Main Bioactive Components and Their Biological Activities from Natural and Processed Rhizomes of Polygonum sibiricum

Polygonatum sibiricum (Asparagaceae) is often used as an herbal drug in the traditional medicine of Southeast Asia. Its rhizome, called “Huang Jing”, is used in traditional Chinese medicine as an immune system stimulant, hypolipidemic agent, anti-aging agent, anti-fatigue agent, and cardiovascular protectant. We investigated the antioxidant, anti-acetylcholinesterase (AChE), anti-inflammatory, and anti-α-glucosidase effects of various solvent extracts and major bioactive components of Polygonatum sibiricum (PS) and processed Polygonatum sibiricum (PPS). Dichloromethane extract of PS showed stronger antioxidant effects by DPPH, ABTS, and FRAP assays, and EtOAc extract displayed relatively high antioxidant activity by a superoxide radical scavenging test. Moreover, acetone, EtOAc, and dichloromethane extracts displayed a significant anti-α-glucosidase effect. EtOH and CH2Cl2 extracts showed effective AChE inhibitory activity. In addition, dichloromethane extract showed the best inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) accumulation in RAW264.7 macrophages. HPLC analysis was used to investigate and compare the content of major active components of various solvent extracts of PS and PPS. Rutin showed the most effective scavenging of DPPH and ABTS free radicals, while scopoletin and isoquercetin displayed the strongest anti-α-glucosidase and anti-AChE effect, respectively. Rutin showed the best inhibition against LPS-induced NO production and also inhibited inducible nitric oxide synthase (iNOS) expression in Western blot. The molecular docking of AChE and iNOS revealed that active components could have a better antagonistic effect than positive controls (common inhibitors). This study shows that the active extracts and components of Polygonatum sibiricum have the potential to be further developed as a natural anti-AChE, anti-α-glucosidase, antioxidant and anti-inflammatory agent.


Preparation of P. sibiricum Extracts
The rhizomes of P. sibiricum were purchased from Nantou, Taiwan, in January 2022, and the processed rhizomes of P. sibiricum were supplied from Changsheng Chinese medicine shop, Taipei city, Taiwan, in August 2021 and identified by Prof. J.-J. Chen. The voucher specimens were stored in the Department of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan. First, the rhizomes of P. sibiricum (15 g) were soaked in 150 mL of different solvents (water, MeOH, EtOH, acetone, EtOAc, and dichloromethane) and shaken on an orbital shaker for 24 h at room temperature. Subsequently, the extracts were filtered through filter paper (Whatman No. 1) and concentrated using a rotary evaporator at 38 °C. Finally, all extracts were stored at −20 °C until further experiments.

Preparation of P. sibiricum Extracts
The rhizomes of P. sibiricum were purchased from Nantou, Taiwan, in January 2022, and the processed rhizomes of P. sibiricum were supplied from Changsheng Chinese medicine shop, Taipei city, Taiwan, in August 2021 and identified by Prof. J.-J. Chen. The voucher specimens were stored in the Department of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan. First, the rhizomes of P. sibiricum (15 g) were soaked in 150 mL of different solvents (water, MeOH, EtOH, acetone, EtOAc, and dichloromethane) and shaken on an orbital shaker for 24 h at room temperature. Subsequently, the extracts were filtered through filter paper (Whatman No. 1) and concentrated using a rotary evaporator at 38 • C. Finally, all extracts were stored at −20 • C until further experiments.

Measurement of Total Phenolic Content (TPC)
The TPC of different solvent extracts was measured using a previously reported method [30].

Measurement of Total Flavonoid Content (TFC)
The TFC of different solvent extracts was determined using a previously reported method [31].

DPPH Radical Scavenging Assay
This assay was determined using the procedure previously reported [32].

ABTS Radical Scavenging Assay
This assay was determined using the procedure previously reported [33].

Superoxide Radical Scavenging Assay
This assay was determined using the method previously reported [32].

Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was determined using the method previously reported [34].

α-Glucosidase Inhibitory Assay
This assay was performed according to the previous method [35].

Acetylcholinesterase Inhibitory Assay
The AChE inhibitory assay was performed according to the previous method, with slight modifications [36]. First, 1 mL of DTNB (15 mM) solution, 1 mL of AChI (15 mM) solution, 1 mL of AChE (0.2 U/mL), and various concentrations of PS (50-400 µg/mL) were prepared using sodium phosphate buffer (0.1 M, pH 8.0). The reaction was started by adding 140 µL PBS, 10 µL DTNB solution, 15 µL AChE solution and 20 µL sample. The reaction mixture was incubated at 20 • C in the dark for 10 min, and 10 µL of AChI solution was added. Finally, the reaction mixture was incubated at 20 • C for 10 min, and the absorbance was measured at 405 nm.

Nitric Oxide Inhibitory Assay
The NO inhibitory assay was performed based on the reference method [37].

MTT Assay
The MTT assay was carried out according to the previous method [37].

Western Blot Analysis
The Western blot analysis was performed using the reference method with slight modifications [37]. RAW264.7 cells were seeded in 6 cm dishes for 24 h. The cells were treated with sample (12.5, 25, and 50 µM) and LPS (100 ng/mL). After treatment with drugs, cells were washed with cold PBS, and proteins were collected with lysis buffer containing protein inhibitors. After quantification, equal amounts of protein samples were separated using 5-8% SDS-polyacrylamide gels. The proteins were transferred to the membrane by electrophoresis. The membranes were soaked in 2% BSA blocking buffer for 2 h and then washed 2-3 times with TBST. Next, the membranes were visualized with primary antibody (iNOS, TNF-α, and IL-6) and soaked for 1 day. The next day, membranes were washed again with TBST 2-3 times and then soaked with secondary antibody for 2 h. Finally, immuno-reactivity was detected with ECL reagents, exposed with a luminometer photometer, and quantified with Image J.

Molecular Modeling Docking Study
The in silico evaluation was performed with AutoDock Vina (ADT ver. 4.0.1) software [38]. Crystal structures of AChE were retrieved from the Protein Data Bank (PDB: 1C2B), and hydrogen atoms were added to prepare the docked receptors. The 3D structures of the ligands were constructed in the Chem3D program. The addition of hydrogen, the Gasteiger charge measurement of the protein atoms and the selection of the flexible twist of the ligand were performed by AutodockTools (ADT ver. 1.5.6). The grid dimensions were designed as 15 Å × 15 Å × 15 Å for isoquercetin, 14 Å × 14 Å × 14 Å for scopoletin, 10 Å × 10 Å × 10 Å for ru-tin, and 20 Å × 20 Å × 20 Å for chlorogenic acid. Finally, the crystal structure of iNOS was retrieved from the Protein Data Bank (PDB: 1M9T). The grid dimensions were designed as 17Å × 17Å × 17Å for rutin, 15Å × 15Å × 16Å for isoquercetin, 20Å × 16Å × 14Å for hyperoside, and 16Å × 16Å × 16Å for quercetin. The binding affinity energy was provided as a docking fraction and measured in kcal/mol. The best interactions were only considered to be the highest fraction of the gestalt. The visualization of the best docking interactions was analyzed in Biovia Discovery Studio Client 2021 [39].

Statistical Analysis
Statistical analysis was carried out using the t-test, and all assays were performed at least three times and expressed as mean ± standard error of the mean (SEM). Less than 0.05 was considered statisti-cally significant.

Measurement of TPC, TFC and Yield in Various Solvent Extracts
We studied the TPC, TFC and yields in different solvent extracts of Polygonatum sibiricum (PS) and processed Polygonatum sibiricum (PPS). Table 1 displays TPC, TFC, and extraction yield of CH 2 Cl 2 , EtOAc, acetone, MeOH, EtOH, and water extracts from PS and PPS. The yields of various solvent extracts were ranged from 13.49 ± 1.53% (methanol extract) to 0.35 ± 0.12% (dichloromethane extract) of PS, and 15.90 ± 2.64% (water extract) to 1.25 ± 0.03% (dichloromethane extract) of PPS. The dichloromethane extract of PS and ethyl acetate extract of PPS showed the highest TPC with 77.50 ± 7.34 and 60.47 ± 1.91 mg/g, respectively. The highest TFC (86.02 ± 1.54 and 98.30 ± 0.47 mg/g) were found in the ethyl acetate extracts of PS and PPS, respectively, among all solvent extracts (Table 1). a TPC was displayed in mg of gallic acid equivalents (GAE) per gram of fresh extract. b TFC was displayed in mg of quercetin equivalents (QCE) per gram of fresh extract. c Yield was calculated as % yield = (weight of fresh extract/initial weight of dry sample) × 100. Values are expressed as means ± SD (n = 3). *** p < 0.001, ** p < 0.01, and * p < 0.05 compared with the control. PS means Polygonatum sibiricum. PPS means processed Polygonatum sibiricum.

DPPH Free-Radical Scavenging Effect of Various Solvent Extracts
The DPPH radical scavenging ability of different solvent extracts is displayed in Table 2. BHT was used as a positive control. From the results, the dichloromethane (SC 50 = 236.14 ± 3.89 µg/mL) and acetone extracts (SC 50 = 278.31 ± 3.26 µg/mL) of PS showed relatively strong antioxidant effects by DPPH radical scavenging assay among all extracts.

Ferric Reducing Antioxidant Power (FRAP) Effect of Various Solvent Extracts
The FRAP activities of all extracts are shown in Table 2, and BHT was used as a positive control. The FRAP assay is expressed as millimoles (mM) of Trolox equivalents (TE) per gram of extract. The dichloromethane extract (667.08 ± 18.56 mM TE/g), ethyl acetate extract (651.03 ± 20.56 mM TE/g) and acetone extract (515.84 ± 24.86 mM TE/g) of PS and ethyl acetate extract (604.38 ± 3.73 mM TE/g) of PPS showed relatively high antioxidant capacity. Based on the aforementioned antioxidant data (DPPH and FRAP assays), the dichloromethane extracts of PS showed relatively high antioxidant effects among all the extracts. As for the ethyl acetate extract of PS, it showed a higher antioxidant effect by the superoxide radical scavenging test.
The dichloromethane, ethyl acetate and acetone extracts of PS were more effective than the positive control, acarbose (IC 50 = 379.07 ± 4.23 µg/mL). These results suggest that the low-polarity solvent extracts of PS had a higher α-glucosidase inhibitory effect (Table 3).

Acetylcholinesterase (AChE) Inhibitory Effect of Various Solvent Extracts
Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by a decline in cognitive and memory functions. Acetylcholinesterase (AChE) inhibitors block the AChE enzyme, thus revitalizing cholinergic action to improve memory and cognitive function. The AChE inhibitory effect of each extract is presented in Table 3, and the AChE inhibitor, chlorogenic acid, is used as a positive control [40]. From our test results, the dichloromethane extract (IC 50 = 60.90 ± 6.18 µg/mL) of PS and the ethyl acetate extract (IC 50 = 32.60 ± 5.27 µg/mL) and dichloromethane extract (IC 50 = 56.27 ± 7.11 µg/mL) of PPS showed the most effective AChE-inhibitory effect among all extracts.

Nitric Oxide Inhibitory (NO) Effect of Various Solvent Extracts
The NO inhibitory assay of each extract is displayed in Table 4 and Figure 2. Quercetin is used as a positive control. From our test results, the dichloromethane extract (IC 50 = 18.84 ± 1.80 µg/mL) of PS and dichloromethane extract (IC 50 = 27.48 ± 6.99 µg/mL) of PPS show the greatest NO inhibitory effect among all solvent extracts. 7.52 ± 0.25 * a The IC 50 value was defined as half-maximal inhibitory concentration, and was expressed as mean ± SD (n = 3). b Quercetin was applied as positive control. ** p < 0.01, and * p < 0.05 compared with the control. PS means Polygonatum sibiricum. PPS means processed Polygonatum sibiricum.

MTT Assay of Various Solvent Extracts
The MTT assay is mainly used to detect the cytotoxicity of tested samples. From the MTT assay (Figure 2), the cell survival rate of various extracts of PS and PPS is higher than 80%, and this suggests that the inhibitory activity of these extracts against LPS-induced NO production does not result from their cytotoxicities.

Quantitation of Bioactive Components in Various Solvent Extracts
Figures S1-S18 showed the quantification of bioactive compounds in various solvent extracts of Polygonatum sibiricum by reversed-phase HPLC analysis and the 1 H-NMR spectra of bioactive compounds. Table S1 displayed retention time, limit of detection (LOD), limit of quantification (LOQ), and regression analysis for five components of P. sibiricum in reversed phase HPLC. The amounts of the five bioactive compounds in different solvent extracts are illustrated in Table 5. The content of five bioactive compounds in different solvent extracts of PS ranged from the highest value of 17.25 ± 1.68 mg/g (dichloromethane extract) to the lowest value of 6.77 ± 0.50 mg/g (water extract), in order of dichloromethane > ethanol > ethyl acetate > methanol > acetone > water extract. In addition, the sum of the five bioactive compounds in different solvent extracts of PPS ranged from a maximum value of 27.49 ± 2.31 mg/g (dichloromethane extract) to a minimum value of 17.19 ± 2.66 mg/g (water extract), in order of dichloromethane > acetone > ethanol > methanol > ethyl acetate > water extract. Both PS and PPS showed the presence of 5-HMF in different solvent extracts, but the content of PPS was much higher than that of PS. However, scopoletin was only present in PS and not in PPS ( Table 5). The five major bioactive compounds are 5-hydroxymethylfurfural (5-HMF) (1), scopoletin (2), isoquercetin (3), hyperoside (4), and rutin (5) (Figure 3). the dichloromethane extract (IC50 = 60.90 ± 6.18 μg/mL) of PS and the ethyl acetate extract (IC50 = 32.60 ± 5.27 μg/mL) and dichloromethane extract (IC50 = 56.27 ± 7.11 μg/mL) of PPS showed the most effective AChE-inhibitory effect among all extracts.

Nitric Oxide Inhibitory (NO) Effect of Various Solvent Extracts
The NO inhibitory assay of each extract is displayed in Table 4 and Figure 2. Quercetin is used as a positive control. From our test results, the dichloromethane extract (IC50 = 18.84 ± 1.80 μg/mL) of PS and dichloromethane extract (IC50 = 27.48 ± 6.99 μg/mL) of PPS show the greatest NO inhibitory effect among all solvent extracts.

MTT Assay of Isolated Components
As shown in Figure 4, the cell survival rate of all isolated compounds at 100 μM was higher than 80%, and this suggested that the inhibitory activity of these isolated compounds against LPS-induced NO production does not result from their cytotoxicities.

MTT Assay of Isolated Components
As shown in Figure 4, the cell survival rate of all isolated compounds at 100 µM was higher than 80%, and this suggested that the inhibitory activity of these isolated compounds against LPS-induced NO production does not result from their cytotoxicities.

Western Blot Analysis of Isolated Components
Rutin (5), isoquercetin (3), and hyperoside (4), with their better inhibitory activity against NO production, were selected for further analysis of their inhibitory effect on iNOS. As shown in Figure 5, compounds 3, 4, and 5 significantly inhibited iNOS production in a concentration-dependent manner. Therefore, the reason for their inhibition of NO production can be verified.  In addition, compounds 3, 4, and 5 were further tested for their activity in inhibiting the production of TNF-α and IL-6. As displayed in Figure 6, compounds 3, 4, and 5 also significantly inhibited the production of TNF-α and IL-6 in a concentration-dependent manner. This verifies that these compounds possess potent anti-inflammatory activity.

Molecular Docking Study
To further investigate the interaction between biologically active compounds and acetylcholinesterase and try to explain how these compounds exert their antagonistic effects, docking models of compounds were generated using the Discovery Studio 2021 (Accelrys, San Diego, CA, USA) modeling program. The crystal structure (PDB: 1C2B) of acetylcholinesterase from Electrophorus electricus was also used in this study [41].
The molecular docking simulations were performed along with the binding affinity calculations for PDB: 1C2B (acetylcholinesterase) and active compounds. The interactions between active compounds and PDB: 1C2B were displayed in the best-docked poses for the calculations. These results demonstrate the high accuracy of the existing simulation system, thereby supporting further calculations. The lowest binding energy for each ligand was considered the optimal conformation, and the binding affinities are shown in Table 9. In this study, chlorogenic acid was used as a positive control. The binding energies of isoquercetin (3) (−7.5 kcal/mol), scopoletin (2) (−7.0 kcal/mol), and rutin (5) (−6.8 kcal/mol) were lower than those of chlorogenic acid (−5.3 kcal/mol), suggesting that these compounds can have better docking ability with the crystal structure of PDB: 1C2B.

Molecular Docking Study
To further investigate the interaction between biologically active compounds and acetylcholinesterase and try to explain how these compounds exert their antagonistic effects, Figure 6. Western blot analysis of TNF-α and IL-6 proteins of rutin (2), isoquercetin (3), and hyperoside (5). Quantification data of TNF-α/β-actin and IL-6/β-actin are expressed as mean ± SEM; Quercetin was applied as positive control. * p < 0.05 vs. control group, # p < 0.05 vs. LPS group. Table 9. Binding energies of active compounds and chlorogenic acid with AChE calculated in silico.

Compounds
Affinity (kcal/mol) a Chlorogenic acid was used as a positive control.
Interactions of isoquercetin (3) with the active sites of E. electricus acetylcholinesterase (AChE) are shown in Figure 7. Isoquercetin is bound to GLU 202, TYR 124, ASP 74, ASN 87 and GLY 120 via conventional hydrogen bonding, with unfavorable donor-donor interactions found on ARG 296. There are other interactions with isoquercetin, such as π-cation, π-lone pair and π-π T-shaped interactions, which can mainly form stable complexes of isoquercetin and protein.  Molecular docking of scoloptein (2) and E. electricus acetylcholinesterase is shown in Figure 8. Scoloptein binds to GLY 448 via carbon-hydrogen bonding and to TRP 86 via conventional hydrogen bonding. In addition, there is a π-π stacked interaction with scoloptein to make a stable complex of scoloptein and AChE.   Interactions of rutin (5) with the active sites of E. electricus AChE are shown in Figure S19. Rutin (5) binds to TYR 124 and GLN 71 via conventional hydrogen bonding and to TRP 286, PRO 88, and SER 125 via carbon-hydrogen bonding. Unfavorable donor-donor and unfavorable acceptor-acceptor interactions were found on PHE 295, TYR 341, and TYR 337. There are other interactions with rutin, such as π-π T-shaped, alkyl, and π-π stacked interactions to make a stable complex of rutin and AChE.
From the above results, it can be concluded that the binding energies of compounds 2, 3, and 5 are better than that of chlorogenic acid, as shown in Table 9. Likewise, the anti-AChE effects of compounds 2, 3, and 5 were confirmed to be superior to that of chlorogenic acid (Figure 9).
According to the results of the NO production inhibition test (Table 8) and the Western blotting results of a related protein, inducible nitric oxide synthase (iNOS) (Figure 6), the three active compounds, rutin (5), isoquercetin (3), and hyperoside (4), all have antiinflammatory potential. Therefore, these three compounds were used in a molecular docking model to see their binding abilities to iNOS. The 3D structure (PDB: 1M9T) of iNOS used as a docking model is from Mus musculus, and the active site consists of four pockets, where the substrate-binding S pocket contains heme [42]. According to the results of the NO production inhibition test (Table 8) and the Western blotting results of a related protein, inducible nitric oxide synthase (iNOS) (Figure 6), the three active compounds, rutin (5), isoquercetin (3), and hyperoside (4), all have antiinflammatory potential. Therefore, these three compounds were used in a molecular docking model to see their binding abilities to iNOS. The 3D structure (PDB: 1M9T) of iNOS used as a docking model is from Mus musculus, and the active site consists of four pockets, where the substrate-binding S pocket contains heme [42].
Interactions of rutin (5) with the active sites of Mus musculus iNOS are shown in Figure  10. Rutin mainly forms a conventional hydrogen bond with GLY 365, and even with GLN 257, ARG 382 and GLU 371, and it also forms π-alkyl interactions with VAL 346 and PRO 344. Rutin forms multiple interactions with HEM 901, such as π-cations, π-π stacked, π-donor hydrogen bonds, carbon-hydrogen bonds, and conventional hydrogen bonds, which increases the parallelism between rutin and HEM 901 and increases the affinity of rutin and iNOS.
The next compound is isoquercetin (3), which mainly forms conventional hydrogen bonds with TRP 366 and even with GLU 371, ASP 376 and ARG 382, and also forms πalkyl interactions with PRO 344 and VAL 346. Carbon hydrogen bonds are observed between isoquercetin and GLN 371 and GLN 257. Most importantly, isoquercetin forms ππ-carbon and π-π stacked interactions with HEM 901 to remain parallel to HEM 901 and increase the affinity of isoquercetin and iNOS. (Figure 11).
The last is hyperoside (4), which mainly forms conventional hydrogen bonds with TRP 366 as well as with GLU 371, ASP 376, GLN 257, SER 256 and ARG 382. Hyperoside also forms π-alkyl interactions with PRO 344 and VAL 346. In addition, hyperoside forms π-cation, π-π stacked, and conventional hydrogen bond interactions with HEM 901 to increase their affinity ( Figure 12). Interactions of rutin (5) with the active sites of Mus musculus iNOS are shown in Figure 10. Rutin mainly forms a conventional hydrogen bond with GLY 365, and even with GLN 257, ARG 382 and GLU 371, and it also forms π-alkyl interactions with VAL 346 and PRO 344. Rutin forms multiple interactions with HEM 901, such as π-cations, π-π stacked, π-donor hydrogen bonds, carbon-hydrogen bonds, and conventional hydrogen bonds, which increases the parallelism between rutin and HEM 901 and increases the affinity of rutin and iNOS.
The next compound is isoquercetin (3), which mainly forms conventional hydrogen bonds with TRP 366 and even with GLU 371, ASP 376 and ARG 382, and also forms π-alkyl interactions with PRO 344 and VAL 346. Carbon hydrogen bonds are observed between isoquercetin and GLN 371 and GLN 257. Most importantly, isoquercetin forms π-π-carbon and π-π stacked interactions with HEM 901 to remain parallel to HEM 901 and increase the affinity of isoquercetin and iNOS. (Figure 11).
The last is hyperoside (4), which mainly forms conventional hydrogen bonds with TRP 366 as well as with GLU 371, ASP 376, GLN 257, SER 256 and ARG 382. Hyperoside also forms π-alkyl interactions with PRO 344 and VAL 346. In addition, hyperoside forms π-cation, π-π stacked, and conventional hydrogen bond interactions with HEM 901 to increase their affinity ( Figure 12).      According to the binding energy data presented in Table 10, the binding affinity of rutin (5) was significantly higher than compounds 1-4 and the positive control quercetin ( Figure 13). This indicated that rutin (5) has the greatest binding affinity with iNOS, which is consistent with the strongest anti-NO activity of rutin (5) presented in Table 8. Table 10. Binding energies of active compounds and quercetin with iNOS calculated in silico.

Discussion
Various approaches have been devoted to extracting natural products (i.e., herbs, plants and fungi) to replace modern medicines. For many years, the most common extraction methods were boiling or decoction, both of which were considered cost-effective and easy to perform. Researching and finding natural ingredients with health benefits from medicinal plants is a hot trend these days. In recent studies, organic solvents have been used to obtain extracts of natural products, including various metabolites, depending on the polarity and properties of these specific compounds [43]. Other exogenous factors may also affect natural product extraction, including the type of solvent used, the temperature altered during extraction, and the property of the plant material [44]. Changes in solvent polarity can lead to differences in phytochemical composition and biological activity. Therefore, the rhizomes of Polygonum sibiricum were extracted using solvents of different polarities, and the biological activities of the extracts and isolated compounds were evaluated. The results show that by using solvents of different polarities, different extracts and components with different contents can be obtained and show different biological activities.
The ABTS and DPPH assays are mainly used to evaluate the antioxidant effects of natural compounds, which are usually related to the proton radical scavenging or hydrogen donating capacity of the compounds [45]. The superoxide radical scavenging effect is assessed by superoxide anion derived from dissolved oxygen by phenazine methosulphate/nicotinamide adenine dinucleotide (PMS/NADH) coupling reaction, which reduces nitroblue tetrazolium (NBT) [46]. The FRAP assay is evaluated by the antioxidant potential of various extracts and samples by reducing iron (Fe 3+ ) complexes to ferrous (Fe 2+ ) complexes [47]. In our study, dichloromethane and acetone extracts of PS show higher antioxidant effects than other extracts in DPPH and ABTS assays, which correlates with their higher TPC than other extracts.

Discussion
Various approaches have been devoted to extracting natural products (i.e., herbs, plants and fungi) to replace modern medicines. For many years, the most common extraction methods were boiling or decoction, both of which were considered cost-effective and easy to perform. Researching and finding natural ingredients with health benefits from medicinal plants is a hot trend these days. In recent studies, organic solvents have been used to obtain extracts of natural products, including various metabolites, depending on the polarity and properties of these specific compounds [43]. Other exogenous factors may also affect natural product extraction, including the type of solvent used, the temperature altered during extraction, and the property of the plant material [44]. Changes in solvent polarity can lead to differences in phytochemical composition and biological activity. Therefore, the rhizomes of Polygonum sibiricum were extracted using solvents of different polarities, and the biological activities of the extracts and isolated compounds were evaluated. The results show that by using solvents of different polarities, different extracts and components with different contents can be obtained and show different biological activities.
The ABTS and DPPH assays are mainly used to evaluate the antioxidant effects of natural compounds, which are usually related to the proton radical scavenging or hydrogen donating capacity of the compounds [45]. The superoxide radical scavenging effect is assessed by superoxide anion derived from dissolved oxygen by phenazine methosulphate/nicotinamide adenine dinucleotide (PMS/NADH) coupling reaction, which reduces nitroblue tetrazolium (NBT) [46]. The FRAP assay is evaluated by the antioxidant potential of various extracts and samples by reducing iron (Fe 3+ ) complexes to ferrous (Fe 2+ ) complexes [47]. In our study, dichloromethane and acetone extracts of PS show higher antioxidant effects than other extracts in DPPH and ABTS assays, which correlates with their higher TPC than other extracts.
A comparative assessment of the TPC, TFC, and antioxidant effects (DPPH, ABTS, superoxide, and FRAP) of various solvent extracts (CH 2 Cl 2 , EtOAc, acetone, EtOH, MeOH, and water) of PS and PPS was demonstrated for the first time in this study. This may give a direction for the choice of suitable solvents for TFC, TPC, and antioxidant extraction applications. According to the antioxidant assay result, rutin (5) (SC 50 = 5.60 ± 0.34 µM) showed the strongest DPPH radical scavenging effect. Compounds 3, 4 and 5 displayed higher superoxide radical scavenging effects than 1 and 2. In addition, compounds 3 and 4 showed a more effective antioxidant effect than BHT by FRAP assay.
Alzheimer's disease (AD) is a progressive degenerative brain disease in which cognitive and memory functions deteriorate. It is primarily treated by AChE inhibitors that increase the cholinergic effects of the brain. In contrast, chlorogenic acid, a phenolic acid derived from food (fruits and vegetables), has been shown to have neuroprotective properties associated with Alzheimer's disease [24,49]. From the previous research data, isoquercetin (3) has attracted the attention of many researchers because of its potential to improve brain memory function through different mechanisms, so it is believed to have a certain role in delaying the progression of AD memory loss [50]. In our study, isoquercetin (3) (IC 50 = 23.13 ± 3.15 µM), scopoletin (2) (IC 50 = 32.35 ± 2.05 µM) and rutin (5) (IC 50 = 33.09 ± 5.43 µM) also show potent anti-AChE effects and deserve further investigation.
Based on the results of the anti-AChE assay, compound 3 exhibited the most significant inhibitory effect among all isolated compounds; therefore, the interaction between AChE and 3 was assessed by molecular docking. Compound 3 showed the best binding energy (−7.5 kcal/mol) compared to chlorogenic acid (−5.3 kcal/mol). This revealed that 3 could dock into the pocket of the crystal structure of AChE from E. electricus more efficiently than chlorogenic acid.
The inflammatory response is a defense mechanism of the body. A general inflammatory response leads to tissue protection and regeneration after injury and, in this way, responds to injury and infection. Under inflammatory conditions, activated macrophages can exhibit detrimental effects involving the overproduction of inflammatory cytokines such as NO, TNF-α and IL-6. Therefore, inhibiting the aberrant activation of macrophages may have therapeutic potential in the treatment of inflammation-related degenerative diseases. In our study, compounds 3 (IC 50 = 17.03 ± 1.28 µM) and 5 (IC 50 = 9.89 ± 1.36 µM) exhibited more effective anti-NO activities than the positive control, quercetin (IC 50 = 18.26 ± 0.54 µM). Compounds 3, 4, and 5 also significantly inhibited the production of iNOS, TNF-α and IL-6 in a concentration-dependent manner.
Based on Western blot analysis of iNOS expression, compound 5 showed the strongest inhibitory effect among all isolated compounds. Therefore, the interaction between iNOS and compound 5 was assessed by molecular docking. Compound 5 showed the best binding energy (−9.5 kcal/mol) compared to quercetin (−7.9 kcal/mol). This revealed that 5 could dock into the pocket of the crystal structure of iNOS from M. musculus more efficiently than quercetin. In addition, our study performed molecular docking for the first time to calculate the binding energy of 5 with M. musculus iNOS.
Bioactivity assays indicated that 5 exhibited the most potent antioxidant effects as measured by DPPH and ABTS assays. In the FRAP experiment, 3 showed the most potent antioxidant effect (3898.88 ± 23.23 mM TE/g). Compounds 2-4 had a stronger anti-αglucosidase effect than acarbose, and compounds 2, 3, and 5 showed better anti-AChE activity than chlorogenic acid. In addition, compounds 3, 4, and 5 obviously inhibited iNOS and NO production in a concentration-dependent manner. Compounds 3, 4, and 5 also significantly inhibited the production of TNF-α and IL-6 in a concentration-dependent manner. This verifies that these compounds possess potent anti-inflammatory activity. Further molecular docking computing results supported that the binding affinity of rutin (5) was significantly higher than compounds 1-4 and the positive control quercetin. This revealed that 5 has the strongest binding affinity with iNOS, which is consistent with the highest anti-NO activity of 5.
The above active extracts and active compounds (especially 3-5) can be applied as herbal antioxidants against oxidative damage, and scopoletin (2) may be used as a natural anti-α-glucosidase agent. Furthermore, rutin (5) and isoquercetin (3) also can be used as natural anti-inflammatory and anti-AChE agents.