*2.9. Data Analysis*

All the experiments were carried out in triplicate. Values were presented as the mean values ± standard deviation (SD). Analyses of variance and significance di fferences were analyzed by SPSS Statistics version 17.0 (IBM SPSS, Chicago, USA).

#### **3. Results and Discussion**

#### *3.1. Total Phenolic Content and Total Flavonoid Content*

The choice of the extraction solvent is very important for the recovering of phenolic compounds in plant matrix [25]. Common solvents including ethanol, methanol, acetone, and ethyl acetate have been widely used for the extraction of nature antioxidants in plant matrix [26,27].

As seen from Figure 1, results of TPC and TFC showed that concentrations significantly varied between the di fferent solvents in various parts of raspberry. Regardless of LE, FPE, or SE, 50% methanol/ethanol showed high e fficiency in the extraction of total phenolic and flavonoid compounds, followed by 100% EtOH and 100% methanol. In contrast, water and ethyl acetate were the least e fficient solvents for extracting the phenolics and flavonoids compounds. It is worth noting that 50% methanol was evidently better than 100% methanol for extracting phenolic compounds (*p* < 0.01), which may be due to that the extraction e fficiency depends on the polarity of the compounds present in the samples [28,29]. P. <sup>L</sup>ópez-Perea et al, (2019) also reported that 80% methanol showed higher e fficiency in the extraction of phenolic compounds from wheat bran and barley husk than 100% methanol [30]. When extracted by 50% methanol solution, the highest TPC (63.79 ± 3.11 mg GAE/g DW) and TFC (38.68 ± 2.4 mg RE/g DW) were found in LE, followed by FPE (42.26 ± 3.11 mg GAE/g DW for TPC, 28.60 ± 2.12 mg RE/g DW for TFC), and comparatively low amounts in SE (20.25 ± 1.79 mg GAE/g DW for TPC, 15.03 ± 1.82 mg RE/g DW for TFC). Wanyo et al. (2014) reported that 64% ethanol was the most e fficient phenolic compounds extraction solvent [31]. Djordjevic et al. (2011) showed that high concentrations of phenolic compounds in barley grain (*Hordeum vulgare*) were obtained with 70% ethanol [32]. Our results also confirmed the choice of solvent varieties and concentrations have very significant influences in the extraction e fficiency of phenolic compounds. Therefore, in the next study, in order to investigate the phenolic compositions and their in vitro biological activities of various parts in raspberry, 50% methanol was chosen as the best extraction solvent.

**Figure 1.** The e ffects of extraction solvents on total phenolic contents ( **A**) and total flavonoid contents (**B**) in various parts of raspberry. LE, leaves extracts; SE, seeds extracts; FPE, fruit pulp extracts; EtOH,

ethanol. Di fferent lowercase letters (a–g) mean statistically significant di fferences following di fferent extraction solvents. Different uppercase letters (A–C) mean statistically significant differences following di fferent samples under same extraction solvents.

#### *3.2. HPLC-ESI-HR-qTOF-MS*/*MS Characterization and Quantification of Phenolic Compositions*

The phenolic compositions in di fferent samples were identified by comparing their retention times and MS spectrum data with the authentic standards and reported data [14]. Table 1 and Figure 2 show the corresponding identification results of each peaks in HPLC chromatograms. Based on the MS/MS spectrum data, compound 1 displayed a [M-H]− in *m*/*z* 169.1221, which was easily identified as gallic acid. Chlorogenic acid (compound 2) and epicatechin (compound 3) were identified by their UV spectrum and MS spectral data, respectively. Compounds 4, 5, and 7 showed the parent ions at 579 [C30 H26 O12+H]<sup>+</sup> and its fragments ions at *m*/*z* 291.0503 [C15 H14 O6+H]<sup>+</sup>, which can be tentatively identified as three isomers of procyanidin dimers. Among them, compound 7 can be identified as procyanidin B2 according to MS spectrum data and the retention time of the standard. Compounds 6 and 8 showed the parent ions [M+H]<sup>+</sup> at 867 and its MS/MS fragments ions at 579.1502 [C30 H26 O12+H]<sup>+</sup> and 291.0155 [C15 H14 O6+H]<sup>+</sup>, respectively, which can be likely identified as two isomers of procyanidin trimer. By comparing the MS fragments information and the retention time of the standard, compound 8 can be identified as procyanidin C3. Ellagic acid pentoside was easily identified by the parent ion [M+H]<sup>+</sup> at *m*/*z* 435 and its ion fragment at *m*/*z* 303.0142 [C14 H6O8+H]<sup>+</sup>. Compound 10 was identified as rutin by the parent ion [M+H]<sup>+</sup> at *m*/*z* 611 and its ions fragments *m*/*z* at 303.0563 [M-glc+H]<sup>+</sup> and 163.1221 [M-C15 H10 O7] +. Compound 11 was kaempferol-galactoside-glucoside by its ions [M+H]<sup>+</sup> at *m*/*z* 611 and two fragments ions at *m*/*z* 449.1338 [M−glc+H]<sup>+</sup> and 287.0716 [C15 H10 O6+H]<sup>+</sup>. Compound 13 with [C14 H6O8+H]<sup>+</sup> ion at *m*/*z* 303 was easily identified as ellagic acid. Compounds 14 and 15 with [M+H]<sup>+</sup> ions at *m*/*z* 465 giving MS/MS fragments ions *m*/*z* at 303 [C15 H10 O7+H]<sup>+</sup> and 161 [M-C15 H10 O7+H]<sup>+</sup> were very likely to be two isomers of quercetin glucosides. By comparing the retention time of two standard compounds, they were identified as quercetin-3-O-galactoside and quercetin-3-O-glucoside, respectively. Compound 16 with [M+H]<sup>+</sup> ion *m*/*z* at 435 giving fragment ion at 303.0500 [C15 H10 O7+H]<sup>+</sup> was identified as avicularin. Kaempferol-7- *O*-glucuronide (Compound 17) was easily identified by the [M+H]<sup>+</sup> ion *m*/*z* at 463 and its MS/MS fragment ion at 287.0546 [C15 H10 O6+H]<sup>+</sup>. Quercetin-7- *O*-glucuronide (Compound 18) was identified by the ion [M+H]<sup>+</sup> at *m*/*z* 479 and its fragment ion at 303.0506 [C15 H10 O7+H]<sup>+</sup>. Compound 19 was easily identified as kaempferol-3- *O*-glucuronide by the [M+H]<sup>+</sup> ion at *m*/*z* 463 and its ion fragment at [C15 H10 O7+H]<sup>+</sup>.

The quantification results of fifteen phenolic compounds are shown in Table 2. The LE possessed the widest range of phenolic compositions, but also with the highest contents of individual phenolics. However, SE included the narrowest range of investigated compounds and the lowest contents of individual phenolics. Regardless of LE, FPE, or SE, gallic acid, ellagic acid, and procyanidin C3 were the most abundant of phenolic compounds. Meanwhile, the contents of gallic acid and ellagic acid in LE reached up to 539.42 ± 2.09 μg/g DW and 527.26 ± 3.27 μg/g DW. Their contents in FPE were 339.45 ± 2.17 μg/g DW and 95.42 ± 0.53 μg/g DW, respectively, which were significantly lower than those in LE. The contents of procyanidin B2 (10.72 ± 0.07 μg/g DW) and procyanidin C3 (252.37 ± 0.05 μg/g DW) in FPE were evidently higher than those in LE or SE. Particularly, some flavonols compounds (quercetin-3-glucoside, kaempferol-7- *O*-glucuronide, quercetin-7- *O*-glucuronide and kaempferol-3- *O*-glucuronide) except for rutin and avicularin were only found in LE and SPE of raspberry. Meanwhile, it can be found that there also existed high contents of some invididual phenolic compounds in SE, such as gallic acid (127.15 ± 3.21 μg/g DW), procyanidin C3 (29.12 ± 0.11 μg/g DW) and ellagic acid (48.32 ± 0.23 μg/g DW), which showed that SE may be used as a good food ingredient enriched in phenolic compounds. Qin et al. (2018) have confirmed that high levels of gallic acid and ellagic acid existing in the form of bound phenolics were found in raspberry fruit and seed extracts, which was consistent with the results of our study [13].

**Figure 2.** HPLC chromatograms (280 nm) of phenolic standards mixtures ( **A**) and various parts extracts in raspberry (**B**). Peaks identification and their MS data are shown in Table 1. PSM, phenolic standard mixtures; 1, Gallic acid; 3, Epicatechin; 7, Procyanidin B2; 8, Procyanidin C3; 9, Ellagic acid pentoside; 10, Rutin; 13, Ellagic acid; 15, Quercetin 3-O-glucoside; 16, Avicularin; 17, Kaempferol-7-O-glucuronide; 18, Quercetin-7-O-glucuronide; 19, Kaempferol-3-O-glucuronide. LE, leaves extracts; SE, seeds extracts; FPE, fruit pulp extracts.




**Table 2.** Contents of the main individual phenolics of various parts in raspberry.

Different lowercase letters (a–c) mean statistically significant differences following different samples (*p* < 0.05). N.D., not detected; LE, leaves extracts; FPE, fruit pulp extracts; SE, seed extracts. \* Procyanidin dimer and # procyanidin trimer were quantified by procyanidin B2 and procyanidin C3, respectively.

#### *3.3. Antioxidant Activities*

In order to fully reflect the antioxidant capacity of the samples extracts, four well-known chemical test methods including DPPH, ABTS<sup>+</sup>, and OH− free radical scavenging activities and ferric reducing antioxidant activity (FRAP) were used to perform the antioxidant activity assays.

It was found that LE exhibited the strongest antioxidant activity, followed by FPE, and SE performed the weakest antioxidant activity. All of the samples' extracts showed the antioxidant activities in a concentration-dependent manner (Figure 3A–D). The insets of Figure 3A–C show the corresponding IC50 values of the samples/controls. Notably, the lower the IC50 values indicated, the stronger the antioxidant activity. For the DPPH assay, the IC50 value of LE (5.60 ± 0.31 μg/mL) was lower than that of Vc (16.80 ± 1.31 μg/mL) and Trolox (39.90 ± 0.67 μg/mL), about one-third of that of FPE (18.71 ± 1.35 μg/mL), and one-sixth of that of SE (32.33 ± 1.42 μg/mL). LE also exhibited the strongest ABTS+ free radical scavenging activity. The IC50 value of LE (3.70 ± 0.17 μg/mL) was lower than that of Trolox (41.50 ± 1.97 μg/mL), FPE (29.43 ± 1.83 μg/mL), and SE (33.15 ± 2.19 μg/mL). Meanwhile, three various parts' extracts in raspberry (LE, FPE and SE) also exhibited the strong OH− free radical scavenging activity, and their corresponding IC50 values were 3.01 ± 0.13 μg/mL, 6.40 ± 0.27 μg/mL, and 8.61 ± 0.52 μg/mL, respectively. The IC50 value of LE was also lower than that of Vc (19.83 ± 0.37 μg/mL) and Trolox (33.91 ± 1.82 μg/mL). The insets of Figure 3A–C showed the results of FRAP assay. As is well known, higher FRAP values represent stronger anti-oxidant activity. The FRAP value of LE (198.32 ± 21.72 mM/g DW) was also higher than that of FPE (100.81 ± 12.31 mM/g DW) and SE (21.92 ± 3.72 mM/g DW).

The correlation co-efficient analysis results between the phenolic contents at different concentrations and antioxidant activities of the three extracts clearly verified that there was a good correlation between these two parameters (DPPH vs. TPC, *r* = −0.807, *p* < 0.01; ABTS vs. TPC, *r* = −0.875, *p* < 0.01; OH− vs. TPC, *r* = −0.792, *p* < 0.01), suggesting that the phenolic compounds significantly contributed to the antioxidant activities of three samples extracts. Qin et al. (2018) reported that the antioxidant activities of raspberry fruits and seeds extracts were strongly correlated with the released phenolic contents during in vitro digestion [13]. Wang et al. (2019) also confirmed that the released bound phenolics of raspberry leaves and seeds treated with different methods were responsible for their antioxidant activities [14]. In the present study, gallic acid, ellagic acid, and procyanidin C3 were the major phenolic compounds in different parts of raspberry. Many researches have verified that these three phenolics

possess strong antioxidant activity [33]. Malinda et al. (2017) reported that gallic acid and ellagic acid showed strong DPPH radical scavenging activity with IC50 values of 2.24 μg/mL and 4.80 μg/mL, respectively [34]. Bialonska et al. (2009) have also verified that ellagic acid displayed no significant differences in the antioxidant activity with Vc by in vivo testing [35]. Because LE possessed higher contents of these main phenolic compounds (especially for gallic acid and ellagic acid) than FPE and SE, thus, LE showed the strongest antioxidant capacity, followed by FPE. The results of the above studies further revealed that the phenolic compounds of various parts in raspberry may contribute evidently to their antioxidant activities.

**Figure 3.** The antioxidant activities of various parts' extracts of raspberry and the positive controls (Vc and Trolox): DPPH ( **A**), ABTS+ (**B**), and OH− radical scavenging activity ( **C**) and Ferric reducing antioxidant power (FRAP) ( **D**). The insets of Figure 3A–D represent the corresponding IC50 values of the samples/controls. LE, leaves extracts; FPE, fruit pulp extracts; SE, seed extracts. Vc, ascorbic acid. Di fferent lowercase letters (a–e) mean statistically significant di fferences following di fferent samples.

#### *3.4. Type II Diabetes Related Enzymes Inhibitory Activities*

Alpha-glucosidase or α-amylase, two key digestive enzymes in the digestive tract, can break down macromolecular carbohydrates into monosaccharide/glucose. As well known, excess glucose accumulates in the blood instead of being used for energy, which may cause type II diabetes. Many researches have confirmed that phenolic compounds may bind the amino acid residues with the active sites of digestive enzymes into complex formation by hydrogen bonding, and thereby inhibit the catalytic reaction of digestive enzymes on carbohydrates [36]. Therefore, the phenolic fractions in various parts of raspberry on the inhibition of digestive enzymes were used to evaluate their potential hypoglycemic e ffect.

Figure 4 exhibits that the various parts of raspberry extracts tended to be strong inhibitors of type II diabetes-related enzymes. It can be found that the samples' extracts or acarbose all showed the inhibition activity of digestive enzymes in a concentration-dependent manner (Figure 4A,B). Figure 4C,D show the IC50 values of the samples extracts or the main individual phenolic compounds on the inhibition effects on digestive enzymes. Similarly, the lower IC50 values indicated the stronger inhibition activity of digestive enzymes. The α-glucosidase inhibition activity of LE (IC50 = 96.50 ± 7.71 μg/mL) was evidently stronger than that of FPE (IC50 = 265.41 ± 20.7 μg/mL) and SE (IC50 = 218.5 ± 17.53 μg/mL) (*p* < 0.01). However, the α-glucosidase inhibition potency of acarbose (IC50 = 267.47 ± 19.72 μg/mL) was lower than that of LE (*p* < 0.05). FPE also possessed good inhibition activity against α-glucosidase, but there is no statistically significant di fference with acarbose (*p* > 0.05). Figure 4C presents that the IC50 value for α-glucosidase inhibition activity of PC was 93.37 ± 5.79 μg/mL, which was significantly lower than that of GA (IC50 = 590.34 ± 15.71 μg/mL) and EA (IC50 = 976.32 ± 41.72 μg/mL) (*p* < 0.01). For α-amylase inhibition activity assay, LE have the lowest IC50 values (IC50 = 118.42 ± 2.79 μg/mL), followed by FPE (IC50 = 388.27 ± 2.47 μg/mL) and SE (IC50 = 891.12 ± 25.71 μg/mL). Moreover, the IC50 value of acarbose (IC50 = 442.23 ± 19.74 μg/mL) was higher than that of LE (*p* < 0.05), which indicates that LE may be used as a potential good anti-diabetic resource. Among the three main phenolic compounds, PC showed the strongest α-amylase inhibition activity (IC50 = 92.31 ± 3.51 μg/mL), followed by EA (IC50 = 516.73 ± 25.29 μg/mL), and GA (IC50 = 397.37 ± 12.37 μg/mL) (*p* < 0.01).

**Figure 4.** The digestive enzymes inhibitory abilities of various parts extracts in raspberry and acarbose: α-glucosidase inhibitory activity ( **A**) and α-amylase inhibitory activity (**B**). Figure 4C,D represents the corresponding IC50 values of the samples/controls. LE, leaves extracts; FPE, fruit pulp extracts; SE, seed extracts; GA, gallic acid; EA, ellagic acid; PC, procyanidin C3. Di fferent lowercase letters (a–e) mean statistically significant di fferences following di fferent samples.

The correlation analysis results between the TPC and two digestive enzymes inhibition potency clearly verified that there was a good positive correlation between these two parameters (α-glucosidase inhibition potency vs. TPC, *r* = 0.781, *p* < 0.05; α-amylase inhibition potency vs. TPC, *r* = 0.854, *p* < 0.01). Many researches have confirmed that phenolic-rich extracts from leaf-tea, edible fruits, and natural products possess good inhibitory ability on digestive enzymes [23,37]. Wang et al. (2018) have confirmed that procyanidin C3 possessed strong α-glucosidase inhibitory capacity [38]. Zhang et al. (2010) reported that gallic acid, anthocyanins, and rutin in raspberry have strong α-glucosidase

inhibitory capacity, but ellagic acid possessed the weakest inhibitory ability of α-glucosidase, which was consistent with the results of our study [39].

#### *3.5. Molecular Docking Results*

Molecular docking analysis was further done to analyze the digestive enzymes inhibitory mechanisms of the main phenolic compounds including GA, EA, and PC. The results clearly revealed that the structures of phenolic compounds significantly a ffect their inhibitory e ffects on α-glucosidase or α-amylase. Table 3 and Figure 5 show the molecular docking results with regard to interactions between α-glucosidase and several main phenolic molecules/acarbose binding. From Table 3, the molecular docking C-Scores values of those several molecules/acarbose were all ≥ 4, which indicates credible docking results. Figure 5 shows that GA interacted with the active sites of α-glucosidase and formed six H-bonds (yellow dotted line) with five amino acid residues (Asp 69, Arg 213, Asp 215, Asp 352, and His 351). The distances of H-bonds ranged from 1.896 Å to 2.600 Å. EA formed six H-bonds (the shortest distance was 1.700 Å and the longest distance was 2.433 Å) with five amino acid residues (Asp 215, Asp 352, Arg 213, Glu 411, and His 351). PC formed thirteen H-bonds within 4 Å (the distances ranged from 1.776 Å to 2.665 Å) with eleven amino acid residues (Asp 69, Asp 215, Asp 242, Glu 411, Gln 279, Gln 353, Leu 313, Lys 156, Tyr 158, The 314 and Pro 312). However, it was found that acarbose formed sixteen H-bonds within 4 Å (distances ranged from 1.776 Å to 2.732 Å) with ten amino acid residues (Asp 69, Asp 215, Asp 352, Arg 442, Glu 273, Glu 411, Gln 279, Tyr 158, Lys 156, and His 280). Some amino acid residues (Asp 69, Asp 215, and Asp 352) of α-glucosidase at least interacted with the above three investigated molecules, suggesting that these amino acid residues may play important roles in exerting the catalytic reaction of α-glucosidase.


**Table 3.** The analysis results of the main phenolic molecules and acarbose dockings into α-glucosidase or α-amylase ligands.

> Notes: GA, gallic acid; EA, ellagic acid; PC, procyanidin C3.

Table 3 and Figure 6 show the molecular docking results of α-amylase with the investigated phenolic molecules. The C-Scores of three main phenolic molecules and acarbose were all ≥ 4. Figure 6 shows that six H-bonds (yellow dotted line) were formed between the active site of α-amylase and gallic acid. The five amino acid residues with the active site were Asp 197, Arg 195, Glu 233, His 299, and His 305, respectively. The average distance of six H-bonds was 2.115 Å. Ellagic acid formed H-bonds with four amino acid residues with the active site, namely Asp 197, Asp 300, Arg 195, and His 299. The distances of H-bonds ranged from 1.937 Å to 2.735 Å. However, procyanidin C3 formed nine H-bonds with the active sites of six amino acid residues (Asp 197, Glu 233, Gly 306, Lys 200, Tyr 155, and His 305). The shortest distance was 1.864 Å and the longest distance was 2.724 Å. It was found that acarbose formed eleven H-bonds (the distances ranged from 1.776 Å to 2.732 Å) with seven amino acid residues, namely, Asp 300, Gln 63, Glu 240, Gly 306, Tyr 151, Lys 200, and His 305.

**Figure 5.** Molecular docking of the main three phenolic compounds and acarbose with the α-glucosidase. The 3D docking structures of three main phenolic compounds and acarbose were inserted into the hydrophobic cavity of the α-glucosidase (blue): gallic acid (**A1**); ellagic acid (**B1**); procyanidin C3 (**C1**); acarbose (**D1**). The conformation of active molecules interactions with amino acid residues in the active site of α-glucosidase: gallic acid (A2), ellagic acid (B2), procyanidin C3 (C2), and acarbose (D2) with residues in the active sites of the α-glucosidase, respectively. The dashed line stands for hydrogen bonds.

**Figure 6.** Molecular docking of the main three phenolic compounds and acarbose with α-amylase. The 3D docking structures of three phenolic compounds and acarbose were inserted into the hydrophobic cavity of α-amylase (blue): gallic acid (**A1**); ellagic acid (**B1**); procyanidin C3 (**C1**); acarbose (**D1**). The conformations of active molecules interactions with amino acid residues in the active site of α-amylase: gallic acid (**A2**), ellagic acid (**B2**), procyanidin C3 (**C2**), and acarbose (**D2**) with residues in the active sites of the α-amylase, respectively. The dashed line stands for hydrogen bonds.

It can be found that the numbers and distances of the H-bonds play important roles in exerting the catalytic reaction of the complex of digestive enzymes and ligands, and thereby cause the differences in inhibitory activity of digestive enzymes. Regardless of acarbose docked with α-glucosidase or α-amylase, the higher numbers of H-bonds and amino acid residues with active site were formed in the complex of digestive enzymes and acarbose. As a result, acarbose showed very good inhibitory effect of α-glucosidase and α-amylase. The numbers of H-bonds and amino acid binding sites formed by the two phenolic molecules (GA and EA) docked with α-glucosidase were equal, but there existed significant differences in inhibitory capacities of α-glucosidase. It may be due to that different interaction sites (amino acid residues) existed in between these molecules and α-glucosidase. Both of GA and EA all interacted with the amino acid residues His 351, Asp 215, Asp 352, and Arg 213 of α-glucosidase. EA also interacted with the amino acid residue Glu 411 of α-glucosidase. Some researchers have confirmed that some active sites (Glu 411) of α-glucosidase may inhibit the catalytic activity of this enzyme [37]. Consequently, ellagic acid showed the weakest α-glucosidase inhibitory effect. At least three investigated molecules formed H-bonds with the amino acid residues of Asp 69, Asp 215, and Asp 352, which may exert its α-glucosidase inhibitory effect. Hua et al. (2018) have also reported that Asp 69, Asp 215, and Arg 442 were the important residues involved in H-bond formation during the binding with α-glucosidase [37]. Zhang et al. (2018) also reported that the binding active sites (Asp 215 and Asp 352) in between the ligands and α-glucosidase played important roles in exert its α-glucosidase inhibitory effect [23]. Similarly, the order of amino acid residues numbers formed by four molecules (GA, EA, PC, and Acarbose) docked with α-amylase was: Acarbose (7) = PC (7) > GA (5) > EA (4). The order of H-bonds number was: Acarbose (11) > PC (9) > GA (6) > EA (5). Consequently, the order of the docking T-Score was: Acarbose > PC > GA > EA, which was consistent with the results of α-amylase inhibition activity. Moreover, at least three investigated molecules interacted with the amino acid residues Asp 197 and His 305 of α-amylase, indicating that these two amino acid residues may play critical roles in the catalytic reaction of α-amylase. Many reports have confirmed that the amino acid residues Asp 197 and His 305 played critical roles in the catalytic reaction of α-amylase [40,41]. The mechanisms of the digestive enzymes inhibitory activities of these compounds possibly involve the binding of compounds with the catalytic sites of digestive enzymes [37]. The results demonstrated that the hydrogen bonds and the binding residues with active sites have important effects on these digestive enzymes activities.
