# LC-HRMS Profiling and Antidiabetic, Antioxidant, and Antibacterial Activities ofAcacia catechu (L.f.) Willd

Authors: Babita Aryal; Bikash Adhikari; Niraj Aryal; Bibek Raj Bhattarai; Karan Khadayat; Niranjan Parajuli
Journal: BioMed Research International
 (2021)

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## Abstract

Acacia catechu (L.f.) Willd is a profoundly used traditional medicinal plant in Asia. Previous studies conducted in this plant are more confined to extract level. Even though bioassay-based studies indicated the true therapeutic potential of this plant, compound annotation was not performed extensively. This research is aimed at assessing the bioactivity of different solvent extracts of the plant followed by annotation of its phytoconstituents. Liquid chromatography equipped with high resolution mass spectrometry (LC-HRMS) is deployed for the identification of secondary metabolites in various crude extracts. On activity level, its ethanolic extract showed the highest inhibition towards α-amylase and α-glucosidase with an IC50 of 67.8±1 μg/mL and 10.3±0.1 μg/mL respectively, inspected through the substrate-based method. On the other hand, the plant extract showed an antioxidant activity of 23.76±1.57 μg/mL, measured through radical scavenging activity. Similarly, ethyl acetate and aqueous extracts of A. catechu showed significant inhibition against Staphylococcus aureus with a zone of inhibition (ZoI) of 13 and 14 mm, respectively. With the LC-HRMS-based dereplication strategy, we have identified 28 secondary metabolites belonging to flavonoid and phenolic categories. Identification of these metabolites from A. catechu and its biological implication also support the community-based usage of this plant and its medicinal value.


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## Body

## 1. Introduction

Acacia catechu (L.f.) Willd is a deciduous and gregarious tree with a light feathery crown native to Nepal, India, and Myanmar and one of the most promising medicinal plants of the family Fabaceae [1]. A. catechu has received attention as a potential source of bioactive secondary metabolites to be used for the formulation of pharmaceutical products [2, 3]. Various parts of plant extracts were known to have strong antioxidant [2], antimicrobial [4], anti-inflammatory [5], antihyperglycemic [6], and immunomodulatory [7] activities. Although several secondary metabolites have been identified from A. catechu, the molecules, catechin, epicatechin, and quercetin, are the principal contributor to therapeutical properties [8]. Nowadays, plant-based secondary metabolites are extensively used in the management of various infectious diseases and achieved clinical benefits in the health care system.Diabetes mellitus (DM) is a leading cause of hyperglycemia, and carbohydrate-hydrolase inhibitors, such as inhibitors forα-amylase and α-glucosidase, offer an effective strategy to lower the level of postprandial hyperglycemia via control of dietary carbohydrates and glycogen breakdown [9]. Moreover, microbial infections have undermined the existing antibiotic-based treatment era and raised the mortality rate in patients with higher medical expenses and extended hospital stays [10]. Around the world, about 28,000 plant taxa have been known for their medicinal values and about 3000 plant species possess ethnopharmacological uses for the management of DM and others [11]. Different explorers had effectively shown the inhibitory abilities of natural products towards digestive enzymes, hence reducing hyperglycemia [12–14].Hyperglycemia stimulates the autooxidation of glucose to generate free radicals [15]. Free radicals are created constantly in the body during metabolism as they are required to serve various essential functions essential for survival [16]. A body with a weak defense system is unable to counteract these increased radicals, which ultimately leads to imbalance, and this condition of more free radicals than antioxidants is known as oxidative stress. Excess free radicals and reactive oxygen species, beyond the scavenging capacities of the cellular antioxidant system, are involved in several human diseases and complications like arteriosclerosis, cancer, the aging process, diabetes, cardiovascular disease, nerve damage, blindness, and nephropathy [15, 17–20]. Antioxidants from natural products especially fruits, vegetables, herbs, and spices are effective in reducing diabetic vascular complications [15] and have diverse pharmaceutical properties [21, 22].Plant products have played the important role in the development of new therapeutics with milder adverse side effects than commercial drugs [23]. Consequently, it is important to recognize and measure all the secondary metabolites to ensure the biological research reliability and reproducibility over the pharmacological benefits and/or hazards. Currently, liquid chromatography with high resolution mass spectrometry (LC-HRMS) has emerged as a leading tool for detecting and identifying pharmacologically active secondary metabolites [24, 25]. Nuclear magnetic resonance spectroscopy and mass spectrometry followed by chemometric tools are the most used analytical methods of annotation [26]. Additionally, LC-HRMS is useful for exact mass measurement as well as for molecular formula generation of any unknown molecules, parent ions, and fragment ions in the plant extracts [27]. LC-HRMS not only allows for the determination of chemical structure but also offers excellent sensitivity to the low amount of sample in a short time and also plays an important role in the screening of flavonoids and other phenolic contents [28, 29]. Thus, this study was carried out for method validation for some biochemical assays and molecular profiling in the ethanolic extract of A. catechu.


## 2. Materials and Methods

### 2.1. Chemicals

Theα-glucosidase from Saccharomyces cerevisiae (CAS No. 9001-42-7), porcine pancreas α-amylase (CAS No. 9000-90-2), 4-nitrophenyl-α-D-glucopyranoside (pNPG) (CAS No. 3767-28-0), 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3) (CAS No.118291-90-0), acarbose (CAS No. 56180-94-0), and quercetin (CAS No. 117-39-5) were purchased from Sigma-Aldrich, Germany. Gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, Folin-Coicalteu reagent, resazurin sodium, and other chemicals and solvents were purchased from Molychem, Himedia, and Fisher Scientific, India.


### 2.2. Collection and Identification of the Plant

A. catechu was collected from Syangja District of Gandaki Province with coordinates 27° 58′ N, 83° 46′ E Nepal, in January 2020. Its taxonomy was verified in voucher specimen TUCH-201011 by the Central Department of Botany, Tribhuvan University, Kirtipur, Nepal. Its major phytoconstituents and ethnopharmacological application are mentioned in Table S1.


### 2.3. Preparation of Crude Extracts and Fractionation

The barks ofA. catechu were shade dried at room temperature and pulverized. The powder was soaked in ethanol for 24 hours and filtered. The same process was repeated for three successive days, and collected filtrate was concentrated in a vacuum in a rotary evaporator below 45°C. Fractionation was carried out by dissolving 50 g of crude ethanolic extract into distilled water and then partitioned three times with hexane, dichloromethane (DCM), and ethyl acetate successively to obtain respective solvent-solvent fractions.


### 2.4. Total Phenolic Content

The Folin-Coicalteu method was employed for the estimation of total phenolic content (TPC) as described by Luet al. [30] with slight modifications. 20 μL of sample (0.5 mg/mL) plant extracts/standard followed by 100 μL of Folin-Coicalteu reagent (1 : 10; v/v) was added in a 96-well microtiter plate, and initial absorbance was taken. Then, 80 μL of 1 M Na2CO3 solution was added to the above mixture making a final volume of 200 μL and incubated for 25 minutes. Absorbance was measured at 765 nm by using Synergy LX Multi-Mode Reader with Gen5 3.08.01 software. The TPC was determined using a calibration curve with gallic acid, and results were expressed as milligrams of gallic acid equivalent per gram of dry weight of the extract (mg GAE/g). Triplicates of each measurement were carried out for validation.


### 2.5. Total Flavonoid Content

Total flavonoid content (TFC) were estimated by the AlCl3 method, based on the formation of a complex between AlCl3 and flavonoid with a maximum absorbance at 415 nm [31]. Briefly, 20 μL of each extract (0.5 mg/mL) was separately mixed with 60 μL ethanol and 5 μL 10% AlCl3. Subsequently, 5 μL of 1 M potassium acetate and 110 μL distilled water were supplemented into each well, and the reaction mixture was allowed to stand for 25 minutes. Triplicates of each measurement were carried out to verify experimental reproducibility. The TFC was determined using a calibration curve with quercetin, and results were expressed as milligrams of quercetin equivalent per gram dry weight of extract (mg QE/g).


### 2.6. Free Radical Scavenging Activity

The radical scavenging activity was observed to demonstrate the antioxidant ability of plant extracts as described previously with slight modifications [32]. The reaction was done in 200 μL volume by mixing 100 μL DPPH (0.1 mM) and 100 μL plant extract. Then, it was incubated for 25 minutes in the dark and absorbance was taken at 517 nm. The percent scavenging was calculated by the following formula:
(1)%scavenging=Ao−AtAo×100,where Ao is absorbance of DPPH radical with 50% DMSO and At is absorbance of DPPH radical with test or reference sample.


### 2.7.In Vitro α-Glucosidase Inhibitory Assay

The competitive inhibition-based assay withα-glucosidase was carried out using a method adopted by Fouotsa et al. [33] and Aryal et al. [34] with slight modifications. Briefly, 20 μL of plant extract prepared on 30% DMSO and 80 μL of the α-glucosidase enzyme (0.5 U/mL) prepared in 50 mM phosphate saline buffer (pH 6.8) were added, and initial absorbance was taken at 410 nm in a microplate reader. Then, the microtiter plate was preincubated at 37°C for 15 minutes. 100 μL of 1.4 mM substrate (pNPG) was added and incubated at 37°C for 25 minutes, and absorbance was measured. Acarbose was used as the reference compound, and each assay was performed in triplicate for validation. The percentage inhibition of α-glucosidase by plant extract was calculated by using the following formula:
(2)%enzymeinhibition=Ac−AtAc×100,where Ac is the absorbance of enzyme-substrate reaction with 30% DMSO and At is the absorbance of enzyme-substrate with plant extract.


### 2.8.In Vitro α-Amylase Inhibition Assay

The competitive inhibition-based assay withα-amylase was carried out by following the method adopted by Khadayat et al. [32] with slight modifications. 20 μL of plant extracts in 50% DMSO and 80 μL of porcine pancreas α-amylase (1.5 U/mL) in phosphate buffer of pH 7.0 were loaded, and initial absorbance was taken at 405 nm. The microtiter plate was incubated at 37°C for 15 minutes. Then, 100 μL of 1 mM CNPG3, the substrate was added to initiate the reaction with incubation for 25 minutes, and the change in absorbance was monitored at 405 nm [32]. Acarbose was used as a reference compound. The percentage inhibition of α-amylase enzyme was estimated using equation (2) mentioned earlier.


### 2.9. Antibacterial Assay

The antibacterial activity ofA. catechu extract against Gram-positive (Staphylococcus aureus ATCC 25923) and Gram-negative (Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 13883, Salmonella typhi ATCC 14028, and Shigella sonnei ATCC 25931) was assayed through Agar well diffusion method in a Mueller Hinton Agar (MHA) plate [35]. The inoculum of microorganisms in Mueller Hinton broth (MHB) adjusted to 0.5 McFarland equivalents was spread on the surface of the MHA plate using a sterile cotton swab. Then, wells were punched aseptically into the agar surface by using a sterile cork borer of 6 mm diameter and filled with 50 μL (50 mg/mL) of plant extract prepared in 50% DMSO. The plates were allowed to diffuse at room temperature for 2 hours. 50 μL of neomycin (1 mg/mL) and DMSO was used as positive and negative control, respectively. The zone of inhibition (ZoI) was determined by measuring the diameters of bacterial growth on plates after incubation at 37°C for 24 hours.The extracts with maximum ZoI were further evaluated for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) via the resazurin-based turbidimetric broth microdilution method [36]. The minimum concentration before the color change (from blue to pink) was taken as MIC value, and higher concentrations were streaked onto the MHA plates at different compartments and incubated overnight at 37°C to observe concentration corresponding to no growth of bacteria, i.e., MBC.


### 2.10. LC-HRMS Analysis

The LC-HRMS analysis of ethyl acetate and the aqueous fraction was performed using an Agilent 6520, Accurate-Mass Q-TOF Mass Spectrometer equipped with a G1311A quaternary pump, G1329A autosampler, and G1315D diode array detector at Sophisticated Analytical Instrument Facility (SAIF), CSIR-Central Drug Research Institute, Lucknow. Source and scan parameter settings include gas temp: 30°C, gas flow: 11.01/min, nebulizer: 40 psi, VCap: 3500, fragmentor: 175, skimmer1: 65.0, and octopoleRF Peak: 750. The solvent elution consists of acetonitrile, 5 mM acetate buffer, and water at the flow rate of 1.5 mL/min. The elution gradient was started from 5% acetonitrile for 0.1 min to 30% acetonitrile for 10 min, 80% acetonitrile for 32 minutes, and back to its initial conditions. During the whole process, column temperature was maintained at 30°C. After passing through the flow cell of the diode array detector, the column elute was directed to Q-TOF HRMS fitted with an electrospray interface. The mass spectrum analysis was carried out using positive electron spray ionization (ESI-positive mode) within the mass range of 100-2000 daltons at a scan rate of 1.03.


### 2.11. Data Analysis

The results were processed by using Gen5 Microplate Data Collection and Analysis Software and then by MS Excel. Inhibition of enzymatic hydrolysis of the substrates (pNPG and CNPG3) by 50% (IC50) was calculated using the GraphPad Prism Software version 8. All the experiments were carried out in triplicate, and data were presented in mean±standarddeviation. Raw data files acquired from the LC-HRMS were processed using MZmine 2 and then Mestre Nova 12.0 for compound annotation using PubChem, Dictionary of Natural Products 2, ChemSpider, and METLIN database.


## 2.1. Chemicals

Theα-glucosidase from Saccharomyces cerevisiae (CAS No. 9001-42-7), porcine pancreas α-amylase (CAS No. 9000-90-2), 4-nitrophenyl-α-D-glucopyranoside (pNPG) (CAS No. 3767-28-0), 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3) (CAS No.118291-90-0), acarbose (CAS No. 56180-94-0), and quercetin (CAS No. 117-39-5) were purchased from Sigma-Aldrich, Germany. Gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, Folin-Coicalteu reagent, resazurin sodium, and other chemicals and solvents were purchased from Molychem, Himedia, and Fisher Scientific, India.


## 2.2. Collection and Identification of the Plant

A. catechu was collected from Syangja District of Gandaki Province with coordinates 27° 58′ N, 83° 46′ E Nepal, in January 2020. Its taxonomy was verified in voucher specimen TUCH-201011 by the Central Department of Botany, Tribhuvan University, Kirtipur, Nepal. Its major phytoconstituents and ethnopharmacological application are mentioned in Table S1.


## 2.3. Preparation of Crude Extracts and Fractionation

The barks ofA. catechu were shade dried at room temperature and pulverized. The powder was soaked in ethanol for 24 hours and filtered. The same process was repeated for three successive days, and collected filtrate was concentrated in a vacuum in a rotary evaporator below 45°C. Fractionation was carried out by dissolving 50 g of crude ethanolic extract into distilled water and then partitioned three times with hexane, dichloromethane (DCM), and ethyl acetate successively to obtain respective solvent-solvent fractions.


## 2.4. Total Phenolic Content

The Folin-Coicalteu method was employed for the estimation of total phenolic content (TPC) as described by Luet al. [30] with slight modifications. 20 μL of sample (0.5 mg/mL) plant extracts/standard followed by 100 μL of Folin-Coicalteu reagent (1 : 10; v/v) was added in a 96-well microtiter plate, and initial absorbance was taken. Then, 80 μL of 1 M Na2CO3 solution was added to the above mixture making a final volume of 200 μL and incubated for 25 minutes. Absorbance was measured at 765 nm by using Synergy LX Multi-Mode Reader with Gen5 3.08.01 software. The TPC was determined using a calibration curve with gallic acid, and results were expressed as milligrams of gallic acid equivalent per gram of dry weight of the extract (mg GAE/g). Triplicates of each measurement were carried out for validation.


## 2.5. Total Flavonoid Content

Total flavonoid content (TFC) were estimated by the AlCl3 method, based on the formation of a complex between AlCl3 and flavonoid with a maximum absorbance at 415 nm [31]. Briefly, 20 μL of each extract (0.5 mg/mL) was separately mixed with 60 μL ethanol and 5 μL 10% AlCl3. Subsequently, 5 μL of 1 M potassium acetate and 110 μL distilled water were supplemented into each well, and the reaction mixture was allowed to stand for 25 minutes. Triplicates of each measurement were carried out to verify experimental reproducibility. The TFC was determined using a calibration curve with quercetin, and results were expressed as milligrams of quercetin equivalent per gram dry weight of extract (mg QE/g).


## 2.6. Free Radical Scavenging Activity

The radical scavenging activity was observed to demonstrate the antioxidant ability of plant extracts as described previously with slight modifications [32]. The reaction was done in 200 μL volume by mixing 100 μL DPPH (0.1 mM) and 100 μL plant extract. Then, it was incubated for 25 minutes in the dark and absorbance was taken at 517 nm. The percent scavenging was calculated by the following formula:
(1)%scavenging=Ao−AtAo×100,where Ao is absorbance of DPPH radical with 50% DMSO and At is absorbance of DPPH radical with test or reference sample.


## 2.7.In Vitro α-Glucosidase Inhibitory Assay

The competitive inhibition-based assay withα-glucosidase was carried out using a method adopted by Fouotsa et al. [33] and Aryal et al. [34] with slight modifications. Briefly, 20 μL of plant extract prepared on 30% DMSO and 80 μL of the α-glucosidase enzyme (0.5 U/mL) prepared in 50 mM phosphate saline buffer (pH 6.8) were added, and initial absorbance was taken at 410 nm in a microplate reader. Then, the microtiter plate was preincubated at 37°C for 15 minutes. 100 μL of 1.4 mM substrate (pNPG) was added and incubated at 37°C for 25 minutes, and absorbance was measured. Acarbose was used as the reference compound, and each assay was performed in triplicate for validation. The percentage inhibition of α-glucosidase by plant extract was calculated by using the following formula:
(2)%enzymeinhibition=Ac−AtAc×100,where Ac is the absorbance of enzyme-substrate reaction with 30% DMSO and At is the absorbance of enzyme-substrate with plant extract.


## 2.8.In Vitro α-Amylase Inhibition Assay

The competitive inhibition-based assay withα-amylase was carried out by following the method adopted by Khadayat et al. [32] with slight modifications. 20 μL of plant extracts in 50% DMSO and 80 μL of porcine pancreas α-amylase (1.5 U/mL) in phosphate buffer of pH 7.0 were loaded, and initial absorbance was taken at 405 nm. The microtiter plate was incubated at 37°C for 15 minutes. Then, 100 μL of 1 mM CNPG3, the substrate was added to initiate the reaction with incubation for 25 minutes, and the change in absorbance was monitored at 405 nm [32]. Acarbose was used as a reference compound. The percentage inhibition of α-amylase enzyme was estimated using equation (2) mentioned earlier.


## 2.9. Antibacterial Assay

The antibacterial activity ofA. catechu extract against Gram-positive (Staphylococcus aureus ATCC 25923) and Gram-negative (Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 13883, Salmonella typhi ATCC 14028, and Shigella sonnei ATCC 25931) was assayed through Agar well diffusion method in a Mueller Hinton Agar (MHA) plate [35]. The inoculum of microorganisms in Mueller Hinton broth (MHB) adjusted to 0.5 McFarland equivalents was spread on the surface of the MHA plate using a sterile cotton swab. Then, wells were punched aseptically into the agar surface by using a sterile cork borer of 6 mm diameter and filled with 50 μL (50 mg/mL) of plant extract prepared in 50% DMSO. The plates were allowed to diffuse at room temperature for 2 hours. 50 μL of neomycin (1 mg/mL) and DMSO was used as positive and negative control, respectively. The zone of inhibition (ZoI) was determined by measuring the diameters of bacterial growth on plates after incubation at 37°C for 24 hours.The extracts with maximum ZoI were further evaluated for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) via the resazurin-based turbidimetric broth microdilution method [36]. The minimum concentration before the color change (from blue to pink) was taken as MIC value, and higher concentrations were streaked onto the MHA plates at different compartments and incubated overnight at 37°C to observe concentration corresponding to no growth of bacteria, i.e., MBC.


## 2.10. LC-HRMS Analysis

The LC-HRMS analysis of ethyl acetate and the aqueous fraction was performed using an Agilent 6520, Accurate-Mass Q-TOF Mass Spectrometer equipped with a G1311A quaternary pump, G1329A autosampler, and G1315D diode array detector at Sophisticated Analytical Instrument Facility (SAIF), CSIR-Central Drug Research Institute, Lucknow. Source and scan parameter settings include gas temp: 30°C, gas flow: 11.01/min, nebulizer: 40 psi, VCap: 3500, fragmentor: 175, skimmer1: 65.0, and octopoleRF Peak: 750. The solvent elution consists of acetonitrile, 5 mM acetate buffer, and water at the flow rate of 1.5 mL/min. The elution gradient was started from 5% acetonitrile for 0.1 min to 30% acetonitrile for 10 min, 80% acetonitrile for 32 minutes, and back to its initial conditions. During the whole process, column temperature was maintained at 30°C. After passing through the flow cell of the diode array detector, the column elute was directed to Q-TOF HRMS fitted with an electrospray interface. The mass spectrum analysis was carried out using positive electron spray ionization (ESI-positive mode) within the mass range of 100-2000 daltons at a scan rate of 1.03.


## 2.11. Data Analysis

The results were processed by using Gen5 Microplate Data Collection and Analysis Software and then by MS Excel. Inhibition of enzymatic hydrolysis of the substrates (pNPG and CNPG3) by 50% (IC50) was calculated using the GraphPad Prism Software version 8. All the experiments were carried out in triplicate, and data were presented in mean±standarddeviation. Raw data files acquired from the LC-HRMS were processed using MZmine 2 and then Mestre Nova 12.0 for compound annotation using PubChem, Dictionary of Natural Products 2, ChemSpider, and METLIN database.


## 3. Results

### 3.1. Total Phenolic and Flavonoid Content

The TPC and TFC were expressed as the GAE/g and QE/g of extract using calibration curves of gallic acid and quercetin, respectively. The TPC ofA. catechu was found to be 175.48±4.67 mg GAE/g which was greater than its TFC examined, i.e., 7.66±1.0 mg QE/g.


### 3.2. Free Radical Scavenging Activity

The antioxidant of ethanolic extracts ofA. catechu was evaluated by using a quick, reliable, and reproducible method through the measurement of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. DPPH radical scavenging results were reported as IC50 and compared with the IC50 value of quercetin (6.3±1.0 μg/mL) as a standard. The radical scavenging ability of plant extract is 23.76±1.57 μg/mL.


### 3.3.α-Glucosidase and α-Amylase Inhibitory Activities

The results ofα-glucosidase and α-amylase inhibitory activity of the extracts are expressed in Table 1. Among the tested fraction, aqueous and ethyl acetate fraction showed the most activity with an IC50 value of 82.6±0.3 and 130.2±0.6 μg/mL against the α-glucosidase as compared to acarbose (IC50=344.23±1.03 μg/mL). In the α-amylase assays, the crude ethanol extract showed antidiabetic activity with an IC50 of 67.8±1.8 μg/mL, compared to acarbose (IC50=6.1±0.1 μg/mL).Table 1
α-Glucosidase and α-amylase inhibitory activity of different fractions of A. catechu.
ExtractsIC50 (μg/mL)α-Glucosidaseα-AmylaseCrude ethanol extract10.3±0.167.8±1.8Hexane fraction<50 %<50 %Dichloromethane fraction174.7±0.6<50 %Ethyl acetate fraction130.2±0.6<50 %Aqueous fraction82.6±0.3<50%Standard (acarbose)344.2±1.036.02±0.1


### 3.4. Antibacterial Activity

The antibacterial activity of plant extracts againstS. aureus ATCC 25923, E. coli ATCC 25922, K. pneumoniae ATCC 13883, S. typhi ATCC 14028, and S. sonnei ATCC 25931 was performed. The antibacterial activity was measured in terms of ZoI diameter in millimeters (mm) as shown in Table 2. Based on the ZoI, MIC and MBC were evaluated against S. aureus ATCC 25923. The MIC and MBC of the aqueous fraction of A. catechu bark extract were 6.25 and 12.5 mg/mL while those of neomycin are 0.0625 and 0.25 mg/mL respectively.Table 2
Zone of inhibition of each fraction of plants.
MicroorganismsZone of inhibition (ZoI) in millimeters (mm)Hexane FractionDichloromethane fractionEthyl acetate fractionAqueous fractionPositive controlStaphylococcus aureus11 mm9 mm13 mm14 mm10 mmEscherichia coli————16 mmKlebsiella pneumonia——8 mm10 mm19 mmSalmonella typhi————18 mmShigella sonnei6 mm7 mm12 mm10 mm25 mmNote: positive control (50 mg/mL neomycin).


### 3.5. LC-HRMS-Driven Molecular Annotation

The raw data of LC-HRMS were processed using MZmine, and the fraction with the best total ion chromatogram (TIC) was proceeding for further analysis using MestreNova 12.0 software. The TIC obtained using MZmine of ethyl acetate and aqueous fraction is shown in FigureS1. Details of identified compounds with their theoretical and observed mass to charge ratio, double bond equivalence (DBE), molecular formula, absolute errors in parts per million (ppm), and retention time (Rt) in positive ion mode in ESI are presented in Table 3. The compounds were identified based on the observed mass spectra and also compared with the literature data (Figure 1). We observed the presence of flavonoids and phenolic compounds in the extract of A. catechu such as catechin/epicatechin (m/z=291.08) (Figure S2), gallocatechin/epigallocatechin (m/z=307.08) (Figure S3), procyanidin B1/procyanidin B3 (m/z=579.15) (Figure S4), emodin (m/z=271.06) (Figure S5), epiafzelechin/afzelechin (m/z=275.08) (Figure S6), maclurin (m/z=263.05) (Figure S7), irisflorentin (m/z=387.10) (Figure S8), naringenin (m/z=273.07) (Figure S9), isoquercetin (m/z=465.10) (Figure S10), diosmetin (m/z=301.07) (Figure S11), chrysin (m/z=255.06) (Figure S12), myricetin (m/z=319.04) (Figure S13), kaempferol (m/z=287.05) (Figure S14), avicularin (m/z=435.09) (Figure S15), prodelphinidin B3 (m/z=595.14) (Figure S16), prodelphinidin B (m/z=611.14) (Figure S17), quercetin (m/z=303.05) (Figure S18), taxifolin (m/z=305.06) (Figure S19), acacetin (m/z=285.07) (Figure S20), aciculatinone (m/z=413.12) (Figure S21), gossypin (m/z=481.09) (Figure S22), pterocarpin (m/z=299.09) (Figure S23), isorhamnetin (m/z=317.06) (Figure S24), and trihydroxy dimethoxyflavone (m/z=331.08) (Figure S25).Table 3
Identified compounds fromA. catechu with names, base peak, double bond equivalence (DBE), retention time (Rt), and m/z values.
Annotated compoundsSystematic nameCalculated massObserved massFormulaDBEAbsolute error (ppm)Rt (minute)Fragment peakReferenceCatechin (1)[(2R,3S)-2-(3,4-Dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol]290.07291.08C15H14O690.1610.4313.07 [M+Na]+ and 139[8, 37, 38]LC/MS and LC/MS/MS, NMR, UVEpicatechin (2)[(2R,3R)-2-(3,4-Dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol]290.07291.08C15H14O690.1610.4313.07 [M+Na]+ and 139.03[8, 37, 38]LC/MS and LC/MS/MS, UV, NMRGallocatechin (3)[(2R,3S)-2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol]306.07307.08C15H14O792.267.15313.07 [M+Na]+, 289.07, and 139.03[8, 38, 39]LC/MS and LC/MS/MS, UV, NMREpigallocatechin (4)[(2R,3R)-2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol]306.07307.08C15H14O792.267.15313.07 [M+Na]+, 289.07, and 139.03[8, 38, 39]LC/MS and LC/MS/MS, UV, NMRProcyanidin B1 (5)(2R,2′R,3R,3′S,4R)-2,2′-Bis(3,4-dihydroxyphenyl)-3,3′,4,4′-tetrahydro-2H,2'H-4,8′-bichromene-3,3′,5,5′,7,7′-hexol578.15579.15C30H26O12182.219.87427.10 [M+H−152]+, 289.07 (kampferol)[8, 40]HPLC, LC/MS, and LC/MS/MSProcyanidin B3 (6)(2R,2′R,3S,3′S,4S)-2,2′-Bis(3,4-dihydroxyphenyl)-3,3′,4,4′-tetrahydro-2H,2′H-4,8′-bichromene-3,3′,5,5′,7,7′-hexol578.15579.15C30H26O12182.219.87427.10 [M+H−152]+, 289.07 (kampferol)[8, 41]NMR, (Q-Trap), LC/MS and LC/MS/MSEmodin (7)1,3,8-Trihydroxy-6-methyl-9,10-anthraquinone270.05271.06C15H10O5114.1818.60253.16, 243.17, 229.14, 225.13, and 197.08[42]UPLC-DAD-MS/MSAfzelechin (8)(2R,3S)-2-(4-Hydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol274.08275.08C15H14O593.5112.77257.17, 233.08[43–45] UHPLC-PDA-HRMS, HPLC/ESI-MS and NMR, HPLC/MS/MSEpiafzelechin (9)(2R,3R)-2-(4-Hydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol274.08275.08C15H14O593.5112.77257.17, 233.08[43–45]UHPLC-PDA-HRMS, HPLC/ESI-MS and NMR, HPLC/MS/MSMaclurin (10)(3,4-Dihydroxyphenyl)-(2,4,6-trihydroxyphenyl)methanone262.04263.05C13H10O694.242.96153.08[46]HPLC/ESI-MSIrisflorentin (11)9-Methoxy-7-(3,4,5-trimethoxyphenyl)-[1,3]dioxolo[4,5-g]chromen-8-one386.09387.1C20H18O8122.455.07357.09 [M+H−CH3×2]+, 372.07 [M+H−CH3]+[47–49]HPLC–DAD–ESI-MS, NMR, LC-MSNaringenin (12)5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman-4-one272.06273.07C15H12O5102.0712.56153.03[50](LC–MS/MS)Isoquercetin (13)2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one464.09465.1C21H20O12123.0212.95303.05 (quercetin), 289.07 (kampferol)[51]Diosmetin (14)5,7-Dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-chromen-4-one300.06301.07C16H12O6114.9413.72289.09, 149.09[52, 53]UHPLC-LTQOrbitrap MS, NMRChrysin (15)5,7-Dihydroxy-2-phenylchromen-4-one254.05255.06C15H10O4112.2616.18153.12[54]UPLC-MS/MSMyricetin (16)3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)chromen-4-one318.03319.04C15H10O8113.5412.64[55]HPLC-PAD, UV/MS+NMRKaempferol (17)3,5,7-Trihydroxy-2-(4-hydroxyphenyl)chromen-4-one286.04287.05C15H10O6111.1617.17259.13, 165.09, 153.12[56]Q-TOF-HRMSAvicularin (18)3-[(2S,3R,4R,5S)-3,4-Dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromen-4-one434.08435.09C21H20O13123.3913.38303.05, 287.06, and 183.10[57]UHPLC-DAD-ESI-HRMS/MS, NMRProdelphinidin B3 (19)(2R,2′R,3S,3′S,4S)-2,2′-Bis(3,4,5-trihydroxyphenyl)-3,3′,4,4′-tetrahydro-2H,2′H-4,8′-bichromene-3,3′,5,5′,7,7′-hexol594.13595.14C30H26O13183.035.78427.08, 169.07, 291.09, 305.07[40, 58]HPLC, ESI-Q-TOFProdelphinidin B (20)2-(3,4,5-Trihydroxyphenyl)-8-[3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H-chromene-3,5,7-triol610.13611.14C30H25O14182.528.05307.08[59]UPLC-ESI-MSQuercetin (21)2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one302.04303.05C15H10O7111.613.96285.15, 257.13, 247.15, 229.13, 201.12, 183.10, 165.08, 153.11, 137.08, 121.03, 111.06[60]FTMS, HPLC, LC MS/MS, and LCMSTaxifolin (22)(2R,3R)-2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dihydrochromen-4-one304.05305.06C15H12O7101.7813.51287.05, 179.09[61, 62]1UHPLC-DAD-FLD, UHPLC-HRMS/MS, and HPLC-ESI-IT-TOF-MSAcacetin (23)5,7-Dihydroxy-2-(4-methoxyphenyl)chromen-4-one284.06285.07C16H12O5112.6416.38245.15 (M+H−CO2)+, 213.13(M−CO2−C0+H)+[63]UHPLC-Q-TOF-MS/MSAciculatinone (24)5-Hydroxy-8-[(2R,5R,6R)-5-hydroxy-6-methyl-4-oxooxan-2-yl]-2-(4-hydroxyphenyl)-7-methoxychromen-4-one412.11413.12C22H20O8133.808.18325.10,296.10, 295.09[64]NMR, HREIMSGossypin (25)2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one480.09481.09C21H20O13122.3211.79319.04,[65, 66]HRMS, LCMS/MSPterocarpin (26)(1R,12R)-16-Methoxy-5,7,11,19-tetraoxapentacyclo[10.8.0.02,10.04,8.013,18]icosa-2,4(8),9,13(18),14,16-hexaene298.08299.09C17H14O5113.5914.59[67] DART-TOF-MSIsorhamnetin (27)3,5,7-Trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one316.05317.06C16H12O7111.0317.70303.21, 274.20, 153.12[68]LC-MS/MSTrihydroxy dimethoxyflavone (28)5,6-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxychromen-4-one330.07331.08C17H1407112.3614.25301.08 and 315.09[69]UHPLC/Q-TOF MSFigure 1
Annotated secondary metabolites in the ethanolicA. catechu bark extracts


## 3.1. Total Phenolic and Flavonoid Content

The TPC and TFC were expressed as the GAE/g and QE/g of extract using calibration curves of gallic acid and quercetin, respectively. The TPC ofA. catechu was found to be 175.48±4.67 mg GAE/g which was greater than its TFC examined, i.e., 7.66±1.0 mg QE/g.


## 3.2. Free Radical Scavenging Activity

The antioxidant of ethanolic extracts ofA. catechu was evaluated by using a quick, reliable, and reproducible method through the measurement of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. DPPH radical scavenging results were reported as IC50 and compared with the IC50 value of quercetin (6.3±1.0 μg/mL) as a standard. The radical scavenging ability of plant extract is 23.76±1.57 μg/mL.


## 3.3.α-Glucosidase and α-Amylase Inhibitory Activities

The results ofα-glucosidase and α-amylase inhibitory activity of the extracts are expressed in Table 1. Among the tested fraction, aqueous and ethyl acetate fraction showed the most activity with an IC50 value of 82.6±0.3 and 130.2±0.6 μg/mL against the α-glucosidase as compared to acarbose (IC50=344.23±1.03 μg/mL). In the α-amylase assays, the crude ethanol extract showed antidiabetic activity with an IC50 of 67.8±1.8 μg/mL, compared to acarbose (IC50=6.1±0.1 μg/mL).Table 1
α-Glucosidase and α-amylase inhibitory activity of different fractions of A. catechu.
ExtractsIC50 (μg/mL)α-Glucosidaseα-AmylaseCrude ethanol extract10.3±0.167.8±1.8Hexane fraction<50 %<50 %Dichloromethane fraction174.7±0.6<50 %Ethyl acetate fraction130.2±0.6<50 %Aqueous fraction82.6±0.3<50%Standard (acarbose)344.2±1.036.02±0.1


## 3.4. Antibacterial Activity

The antibacterial activity of plant extracts againstS. aureus ATCC 25923, E. coli ATCC 25922, K. pneumoniae ATCC 13883, S. typhi ATCC 14028, and S. sonnei ATCC 25931 was performed. The antibacterial activity was measured in terms of ZoI diameter in millimeters (mm) as shown in Table 2. Based on the ZoI, MIC and MBC were evaluated against S. aureus ATCC 25923. The MIC and MBC of the aqueous fraction of A. catechu bark extract were 6.25 and 12.5 mg/mL while those of neomycin are 0.0625 and 0.25 mg/mL respectively.Table 2
Zone of inhibition of each fraction of plants.
MicroorganismsZone of inhibition (ZoI) in millimeters (mm)Hexane FractionDichloromethane fractionEthyl acetate fractionAqueous fractionPositive controlStaphylococcus aureus11 mm9 mm13 mm14 mm10 mmEscherichia coli————16 mmKlebsiella pneumonia——8 mm10 mm19 mmSalmonella typhi————18 mmShigella sonnei6 mm7 mm12 mm10 mm25 mmNote: positive control (50 mg/mL neomycin).


## 3.5. LC-HRMS-Driven Molecular Annotation

The raw data of LC-HRMS were processed using MZmine, and the fraction with the best total ion chromatogram (TIC) was proceeding for further analysis using MestreNova 12.0 software. The TIC obtained using MZmine of ethyl acetate and aqueous fraction is shown in FigureS1. Details of identified compounds with their theoretical and observed mass to charge ratio, double bond equivalence (DBE), molecular formula, absolute errors in parts per million (ppm), and retention time (Rt) in positive ion mode in ESI are presented in Table 3. The compounds were identified based on the observed mass spectra and also compared with the literature data (Figure 1). We observed the presence of flavonoids and phenolic compounds in the extract of A. catechu such as catechin/epicatechin (m/z=291.08) (Figure S2), gallocatechin/epigallocatechin (m/z=307.08) (Figure S3), procyanidin B1/procyanidin B3 (m/z=579.15) (Figure S4), emodin (m/z=271.06) (Figure S5), epiafzelechin/afzelechin (m/z=275.08) (Figure S6), maclurin (m/z=263.05) (Figure S7), irisflorentin (m/z=387.10) (Figure S8), naringenin (m/z=273.07) (Figure S9), isoquercetin (m/z=465.10) (Figure S10), diosmetin (m/z=301.07) (Figure S11), chrysin (m/z=255.06) (Figure S12), myricetin (m/z=319.04) (Figure S13), kaempferol (m/z=287.05) (Figure S14), avicularin (m/z=435.09) (Figure S15), prodelphinidin B3 (m/z=595.14) (Figure S16), prodelphinidin B (m/z=611.14) (Figure S17), quercetin (m/z=303.05) (Figure S18), taxifolin (m/z=305.06) (Figure S19), acacetin (m/z=285.07) (Figure S20), aciculatinone (m/z=413.12) (Figure S21), gossypin (m/z=481.09) (Figure S22), pterocarpin (m/z=299.09) (Figure S23), isorhamnetin (m/z=317.06) (Figure S24), and trihydroxy dimethoxyflavone (m/z=331.08) (Figure S25).Table 3
Identified compounds fromA. catechu with names, base peak, double bond equivalence (DBE), retention time (Rt), and m/z values.
Annotated compoundsSystematic nameCalculated massObserved massFormulaDBEAbsolute error (ppm)Rt (minute)Fragment peakReferenceCatechin (1)[(2R,3S)-2-(3,4-Dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol]290.07291.08C15H14O690.1610.4313.07 [M+Na]+ and 139[8, 37, 38]LC/MS and LC/MS/MS, NMR, UVEpicatechin (2)[(2R,3R)-2-(3,4-Dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol]290.07291.08C15H14O690.1610.4313.07 [M+Na]+ and 139.03[8, 37, 38]LC/MS and LC/MS/MS, UV, NMRGallocatechin (3)[(2R,3S)-2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol]306.07307.08C15H14O792.267.15313.07 [M+Na]+, 289.07, and 139.03[8, 38, 39]LC/MS and LC/MS/MS, UV, NMREpigallocatechin (4)[(2R,3R)-2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol]306.07307.08C15H14O792.267.15313.07 [M+Na]+, 289.07, and 139.03[8, 38, 39]LC/MS and LC/MS/MS, UV, NMRProcyanidin B1 (5)(2R,2′R,3R,3′S,4R)-2,2′-Bis(3,4-dihydroxyphenyl)-3,3′,4,4′-tetrahydro-2H,2'H-4,8′-bichromene-3,3′,5,5′,7,7′-hexol578.15579.15C30H26O12182.219.87427.10 [M+H−152]+, 289.07 (kampferol)[8, 40]HPLC, LC/MS, and LC/MS/MSProcyanidin B3 (6)(2R,2′R,3S,3′S,4S)-2,2′-Bis(3,4-dihydroxyphenyl)-3,3′,4,4′-tetrahydro-2H,2′H-4,8′-bichromene-3,3′,5,5′,7,7′-hexol578.15579.15C30H26O12182.219.87427.10 [M+H−152]+, 289.07 (kampferol)[8, 41]NMR, (Q-Trap), LC/MS and LC/MS/MSEmodin (7)1,3,8-Trihydroxy-6-methyl-9,10-anthraquinone270.05271.06C15H10O5114.1818.60253.16, 243.17, 229.14, 225.13, and 197.08[42]UPLC-DAD-MS/MSAfzelechin (8)(2R,3S)-2-(4-Hydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol274.08275.08C15H14O593.5112.77257.17, 233.08[43–45] UHPLC-PDA-HRMS, HPLC/ESI-MS and NMR, HPLC/MS/MSEpiafzelechin (9)(2R,3R)-2-(4-Hydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol274.08275.08C15H14O593.5112.77257.17, 233.08[43–45]UHPLC-PDA-HRMS, HPLC/ESI-MS and NMR, HPLC/MS/MSMaclurin (10)(3,4-Dihydroxyphenyl)-(2,4,6-trihydroxyphenyl)methanone262.04263.05C13H10O694.242.96153.08[46]HPLC/ESI-MSIrisflorentin (11)9-Methoxy-7-(3,4,5-trimethoxyphenyl)-[1,3]dioxolo[4,5-g]chromen-8-one386.09387.1C20H18O8122.455.07357.09 [M+H−CH3×2]+, 372.07 [M+H−CH3]+[47–49]HPLC–DAD–ESI-MS, NMR, LC-MSNaringenin (12)5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman-4-one272.06273.07C15H12O5102.0712.56153.03[50](LC–MS/MS)Isoquercetin (13)2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one464.09465.1C21H20O12123.0212.95303.05 (quercetin), 289.07 (kampferol)[51]Diosmetin (14)5,7-Dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-chromen-4-one300.06301.07C16H12O6114.9413.72289.09, 149.09[52, 53]UHPLC-LTQOrbitrap MS, NMRChrysin (15)5,7-Dihydroxy-2-phenylchromen-4-one254.05255.06C15H10O4112.2616.18153.12[54]UPLC-MS/MSMyricetin (16)3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)chromen-4-one318.03319.04C15H10O8113.5412.64[55]HPLC-PAD, UV/MS+NMRKaempferol (17)3,5,7-Trihydroxy-2-(4-hydroxyphenyl)chromen-4-one286.04287.05C15H10O6111.1617.17259.13, 165.09, 153.12[56]Q-TOF-HRMSAvicularin (18)3-[(2S,3R,4R,5S)-3,4-Dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromen-4-one434.08435.09C21H20O13123.3913.38303.05, 287.06, and 183.10[57]UHPLC-DAD-ESI-HRMS/MS, NMRProdelphinidin B3 (19)(2R,2′R,3S,3′S,4S)-2,2′-Bis(3,4,5-trihydroxyphenyl)-3,3′,4,4′-tetrahydro-2H,2′H-4,8′-bichromene-3,3′,5,5′,7,7′-hexol594.13595.14C30H26O13183.035.78427.08, 169.07, 291.09, 305.07[40, 58]HPLC, ESI-Q-TOFProdelphinidin B (20)2-(3,4,5-Trihydroxyphenyl)-8-[3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H-chromene-3,5,7-triol610.13611.14C30H25O14182.528.05307.08[59]UPLC-ESI-MSQuercetin (21)2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one302.04303.05C15H10O7111.613.96285.15, 257.13, 247.15, 229.13, 201.12, 183.10, 165.08, 153.11, 137.08, 121.03, 111.06[60]FTMS, HPLC, LC MS/MS, and LCMSTaxifolin (22)(2R,3R)-2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dihydrochromen-4-one304.05305.06C15H12O7101.7813.51287.05, 179.09[61, 62]1UHPLC-DAD-FLD, UHPLC-HRMS/MS, and HPLC-ESI-IT-TOF-MSAcacetin (23)5,7-Dihydroxy-2-(4-methoxyphenyl)chromen-4-one284.06285.07C16H12O5112.6416.38245.15 (M+H−CO2)+, 213.13(M−CO2−C0+H)+[63]UHPLC-Q-TOF-MS/MSAciculatinone (24)5-Hydroxy-8-[(2R,5R,6R)-5-hydroxy-6-methyl-4-oxooxan-2-yl]-2-(4-hydroxyphenyl)-7-methoxychromen-4-one412.11413.12C22H20O8133.808.18325.10,296.10, 295.09[64]NMR, HREIMSGossypin (25)2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one480.09481.09C21H20O13122.3211.79319.04,[65, 66]HRMS, LCMS/MSPterocarpin (26)(1R,12R)-16-Methoxy-5,7,11,19-tetraoxapentacyclo[10.8.0.02,10.04,8.013,18]icosa-2,4(8),9,13(18),14,16-hexaene298.08299.09C17H14O5113.5914.59[67] DART-TOF-MSIsorhamnetin (27)3,5,7-Trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one316.05317.06C16H12O7111.0317.70303.21, 274.20, 153.12[68]LC-MS/MSTrihydroxy dimethoxyflavone (28)5,6-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxychromen-4-one330.07331.08C17H1407112.3614.25301.08 and 315.09[69]UHPLC/Q-TOF MSFigure 1
Annotated secondary metabolites in the ethanolicA. catechu bark extracts


## 4. Discussion

The study was aimed at making a comparative study of ethanolic and methanolic extract ofA. catechu in terms of antidiabetic, antioxidant, antibacterial, and secondary metabolite profiling. Figure 2 shows the schematic workflow of the study. The TPC, TFC, and antioxidant activity of the crude ethanol extract of A. catechu were determined and compared with our previous work [34]. Studies have shown the valuable uses of plant-based bioactive secondary metabolites to mankind [8]. Plant extract contains many phenolic compounds with inhibitory activity against α-glucosidase supported by TPC and TFC of crude extract 175.48±4.67 mg GAE/g and TFC 7.66±1.0 mg QE/g respectively. The methanolic extract of A. catechu exhibited TPC and TFC of 186.675±2.021 mg GAE/g and 10.24±0.69 mg QE/g respectively [34]. Previous studies suggest that variation in TPC and TFC is due to differences in extracting solvents [70]. Likewise, the difference between antioxidant activity among the extracts might be due to the presence of metabolites at a different level which stabilizes the free radicals by donating hydrogen atoms [71, 72].Figure 2
Schematic workflow of the studyCompounds such as caprylic acid, methyl ester, lauric acid methyl ester, 2-ethyl-3-methyl-1-butene, myristic acid methyl ester [73], catechin, acacatechin, catechutannic acid, 4-hydroxybenzoic acid, afzelechin, epiafzelechin, mesquitol, ophioglonin, aromadendrin, kaempferol, baicalin, baicalein, and quercetin [74–77] and 5-hydroxy-2-[2-(4-hydroxyphenyl)acetyl]-3-methoxylbenzoic acid, (2S,3S)-3,7,8,3′,4′-pentahydroxyflavane, rhamnetin, 4-hydroxyphenyl ethanol, 3,3′,5,5′,7-pentahydroxyflavane, and fisetinidol [78] were isolated from A. catechu. Phytochemicals such as phenolic and flavonoid isolated from various parts of the plants are used as antioxidant, antimicrobial, antiulcer, antidiabetic, anticancer, antihyperlipidemic, antidiabetic, and hepatoprotective candidates [79, 80]. Previously, the IC50 value for inhibition of α-amylase activity by A. catechu ethanol extract was reported as 341.20±15.30 μg/mL by Lakshmi et al. [2], while ethanol extract showed an inhibition of 67.8±1.8 μg/mL in this study. Likewise, the methanolic extract of the A. catechu bark showed an IC50 of 115±4.0 mg/mL and 23.7±0.7 mg/mL against α-glucosidase and α-amylase, respectively [34], whereas leaf extracts showed an IC50 of 0.4977 mg/mL against α-glucosidase [81]. The difference in activities might be due to expression levels of secondary metabolites, which are greatly influenced by the regulation of biosynthetic gene clusters in climatic variation. Here, in our study, the crude extracts showed a potent inhibitory activity against carbohydrate hydrolases than that of the solvent fractions, which might be due to the synergistic effect of metabolites [82].The phytochemicals exhibit a wide range of antioxidant, antidiabetic, and antimicrobial activity [83, 84]. The antimicrobial activity of extracts from different parts of A. catechu [85–87] has been shown previously. A. catechu resin exhibits an inhibitory effect against B. subtilis (MIC: 20 μg/mL), S. aureus (MIC: 40 μg/mL), P. aeruginosa (MIC: 220 μg/mL), and E. coli (MIC: 330 μg/mL) [87]. In our study, the fractions of A. catechu from ethanol extract showed comparable antibacterial activities to methanol extract fraction as reported by Aryal et al. [34]. It might be due to the same experimental conditions, the only difference in the extracting solvent.The antioxidant mechanisms of flavonoid and phenolic compounds play an important role in protecting humans against infections and degenerative diseases [88]. Polyphenols with the number of hydroxyl groups can act as a source of hydrogen and electron donor to radicals to stabilize them and hence reduce the oxidative stress which plays an important role in the management of diabetes [89, 90]. Hydroxyl groups at 3′, 4′, and 5′of ring B (a pyrogallol group) and the double bond between carbon-2 and carbon-3 conjugated with the 4-oxo (=O) and 3-hydroxyl (-OH) group in ring C enhance the radical scavenging activity of flavonoids [91]. From the study of a structure-activity relationship, hydroxyl groups and their configuration, a ketonic functional group at carbon-4, and a double bond at carbons (C2–C3) are quite important structural features on flavonoids to show their antioxidant and antidiabetic ability [92, 93].Isolation of plant metabolites is considerably challenging due to the lack of standardized instruments, income sources, and the availability of the laboratory in our context. To identify the metabolites within a short instance of time with a small number of samples, LC-HRMS of ethyl acetate and aqueous fractions ofA. catechu bark extract were carried out. To further evaluate the metabolites, MZmine 2 was used to study the peak features of the raw MS files. The TIC obtained by overlaying through MZmine 2 is shown in Figure S1. Then, Mesternova 12.0 software is utilized to annotate the metabolites based on m/z, retention time, and molecular formula, and other databases are used to search and assign formulas and compound structures. Twenty-eight phenolic and flavonoids were annotated during our study by comparing our spectral data with the literature. The base peak atm/z 291.08, molecular formula C15H14O6, and DBE 9 and fragment peaks at 313.07 [M+Na]+ and 139.03 are speculated as catechin (1) or epicatechin (2). This data is consistent with Shen et al. [8] and Ibrahim et al. [37]. The fragmentation pattern of catechin and epicatechin is shown in Figure S26. Catechin/epicatechin was already reported with antidiabetic [94] (IC50 of 160±67 μg/mL against α-amylase; 31 μg/mL and >290 μg/mL against α-glucosidase), antimicrobial [95], anti-inflammatory [96], and antioxidant [97] abilities and also reduces the risk of ischemic heart disease [98]. Likewise, [M+H]+ at m/z 307.08, molecular formula C15H14O7, DBE 9, and along with fragment peak at 289.07 and 139.03 is considered as gallocatechin (3) or epigallocatechin (4) based on result analysis from Shen et al. [8]. The fragmentation pattern of gallocatechin/epigallocatechin is shown in Figure S27. It has shown different pharmacological activities such as antiviral [99, 100], antioxidant [101], and antibacterial [102] activities. Compounds with characteristic fragment ions 427.10 [M+H−152]+ and 289.07 (kaempferol) and base peak [M+H]+ at m/z 579.15, molecular formula C30H26O12, and DBE 18 are annotated as procyanidin B1 (5) or procyanidin B (6). The spectral data coincides with data from Shen et al. [8]. Likewise, it is reported to be used as antioxidant, antibacterial, antiviral, anticarcinogenic, anti-inflammatory, antiallergic, and vasodilatory candidate. They can inhibit lipid peroxidation, platelet aggregation, and capillary hyperpermeability [62, 103]. The base peak [M+H]+ at m/z 271.06, molecular formula C30H26O12, and DBE 11 with characteristic fragment ions 253.16, 243.17, 229.14, 225.13, and 197.08 is explicated as emodin (7). The data is in agreement with Zhan et al. [42]. The fragmentation pattern of emodin is shown in Figure S28. Emodin also exhibits a wide spectrum of pharmacological properties including antiallergic, antiosteoporotic, antidiabetic, anti-inflammatory, anticancer, antiviral, antimicrobial, antioxidant, hepatoprotective, and immunosuppressive activities [104]. The compound with base peak 275.08, molecular formula C15H14O5, and DBE 9 and fragment peaks at 257.17, 233.08, 191.07, and 169.11 is expected to be afzelechin (8) or epiafzelechin (9). The data is consistent with Mittal et al. [43]. It has been reported to have antidiabetic [105], antimicrobial [106], and antioxidant [107] abilities.The base peak atm/z 263.05 with molecular formula C13H10O6 and DBE 9 and fragment peaks at 153.08 are assigned as maclurin (10), consistent with the result of Berardini et al. [46]. Maclurin (10) has been reported to have antioxidant [108] and anticancer activities [109]. Similarly, the compound at base peak m/z 387.1 and fragment peaks at 357.09 (M+H−CH3×2)+, 372 [M+H−CH3]+ is assigned as irisflorentin (11) based on result analysis by Zhang et al. [49]. It is used as anti-inflammatory [110], antiallergic [111], anticholinesterase [112], and antimicrobial agent [113]. Likewise, mass spectrum with a base peak at m/z 273.07, molecular formula C15H12O, and DBE 10 and fragment peak at 153.10 is predicted to be naringenin (12) corresponding to fragment pattern analyzed by Tong et al. [50]. The fragmentation pattern of naringenin is shown in Figure S29. It shows antibacterial [114], anticancer [115], antioxidant, anti-inflammatory, hepatoprotective, nephroprotective, immunomodulatory, and antidiabetic properties [116]. Similarly, a base peak with m/z 465.1 and molecular formula C21H20O12 and the fragment ion at 303.05 and 289.07 are interpreted as isoquercetin (13) comparing our spectra with Liu et al. [51]. The fragmentation pattern of isoquercetin is shown in Figure S30. It acts as antioxidation, anticancer, anticardiovascular, antidiabetes, and antiallergic candidate [117]. Likewise, the compound with fragment peaks at 289.09 and 149.09 is annotated as diosmetin (14) having a base peak at m/z 301.07. The spectral data matches with data from Chen et al. [52] and Park et al. [53]. The fragmentation pattern of diosmetin is shown in Figure S31. It is used as an anticancer, anti-inflammatory, antioxidant, and antimicrobial agent [118]. The base peak at m/z 255.06, molecular formula C15H10O4, and DBE 11 and characteristic fragment ion at 153.12 are expected to be chrysin (15) with reference from the data collected from our spectra and Zhao et al. [54]. The fragmentation pattern of diosmetin is shown in Figure S32. It shows anticancer, antidiabetic, neuroprotective, antiallergic, antihyperlipidemic, antimicrobial, antiobesity, anti-inflammatory, hepatoprotective, cardiovascular, reproductive, and antioxidant activities [119]. The other flavonoid is expected to be myricetin (16) with base peak m/z 319.04, molecular formula C15H10O8, and DBE 11 with reference from the data collected from our spectra and Saldanha et al. [55]. It is used as antimicrobial, antioxidant, and anti-inflammatory agent [120, 121]. Likewise, a base peak with m/z 287.05, molecular formula C15H10O6, and DBE 11 and fragment peak at m/z 259.13, 165.09, and 153.12 were predicted to be kaempferol (17) as spectral figures match with the literature of March and Miao [56]. The fragmentation pattern of kaempferol is shown in Figure S33. It has neuroprotective, antimicrobial, antioxidant, anti-inflammatory, and anticancer effects [122]. Moreover, in our spectra, a base peak with m/z 435.09, molecular formula C21H20O13, and DBE 12 and fragment peak at m/z 303.05, 287.06, and 183.10 were annotated as avicularin (18) taking reference of Santos et al. [57]. The fragmentation pattern of avicularin is shown in Figure S34. It is used as an anticancer, anti-inflammatory, and anti-infectious candidate [123].The molecular ion peak atm/z 595.14, molecular formula C30H26O13, and DBE 18 and fragment ions at m/z 427.08, 169.07, 291.09, and 305.06 were annotated as prodelphinidin B3 (19). These spectral data are consistent with Friedrich et al. [40] and Pinto et al. [58]. The fragmentation pattern of prodelphinidin B3 is shown in Figure S35. It is used as antidiabetic, antiviral, and anti-inflammatory activities [124]. Additionally, [M+H]+ at m/z 611.14, molecular formula C30H26O14, and DBE 18 and fragment ions at m/z 307.08 are considered as prodelphinidin B (20) relying on result analysis from Navarro et al. [59]. The fragmentation pattern of prodelphinidin B is shown in Figure S36. It is used as antidiabetic, antiviral, and anti-inflammatory candidate [124]. Base peak m/z 303.05, molecular formula C15H10O7, DBE 11, and fragments ions at m/z 285.15, 257.13, 247.15, 229.13, 201.12, 183.10, 165.08, 153.11, 137.08, 121.03, and 111.06 were annotated as quercetin (21) whose spectra exactly match with the spectra data of Scigelova et al. [60]. It is used as an anti-inflammatory, antioxidant, antiviral, antimicrobial, and anticancer agent [125]. A. catechu may contain taxifolin (22) with the base peak at m/z 305.06, molecular formula C15H12O7, and DBE 10, and characteristic fragment ions are 287.05 (M+H−H2O)+ and 177.0253 (M+H−H2O−C3O2−C2H2O]+ comparing our result with Michel et al. [61] and Yang et al. [62]. It is known to have potential antibacterial, antifungal, anti-inflammatory, analgesic, antioxidant, antipyretic, platelet inhibitory, and even anticancer actions [126, 127]. Likewise, a base peak at m/z 284.06, molecular formula C16H12O5, and DBE 11 and characteristic fragment ions at m/z 245.15 (M+H−CO2)+, 213.13(M−CO2−CO+H)+ are annotated as acacetin (23). The data is the same as the spectral data of Yin et al. [63]. The molecular ion at m/z 413.12, molecular formula C22H20O8, and DBE 13 and fragments ions at m/z 325.10, 296.10, and 295.09 are characterized as aciculatinone (24). The spectral data exactly match with Shen et al.[64]. The base peak at m/z 481.09 and fragment peaks 319.04 which are in agreement with previous studies done by Giorio et al. [65] and Petsalo et al. [66] are speculated as gossypin (25). The base peak at m/z 299.09, molecular formula C17H14O5, and DBE 11 manifested that it could be pterocarpin (26) comparing spectral data with Geiger et al. [67]. Moreover, another annotated compound is isorhamnetin (27) with the base peak at m/z 317.06, molecular formula C16H12O7, and DBE 11. The data coincides with Chen et al. [68]. The fragmentation pattern of isorhamnetin is shown in Figure S37. Likewise, [M+H]+ at 331.08 along with fragment peak at 315.09, and 301.08 is considered as trihydroxy dimethoxyflavone (28) relying on result analysis from Zhang et al. [69]. The fragmentation pattern of trihydroxy dimethoxyflavone is shown in Figure S38.In our study, we have annotated 28 secondary metabolites from the ethanol extract ofA. catechu where seven of the secondary metabolites, namely, catechin, epicatechin, gallocatechin, epigallocatechin, procyanidin, emodin, and quercetin, were already annotated by Aryal et al. [34] in methanol extract. To support our annotation, more spectroscopic data are required. Hence, further investigation is required for the separation of potential enzyme inhibitors, antioxidant, and antibacterial compounds and hence the inhibitory activities and enzymatic kinetics of such compounds to develop them as future drug candidates or food supplements.


## 5. Conclusion

The bark ofA. catechu has significant potential in inhibiting carbohydrate hydrolases due to the abundance of flavonoids and polyphenols. Our findings open the entryway for better utilization of phytoconstituents of A. catechu for the management of diabetes and pathogens. Further research on the isolation of potential inhibitors from solvent fraction, the study of pharmacokinetic parameters, kinetics, and in vivo experiments are required to develop an influential therapeutic approach against diabetes.



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*Source: 7588711-2021-08-15.xml*