, Nepal. Acknowledgments is project was supported by the Higher Education Reform Project (HERP), Tribhuvan University, to Niranjan Parajuli. Grateful acknowledgment is made to the Central Department of Botany, Tribhuvan University, Nepal, for plant identification.