Antioxidant and Anti-α-Glucosidase Activities of Various Solvent Extracts and Major Bioactive Components from the Fruits of Crataegus pinnatifida

Crataegus pinnatifida is used to treat various diseases, including indigestion, congestive heart failure, hypertension, atherosclerosis, and myocardial dysfunction. We evaluated antioxidant and anti-α-glucosidase activities of various solvent extracts and major bioactive components from the fruit of C. pinnatifida. Ethyl acetate extracts showed potent antioxidant activities with IC50 values of 23.26 ± 1.97 and 50.73 ± 8.03 μg/mL, respectively, in DPPH and ABTS radical scavenging assays. Acetone extract exhibited significant anti-α-glucosidase activity with IC50 values of 42.35 ± 2.48 μg/mL. HPLC analysis was used to examine and compare the content of active components in various solvent extracts. We isolated four active compounds and evaluated their antioxidant and anti-α-glucosidase properties. Among the isolated compounds, chlorogenic acid and hyperoside showed potential antioxidant activities in ABTS and superoxide radical scavenging assays. Moreover, hyperoside also displayed stronger anti-α-glucosidase activity than other isolates. The molecular docking model and the hydrophilic interactive mode of anti-α-glucosidase assay revealed that hyperoside might have a higher antagonistic effect than positive control acarbose. The present study suggests that C. pinnatifida and its active extracts and components are worth further investigation and might be expectantly developed as the candidates for the treatment or prevention of oxidative stress-related diseases and hyperglycemia.


Introduction
Free radicals are generated from normal biochemical reactions in the body and increase oxidative stress and probably damage biological molecules such as lipids, proteins, and DNA [1]. Free radicals are associated with cancer, atherosclerosis, aging, inflammation, ischemic heart disease, diabetes, and neurodegenerative disorders [2]. To diminish the

Reverse-Phase HPLC
Reversed-phase separations were performed using a LiChrospher ® 100 RP-18 Endcapped (5 µm; column of dimensions 4.6 × 250 mm) purchased from Merck KGaA, Darmstadt, Germany. HPLC-PDA chromatographic fingerprints were obtained with an Agilent 1260 Infinity II HPLC instrument equipped with a 1260 Infinity II quaternary pump, a 1260 Infinity II degasser, a 1260 Infinity II vialsampler, a 1260 Infinity II column thermostat, a 1260 Infinity II diode array detector HS, and a PC with the Agilent Chemstation software. All of them are from Agilent Technologies (Waldbronn, Germany). Gradient separation using 0.2% acetic acid in water (v/v) (solvent A) and water:methanol:acetonitrile = 3:2:5 (solvent B) as mobile phase was as follows: 0-20 min, linear gradient from 0 to 15% B; 20-65 min, 15% B with isocratic elution; 65-110 min, linear gradient from 15 to 40% B; 110-120 min, linear gradient from 40 to 100% B; 120-130 min, back to initial conditions at 0% B; and 130-135 min, at 0% B. The flow rate was 1.0 mL/min, and the injection volume was 10 µL. Peaks were detected at 280 nm. Different compounds were identified by retention time. To guarantee peak purity, DAD acquisition from 200 to 650 nm was conducted to register UV-spectra. For the quantitative analysis of four compounds in the extracts, aliquots of samples were dispersed in 10 mL of a methanol solution by sonication for 5 min. Then, the samples were centrifuged for 15 min at 3500 rpm, and the supernatant extracts were filtered through 0.45 µm PTFE syringe filters (Zhejiang Sorfa Medical Plastic Co., Ningbo, China).

Determination of Total Phenolic Content
Total phenolic content (TPC) of different extracts was determined by Folin-Ciocalteau's method with a slight modification of [19]. Briefly, 0.5 N Folin-Ciocalteu reagent (50 µL) was diluted by ddH 2 O. Gallic acid was diluted by MeOH (0-100 µg/mL) as standard. Each extract or gallic acid (50 µL) was added to a 96-well microplate. The mixture was incubated for 5 min. Subsequently, 20% Na 2 CO 3 solution (100 µL) was added. After Antioxidants 2022, 11, 320 4 of 15 40 min, it was incubated in darkness at room temperature before measuring the absorbance of the supernatant at 750 nm. The TPC of the extracts was determined from a standard calibration curve using gallic acid in the range of 0-100 µg/mL with R 2 value of 0.9992. The concentration of TPC was expressed in milligram (mg) gallic acid equivalents (GAE) per gram of dried extract. All measurements were conducted in triplicate.

Determination of Total Flavonoid Content
The total flavonoid content (TFC) of each extract was measured using the aluminum chloride colorimetry method described by Do et al. [20] with slight modifications. In short, the extracted sample was diluted with MeOH to reach a concentration of 100 µg/mL. Quercetin was diluted in MeOH (0-100 µg/mL) as standard. The diluted extract sample or quercetin (400 µL) was mixed with 10% (w/v) aluminum chloride solution (200 µL) and 0.1 mM potassium acetate solution (200 µL). The mixture was reacted at room temperature for 30 min. Then, the absorbance of the mixture was measured at 415 nm. The TFC of the extracts was determined from a standard calibration curve using quercetin with R 2 value of 0.9997. The concentration of TFC was expressed in milligram quercetin equivalents (GAE) per gram of dried extract. All measurements were conducted in triplicate.

DPPH Radical Scavenging Activity
The scavenging activity of DPPH radical was investigated as described with slight modification [21]. DPPH solution was prepared diluted by EtOH to reach the final concentration of 200 µM. Different concentrations (200, 100, 50, 25, and 12.5 µg/mL) of extracts or compounds (100 µL) were mixed with DPPH solution (100 µL) and incubated for 30 min at room temperature and in darkness, and the absorbance of the mixture was measured at 520 nm. DPPH radical scavenging activity was calculated with the following formula: where A t is the absorbance of the test sample, and A c is the absorbance of the control (untreated group). BHT was used as the positive control. All half-maximal inhibitory concentration (IC 50 ) values of tested activities were determined by the linear regression of the percentage of remaining DPPH radical against the sample's concentration.

ABTS Radical Scavenging Activity
ABTS radical scavenging activity of each extract was measured based on the method with slight changes [22]. In brief, the stock of working solution was prepared by the mix of 28 mM ABTS solution and 9.6 mM potassium persulfate with ddH 2 O (final concentration, 1/1, v/v) and leaving the mixture in the dark for about 16 h at room temperature before use. The working solution was diluted by EtOH to reach the absorbance of 0.70 ± 0.02 at 740 nm for further experiments. Different concentrations (200, 100, 50, 20, and 12.5 µg/mL) of extracts or compounds (10 µL) were mixed with the working solution (190 µL). The mixture was incubated at room temperature for 6 min, the antioxidant activity of the mixture was determined by calculating the decrease in absorbance measured at 740 nm by the following equation: Where A c and A t are the absorbance of the control (untreated group) and test sample, respectively. The IC 50 values of all tested activities were determined by the liner regression of the percentage of remaining ABTS radical against the sample's concentration.

Superoxide Radical Scavenging Activity
The scavenging activity of superoxide anion radical (O 2 •− ) was evaluated using a modified method [23]. NBT (300 µM), NADH (468 µM), and PMS (120 µM) were prepared with dilution by Tris-HCl buffer (16 mM, pH 8.0), respectively. An amount of 50 µL of Antioxidants 2022, 11, 320 5 of 15 NBT, PMS, and different concentrations (400, 200, 10, 50, and 25 µg/mL) of extracts or compounds were mixed. An amount of 50 µL NADH solution was added, and the reaction was initiated to produce superoxide radicals. The absorbance was measured at 560 nm after incubating at room temperature for 5 min. The scavenging activity was measured by the following equation: where A t is the absorbance of the test sample, and A c is the absorbance of the control (untreated group). The IC 50 values of all tested activities were determined by the linear regression of the percentage of remaining superoxide radical against the sample concentration.

Hydroxyl Radical Scavenging Activity
The scavenging activity of hydroxyl radical was measured based on the method described by Mathew and Abraham with slight revision [24]. In short, different concentrations (400, 200, 10, 50, and 25 µg/mL) of extracts or compounds (200 µL) were mixed with working solution (200 µL) contained 1 mM FeCl 3 , 1 mM EDTA, and 2.8 mM deoxyribose. Then, 20 µL ascorbic acid (1 mM) and 100 µL H 2 O 2 (20 mM) in sodium phosphate buffer (20 mM pH 7.4) was added. The mixture was incubated in a water bath at 37 • C for 1 h to trigger the Fenton reaction and form a hydroxyl radical. After incubation, 2.8% TCA (200 µL) and 1% TBA (200 µL) were added and boiled in the dry bath at 100 • C for 1 h. The mixture was measured at 532 nm by the following equation: where A t and A c are the absorbance of the test sample and the control (untreated group), respectively. The IC 50 values of all tested activities were determined by the linear regression of the percentage of remaining hydroxyl radical against the sample's concentration.

Ferric Reducing Antioxidant Power (FRAP)
Ferric reducing antioxidant power was measured based on previous study with slight modification [25]. The working solution was mixed with acetate buffer (pH 3.6), ferric chloride solution (20 mM), and TPTZ solution (10 mM TPTZ in 40 mM HCl) in a proportion of 10:1:1, respectively, and freshly prepared before used. An amount of 900 µL of the working solution was warmed to 37 • C and then mixed with 100 µL of the diluted sample, blank or standard in a microcentrifuge tube. The tubes were vortexed and in the dry bath at 37 • C for 40 min. Absorbance was measured at 593 nm. The standard curve was linear between 0 and 100 mM Trolox with an R 2 value of 0.998. Results are expressed in mM TE/g dry weight. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve.

α-Glucosidase Inhibitory Activity Assay
The inhibition assay of α-glucosidase was conducted using the conditions previously reported with slight modifications [26]. The α-Glucosidase solution was diluted by 0.1 M sodium phosphate buffer (pH 6.8) to 1 U/mL. Different concentrations (400, 200, 10, 50, and 25 µg/mL) of extracts or compounds (100 µL) were mixed with α-glucosidase solution (20 µL) in a microcentrifuge tube. Subsequently, p-NPG (0.53 mM; 380 µL), the substrate, was added and incubated in dry baths at 37 • C for 40 min after the mixture vortexed. The reaction was terminated after adding 0.1 M Na 2 CO 3 solution (500 µL). The absorbance of released p-nitrophenol (p-PNP) was measured by calculating the decrease in absorbance measured at 400 nm by the following equation: where A c is the absorbance of the control (untreated group), and A t is the absorbance of the test sample. The IC 50 values of all tested activities were determined by the linear regression of the percentage of remaining α-glucosidase against the sample concentration.

Molecular Modeling Docking Study
All calculations are performed by Discovery Studio 2019 (San Diego, CA, USA) software. Primarily, the structure of hyperoside or acarbose is energy minimized until the default derivative convergence criterion of 0.01 kcal/mol is met. The crystal structure (PDB: 3A4A) is retrieved from the Protein Databank, and hydrogen atoms are added to prepare the docked receptor. This protein structure is subsequently used in the CDocker program to dock hyperoside or acarbose into the active site. Ten different docking poses are calculated and ranked by using the PLP-Score scoring function. The top-ranked docking solution is visually analyzed to determine the binding mode of hyperoside or acarbose.

Statistical Analysis
All data are expressed as mean ± SEM. Statistical analysis was carried out using Student's t-test. A probability of 0.05 or less was considered statistically significant. All experiments were performed at least 3 times.

Determination of Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and Yields in Each Solvent Extract
TPCs, TFCs, and yields of various solvent extracts from C. pinnatifida were evaluated. TPCs, TFCs, and extraction yields of n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH extracts from C. pinnatifida are shown in Table 1. 28.0 ± 0.75 a TPC was expressed in mg of gallic acid equivalents (GAE) per gram of extract. b TFC was expressed in milligram of quercetin equivalents (QE) per gram of extract. Values are expressed as means ± standard error. c Yield was calculated as % yield = (weight of extract/initial weight of dry sample) × 100; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control.
The yields of various solvent extracts from C. pinnatifida ranged from 2.3 ± 1.67% (n-hexane extract) to 28.0 ± 0.75% (MeOH extract). Higher polar solvent (including MeOH and EtOH) extracts had higher yields. The results indicated that C. pinnatifida is abundant in high polar components.
The comparative assessment of total phenolic content (TPC) and total flavonoid content (TFC) of various solvent extracts (n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH) from the fruits of C. pinnatifida was first mentioned in this study. This can supply a guide for the option of suitable solvents in TPC and TFC extraction applications.

Superoxide Radical Scavenging Activity
This assay is measured by the PMS/NADH-NBT system. Superoxide anion radicals generated from dissolved oxygen by PMS/NADH coupling reaction reduce NBT. The decrease in absorbance at 560 nm with antioxidants indicates a reduction in superoxide anion radicals in the reaction mixture [29]. The results are shown in Table 2, and only EtOAc extract exerted a significant effect (IC 50 = 122.95 ± 9.07 µg/mL) on the superoxide radical scavenging assay.
Based on the above results of the antioxidant assays, the EtOAc extract from C. pinnatifida possessed the highest phenolic content and the highest hydrogen atom or electron donation abilities in DPPH and ABTS radical scavenging assays. In addition, the EtOAc extract also showed potent superoxide and hydroxyl radical scavenging effects. Moreover, acetone extract showed the highest reducing ability in the FRAP assay.
The comparative evaluation of antioxidant assays (DPPH, ABTS, superoxide, hydroxyl, and FRAP) of various solvent extracts (n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH) from the fruits of C. pinnatifida was first proposed in our study. This can provide an indication for the selection of appropriate solvents in antioxidant extraction applications.

Quantitation of Active Components in Different Solvent Extracts
Figures S2-S9 displayed the quantification of active components in different solvent extracts from C. pinnatifida by HPLC analysis with reverse-phase. The contents of four active compounds in each solvent extract was shown in Table 4. Total amount of four active compounds in each extract ranged from a maximum of 33.62 ± 0.49 µg/kg (EtOH extract) to a minimum of 1.92 ± 0.25 µg/kg (n-hexane extract) in succeeding order of EtOH > MeOH > EtOAc > acetone > dichloromethane > chloroform > n-hexane extract. EtOH (33.62 ± 0.49 µg/kg) and MeOH (27.76 ± 0.45 µg/kg) extracts exhibited higher amounts of four active compounds compared with other extracts. Procyanidin B2 was the most abundant among the four active compounds in organic solvent extract followed by chlorogenic acid, epicatechin, and hyperoside.
The comparative evaluation for identification and quantification of the major active components (epicatechin, chlorogenic acid, hyperoside, and procyanidin B2) of different solvent extracts (n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH) from the fruits of C. pinnatifida by HPLC analysis is first conducted in our study.

Molecular Modeling Docking
According to the results of anti-α-glucosidase assay, hyperoside exhibited the most potent anti-α-glucosidase activity among all isolated compounds. Thus, the interaction between α-glucosidase and hyperoside was evaluated by molecular modeling docking. The 3D crystal structure of α-glucosidase showed that it mainly contains numerous structural domains including the N-terminal domain, the barrel domain in which the active site is located, and the C-terminal domain. The α-glucosidase active site is primarily formed by numerous β-sheets and several loops or α-helices. Importantly, the active site from different species shows certain conformational similarities. It mainly contains hydrophilic residues and can accept compounds with distinct sizes to enter. The crystal structure of α-glucosidase from beta vulgaris complexed with acarbose (Figure 2a; PDB: 3W37) reveals that its active site is not deep but wide enough to allow acarbose to enter in the flat conformation [34]. Due to containing four glucosyl-like groups, acarbose mainly interacts with the active site by significant H-bond interactions including (1) the 2-hydroxyl group on the A ring interacting with Arg 552 by acting as the H-bond donor; (2) the amino linker between A and B rings makes H-bond contact with Asp 568 and Arg 552; (3) on the B ring, the 3 -hydroxyl group interacts with Arg 552 by acting as the H-bond acceptor and the 2 -hydroxyl group makes hydrophilic contact with Asp 232 by acting as the H-bond donor; (4) the oxygen atom on the ether moiety of the D ring makes H-bond interactions with Asn 237. To further study how hyperoside (Figure 2b) might interact with α-glucosidase of Saccharomyces cerevisiae to exhibit its antagonistic effect, the docking models of hyperoside were generated by Discovery Studio 2019 (Accelrys, San Diego, USA) CDocker modeling program. The 3D crystal structure for α-glucosidase of Saccharomyces cerevisiae is not available at this moment; thus, the crystal structure (PDB: 3A4A) of Saccharomyces cerevisiae (PDB: 3A4A) containing 72% sequence homology with α-glucosidase from Saccharomyces cerevisiae is usually used to perform the docking study and is also employed in this study. In the crystal structure (PDB: 3A4A), the configuration of its active site is quite similar to that of α-glucosidase from beta vulgaris, but it is deep and narrow. The crystal structure of this active site reveals that its co-crystallized ligand, α-D-glucopyranose, locates deeply in the ligand-binding pocket and makes three essential H-bond interactions including (1) the 4-hydroxyl group interacting with His 351 and Asp 352 by acting as the H-bond donor; (2) the 5-hydroxyl group that makes H-bond contact with Glu 277 as well as Asp 352 by acting as the H-bond donor and also serves as the H-bond acceptor to interact with Arg 213; and (3) the 6-hydroxymethyl group that acts as the H-bond donor to interact with Glu 277 and Asp 352.
As shown in Figure 3a, the docking model of hyperoside indicated that hyperoside did not enter the active site in the flat conformation due to the narrow entrance. Alternatively, the A ring of hyperoside leaned toward the binding position of α-D-glucopyranose and the D ring of hyperoside located nearby the entrance of the active site. Importantly, hyperoside made some significant hydrophilic interactions, including the following: (1) the 5-hydroxyl and 7-hydroxyl groups on the A ring both served as the H-bond donors to interact with Asp 69 and Asp 215, respectively; (2) the 4-keto group on the B ring behaved as the H-bond acceptor to make contact with Arg 442; (3) both 3′-hydroxyl and 4′-hydroxyl groups on the A ring acted as the H-bond donors to interact with His 280 and Asp 307 respectively; and (4) the 5′′-hydroxyl group on the D ring acted as the H-bond donor to interact with Glu 411 and the 6′′-hydroxymehtyl group also on the D ring behaved as the H-bond acceptor to contact with the amido side chain of Asn 415. Apart from hydrophilic interaction, hyperoside also made important hydrophobic contact: the π-π interaction between its B ring and Tyr 158. To further study how hyperoside (Figure 2b) might interact with α-glucosidase of Saccharomyces cerevisiae to exhibit its antagonistic effect, the docking models of hyperoside were generated by Discovery Studio 2019 (Accelrys, San Diego, USA) CDocker modeling program. The 3D crystal structure for α-glucosidase of Saccharomyces cerevisiae is not available at this moment; thus, the crystal structure (PDB: 3A4A) of Saccharomyces cerevisiae (PDB: 3A4A) containing 72% sequence homology with α-glucosidase from Saccharomyces cerevisiae is usually used to perform the docking study and is also employed in this study. In the crystal structure (PDB: 3A4A), the configuration of its active site is quite similar to that of α-glucosidase from beta vulgaris, but it is deep and narrow. The crystal structure of this active site reveals that its co-crystallized ligand, α-D-glucopyranose, locates deeply in the ligand-binding pocket and makes three essential H-bond interactions including (1) the 4-hydroxyl group interacting with His 351 and Asp 352 by acting as the H-bond donor; (2) the 5-hydroxyl group that makes H-bond contact with Glu 277 as well as Asp 352 by acting as the H-bond donor and also serves as the H-bond acceptor to interact with Arg 213; and (3) the 6-hydroxymethyl group that acts as the H-bond donor to interact with Glu 277 and Asp 352.
As shown in Figure 3a, the docking model of hyperoside indicated that hyperoside did not enter the active site in the flat conformation due to the narrow entrance. Alternatively, the A ring of hyperoside leaned toward the binding position of α-D-glucopyranose and the D ring of hyperoside located nearby the entrance of the active site. Importantly, hyperoside made some significant hydrophilic interactions, including the following: (1) the 5-hydroxyl and 7-hydroxyl groups on the A ring both served as the H-bond donors to interact with Asp 69 and Asp 215, respectively; (2) the 4-keto group on the B ring behaved as the H-bond acceptor to make contact with Arg 442; (3) both 3 -hydroxyl and 4 -hydroxyl groups on the A ring acted as the H-bond donors to interact with His 280 and Asp 307 respectively; and (4) the 5 -hydroxyl group on the D ring acted as the H-bond donor to interact with Glu 411 and the 6 -hydroxymehtyl group also on the D ring behaved as the H-bond acceptor to contact with the amido side chain of Asn 415. Apart from hydrophilic interaction, hyperoside also made important hydrophobic contact: the π-π interaction between its B ring and Tyr 158. As the positive control, the docking model for acarbose is also generated to compare its binding mode with hyperoside. Acarbose was bound to the catalytic site similarly as hyperoside by leaning its A ring toward the binding position of α-D-glucopyranose and located its D ring to protrude out of the entrance of the active site. Since it contains a lot of hydrophilic moieties, acarbose mainly interacted with the active site by the H-bond interactions, including the following: (1) the 5-hydroxymethyl group on the A ring For the comparison of the binding modes of hyperoside and acarbose, hyperoside can reside in the middle of the substrate binding pocket and its D ring is located at the entrance of the pocket to block the entrance and enhance its inhibitory effect. For acarbose, its A ring can be located in a similar position as the A ring of hyperoside but its D ring partially protrudes out of the substrate-binding pocket. For their hydrophilic interactive binding mode (Figure 3b), the A rings of hyperoside and acarbose exert similar binding modes. The B and C rings of hyperoside not only make significant hydrophilic but also hydrophobic interactions with the residues in the middle of the substrate-binding pocket. The B and C rings of acarbose exert only one hydrophilic interaction and they are surrounded by some hydrophobic residues including Tyr 158, Phe 159, and Phe 178; thus, their numerous hydroxyl moieties might not be able to exhibit significant hydrophobic interactions as the B ring of hyperoside. Moreover, the D ring of hyperoside resides at the entrance of the substrate-binding pocket to block the entrance through two essential H-bond interactions but the D ring of acarbose protrudes out of the pocket and exerts its interactions with the residues outside of the pocket. The interaction As the positive control, the docking model for acarbose is also generated to compare its binding mode with hyperoside. Acarbose was bound to the catalytic site similarly as hyperoside by leaning its A ring toward the binding position of α-D-glucopyranose and located its D ring to protrude out of the entrance of the active site. Since it contains a lot of hydrophilic moieties, acarbose mainly interacted with the active site by the Hbond interactions, including the following: (1) the 5-hydroxymethyl group on the A ring For the comparison of the binding modes of hyperoside and acarbose, hyperoside can reside in the middle of the substrate binding pocket and its D ring is located at the entrance of the pocket to block the entrance and enhance its inhibitory effect. For acarbose, its A ring can be located in a similar position as the A ring of hyperoside but its D ring partially protrudes out of the substrate-binding pocket. For their hydrophilic interactive binding mode (Figure 3b), the A rings of hyperoside and acarbose exert similar binding modes. The B and C rings of hyperoside not only make significant hydrophilic but also hydrophobic interactions with the residues in the middle of the substrate-binding pocket. The B and C rings of acarbose exert only one hydrophilic interaction and they are surrounded by some hydrophobic residues including Tyr 158, Phe 159, and Phe 178; thus, their numerous hydroxyl moieties might not be able to exhibit significant hydrophobic interactions as the B ring of hyperoside. Moreover, the D ring of hyperoside resides at the entrance of the substrate-binding pocket to block the entrance through two essential H-bond interactions but the D ring of acarbose protrudes out of the pocket and exerts its interactions with the residues outside of the pocket. The interaction with the residue in the pocket should contribute more to the antagonistic effect than that with the residue out of the pocket. All modeling results shown above highly indicate that acarbose might have a lesser antagonistic effect than hyperoside due to the difference in the binding mode. However, to generate the crystal structure of the α-glucosidase from Saccharomyces cerevisiae complexed with hyperoside or acarbose is needed to further establish the nature of their respective interaction.

Conclusions
Various solvent extracts of C. pinnatifida and its isolated compounds were investigated with different antioxidant systems and anti-α-glucosidase activity assay. Ethyl acetate extract from C. pinnatifida showed significant antioxidant activities through DPPH, ABTS, superoxide, hydroxyl radical scavenging tests, and FRAP assay. TPC in the extract of ethyl acetate also showed the highest value among all solvent extracts, indicating that the ethyl acetate extract possessed the largest amounts of polyphenols, and it corresponded to the results of antioxidant activities. The acetone extract possessed relatively high ferric reducing antioxidant power among all solvent extracts. In addition, acetone extract also showed the strongest α-glucosidase inhibitory property.
Four isolated compounds from C. pinnatifida were quantified by HPLC and identified as epicatechin, chlorogenic acid, hyperoside, and procyanidin B2. The bioactivity assays demonstrated that all isolates displayed antioxidant activities. In addition, hyperoside showed strong anti-α-glucosidase property. As the result of molecular modeling docking, hyperoside also exhibited the high affinity with α-glucosidase and, thus, displayed potential inhibitory activity against α-glucosidase.
The comparative evaluation of antioxidant and anti-α-glucosidase activities in various solvent extracts and bioactive compounds from the fruits of C. pinnatifida is first mentioned in this study. The results are enough to support the importance of the suitable solvents to extract bioactive compounds. The bioactive extracts and their isolated compounds mentioned above can be used as natural antioxidants in the food industry and dietary supplements against oxidative damage. Furthermore, the acetone extract and hyperoside can also be used as natural α-glucosidase inhibitors.