Bioactive Naphthoquinone and Phenazine Analogs from the Endophytic Streptomyces sp. PH9030 as α-Glucosidase Inhibitors

A talented endophytic Streptomyces sp. PH9030 is derived from the medicinal plant Kadsura coccinea (Lem.) A.C. Smith. The undescribed naphthoquinone naphthgeranine G (5) and seven previously identified compounds, 6–12, were obtained from Streptomyces sp. PH9030. The structure of 5 was identified by comprehensive examination of its HRESIMS, 1D NMR, 2D NMR and ECD data. The inhibitory activities of all the compounds toward α-glucosidase and their antibacterial properties were investigated. The α-glucosidase inhibitory activities of 5, 6, 7 and 9 were reported for the first time, with IC50 values ranging from 66.4 ± 6.7 to 185.9 ± 0.2 μM, as compared with acarbose (IC50 = 671.5 ± 0.2 μM). The molecular docking and molecular dynamics analysis of 5 with α-glucosidase further indicated that it may have a good binding ability with α-glucosidase. Both 9 and 12 exhibited moderate antibacterial activity against methicillin-resistant Staphylococcus aureus, with minimum inhibitory concentration (MIC) values of 16 μg/mL. These results indicate that 5, together with the naphthoquinone scaffold, has the potential to be further developed as a possible inhibitor of α-glucosidase.


Introduction
Diabetes mellitus refers to a group of metabolic illnesses that are characterized by abnormally high amounts of glucose in the bloodstream [1].The global diabetes population is anticipated to exceed 1.3 billion by 2050 [2].Type 2 diabetes mellitus (T2DM) is the most prevalent type of diabetes and is found on every continent [3].α-Glucosidase inhibitors are the most effective medications for treating type 2 diabetes mellitus [4].Currently, the genus Streptomyces is the leading developer of novel and advanced secondary metabolites [5].Acarbose, derived from a strain of the Streptomyces genus, was the first α-glucosidase inhibitor to receive approval in Europe and the U.S. for the treatment of type 2 diabetes [6][7][8].Voglibose is a synthetic derivative of N-substituted valiolamine produced by Streptomyces hygroscopicus and was chosen as a possible α-glucosidase inhibitor in 1994 [9].In 1996, miglitol, a synthetic form of nojirimycin produced by Streptomyces roseochromogenes, was discovered to be a possible inhibitor of α-glucosidase [10].It is believed that Streptomyces is a major source of α-glucosidase inhibitors.
roseochromogenes, was discovered to be a possible inhibitor of α-glucosidase [10].It is believed that Streptomyces is a major source of α-glucosidase inhibitors.
Naphthoquinone, an important class of natural products derived from microorganisms, exhibits interesting biological activities [11][12][13][14].The naphthgeranines, naphterpins and marinones are meroterpenoid families that share a similar naphthoquinone ring structure and have cyclized C-3 geranyl or farnesyl side chains [15].For example, naphthgeranine B (Figure 1, 1), which was isolated in 1990 from soil-derived Streptomyces sp KO-3988, exhibited a potent cytotoxic effect on HeLa S3 cells (IC50 = 1.6 µg/mL) [16].In 1992, marinone (Figure 1, 2), which is isolated from the marine-derived Streptomyces sp CNB-632, was demonstrated to exhibit inhibitory effects against Bacillus subrifis, with MIC of 1 µg/mL [17].Phenazines are a diverse collection of secondary metabolites with redox activity that are synthesized by several types of bacteria, such as Streptomyces and Pseudomonas, as well as by Methanosarcina species [18].The fundamental composition of phenazines consists of a pyrazine ring (1,4-diazabenzene) that contains two interconnected benzene rings [19].Kankanamge [27].5-Methyl phenazine-1-carboxylic acid (Figure 1, 4) which is obtained from the bacterium PUW5, showed a specific ability to kill lung (A549) and breast (MDA MB-231) cancer cells with IC50 values of 488.7 ± 2.52 nM and 458.6 ± 2.48 nM for lung and breast cancer cells, respectively [28].Kadsura coccinea (Lem.)A.C. Smith (Figure 2A), "黑老虎" in Chinese, is a perennia climbing shrub of the Schisandraceae family, known for its medicinal properties.The roots and stems of this plant are used in traditional Chinese medicine for the treatment of gastroenteric diseases, rheumatism, trauma and pain [29,30].To the best of our knowledge only a few reports of endophytes and the natural products that they produce from K. coccinea.In our continuous exploration of Streptomyces natural products [31][32][33], we have started a project to isolate natural products from endophytic Streptomyces strains found in the rhizosphere soil and roots of K. coccinea.In this study, we report the isolation of Streptomyces from K. coccinea (Figure 2B), and that bioactivity-guided natural product isolation Kadsura coccinea (Lem.)A.C. Smith (Figure 2A), "黑老虎" in Chinese, is a perennial climbing shrub of the Schisandraceae family, known for its medicinal properties.The roots and stems of this plant are used in traditional Chinese medicine for the treatment of gastroenteric diseases, rheumatism, trauma and pain [29,30].To the best of our knowledge, only a few reports of endophytes and the natural products that they produce from K. coccinea.In our continuous exploration of Streptomyces natural products [31][32][33], we have started a project to isolate natural products from endophytic Streptomyces strains found in the rhizosphere soil and roots of K. coccinea.In this study, we report the isolation of Streptomyces from K. coccinea (Figure 2B), and that bioactivity-guided natural product isolation has yielded eight compounds including a new naphthoquinone derivative, naphthgeranine G (Figure 3A, 5), together with two known naphthoquinone derivatives (Figure 3A, 6-7) and five known phenazine derivatives (Figure 3A,(8)(9)(10)(11)(12).The ability of these compounds to inhibit α-glucosidase was tested.The data obtained suggest that the majority of these compounds exhibited significant inhibitory effects on α-glucosidase.Among these, 5 exhibited the strongest inhibition, with an IC 50 value of 66.4 ± 6.7 µM.Molecular docking and molecular dynamics studies were performed to further investigate the interaction, orientation and conformation of 5 over the active site of α-glucosidase.Therefore, 5 is a potential α-glucosidase inhibitor.Furthermore, the evaluation of antibacterial activity revealed that both 9 and 12 had moderate antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with MIC values of 16 µg/mL.3A, 5), together with two known naphthoquinone derivatives (Figure 3A 6-7) and five known phenazine derivatives (Figure 3A, 8-12).The ability of these com pounds to inhibit α-glucosidase was tested.The data obtained suggest that the majority of these compounds exhibited significant inhibitory effects on α-glucosidase.Among these, 5 exhibited the strongest inhibition, with an IC50 value of 66.4 ± 6.7 µM.Molecula docking and molecular dynamics studies were performed to further investigate the inter action, orientation and conformation of 5 over the active site of α-glucosidase.Therefore 5 is a potential α-glucosidase inhibitor.Furthermore, the evaluation of antibacterial activ ity revealed that both 9 and 12 had moderate antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA), with MIC values of 16 µg/mL.

Actinomycete Isolation, Small-Scale Fermentation and Antibacterial Activity Assay
Thirty isolates, designated PH9001-PH9030 (Figure 2B), were obtained from the rhizosphere soil and roots of the medicinal plant K. coccinea.The plant samples were collected from a mountain ditch in Tongdao County, Huaihua City, Hunan Province, China (Figure 2A).The isolates were obtained through a series of repeated pure cultures on three different agar media (Table S1, MH14, MH15 and MH16).The morphological characteristics of most of the selected isolates are shown in Figure 2B.For further fermentation and antibacterial activity testing, 18 isolates were selected on the basis of their unique characteristics.The most prevalent secondary metabolites of PH9030 in MH18 medium (Table S1) are shown in Figure S1, where they also strongly inhibited MRSA and Staphylococcus aureus ATCC 29213 (Table S2, Figures S1 and S2).As a result, the strain PH9030 was chosen for further study.

Identification and Phylogenetic Analysis of Strain PH9030
The partial 16S rRNA gene sequences (Figure S3) were identified, and a phylogenetic tree was constructed via the BLAST tool of the NCBI for the molecular identification of PH9030.These findings indicate that PH9030 is a member of the Streptomyces genus.The phylogenetic tree constructed using 16S rRNA sequences (GenBank ID: PP593435) indicates that Streptomyces sp.PH9030 exhibits a significant resemblance to Streptomyces sp.DS-SO-17 (OQ438799) and Streptomyces sp.DS-SO-7 (OQ438790) (Figure 2C).Consequently, larger-scale liquid fermentation of S. sp.PH9030 (123 L) was subsequently used to extract natural compounds and assess their effectiveness in inhibiting α-glucosidase and combating bacterial growth.

Actinomycete Isolation, Small-Scale Fermentation and Antibacterial Activity Assay
Thirty isolates, designated PH9001-PH9030 (Figure 2B), were obtained from the rhizosphere soil and roots of the medicinal plant K. coccinea.The plant samples were collected from a mountain ditch in Tongdao County, Huaihua City, Hunan Province, China (Figure 2A).The isolates were obtained through a series of repeated pure cultures on three different agar media (Table S1, MH14, MH15 and MH16).The morphological characteristics of most of the selected isolates are shown in Figure 2B.For further fermentation and antibacterial activity testing, 18 isolates were selected on the basis of their unique characteristics.The most prevalent secondary metabolites of PH9030 in MH18 medium (Table S1) are shown in Figure S1, where they also strongly inhibited MRSA and Staphylococcus aureus ATCC 29213 (Table S2, Figures S1 and S2).As a result, the strain PH9030 was chosen for further study.

Identification and Phylogenetic Analysis of Strain PH9030
The partial 16S rRNA gene sequences (Figure S3) were identified, and a phylogenetic tree was constructed via the BLAST tool of the NCBI for the molecular identification of PH9030.These findings indicate that PH9030 is a member of the Streptomyces genus.The phylogenetic tree constructed using 16S rRNA sequences (GenBank ID: PP593435) indicates that Streptomyces sp.PH9030 exhibits a significant resemblance to Streptomyces sp.DS-SO-17 (OQ438799) and Streptomyces sp.DS-SO-7 (OQ438790) (Figure 2C).Consequently, larger-scale liquid fermentation of S. sp.PH9030 (123 L) was subsequently used to extract natural compounds and assess their effectiveness in inhibiting α-glucosidase and combating bacterial growth.

In Vitro α-Glucosidase Inhibitory Activity
The in vitro α-glucosidase inhibitory activities of the compounds 5-12 were assessed.The α-glucosidase inhibitory activities of 5, 6, 7 and 9 were reported for the first time, with IC50 values ranging from 66.4 ± 6.7 to 185.9 ± 0.2 µM.Acarbose was used as a positive control, and the results are summarized in Table 2.These findings demonstrated that the majority of the compounds had a very promising α-glucosidase inhibitory activity.Notably, 5 exhibited the highest potency (IC50 = 66.4 ± 6.7 µM), surpassing the activity of acarbose (IC50 = 671.5 ± 0.2 µM).The investigation of the structure-activity connection revealed that the presence of C12-OH greatly enhances the molecular framework of α-glucosidase inhibitory activity.Furthermore, the 12S-conformation exhibits greater strength than the 12R-conformation.

Molecular Docking Simulations of 5 with α-Glucosidase
The software AutoDock Vina 1.1.2was used for molecular docking research to investigate the interactions between 5-12 and α-glucosidase.These compounds, with binding

In Vitro α-Glucosidase Inhibitory Activity
The in vitro α-glucosidase inhibitory activities of the compounds 5-12 were assessed.The α-glucosidase inhibitory activities of 5, 6, 7 and 9 were reported for the first time, with IC 50 values ranging from 66.4 ± 6.7 to 185.9 ± 0.2 µM.Acarbose was used as a positive control, and the results are summarized in Table 2.These findings demonstrated that the majority of the compounds had a very promising α-glucosidase inhibitory activity.Notably, 5 exhibited the highest potency (IC 50 = 66.4 ± 6.7 µM), surpassing the activity of acarbose (IC 50 = 671.5 ± 0.2 µM).The investigation of the structure-activity connection revealed that the presence of C12-OH greatly enhances the molecular framework of α-glucosidase inhibitory activity.Furthermore, the 12S-conformation exhibits greater strength than the 12R-conformation.

Molecular Docking Simulations of 5 with α-Glucosidase
The software AutoDock Vina 1.1.2was used for molecular docking research to investigate the interactions between 5-12 and α-glucosidase.These compounds, with binding energies greater than that of acarbose, had blocking effects, whereas the compounds with binding energies lower than acarbose had no activity.These findings matched the results of the experiments (Tables 2 and S6).Owing to its outstanding α-glucosidase inhibitory action, 5 was our primary focus.Figure 5 shows the molecular docking models of 5.The docking results revealed that 5 formed four hydrogen bonds with Asp-203, Arg-202, Thr-205 and Asn-449 and three hydrophobic interactions with Asp-542, Phe-575 and Tyr-299 (Figure 5).Additionally, to compare the various interactions, we performed molecular docking of acarbose (Figure S63).The affinities of the mentioned inhibitors were calculated, and the results revealed that acarbose has a binding energy of 6.7 kcal/mol and that 5 has a binding energy of 7.2 kcal/mol (Table 3).The docking findings suggested that, compared with acarbose, 5 had a stronger influence on the binding contacts with the active pocket of α-glucosidase, impacting its inhibitory activity.
Molecules 2024, 29, x FOR PEER REVIEW 7 of 15 energies greater than that of acarbose, had blocking effects, whereas the compounds with binding energies lower than acarbose had no activity.These matched the results of the experiments (Tables 2 and S6).Owing to its outstanding α-glucosidase inhibitory action, 5 was our primary focus.Figure 5 shows the molecular docking models of 5.The docking results revealed that 5 formed four hydrogen bonds with Asp-203, Arg-202, Thr-205 and Asn-449 and three hydrophobic interactions with Asp-542, Phe-575 and Tyr-299 (Figure 5).Additionally, to compare the various interactions, we performed molecular docking of acarbose (Figure S63).The affinities of the mentioned inhibitors were calculated, and the results revealed that acarbose has a binding energy of 6.7 kcal/mol and that 5 has a binding energy of 7.2 kcal/mol (Table 3).The docking findings suggested that, compared with acarbose, 5 had a stronger influence on the binding contacts with the active pocket of α-glucosidase, impacting its inhibitory activity.

Molecular Dynamics Simulations
A molecular dynamics simulation was subsequently conducted under physiologically simulated circumstances to elucidate the binding pattern, stability and molecular interaction mode of 5 with the α-glucosidase protein complex.Structural stability is often assessed on the basis of the presence of low root-mean-square deviation (RMSD) and rootmean-square fluctuation (RMSF) values [40].The RMSD fluctuation graphs throughout the simulation display the RMSD of the two systems, α-glucosidase/acarbose and α-glucosidase/naphthgeranine G (5), as shown in Figure 6A.In the first five ns of the simulation, the two systems converge gradually.In the subsequent simulations, the systems maintain relatively stable fluctuations, with the RMSD keeping the fluctuations between 1 and 2 Å.On the basis of their steady fluctuations, the two systems are stable together.As shown in Figure 6B, all proteins had minimal RMSF values after binding tiny ligands, indicating a solid core structure.Consequently, these proteins are more rigid when bound to small molecules, resulting in the inhibitory action of these small molecules.Significantly, there

Molecular Dynamics Simulations
A molecular dynamics simulation was subsequently conducted under physiologically simulated circumstances to elucidate the binding pattern, stability and molecular interaction mode of 5 with the α-glucosidase protein complex.Structural stability is often assessed on the basis of the presence of low root-mean-square deviation (RMSD) and rootmean-square fluctuation (RMSF) values [40].The RMSD fluctuation graphs throughout the simulation display the RMSD of the two systems, α-glucosidase/acarbose and α-glu cosidase/naphthgeranine G (5), as shown in Figure 6A.In the first five ns of the simulation, the two systems converge gradually.In the subsequent simulations, the systems maintain relatively stable fluctuations, with the RMSD keeping the fluctuations between 1 and 2 Å.On the basis of their steady fluctuations, the two systems are stable together.As shown in Figure 6B, all proteins had minimal RMSF values after binding tiny ligands, indicating a solid core structure.Consequently, these proteins are more rigid when bound to small molecules, resulting in the inhibitory action of these small molecules.Significantly, there is a substantial overlap between the red line and the blue line, suggesting that the two tiny chemicals have comparable impacts on the proteins.The radius of gyration (RoG) is a mea-sure of the compactness of a system and may indicate the degree of densification. Figure 6C clearly shows that the α-glucosidase/acarbose and α-glucosidase/naphthgeranine G (5) systems exhibit similar binding effects and vacillate accordingly.A thorough investigation revealed that the RoG of α-glucosidase/naphthgeranine G (5) mostly decreased during the simulation.This finding indicates that the system became more condensed, suggesting a higher level of binding affinity.
systems are stable together.As shown in Figure 6B, all proteins had minimal RMSF values after binding tiny ligands, indicating a solid core structure.Consequently, these proteins are more rigid when bound to small molecules, resulting in the inhibitory action of these small molecules.Significantly, there is a substantial overlap between the red line and the blue line, suggesting that the two tiny chemicals have comparable impacts on the proteins.The radius of gyration (RoG) is a measure of the compactness of a system and may indicate the degree of densification. Figure 6C clearly shows that the αglucosidase/acarbose and α-glucosidase/naphthgeranine G (5) systems exhibit similar binding effects and vacillate accordingly.A thorough investigation revealed that the RoG of α-glucosidase/naphthgeranine G (5) mostly decreased during the simulation.This finding indicates that the system became more condensed, suggesting a higher level of binding affinity.To better represent the binding modalities of small molecules and target proteins, we computed the binding energies via the MM-GBSA approach, which is based on the trajectories of the molecular dynamic simulations.According to Table S5, the binding energy of the α-glucosidase/acarbose complex was −11.8 ± 4.0 kcal/mol, and that of the αglucosidase/naphthgeranine G (5) complex was −16.6 ± 1.4 kcal/mol.Smaller values suggest stronger binding, and negative values suggest that the two molecules may bind to the target proteins.Our calculations indicate that α-glucosidase/naphthgeranine G (5) binds more effectively and has a marginally lower value than acarbose.One of the stronger noncovalent ways to bind is through hydrogen bonds, and having more hydrogen bonds results in better binding.Figure 6D shows that the number of hydrogen bonds between α-glucosidase and acarbose remained between one and nine and mostly To better represent the binding modalities of small molecules and target proteins, we computed the binding energies via the MM-GBSA approach, which is based on the trajectories of the molecular dynamic simulations.According to Table S5, the binding energy of the α-glucosidase/acarbose complex was −11.8 ± 4.0 kcal/mol, and that of the α-glucosidase/naphthgeranine G (5) complex was −16.6 ± 1.4 kcal/mol.Smaller values suggest stronger binding, and negative values suggest that the two molecules may bind to the target proteins.Our calculations indicate that α-glucosidase/naphthgeranine G (5) binds more effectively and has a marginally lower value than acarbose.One of the stronger noncovalent ways to bind is through hydrogen bonds, and having more hydrogen bonds results in better binding.Figure 6D shows that the number of hydrogen bonds between α-glucosidase and acarbose remained between one and nine and mostly changed between three and four.These findings suggest that hydrogen bonds are important for keeping the binding of acarbose stable.Hydrogen bonding is among the strongest noncovalent binding interactions, and a greater number of hydrogen bonds indicates better binding.The number of hydrogen bonds in the α-glucosidase/naphthgeranine G (5) complex changed considerably over the simulation period (0-5) but mostly remained at 1-2.These findings suggest that hydrogen bonding plays a minor role in the interaction between α-glucosidase and naphthgeranine G (5).In summary, naphthgeranine G (5) binds to α-glucosidase more effectively than acarbose.This is in line with the observed experimental findings.

Antibacterial Activities of 5-12
The MIC values of 5-12 against Staphylococcus aureus ATCC 29213, MRSA, Klebsiella pneumoniae ATCC 13883 and Pseudomonas aeruginosa ATCC 9027 were determined via a broth dilution assay in 96-well plates, with levofloxacin as a control (Table S7).Both 9 and 12 demonstrated modest levels of antibacterial activity against MRSA, with MIC values of 16 µg/mL (Figure S64).

Sample Collection
The medicinal plant K. coccinea was collected from a mountain ditch in Tongdao County, Huaihua City, Hunan Province, China (E109 • 25 ′ 53 ′′ , N25 • 52 ′ 00 ′′ ).The plant was identified by the Department of Chinese Pharmacy of the School of Pharmaceutical Sciences, Hunan University of Medicine.

Isolation of Endophytes
The separation methods used for endophytic actinomycetes are detailed in the Supplementary Materials.

Genomic DNA Extraction, 16S rRNA Gene Sequencing and Phylogenetic Tree Construction
S. sp.PH9030 was selected for cultivation in 50 mL of TSB medium.The mixture was agitated at 220 rpm for two days at 30 • C. The resulting mycelium biomass was then obtained by separating it via centrifugal precipitation.In accordance with the instructions provided by the manufacturer, the mycelium biomass that was collected was used in the process of extracting genomic DNA via the Ezup Column Bacteria Genomic DNA Purification Kit.Genomic DNA was extracted via conventional procedures [41].The verified DNA was preserved at a temperature of −20 • C for future use.The 16S rRNA gene was amplified via the universal primer pair 27F/1492R under these conditions [42].The resulting PCR products were subsequently cloned and sequenced [42].The NCBI-BLAST database was used to perform sequence similarity searches and ascertain pairwise similarity values.The GenBank database has been updated with the partial sequences of the 16S rRNA gene that were obtained from S. sp.PH9030.Additionally, the accession code PP593435 was allocated to this sequence.A phylogenetic tree was created via the neighbor joining technique via MEGA 11.0 software.The 13 strains that were closest to each other at the genus level were chosen on the basis of the 16S rRNA sequence, which was compared with the database [43].Bootstrap values (expressed as percentages of 1000 replications) over 50% are shown at branching nodes.The bar was 0.20 substitutions per nucleotide position.

General Methods
The equipment, including those used for optical rotation, HRESIMS, NMR and ECD, as well as the usual reagents used for chemical separation and biological assessment, were identical to those previously reported [32].The details are listed in the Supplementary Materials.

Large-Scale Fermentation and Extraction
The S. sp.PH9030 strain was grown on MH16 (Table S1) agar plates and incubated at 30 • C to obtain spores.S. sp.PH9030 was subsequently grown in 250 mL Erlenmeyer flasks containing 50 mL sterile seed medium TSB and incubated at 30 • C on a rotary shaker (220 rpm) for 48 h.Finally, the seed culture (50 mL) was transferred into 2 L baffled Erlenmeyer flasks containing 500 mL sterile seed medium MH13 (Table S1) containing 4% microporous resins D1300 at 30 • C for 7 days.After fermentation, the culture (123 L) was filtered with EtOAc/MeOH (v/v, 1:1) (5 × 3 L) to yield D1300 resins.The EtOAc/MeOH extract was subsequently evaporated in vacuo to afford 159.4 g of crude extract.

ECD Calculation Methods
The ECD spectrum of 5 was calculated via the Gaussian 09 program [44].The B3LYP/6-31G (d) level was used to optimize those configurations.With the CPCM model in methanol solution, the ECD spectrum was computed via TDDFT at the B3LYP/6-311+ +G (2d, p) level [45].The details are provided in the Supplementary Materials.

α-Glucosidase Inhibition Assay
The Worawalai technique was employed to evaluate the inhibitory activity of 5-12 against α-glucosidase [Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China, Product No. G5003] with minor modifications [46].The levels of α-glucosidase were detected at 405 nm for a spectrophotometric in vitro α-glucosidase inhibitory activity test.The Supplementary Materials provide a detailed description of the reaction system.

Molecular Docking Analysis
The approach is outlined in the Supplementary Materials.

Molecular Dynamic Simulations
The approach is described in the Supplementary Materials.

Antibacterial Assay
The broth dilution method was used to determine the minimum inhibitory concentrations (MICs) [47].The details are described in the Supplementary Materials.

Figure 2 .
Figure 2. Source of Streptomyces.(A) The whole plant of K. coccinea.(B) Morphological characteriza tion of actinobacterial isolates.Colony morphology of different actinobacterial isolates derived from the medicinal plant K. coccinea.(C) Phylogenetic tree analysis of S. sp.PH9030 (red).

Figure 2 .
Figure 2. Source of Streptomyces.(A) The whole plant of K. coccinea.(B) Morphological characterization of actinobacterial isolates.Colony morphology of different actinobacterial isolates derived from the medicinal plant K. coccinea.(C) Phylogenetic tree analysis of S. sp.PH9030 (red).

Figure 4 .
Figure 4. (A) Key 1 H-1 H COSY and HMBC correlations of compound 5. (B) Experimental and calculated ECD spectra of the compound 5 in MeOH.

Figure 4 .
Figure 4. (A) Key 1 H-1 H COSY and HMBC correlations of compound 5. (B) Experimental and calculated ECD spectra of the compound 5 in MeOH.

Figure 6 .
Figure 6.Molecular dynamics of acarbose and 5 with residues in the active pocket of α-glucosidase.(A) The RMSD values of the complex and protein backbone systems were calculated throughout the dynamic simulation.(B) RMSF changes the shapes of the residues that bond in both free proteins and complicated systems.(C) The gyration radius of the four systems was measured during the molecular dynamics simulation.(D) The quantity of hydrogen bonds present in the molecular dynamics simulation.

Figure 6 .
Figure 6.Molecular dynamics of acarbose and 5 with residues in the active pocket of α-glucosidase.(A) The RMSD values of the complex and protein backbone systems were calculated throughout the dynamic simulation.(B) RMSF changes the shapes of the residues that bond in both free proteins and complicated systems.(C) The gyration radius of the four systems was measured during the molecular dynamics simulation.(D) The quantity of hydrogen bonds present in the molecular dynamics simulation.
a Data are presented as means ± SDs; b NA: not active.
a Data are presented as means ± SDs; b NA: not active.