Inhibitory Mechanism of Tangzhiqing on Sucrase: Changed inhibitory kinetics and secondary molecular conformation of sucrase and chemical components from Tangzhiqing

Inhibitory Mechanism of Tangzhiqing on Sucrase: Changed inhibitory kinetics and secondary molecular conformation of sucrase and chemical components from Tangzhiqing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibitory Mechanism of Tangzhiqing on Sucrase: Changed inhibitory kinetics and secondary molecular conformation of sucrase and chemical components from Tangzhiqing Yanfen Li, Mengyao Zhu, Ruihua Wang, Xin Xia, Ting Jiang, Fengying Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5255841/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background Tangzhiqing (TZQ), a component-based herbal medicine, has been reported to have postprandial hypoglycemic effects on sucrose in humans. Sucrase, a type of α-glucosidase enzyme, breaks down sucrose into its monomers, glucose and fructose. This study investigated the inhibitory activity of TZQ against sucrase and the conformational changes in sucrase induced by interactions between sucrase and TZQ. Methods Sucrose was used as an indicator substrate. After confirming the suitable reaction concentrations and time, an assay of the inhibitory activity of TZQ against sucrase was performed with a series of TZQ concentrations ranging from 0.7 to 22 μg/mL. The inhibition kinetics were analyzed in the presence and absence of TZQ or acarbose. Acarbose was used as a positive control drug. The changes in the secondary structure changes of sucrase in the presence and absence of TZQ were determined by circular dichroism spectroscopy. To investigate the pharmacological basis of the inhibitory effect of TZQ, we used a UPLC-Q-TOF/MS method to identify TZQ components. Molecular docking studies were carried out to explore the binding ability of sucrase and potential active ingredients in TZQ. Results TZQ showed a notable inhibitory activity against sucrase in a reversible and uncompetitive manner. The half-maximal inhibitory concentration (IC 50 ) values of TZQ and acarbose were 1.49 ± 0.07 and 3.94 ± 0.07 μg/mL, respectively, indicating that TZQ possesses has a considerable inhibitory effect similar to that of acarbose. Circular dichroism results revealed that the binding of TZQ to sucrase causes a rearrangement of the secondary structure of sucrase , with an increase in the content of α-helix components and a decrease in the content of β-sheet components, therefore inhibiting enzyme activity. A total of 64 chemical ingredients were identified from TZQ via UPLC-Q-TOF/MS. In addition to having a binding energy of less than -5 kcal/mol, the potential active ingredients of TZQ, including paeoniflorin, vitexin, chlorogenic acid, and salvianolic acid A, strongly affect the structure of sucrase through π accumulation or salt bridge formation, thus inhibiting its activity and lowering glucose. Conclusion These findings provide a new insight into the inhibitory mechanism of TZQ on sucrase, which may be beneficial for the reasonable use of component-based Chinese medicine to prevent type 2 diabetes mellitus as a novel α-glycosidase inhibitor. Clinical Trial Number ：Not applicable. Tangzhiqing (TZQ) Sucrase α-Glucosidase Inhibitory kinetics Molecular conformation Circular dichroism UPLC-Q-TOF/MS Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Diabetes mellitus (DM), namely, “xiaoke” of traditional Chinese medicine, is a metabolic disease associated with significant morbidity and mortality[1]. In China, the morbidity of DM is approximately 10%, and the number of diabetic patients has reached 114 million, accounting for approximately one-third of the number of diabetic patients worldwide. Among them, type 2 diabetes mellitus (T2DM) patients accounts for more than 90% of the amount of DM[2]. Controlling postprandial hyperglycemia could be considered an effective strategy to treat and prevent T2DM[3]. α-Glucosidase inhibitors are highly important therapeutic strategies. Currently prescribed α-glucosidase inhibitors, such as acarbose, voglibose and miglitol, have shown various side effects, including bloating, diarrhea, flatulence, pain and abdominal discomfort. Since complementary and alternative medicine is effective in the management of T2DM, the search for novel and safer α-glucosidase inhibitors from herbal medicine is considered a hot topic of research. Tangzhiqing (TZQ), a component-based Chinese medicine, has the potential to be an α-glycosidase inhibitor for the prevention and treatment of prediabetes or mild T2DM. It is mainly composed of five kinds of Chinese medicinal herbs, including danshen root, red peony root, hawthorn leaves, mulberry leaves, and lotus leaves (Table 1). TZQ consists of Mulberry leaf alkaloids, Mulberry leaf flavonoids, Mulberry leaf polysaccharide, Lotus leaf alkaloids, Lotus leaf flavonoids, Hawthorn leaf flavonoids, Danshen polyphenols, and red Paeony saponins in a fixed proportion (0.8: 0.1: 14: 2.9: 1.8: 0.2: 0.8: 3, w/w) [4]. In 2010, TZQ tablets were approved for clinical trials by the official National Medical Products Administration (NMPA). Our previous studies revealed that TZQ has the same effect as acarbose in reducing postprandial plasma glucose without any gastrointestinal adverse events in healthy Chinese volunteers[5]. Moreover, the hypoglycemic effect of TZQ increases with increasing dose according to dose-exposure-response analysis[6,7]. Additionally, TZQ can decrease fasting insulin and glycosylated hemoglobin levels in T2DM patients[8]. Table 1. Latin name for the Traditional Chinese medicine of TZQ English name Latin name Chinese name Danshen root Salvia miltiorrhiza Bge. Danshen Red Paeony root Paeonia veitchii Lynch Chishao Hawthorn leaf Crataegu spinnatifida Bge. Shanzhaye Mulberry leaf Morus alba L. Sangye Lotus leaf Nelumbo nucifera Gaertn. Heye Inhibition of brush border α-glucosidase may be one of the mechanisms by which TZQ lowers postprandial plasma glucose. α-Glucosidase inhibitors can inhibit the hydrolysis of disaccharides or oligosaccharides on the small intestinal mucosa to reduce the absorption of carbohydrates, thereby resulting in a lower blood sugar level after dinner. Our previous eight-period crossover studies preliminarily revealed that TZQ has evident effects on postprandial glycemia resulting from sucrose and maltose by inhibiting sucrase and maltase, respectively [9]. Further studies confirmed that the interaction of TZQ and maltase induces enzyme inhibition and conformational changes in maltase [10]. Sucrase, a hydrolytic enzyme, breaks down sucrose into its monomers, glucose and fructose. The secondary structure of sucrase has been studied, and three acidic amino acids play a vital role in its active site. However, the effects of TZQ on the activity and structural changes of sucrase are still unclear. Herein, we studied the enzyme inhibition and conformational changes in sucrase induced by the interaction of TZQ and sucrase. Acarbose was selected as a positive control in this study. This work will be highly important for providing a scientific basis for the utilization and development of TZQ, a component-based Chinese medicine. Methods Materials Brush edge sucrase was from Yishengyuan Gene Technology (Tianjin) Co., LTD.. Baker’s yeast sucrase, sucrose, and acarbose were obtained from Sigma‒Aldrich (Shanghai, China), TS Corporation (Seoul, Korea), and the National Institutes for Food and Drug Control (Beijing, China), respectively. TZQ, a component-based Chinese medicine, was provided by Buchang Shenzhou Pharmaceutical Co., Ltd. (Shandong, China). The Q-marker component of TZQ was preferable to nuciferine, with a content of 5.52 mg/g. Suitable concentrations and reaction times Purified sucrose was used as an indicator substrate. The tested concentrations of sucrose were 0.007, 0.014, 0.028, 0.056, 0.112, 0.224, 0.448, and 0.896 mol/L. Fifty microliters of 10 mg/mL sucrase was premixed with 0.5 mL of the test sample in 0.5 mL of phosphate buffer (pH 6.8) and incubated at 37°C for 5 min. Then, 20 μL of sucrose as the substrate was added to the mixture to start the reaction. The mixture was incubated at 37°C for 16 min and stopped by the addition of 0.1 mL of sodium carbonate (Na 2 CO 3 , 0.2 mol/L) solution. The optimized concentration of sucrose was used to determine a suitable concentration of sucrase. The candidate concentrations of sucrase were 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mg/mL. These reactions were stopped every 2 min to optimize the reaction time. The sucrase activity was determined by measuring the release of glucose at 492 nm via a DNM-9602 automatic microplate reader (PuLang, China). Triplicate samples were created for each test. Enzyme kinetic equation Under the conditions of the optimum reaction time and enzyme concentration determined in section 2.3 with a constant enzyme concentration, the initial rate was determined when the substrate concentration was 0.028, 0.056, 0.112, 0.224, 0.448, and 0.896 mol/L, respectively, and three times for each concentration. The Michaelis constant ( K m ) and the maximum reaction rate ( V max ) were calculated via the double reciprocal method. Inhibitory effect of TZQ against sucrase The assay of the inhibitory activity of TZQ against sucrase was performed according to the methods in the literature with minor modifications [11, 12]. Briefly, mixtures of 20 μL of sodium phosphate buffer (pH=6.8), 70 µL of sucrase solution (10 mg/mL) and 10 µL of TZQ or acarbose solution were prepared and incubated for 5 min in 96-well plates at a constant temperature of 37°C. The reaction was then initiated by adding 20 μL of sucrose at concentrations ranging from 0.028 to 0.896 mol/L for 14 min at 37°C. The tested concentrations of TZQ were 0.7, 1.4, 2.8, 5.6, 11, and 22 μg/mL. Moreover, acarbose was used as a positive control at different concentrations of 10, 20, 40, 80, 160, 320, and 640 μg/mL. After another 15 min of incubation at 37°C, 100 μL of Na 2 CO 3 solution (0.2 mol/L) was added to terminate the reaction, and changes in the absorbance were measured at 492 nm via a DNM-9602 automatic microplate reader. The half-maximal inhibitory concentration (IC 50 ) was used to compare the inhibitory effects of the samples. The relative enzyme activity of the test samples toward sucrase was calculated as follows (Eq. 1): where A sample , A control , and A blank are the absorbance of the sample, the absorbance of the blank control without sample, and the absorbance of the samples without sucrase, respectively. Determination of the inhibition kinetics of TZQ against sucrase To determine the inhibitory characteristics of TZQ, the mechanisms by which the active components of TZQ bind to the sucrase were further explored by analyzing the enzyme kinetics. This inhibition kinetics study was carried out in the presence and absence of TZQ with varying concentrations of sucrose as the substrate. The inhibition type and mechanism were analysed via Lineweaver–Burk plots, which are plots of the enzyme reaction velocity (V) versus the substrate concentration, and the relevant parameters were calculated via the following equation (Eq. 2): where V (mol/L/min) is the enzyme reaction rate and Vmax (mol/L/min) and Km (mg/mL) represent the maximum reaction rate and Michaelis constant, respectively. [I] and [S] are the concentrations of the inhibitor and sucrose, respectively. Ki is the inhibition constant. α denotes the ratio of the noncompetitive inhibition constant to the competitive inhibition constant, and the value of α for noncompetitive inhibition is 1. Circular dichroism spectroscopy The changes in the secondary structure of sucrase in the presence and absence of TZQ were determined by circular dichroism (CD) spectroscopy. The CD spectra were obtained with a 1 mm quartz cell on a J-810 CD spectrophotometer (American OLIS Company, Carlsbad, CA, USA). The spectra were recorded in the far-UV range of 190–250 nm under constant nitrogen flush. The concentrations of sucrase and acarbose were both maintained at 0.5 g/L. The concentrations of TZQ were varied from 0 to 0.28 g/L. The signal of sodium phosphate buffer (0.1 mol/L, pH=7.0) was subtracted to obtain the actual CD spectra of sucrase. The CD spectra were scanned three times to obtain the average value. All the data were expressed as CD ellipticity in mdeg. The contents of secondary structures such as α-helices, β-sheets, β-turns, and random coils of sucrase were analyzed via the online SELCON3 program[13]. Determination of compounds contained in TZQ The identification of components was performed on an Acquity UPLC I-Class instrument coupled with a Xevo G2-SQ-TOF mass spectrometer via an ESI interface (Waters, Milford, MA, USA). Sample separation was carried out at 35°C on a Waters ACQUITY UPLC BEH C18 column (2.7×100 mm, 1.7 μm), and the flow rate was 0.3 mL/min. The mobile phases consisted of acetonitrile (solution B) and 0.1% formic acid water (solution A), and the gradient program was as follows: 10% B from 0-2 min, 10-20% B from 2-6 min, 20-30% B from 6-20 min, 30-80% B from 20-30 min, 80-30% B from 30-40 min, and 30-10% B from 40-45 min. The injection volume was 2 μL for all the samples. The mass parameters were set as follows: capillary voltage, 2.0 kV; sampling cone voltage, 40 V; source offset, 80 V; source temperature, 100°C; desolvation temperature, 400°C; cone gas flow, 50 L/h; and desolvation gas flow, 600 L/h. The scan MS data ranged from 50 to 1200 Da. In MSE mode, the trap collision energy of the low-energy function was set at 6 eV, and the trap collision energy of the high-energy function was increased from 15 to 60 eV. Sample preparation An easy-to-implement ultrasonic extraction method was utilized. In detail, 200 mg of the accurately weighed powder of TZQ was soaked in 5 mL of methanol (v/v), which was further extracted in a water bath at 25℃ with ultrasound assistance for 30 min. After 10 min of centrifugation at a rotation speed of 14,000 rpm, the supernatant was diluted to 400 μg/ml with 50% aqueous methanol, and the final concentration was determined. Molecular Docking To explore the effects of TZQ compounds on the structure of sucrase, we performed molecular docking studies. The small molecule compound structure files were searched through the Pubchem website (https://pubchem.ncbi.nlm.nih.gov/). The SDF files were converted into PDB files using Open Babel 2.3.2 software. The sucrase receptor proteins were retrieved from the pdb database. PYMOL 2.3.4 software was used to dewater the ligands, and AutoDockTools software was used to convert the receptor protein and ligand small molecules into pdbqt format. The molecular docking of the receptor proteins with the ligand small molecules was performed with AutoDock Vina 1.1.2. The docking results were visualized via Pymol. Statistical analysis The results are expressed as the mean values ± standard deviations. The statistical analysis was performed with GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA). One-way analysis of variance (ANOVA) and the post hoc Tukey test were used for statistical comparisons among groups. For comparisons within groups in the visual chamber test, a paired t test was used. Statistical significance was assumed at a confidence level of at least 90% (p < 0.05). Results Confirmation of suitable concentrations and reaction times The results revealed that the enzyme reaction rate was directly proportional to the substrate concentration, ranging from 0.007 to 0.896 mol/L (Figure 1). Especially in the low-concentration range from 0.007 to 0.224 mol/L, the first-order kinetic process was displayed with an exponential equation (Y = 0.19lnX + 1.17, r²=0.996). However, the pattern of the reaction changed to a mixed-order kinetics when the concentration ranged between 0.224 and 0.448 mol/L and tended towards zero-order kinetics when the substrate concentration increased above 0.448 mol/L. Therefore, the excessive concentration of the substrate sucrose was set at 0.448 mol/L. The profiles of the reaction rate versus time at a series of sucrase concentrations from 1 to 8 mg/mL are shown in Figure 2. To achieve the maximum reaction rate of sucrase in the initial reaction, it is critical to optimize the most suitable sucrase concentration and reaction time. When the concentration of sucrase was 7.0 mg/mL or less, the reaction was linear within 14 min. Therefore, the optimal sucrase concentration and reaction time were confirmed to be 7.0 mg/mL and 14 min, respectively.. Michaelis constant and maximum reaction rate According to the intermediate complex hypothesis, the enzyme-catalyzed reaction can be expressed as E+S⇋E.S→P+E. In Michaelis–Menten kinetics, V and 1/ V are calculated from V max ·[S]/( K m +[S]) and K m / V max ·1/[S]+1/ V max , respectively. Profiles were obtained via the double reciprocal method, using 1/ V as the longitudinal coordinate and 1/[S]used as the transverse coordinate. As shown in Figure 3, the V max and K m values were 0.53 mol/L/min and 0.066 mol/L, respectively, which indicates that the enzyme-catalyzed reaction is consistent with Michaelis–Menten kinetics and that the equation can be used to analyze and evaluate the catalytic reaction. Inhibitory effect of TZQ against sucrase The inhibitory effects of TZQ and acarbose against sucrase were both concentration dependent and gradually increased with the increasing concentrations of TZQ or acarbose (Figure 4). The IC 50 was calculated by fitting the data via logistic regression. The IC 50 values of TZQ and acarbose were 1.49 ± 0.07 (Figure 4A) and 3.94 ± 0.07 μg/mL (Figure 4B), respectively, indicating that TZQ has a considerable inhibitory effect on the standard inhibitor acarbose. Inhibition kinetics analysis of TZQ against sucrase The reversibility of the inhibitory effect of TZQ on sucrase was confirmed by the reaction velocity between different concentrations of sucrase and TZQ (Figure 5). As shown in Figure 5A, all of the straight lines passed through the origin point, and the line slopes decreased with increasing TZQ concentration, which indicated that the presence of TZQ did not reduce the amount of sucrase but rather down-regulated the activity of sucrase. The inhibition of TZQ against sucrase was reversible, which was the same as the inhibition mode of acarbose (Figure 5B). Therefore, we speculated that a noncovalent intermolecular interaction exists between sucrase and the active ingredients of TZQ. Lineweaver-Burk plots in the presence and absence of TZQ were obtained at different substrate concentrations to determine the interaction mechanism between TZQ and sucrase. Figure 6 presents good linear relationships between 1/ S and 1/ V in the double reciprocal curves. As the concentration of TZQ increased, the intercepts of the enzyme reaction rate line on the X-axis (-1/Km) and Y-axis (1/Vmax) increased, whereas the slope remained unchanged. As the TZQ concentration increased from 0 to 2.8 and then to 22 μg/mL, the Vmax decreased from 0.54 to 0.15 and then to 0.06 mol/L/min, and the Km decreased from 0.064 to 0.022 and then to 0.009 mol/L, respectively. The reduced V max and K m values indicated that the inhibitory pattern of TZQ against sucrase was uncompetitive; that is, TZQ could only bind to the enzyme-substrate complex (E+S) rather than the substrate-interacting residues. Figure 6B presents an invariant intercept at the Y -axis (1/ V max ), a decrease in the intercept at the X -axis (-1/ K m ), and an increase in the slope of the reaction rate lines of acarbose against sucrase. These results revealed that the positive control acarbose could compete with sucrose for the same active site of the enzyme, which is competitive inhibition. Influences of TZQ on the secondary structure of sucrase The conformational changes in the secondary structure of sucrase in the presence of TZQ and acarbose are displayed in Figure 7A and Figure 7B, respectively. The CD spectrum of sucrase exhibited a single negative peak at approximately 210 nm and a strong positive peak at 193 nm, which is characteristic of a β-sheet structure. Following the addition of TZQ, the far UV-CD spectra of sucrase presented two negative peaks at 210 nm and 220 nm, which are the characteristic of n→π* and π→π* electron transfer for the peptide bonds of the α-helix (Figure 7A). The negative ellipticity of sucrase increased with increasing TZQ concentration, which indicated that the secondary structure of sucrase had partially changed. For the positive control acarbose, a similar tendency as that of TZQ was observed in terms of reducing the β-sheet content and increasing the α-helix content of sucrase (Figure 7B). The contents of different secondary structures of sucrase were quantitatively determined via the SELCON3 program, and the corresponding results for TZQ and acarbose are listed in Table 2 and Table 3, respectively. Similar patterns of changes were observed in the β-sheet and α-helix contents of sucrase. In the TZQ group, the proportion of α-helices increased from 16.5% to 23.4%, whereas the proportion of β-sheets decreased from 43.4% to 25.6% when the concentration increased from 0.07 to 0.28 g/L. In the acarbose group, the proportion of α-helices increased from 16.5% to 18.2%. In contrast, the proportion of β-sheets decreased from 43.4% to 38.6%. These findings indicated that TZQ interacted with sucrase and influenced its enzymatic activity by inducing structural changes such as those in acarbose. Table 2. Secondary structure of sucrase in the absence and presence of TZQ Secondary structures 0 0.07 g/L 0.14 g/L 0.28 g/L α-helix (%) 16.5 17.7 23.4 22.8 β-sheet (%) 43.4 39.4 37.2 25.6 β-turn (%) 11.1 14.4 14.2 21.6 Random coil (%) 29.0 28.4 25.2 30.0 Total (%) 100.0 100.0 100.0 100.0 Table 3 . Secondary structure of sucrase in the absence and presence of acarbose Secondary structures 0 0.5 g/L (0.5 h) 0.5 g/L (1 h) α-helix (%) 16.5 17.0 18.2 β-sheet (%) 43.4 43.3 38.6 β-turn (%) 11.1 10.9 13.0 Random coil (%) 29.0 28.8 30.3 Total (%) 100.0 100.0 100.0 Chemical components identification of TZQ The base peak ion chromatograms of TZQ in positive and negative ion modes are shown in Figure 8. Table 4 summarizes the characterization of the chemical constituents of TZQ by UPLC-Q-TOF/MS. On the basis of the precise relative molecular mass data and multistage mass spectrum fragment ions, combined with control articles, databases and literature reports, 64 chemical components in TZQ were identified, among which 42 chemical components were identified in negative ion mode and 22 chemical components were identified in positive ion mode. Among them, 14 components originated from mulberry leaves, 21 components originated from red paeony roots, 16 from danshen root , 14 from hawthorn leaves, and 11 from lotus leaves. Table 4. Characterization of the chemical constituents of TZQ by UPLC-Q-TOF-MS. No. t R /min Compound Formula Ion mode Precursor ion（m/z） MS/MS（m/z） 1 0.78 2-O-α-D-galactosyranosid-1-deoxynopyrimycin C 12 H 23 NO 9 [M+H] + 326.1420 266.12117、248.10992、230.09875、205.06603、104.10495 2 0.80 Paeonol C 9 H 10 O 3 [M+H] + 167.0709 138.05245、118.08362、109.02500 3 0.80 Ethyl 2-hydroxybenzoate C 9 H 10 O 3 [M+H] + 167.0709 148.05807、138.05245、122.05709 4 0.83 Verbascose C 30 H 52 O 26 [M+HCOO] - 873.2719 665.21580、503.1617、377.08501、341.10803、211.06540、179.05462 5 0.83 Sorbitol C 6 H 14 O 6 [M-H] - ,[M+HCOO] - 181.0702 135.06628、146.05846、149.04555、165.07685 6 0.88 (-)-Catechin C 15 H 14 O 6 [M-H] - 289.0690 125.02252、132.02887、219.04418、245.04255 7 0.89 Quinic acid C 7 H 12 O 6 [M-H] - 191.0547 101.02299、127.03774、149.04381、173.04277 8 0.90 Dehydroroemerine C 18 H 15 NO 2 [M+H] + 278.1202 260.10931、203.04914、174.07348 9 1.18 8-Debenzoylpaeoniflorin C 16 H 24 O 10 [M+HCOO] - 421.1352 345.11965、150.04100、128.03377 10 3.13 Danshensu C 9 H 10 O 5 [M-H] - 197.0439 135.0435 11 3.13 Ethyl gallate C 9 H 10 O 5 [M-H] - 197.0439 135.0435 12 3.13 Anisic acid C 8 H 8 O 3 [M+HCOO] - 197.0439 135.0435 13 4.73 Neochlorogenic acid C 16 H 18 O 9 [M-H] - 353.0869 179.03421、135.04334 14 5.86 Demethyl-Coclaurine C 16 H 17 NO 3 [M-H] - 270.1130 162.0542 15 6.87 Chlorogenic acid C 16 H 18 O 9 [M-H] - 353.0872 191.05441、133.02824 16 7.48 Scopolin C 16 H 18 O 9 [M-H] - 353.0865 223.05997、191.05387、179.03270、173.04415 17 8.10 Quercetin 3, 7-o-beta-d-dipyranoside C 27 H 30 O 17 [M-H] - 625.1407 462.08101、301.03342、299.01916 18 9.57 Paeonol glucoside C 20 H 28 O 12 [M+HCOO] - 505.1565 447.13117、445.13390、285.07496、165.05419、121.02814 19 10.13 Albiflorin C 23 H 28 O 11 [M+H] + 481.1669 319.11483、197.07893、133.06243、105.03120 20 10.15 AlbiflorinR1 C 23 H 28 O 11 [M+HCOO] - , [M-H] - 525.1607 357.11805、283.08201、146.96483、121.02795 21 11.27 L-Asparagine C 4 H 8 N 2 O 3 [M+H] + 133.0625 115.05179、103.05176 22 11.28 paeoniflorin C 23 H 28 O 11 [M+HCOO] - , [M-H] - 525.1617 449.14490、431.13458、327.10776、283.08152、165.05421、121.02812 23 11.28 Oxypaeoniflora C 23 H 28 O 12 [M-H] - 495.1503 449.14490、431.13458、327.10776、283.08152、165.05421、121.02812 24 12.18 Armepavine C 19 H 23 NO 3 [M+H] + 314.1718 283.13001、268.10534、252.11160、189.07843、107.04914 25 12.42 Quercetin 3-sambubioside C 26 H 28 O 16 [M-H] - 595.1313 300.02585、271.02455、243.02887、146.96484 26 12.70 N-Methylcoclaurine C 18 H 21 NO 3 [M+H] + 300.1563 283.12993、237.08776、189.08789、174.06452、107.04683 27 13.16 Rutin C 27 H 30 O 16 [M+H] + 611.1628 303.0499 28 14.14 Isoquercitrin C 21 H 20 O 12 [M+H] + 465.0993 303.04681、229.04758、115.05193、105.03101 29 14.20 Paeoniolide C C 17 H 18 O 6 [M+H] + 319.1160 133.00989、105.03101 30 14.20 Paeoniflorigenone C 17 H 18 O 6 [M+H] + 319.1160 133.00989、105.03101 31 14.43 2-O-Rhamnosylvitexin C 27 H 30 O 14 [M-H] - 577.1562 457.11388、413.08698、301.03406、293.04416、271.02405、151.00192 32 14.43 Hawthorn A C 21 H 18 O 9 [M-H] - 413.0874 301.03406、293.04416、271.02405、151.00192 33 14.74 Salvianolic acid D C 20 H 18 O 10 [M+HCOO] - 463.0880 300.02629、271.02356、255.02835、151.00202 34 14.91 O-Nornuciferine C 20 H 18 O 10 [M+H] + 282.1457 251.10369、219.07753、208.08535、189.06710、165.06714 35 16.65 Salvianolic acid C C 26 H 20 O 10 [M+HCOO] - 537.1045 339.04999、295.05872、146.96452、109.02801 36 19.31 Rosmarinic acid C 18 H 16 O 8 [M-H] - 359.0764 255.02848、179.03348、161.02275、133.02787、121.02786 37 19.31 7-Hydroxycoumarin C 9 H 6 O 3 [M-H] - 161.0229 151.03795、135.04361、133.02787、121.02786 38 20.74 Salvianolic acid A C 26 H 22 O 10 [M-H] - 493.1132 311.05425、293.04487、146.96438、109.02784 39 22.08 Nuciferin C 19 H 21 NO 2 [M+H] + 296.1615 265.11951、235.07248、219.07729、191.15789、178.07505、165.06722、122.01285 40 22.18 Salvianolic acid G C 18 H 12 O 7 [M-H] - 339.0498 321.03913、293.04405、185.02270、109.02798 41 22.20 MudanpiosideC C 30 H 32 O 13 [M-H] - 599.1751 519.09301、339.04981、321.03913、293.04405、185.02270、109.02798 42 22.20 Benzoyloxypaeoniflorin C 30 H 32 O 13 [M-H] - 599.1751 519.09301、339.04981、321.03913、293.04405、185.02270、109.02798 43 22.64 Luteolin C 15 H 10 O 6 [M-H] - 285.0392 133.02771、271.98653 44 22.94 Salvianolic acid B C 36 H 30 O 16 [M-H] - 717.1457 599.17763、519.09260、473.08708、399.04933、321.03929、293.04397、185.02285、109.02793 45 23.21 Isorhamnetin C 16 H 12 O 7 [M+H] + 317.0633 250.09586、237.08798、207.07754、189.06716、178.07511 46 23.21 Isorhamnetin3-O-glucoside C 22 H 22 O 12 [M-H] - 477.1033 315.04952、313.03569、300.02815、271.02483、165.01766、146.96444 47 23.21 Vitexin C 21 H 20 O 10 [M+HCOO] - 477.1033 315.04952、300.02815、271.02482、165.01766、146.96444 48 23.29 Monomethyl lithospermate C 28 H 24 O 12 [M-H] - 551.1198 519.09359、353.06552、321.03942、293.04469、231.02809、146.96444 49 23.67 Przewaquinone E C 18 H 16 O 5 [M+H] + 313.1039 299.10886、189.06848、178.07455 50 24.36 Kaempferol C 15 H 10 O 6 [M-H] - 285.0394 211.07459、146.96431 51 24.55 Mudanpioside J C 31 H 34 O 14 [M-H] - 629.1877 583.18139、553.17095、431.13560、284.03089、146.96436、121.02785 52 24.55 Benzoyl paeoniflorin C 30 H 32 O 12 [M+HCOO] - , [M-H] - 629.1877 583.18139、553.17095、431.13560、284.03089、146.96436、121.02785 53 28.01 Miltionone Ⅰ C 19 H 20 O 4 [M+H] + 313.1423 300.28683、212.23423、141.06794、129.06746 54 28.43 Moracin C C 19 H 18 O 4 [M-H] - 309.1112 147.9642 55 28.89 Paeonenolide F C 32 H 46 O 5 [M+HCOO] - 555.3317 487.34280、437.30452、423.32549、405.31648、393.31590 56 28.98 Neocryptotanshinone C 19 H 22 O 4 [M-H] - 313.1434 269.15325、226.10103、213.12609 57 28.98 4-Prenylresveratrol C 19 H 20 O 3 [M+H] + 297.1457 179.08266、165.06818、152.05940 58 28.98 Isocryptotanshinone C 19 H 20 O 3 [M+H] + 297.1457 211.14450、195.11873、189.06818、141.06762 59 31.21 Tanshinone I C 18 H 12 O 3 [M+H] + 277.0832 249.08887、196.10914、152.05954 60 31.27 TanshinoneIIB C 19 H 20 O 3 [M+H] + 297.1456 254.09120、178.07516、115.05180 61 31.53 Maslinic acid C 30 H 48 O 4 [M-H] - , [M+HCOO] - 471.3479 453.33668、299.20013、227.10574 62 32.08 Hederagenin C 30 H 48 O 4 [M+HCOO] - , [M-H] - 517.3529 423.32552、393.31559 63 32.35 Oplopanaxogenin C C 30 H 48 O 4 [M+HCOO] - , [M-H] - 517.3535 295.22646、277.21606、227.07053、177.08926 64 35.05 Microstegiol C 20 H 26 O 2 [M-H] - 297.1856 281.19109、205.12340 Molecular docking results The ability of TZQ active ingredients to bind to sucrase was investigated through molecular docking studies. The representative docking information of the TZQ active ingredients and sucrase is shown in Figure 9. The sucrase residues GLN-232, LEU-233, LYS-509, ARG-555, and SER-631 formed hydrogen bonds with paeoniflorin, while the surrounding important residues TRP-327, TRP-435, and PHE-604 were hydrophobic with paeoniflorin, in which TRP-327 also formed P-type π stacking with the ligands (Figure 9A). The same docking revealed hydrogen bonds between the sucrase residues ASP-231, SER-631 and the vitexin ligand (Figure 9B). Hydrophobic interactions occur between residues TRP-327 and PHE-479, in which T-type π stacking is formed between the residue TRP-435 ligands (Figure 9B). There are also residues LYS-509, ASP-571, and HIS-629 that form hydrogen bonds with chlorogenic acid ligands, while the hydrophobic interactions are residues LEU-233, TRP-327, TRP-435, and a salt bridge is also formed between LYS-509 (Figure 9C). Salvianolic acid A residues ASP-231, ASP-355, ASP-472, LYS-509, and ASP-632 formed hydrogen bonds with the ligand (Figure 9D). Hydrophobic interactions with the sucrase residues LEU-233, TRP-327, TRP-435, meanwhile TRP-327 also formed P type π stacking. The remaining docking results were shown in Supplementary Material S1. Table 5 summarizes the binding energies of these TZQ active ingredients and sucrase. The docking data revealed that the interaction between the TZQ compounds and sucrase was relatively stable.. Among them, paeoniflorin, vitexin, chlorogenic acid, and salvianolic acid A may have a greater effects on the structure of sucrase. The sucrase structure is affected by π accumulation or salt bridge formation, thus inhibiting its activity and playing a role in lowering glucose. Table 5 . The binding energy of sucrase and small-molecule compounds Chemical compound Binding energy（kcal/mol） Salvianolic acid A -7.8 Salvianolic acid C -8.4 DNJ -5.0 Chlorogenic acid -7.1 Nuciferine -6.6 Maslinic acid -7.3 Vitexin -6.7 Quercetin -7.9 Paeoniflorin -7.5 Discussion TZQ has a certain inhibitory effect on glycosidases[4], but the specific type and mechanism of inhibition are still unclear, which is not conducive to its rational application and clinical promotion. Therefore, the inhibitory ability of TZQ to invertase activity was investigated in the present study. The inhibitory effect of TZQ on sucrase activity (IC 50 = 1.49 ± 0.07 μg/mL) was superior to that of acarbose (IC 50 = 3.94 ± 0.07 μg/mL). As a disaccharide, sucrose is a common carbohydrate eaten by the human body and consists of an α-glucose molecule and a β-fructose molecule connected by α-(1,2)-β-glycosidic bonds [14]. When encountering either alpha-glucosidase or a beta-fructofuronidase, sucrose can be hydrolyzed into an alpha-D-glucose molecule and a beta-D-fructose molecule [15, 16]. The hydrolysis reactions of sucrose are catalyzed by these two enzymes. These results indicate that the effect of TZQ on α - (1,2) - β - glycosidic bonds is similar to that of acarbose [17, 18].In terms of enzyme kinetics, there are four types of inhibition, namely, competitive, noncompetitive, anti-competitive, and mixed. Noncompetitive inhibitors present the same affinity for enzymes and enzyme‒substrate complexes. Anti-competitive inhibitors bind only to enzyme‒substrate complexes, whereas competitive inhibitors compete with substrates for the same active site of the enzyme. Regardless of reversibility, the inhibitory effects of TZQ and acarbose on sucrase are the same, with a straight line passing through the origin (Figure 5). The slope of the straight line with TZQ is smaller than that without TZQ, and the slope gradually decreases with the increasing of concentration, which is a typical feature of reversible inhibition [19]. The kinetics of enzymes can be used to determine enzyme modification pathways and provide a reference for clinical treatment. According to the results of the double reciprocal kinetic curve of the sucrase reaction rate, TZQ was determined to have a noncompetitive inhibitory effect, similar to white tea extract[20], whereas acarbose was determined to have a competitive inhibitory effect (Figure 6). These results suggested that TZQ and acarbose bind to different targets of sucrase. An open-system study on enzyme inhibition revealed that anti-competitive inhibitors (expected to produce permanent inhibitory effects) may have more effective hypoglycemic effects than competitive inhibitors (expected to have only temporary inhibitory effects) [21]. These findings may provide some reference for explaining the mechanism by which TZQ inhibits sucrase activity more effectively than acarbose does. Circular dichroism (CD) spectroscopy is an effective method for analyzing structural changes after interactions between proteins and active molecules. An analysis of the CD spectrum changes in the wavelength range of 190–250 nm revealed that the molecular conformation of sucrase changed after the addition of TZQ, and the α-helix structure gradually decreased, whereas the β-sheet gradually increased. As the concentration increases, the ratio of the two contents gradually approaches one another. For acarbose, the α-helix structure and β-sheet ratio changed similarly with increasing reaction time, but its degree was not as significant as that of TZQ. Moreover, the β-turn content is similar to the changes in the α-helix content. The change in the CD spectrum of the protein secondary structure of the enzyme suggested that some active components of TZQ may combine with sucrase, which promotes the unfolding of the protein polypeptide chain and destroys the α-(1,2)-β-glycosidic bond hydrogen bond network structure of sucrase. The secondary structure of the enzyme becomes loose, which affects enzyme activity and prevents the substrate from binding to the active site of the enzyme[22, 23]. To investigate the pharmacological basis of the inhibitory effect of TZQ on sucrase, we employed high-resolution mass spectrometry to identify the main components of TZQ. A total of 64 chemical ingredients were identified from TZQ via the UPLC‒Q-TOF‒MS method (Table 3). On the basis of previous reports in the literature 24-30, potential compounds with a certain degree of sucrose enzyme inhibition from the 64 compounds were selected. Salvianolic acid C and salvianolic acid A have been reported to have potent α-glucosidase inhibitory activities, with IC 50 values of 4.31 and 19.29 μM, respectively. Additionally, salvianolic acid C exhibited mixed-competitive inhibition when it was bound to α-glucosidase [24]. DNJ and fagomine significantly inhibited the activity of sucrase-mediated intestinal acetone powder in rats, with IC 50 values of 0.14 and 35 μg/mL, respectively [25]. Chlorogenic acid has also been reported to inhibit α-glycosidase activity, and the IC 50 values of chlorogenic acid and its isomer were found to be 2.99 and 3.12 mM, respectively[26]. Nuciferine and Paeoniflorin have been reported to have hypoglycemic effects and beneficial effects on the treatment of type 2 diabetes[27, 28]. Vitexin also has an inhibitory effect on a-glycosidase activity，with an IC 50 value of 4.1 μg/mL [29]. Meanwhile animal experiments have confirmed that maslinic acid has a good hypoglycemic effect[30]. Therefore, 9 of the potential compounds were subjected to molecular docking with sucrase (Table 4). The compound ligands and sucrase interact mostly through hydrophobic interactions, hydrogen bonds, π stacking and salt bridges, thereby altering the structure of sucrase and inhibiting its activity. Further research on the composition and hypoglycaemic mechanism of TZQ compounds is instructive. Compounds with specific hypoglycemic compounds were screened for molecular docking, and the molecular structure changes between the remaining residual compounds and sucrase need to be explored. In this study, the structural changes in and molecular mechanism underlying the inhibitory activity of TZQ against sucrase were investigated via multiple spectroscopic methods. TZQ notably inhibited sucrase in a reversible and uncompetitive manner. Additionally, the inhibitory activity of TZQ against sucrase was considerable compared with that of the positive control acarbose. Circular dichroism revealed that the binding of TZQ to sucrase causes a rearrangement of the secondary structure of sucrase, with an increase in the content of α-helix components and a decrease in the content of β-sheet components, therefore inhibiting enzyme activity. The structural relationships between the compounds and sucrase were initially explored via molecular docking. This study provides a scientific basis for the mechanism of interaction between TZQ and sucrase, which may be useful for the potential application of TZQ as an effective α-glycosidase inhibitor to treat type 2 diabetes. Conclusion In summary, TZQ showed notable inhibitory activity against sucrase in a reversible and uncompetitive manner. Circular dichroism results revealed that the binding of TZQ to sucrase causes a rearrangement of the secondary structure of sucrase, with an increase in the content of α-helix components and a decrease in the content of β-sheet components, therefore inhibiting enzyme activity. A total of 64 chemical ingredients were identified from TZQ via UPLC-Q-TOF/MS. In addition to having a binding energy of less than -5 kcal/mol, the potential active ingredients of TZQ, including paeoniflorin, vitexin, chlorogenic acid, and salvianolic acid A, strongly affect the structure of sucrase through π accumulation or salt bridge formation, thus inhibiting its activity and lowering glucose. These findings provide new insight into the inhibitory mechanism of TZQ on sucrase, which may be beneficial for the reasonable use of component-based Chinese medicine to prevent type 2 diabetes mellitus as a novel α-glycosidase inhibitor. Abbreviations TZQ Tangzhiqing UPLC-Q-TOF/MS Ultraperformance liquid chromatography-quadrupole-time of flight-mass spectrometry IC 50 The half-maximal inhibitory concentration DM Diabetes mellitus T2DM Type 2 diabetes mellitus NMPA National Medical Products Administration PBS Phosphate-buffered saline PE Polyethylene CD Circular dichroism UV Ultraviolet UPLC Ultra performance liquid chromatography SDF Spatial Data Format PDB Protein Data Bank DNJ 1-Deoxynojirimycin Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research was supported by the National Natural Science Foundation of China (No. 81573741) and the Tianjin Municipal Education Commission Research Project (No. 2021KJ159). Authors' contributions YL and ZL designed the experiments. YL, MZ, RW, and ZL wrote the main manuscript text. XX, TJ, FW, and XG conducted the experiments. ST and ZL prepared the figures and analyzed the data. SZ and YH supervised the experiments. All the authors read and approved the final manuscript. Acknowledgements Not applicable. Authors' information 1 Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300250, China. 2 Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China. 3 School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China. 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Tian","suffix":""},{"id":391832513,"identity":"4a2736bc-c0b0-4a1b-82eb-5561e8e66788","order_by":8,"name":"Ziqiang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYBACPiBmBjH4mZkPPiBKCxtMi2Q7W7IBaVoMzvOYCRCnhf/wtscFNfeijQ8zmDEw1NhEE2HLsXLjGceKc7cdZkh7wHAsLbeBoBbGHjNpHrYEkJbjBowNh4nQwswD1PIvIXdzM2ObBHFa2IBaeNsScjcwM7MRqYWHrdyYty8hd8ZhNmaDBGL8wg8KMZ5vCbn9/ec/PvhQY0NYCxCYIZgJRChH0zIKRsEoGAWjABsAAAI/NtnUP9VRAAAAAElFTkSuQmCC","orcid":"","institution":"Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Ziqiang","middleName":"","lastName":"Li","suffix":""},{"id":391832514,"identity":"1a397cb9-78b6-4e68-b3b8-8b3aafc1e49f","order_by":9,"name":"Yuhong Huang","email":"","orcid":"","institution":"Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuhong","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-10-13 14:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5255841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5255841/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72299680,"identity":"1035cdf7-2d42-4a27-be11-96a88b4f2611","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4865219,"visible":true,"origin":"","legend":"\u003cp\u003eSemi-logarithmic (A) and linear (B) plots of the reaction rate versus sucrose concentration from 0.007 to 0.896 mol/L.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/80f0dbc325a92a050a0103fc.png"},{"id":72299685,"identity":"a658b827-fcb8-49b4-8dd6-86478d5339e0","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1370565,"visible":true,"origin":"","legend":"\u003cp\u003eProfiles of the reaction rate versus time at different concentrations of sucrase from 1 to 8 mg/mL.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/9500018043671ed36122ab5a.png"},{"id":72300149,"identity":"321429c9-2c0c-4df0-9b1e-579a23f1c55e","added_by":"auto","created_at":"2024-12-25 01:18:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":893123,"visible":true,"origin":"","legend":"\u003cp\u003eMichaelis-Menten kinetic curves of sucrase.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/3aeea003a1392ac0c217ad6b.png"},{"id":72299682,"identity":"4d825a5d-b1fd-44fe-adc1-a0fdc73f253d","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1885905,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effects of TZQ (A) and acarbose (B) against sucrose.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/0e7da08205800fc5cc1bb2f1.png"},{"id":72299688,"identity":"e67788d7-f6df-4276-abba-a88f7fa7c37f","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5890727,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition kinetic plots of TZQ (A) and acarbose (B) against sucrase.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/4411859869f29ee7ca842562.png"},{"id":72299687,"identity":"c398b6e0-a1fd-46d8-abb2-bc410df745c1","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6102201,"visible":true,"origin":"","legend":"\u003cp\u003eLineweaver-Burk plots of the reversible inhibitory effects of TZQ (A) and acarbose (B) against sucrase.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/40f3d366c51c61ee92f81115.png"},{"id":72299683,"identity":"fce7a1e0-5661-41c6-8a25-fb286c6422ee","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2195711,"visible":true,"origin":"","legend":"\u003cp\u003eFar-UV circular dichroism (CD) spectra of sucrase in the absence and presence of TZQ (A) and acarbose (B)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/38f6ce1877c6e1f5d85094cc.png"},{"id":72299684,"identity":"005d733f-9420-4aa2-bf16-7df6e17925c7","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":203952,"visible":true,"origin":"","legend":"\u003cp\u003eBase peak ion chromatograms of TZQ in positive ion (A) and negative ion (B) modes.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/d5c58ea81782ef0c2821eb94.png"},{"id":72299686,"identity":"a1860d34-f603-420f-b93b-1aa7b84209c9","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":842529,"visible":true,"origin":"","legend":"\u003cp\u003eDetailed results of molecular docking. The blue solid line represents hydrogen bonding; the gray dashed line represents hydrophobic bonding; yellow represents carbon and red represents oxygen in the small-molecule ligands; dark blue represents carbon; red represents oxygen; and blue represents nitrogen in the macromolecular protein.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/b017870250ee3ba380640049.png"},{"id":72301019,"identity":"fddd7138-3294-4bbd-ac1d-cee92a83efef","added_by":"auto","created_at":"2024-12-25 01:27:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25558751,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/ef0dc681-805f-4419-9633-f0de01fd1d1b.pdf"},{"id":72299690,"identity":"56b0127c-e12b-4ff1-8d67-bca39cb921be","added_by":"auto","created_at":"2024-12-25 01:10:40","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5824098,"visible":true,"origin":"","legend":"","description":"","filename":"MaterialS1.png","url":"https://assets-eu.researchsquare.com/files/rs-5255841/v1/7c0551fd88124e7180f36144.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibitory Mechanism of Tangzhiqing on Sucrase: Changed inhibitory kinetics and secondary molecular conformation of sucrase and chemical components from Tangzhiqing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetes mellitus (DM), namely, \u0026ldquo;xiaoke\u0026rdquo; of traditional Chinese medicine, is a metabolic disease associated with significant morbidity and mortality[1]. In China, the morbidity of DM is approximately 10%, and the number of diabetic patients has reached 114 million, accounting for approximately one-third of the number of diabetic patients worldwide. Among them, type 2 diabetes mellitus (T2DM) patients accounts for more than 90% of the amount of DM[2]. Controlling postprandial hyperglycemia could be considered an effective strategy to treat and prevent T2DM[3]. \u0026alpha;-Glucosidase inhibitors are highly important therapeutic strategies. Currently prescribed \u0026alpha;-glucosidase inhibitors, such as acarbose, voglibose and miglitol, have shown various side effects, including bloating, diarrhea, flatulence, pain and abdominal discomfort. Since complementary and alternative medicine is effective in the management of T2DM, the search for novel and safer \u0026alpha;-glucosidase inhibitors from herbal medicine is considered a hot topic of research.\u003c/p\u003e\n\u003cp\u003eTangzhiqing (TZQ), a component-based Chinese medicine, has the potential to be an \u0026alpha;-glycosidase inhibitor for the prevention and treatment of prediabetes or mild T2DM. It is mainly composed of five kinds of Chinese medicinal herbs, including danshen root, red peony root, hawthorn leaves, mulberry leaves, and lotus leaves (Table 1). TZQ consists of Mulberry leaf alkaloids, Mulberry leaf flavonoids, Mulberry leaf polysaccharide, Lotus leaf alkaloids, Lotus leaf flavonoids, Hawthorn leaf flavonoids, Danshen polyphenols, and red Paeony saponins in a fixed proportion (0.8: 0.1: 14: 2.9: 1.8: 0.2: 0.8: 3, w/w)\u0026nbsp;[4]. In 2010, TZQ tablets were approved for clinical trials by the official National Medical Products Administration (NMPA). Our previous studies revealed that TZQ has the same effect as acarbose in reducing postprandial plasma glucose without any gastrointestinal adverse events in healthy Chinese volunteers[5]. Moreover, the hypoglycemic effect of TZQ increases with increasing dose according to dose-exposure-response\u0026nbsp;analysis[6,7]. Additionally, TZQ can decrease fasting insulin and glycosylated hemoglobin levels in T2DM patients[8].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eLatin name for the Traditional Chinese medicine of TZQ\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"546\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eEnglish name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eLatin name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 161px;\"\u003e\n \u003cp\u003eChinese name\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eDanshen root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cem\u003eSalvia miltiorrhiza\u0026nbsp;\u003c/em\u003eBge.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 161px;\"\u003e\n \u003cp\u003eDanshen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eRed Paeony root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cem\u003ePaeonia veitchii\u0026nbsp;\u003c/em\u003eLynch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 161px;\"\u003e\n \u003cp\u003eChishao\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eHawthorn leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cem\u003eCrataegu spinnatifida\u0026nbsp;\u003c/em\u003eBge.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 161px;\"\u003e\n \u003cp\u003eShanzhaye\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eMulberry leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cem\u003eMorus alba\u003c/em\u003e L.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 161px;\"\u003e\n \u003cp\u003eSangye\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eLotus leaf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cem\u003eNelumbo nucifera\u003c/em\u003e Gaertn.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 161px;\"\u003e\n \u003cp\u003eHeye\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eInhibition of brush border \u0026alpha;-glucosidase may be one of the mechanisms by which TZQ lowers postprandial plasma glucose. \u0026alpha;-Glucosidase inhibitors can inhibit the hydrolysis of disaccharides or oligosaccharides on the small intestinal mucosa to reduce the absorption of carbohydrates, thereby resulting in a lower blood sugar level after dinner. Our previous eight-period crossover studies preliminarily revealed that TZQ has evident effects on postprandial glycemia resulting from sucrose and maltose by inhibiting sucrase and maltase, respectively\u0026nbsp;[9]. Further studies confirmed that the interaction of TZQ and maltase induces enzyme inhibition and conformational changes\u0026nbsp;in maltase\u0026nbsp;[10].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSucrase, a hydrolytic enzyme, breaks down sucrose into its monomers, glucose and fructose. The secondary structure of sucrase has been studied, and three acidic amino acids play a vital role in its active site. However, the effects of TZQ on the activity and structural changes of sucrase are still unclear. Herein, we studied the enzyme inhibition and conformational changes in sucrase induced by the interaction of TZQ and sucrase. Acarbose was selected as a positive control in this study. This work will be highly important for providing a scientific basis for the utilization and development of TZQ, a component-based Chinese medicine.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eBrush edge sucrase was from Yishengyuan Gene Technology (Tianjin) Co., LTD.. \u0026nbsp;Baker\u0026rsquo;s yeast sucrase, sucrose, and acarbose were obtained from Sigma‒Aldrich (Shanghai, China), TS Corporation (Seoul, Korea), and the National Institutes for Food and Drug Control (Beijing, China), respectively. TZQ, a component-based Chinese medicine, was provided by Buchang Shenzhou Pharmaceutical Co., Ltd. (Shandong, China). The Q-marker component of TZQ was preferable to nuciferine, with a content of 5.52 mg/g.\u003c/p\u003e\n\u003ch2\u003eSuitable concentrations and reaction times\u003c/h2\u003e\n\u003cp\u003ePurified sucrose was used as an indicator substrate. The tested concentrations of sucrose were 0.007, 0.014, 0.028, 0.056, 0.112, 0.224, 0.448, and 0.896 mol/L. Fifty microliters of 10 mg/mL sucrase was premixed with 0.5 mL of the test sample in 0.5 mL of phosphate buffer (pH 6.8) and incubated at 37\u0026deg;C for 5 min. Then, 20 \u0026mu;L of sucrose as the substrate was added to the mixture to start the reaction. The mixture was incubated at 37\u0026deg;C for 16 min and stopped by the addition of 0.1 mL of sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, 0.2 mol/L) solution. The optimized concentration of sucrose was used to determine a suitable concentration of sucrase. The candidate concentrations of sucrase were 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mg/mL.\u0026nbsp;These reactions were stopped every 2 min to optimize the reaction time. The sucrase activity was determined by measuring the release of glucose at 492 nm via a DNM-9602 automatic microplate reader (PuLang, China). Triplicate samples were created for each test.\u003c/p\u003e\n\u003ch2\u003eEnzyme kinetic equation\u003c/h2\u003e\n\u003cp\u003eUnder the conditions of the optimum reaction time and enzyme concentration determined in section 2.3 with a constant enzyme concentration, the initial rate was determined when the substrate concentration was 0.028, 0.056, 0.112, 0.224, 0.448, and 0.896 mol/L, respectively, and three times for each concentration. The Michaelis constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) and the maximum reaction rate (\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) were calculated via the double reciprocal method.\u003c/p\u003e\n\u003ch2\u003eInhibitory effect of TZQ against sucrase\u003c/h2\u003e\n\u003cp\u003eThe assay of the inhibitory activity of TZQ against sucrase was performed according to the methods in the literature with minor modifications [11, 12]. Briefly, mixtures of 20 \u0026mu;L of sodium phosphate buffer (pH=6.8), 70 \u0026micro;L of sucrase solution (10 mg/mL) and 10 \u0026micro;L of TZQ or acarbose solution were prepared and incubated for 5 min in 96-well plates at a constant temperature of 37\u0026deg;C. The reaction was then initiated by adding 20 \u0026mu;L of sucrose at concentrations ranging from 0.028 to 0.896 mol/L for 14 min at 37\u0026deg;C. The tested concentrations of TZQ were 0.7, 1.4, 2.8, 5.6, 11, and 22 \u0026mu;g/mL. Moreover, acarbose was used as a positive control at different concentrations of 10, 20, 40, 80, 160, 320, and 640 \u0026mu;g/mL. After another 15 min of incubation at 37\u0026deg;C, 100 \u0026mu;L of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution (0.2 mol/L) was added to terminate the reaction, and changes in the absorbance were measured at 492 nm via a DNM-9602 automatic microplate reader. The half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) was used to compare the inhibitory effects of the samples. The relative enzyme activity of the test samples toward sucrase was calculated as follows (Eq. 1):\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"551\" height=\"92\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere A\u003csub\u003esample\u003c/sub\u003e, A\u003csub\u003econtrol\u003c/sub\u003e, and A\u003csub\u003eblank\u003c/sub\u003e are the absorbance of the sample, the absorbance of the blank control without sample, and the absorbance of the samples without sucrase, respectively.\u003c/p\u003e\n\u003ch2\u003eDetermination of the inhibition kinetics of TZQ against sucrase\u003c/h2\u003e\n\u003cp\u003eTo determine the inhibitory characteristics of TZQ, the mechanisms by which the active components of TZQ bind to the sucrase were further explored by analyzing the enzyme kinetics. This inhibition kinetics study was carried out in the presence and absence of TZQ with varying concentrations of sucrose as the substrate. The inhibition type and mechanism were analysed via Lineweaver\u0026ndash;Burk plots, which are plots of the enzyme reaction velocity (V) versus the substrate concentration, and the relevant parameters were calculated via the following equation (Eq. 2):\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"568\" height=\"144\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere V (mol/L/min) is the enzyme reaction rate and Vmax (mol/L/min) and Km (mg/mL) represent the maximum reaction rate and Michaelis constant, respectively. [I] and [S] are the concentrations of the inhibitor and sucrose, respectively. Ki is the inhibition constant. \u0026alpha; denotes the ratio of the noncompetitive inhibition constant to the competitive inhibition constant, and the value of \u0026alpha; for noncompetitive inhibition is 1.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCircular dichroism spectroscopy\u003c/h2\u003e\n\u003cp\u003eThe changes in the secondary structure of sucrase in the presence and absence of TZQ were determined by circular dichroism (CD) spectroscopy. The CD spectra were obtained with a 1 mm quartz cell on a J-810 CD spectrophotometer (American OLIS Company, Carlsbad, CA, USA). The spectra were recorded in the far-UV range of 190\u0026ndash;250 nm under constant nitrogen flush. The concentrations of sucrase and acarbose were both maintained at 0.5 g/L. The concentrations of TZQ were varied from 0 to 0.28 g/L. The signal of sodium phosphate buffer (0.1 mol/L, pH=7.0) was subtracted to obtain the actual CD spectra of sucrase. The CD spectra were scanned three times to obtain the average value. All the data were expressed as CD ellipticity in mdeg. The contents of secondary structures such as \u0026alpha;-helices, \u0026beta;-sheets, \u0026beta;-turns, and random coils of sucrase were analyzed via the online SELCON3 program[13].\u003c/p\u003e\n\u003ch2\u003eDetermination of compounds contained in TZQ\u003c/h2\u003e\n\u003cp\u003eThe identification of components was performed on an Acquity UPLC I-Class instrument coupled with a Xevo G2-SQ-TOF mass spectrometer via an ESI interface (Waters, Milford, MA, USA). Sample separation was carried out at 35\u0026deg;C on a Waters ACQUITY UPLC BEH C18 column (2.7\u0026times;100 mm, 1.7 \u0026mu;m), and the flow rate was 0.3 mL/min. The mobile phases consisted of acetonitrile (solution B) and 0.1% formic acid water (solution A), and the gradient program was as follows: 10% B from 0-2 min, 10-20% B from 2-6 min, 20-30% B from 6-20 min, 30-80% B from 20-30 min, 80-30% B from 30-40 min, and 30-10% B from 40-45 min. The injection volume was 2 \u0026mu;L for all the samples. The mass parameters were set as follows: capillary voltage, 2.0 kV; sampling cone voltage, 40 V; source offset, 80 V; source temperature, 100\u0026deg;C; desolvation temperature, 400\u0026deg;C; cone gas flow, 50 L/h; and desolvation gas flow, 600 L/h. The scan MS data ranged from 50 to 1200 Da. In MSE mode, the trap collision energy of the low-energy function was set at 6 eV, and the trap collision energy of the high-energy function was increased from 15 to 60 eV.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eSample preparation\u003c/h2\u003e\n\u003cp\u003eAn easy-to-implement ultrasonic extraction method was utilized. In detail, 200 mg of the accurately weighed powder of TZQ was soaked in 5 mL of methanol (v/v), which was further extracted in a water bath at 25℃ with ultrasound assistance for 30 min. After 10 min of centrifugation at a rotation speed of 14,000 rpm, the supernatant was diluted to 400 \u0026mu;g/ml with 50% aqueous methanol, and the final concentration was determined.\u003c/p\u003e\n\u003ch2\u003eMolecular Docking\u003c/h2\u003e\n\u003cp\u003eTo explore the effects of TZQ compounds on the structure of sucrase, we performed molecular docking studies. The small molecule compound structure files were searched through the Pubchem website (https://pubchem.ncbi.nlm.nih.gov/). The SDF\u0026nbsp;files were converted into PDB files using Open Babel 2.3.2 software. The sucrase receptor proteins were retrieved from the pdb database. PYMOL 2.3.4 software was used to dewater the ligands, and AutoDockTools software was used to convert the receptor protein and ligand small molecules into pdbqt format. The molecular docking of the receptor proteins with the ligand small molecules was performed with AutoDock Vina 1.1.2. The docking results were visualized via Pymol.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eThe results are expressed as the mean values \u0026plusmn; standard deviations. The statistical analysis was performed with GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA). One-way analysis of variance (ANOVA) and the post hoc Tukey test were used for statistical comparisons among groups. For comparisons within groups in the visual chamber test, a paired t test was used. Statistical significance was assumed at a confidence level of at least 90% (p \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eConfirmation of suitable concentrations and reaction times\u003c/h2\u003e\n\u003cp\u003eThe results revealed that the enzyme reaction rate was directly proportional to the substrate concentration, ranging from 0.007 to 0.896 mol/L (Figure 1). Especially in the low-concentration range from 0.007 to 0.224 mol/L, the first-order kinetic process was displayed with an exponential equation (Y = 0.19lnX + 1.17, r\u0026sup2;=0.996). However, the pattern of the reaction changed to a mixed-order kinetics when the concentration ranged between 0.224 and 0.448 mol/L and tended towards zero-order kinetics when the substrate concentration increased above 0.448 mol/L. Therefore, the excessive concentration of the substrate sucrose was set at 0.448 mol/L.\u003c/p\u003e\n\u003cp\u003eThe profiles of the reaction rate versus time at a series of sucrase concentrations from 1 to 8 mg/mL are shown in Figure 2. To achieve the maximum reaction rate of sucrase in the initial reaction, it is critical to optimize the most suitable sucrase concentration and reaction time. When the concentration of sucrase was 7.0 mg/mL or less, the reaction was linear within 14 min. Therefore, the optimal sucrase concentration and reaction time were confirmed to be 7.0 mg/mL and 14 min, respectively..\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eMichaelis constant and maximum reaction rate\u003c/h2\u003e\n\u003cp\u003eAccording to the intermediate complex hypothesis, the enzyme-catalyzed reaction can be expressed as E+S⇋E.S\u0026rarr;P+E. In Michaelis\u0026ndash;Menten kinetics, \u003cem\u003eV\u003c/em\u003e and 1/\u003cem\u003eV\u003c/em\u003e are calculated from \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e\u0026middot;[S]/(\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e+[S]) and \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e/\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e\u0026middot;1/[S]+1/\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e,\u0026nbsp;respectively. Profiles were\u0026nbsp;obtained\u0026nbsp;via\u0026nbsp;the\u0026nbsp;double reciprocal method, using 1/\u003cem\u003eV\u003c/em\u003e as the longitudinal coordinate and 1/[S]used as the transverse coordinate. As shown in Figure 3, the \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values were 0.53 mol/L/min and 0.066 mol/L, respectively, which indicates that the enzyme-catalyzed reaction is consistent with Michaelis\u0026ndash;Menten kinetics and that the equation can be used to analyze and evaluate the catalytic reaction.\u003c/p\u003e\n\u003ch2\u003eInhibitory effect of TZQ against sucrase\u003c/h2\u003e\n\u003cp\u003eThe inhibitory effects of TZQ and acarbose against sucrase were both concentration dependent and gradually increased with the increasing concentrations of TZQ or acarbose (Figure 4). The IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003ewas calculated by fitting the data via logistic regression. The IC\u003csub\u003e50\u003c/sub\u003e values of TZQ and acarbose were 1.49 \u0026plusmn; 0.07 (Figure 4A) and 3.94 \u0026plusmn; 0.07 \u0026mu;g/mL (Figure 4B), respectively, indicating that TZQ has a considerable inhibitory effect on the standard inhibitor acarbose.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eInhibition kinetics analysis of TZQ against sucrase\u003c/h2\u003e\n\u003cp\u003eThe reversibility of the inhibitory effect of TZQ on sucrase was confirmed by the reaction velocity between different concentrations of sucrase and TZQ (Figure 5). As shown in Figure 5A, all of the straight lines passed through the origin point, and the line slopes decreased with increasing TZQ concentration, which indicated that the presence of TZQ did not reduce the amount of sucrase but rather down-regulated the activity of sucrase. The inhibition of TZQ against sucrase was reversible, which was the same as the inhibition mode of acarbose (Figure 5B). Therefore, we speculated that a noncovalent intermolecular interaction exists between sucrase and the active ingredients of TZQ.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLineweaver-Burk plots in the presence and absence of TZQ were obtained at different substrate concentrations to determine the interaction mechanism between TZQ and sucrase. Figure 6 presents good linear relationships between 1/\u003cem\u003eS\u003c/em\u003e and 1/\u003cem\u003eV\u003c/em\u003e in the double reciprocal curves. As the concentration of TZQ increased, the intercepts of the enzyme reaction rate line on the X-axis (-1/Km) and Y-axis (1/Vmax) increased, whereas the slope remained unchanged. As the TZQ concentration increased from 0 to 2.8 and then to 22 \u0026mu;g/mL, the Vmax decreased from 0.54 to 0.15 and then to 0.06 mol/L/min, and the Km decreased from 0.064 to 0.022 and then to 0.009 mol/L, respectively. The reduced \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u0026nbsp;\u003c/sub\u003eand \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values indicated that the inhibitory pattern of TZQ against sucrase was uncompetitive; that is, TZQ could only bind to the enzyme-substrate complex (E+S) rather than the substrate-interacting residues. Figure 6B presents an invariant intercept at the \u003cem\u003eY\u003c/em\u003e-axis (1/\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e), a decrease in the intercept at the \u003cem\u003eX\u003c/em\u003e-axis (-1/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e), and an increase in the slope of the reaction rate lines of acarbose against sucrase. These results revealed that the positive control acarbose could compete with sucrose for the same active site of the enzyme, which is competitive inhibition.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eInfluences of TZQ on the secondary structure of sucrase\u003c/h2\u003e\n\u003cp\u003eThe conformational changes in the secondary structure of sucrase in the presence of TZQ and acarbose are displayed in Figure 7A and Figure 7B, respectively. The CD spectrum of sucrase exhibited a single negative peak at approximately 210 nm and a strong positive peak at 193 nm, which is characteristic of a \u0026beta;-sheet structure. Following the addition of TZQ, the far UV-CD spectra of sucrase presented two negative peaks at 210 nm and 220 nm, which are the characteristic of n\u0026rarr;\u0026pi;* and \u0026pi;\u0026rarr;\u0026pi;* electron transfer for the peptide bonds of the \u0026alpha;-helix (Figure 7A). The negative ellipticity of sucrase increased with increasing TZQ concentration, which indicated that the secondary structure of sucrase had partially changed. For the positive control acarbose, a similar tendency as that of TZQ was observed in terms of reducing the \u0026beta;-sheet content and increasing the \u0026alpha;-helix content of sucrase (Figure 7B).\u003c/p\u003e\n\u003cp\u003eThe contents of different secondary structures of sucrase were quantitatively determined via the SELCON3 program, and the corresponding results for TZQ and acarbose are listed in Table 2 and Table 3, respectively. Similar patterns of changes were observed in the \u0026beta;-sheet and \u0026alpha;-helix contents of sucrase. In the TZQ group, the proportion of \u0026alpha;-helices increased from 16.5% to 23.4%, whereas the proportion of \u0026beta;-sheets decreased from 43.4% to 25.6% when the concentration increased from 0.07 to 0.28 g/L. In the acarbose group, the proportion of \u0026alpha;-helices increased from 16.5% to 18.2%. In contrast, the proportion of \u0026beta;-sheets decreased from 43.4% to 38.6%. These findings indicated that TZQ interacted with sucrase and influenced its enzymatic activity by inducing structural changes such as those in acarbose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Secondary structure of sucrase in the absence and presence of TZQ\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eSecondary structures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e0.07 g/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e0.14 g/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.28 g/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026alpha;-helix (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e16.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e23.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e22.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026beta;-sheet (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e43.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e39.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e37.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e25.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026beta;-turn (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e14.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e21.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eRandom coil (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e28.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e25.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eTotal (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e. Secondary structure of sucrase in the absence and presence of acarbose\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eSecondary structures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.5 g/L (0.5 h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e0.5 g/L (1 h)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026alpha;-helix (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e16.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e17.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e18.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026beta;-sheet (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e43.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e43.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e38.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026beta;-turn (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e10.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e13.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eRandom coil (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e28.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e30.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eTotal (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003eChemical components identification of TZQ\u003c/h2\u003e\n\u003cp\u003eThe base peak ion chromatograms of TZQ in positive and negative ion modes are shown in Figure 8. Table 4 summarizes the characterization of the chemical constituents of TZQ by UPLC-Q-TOF/MS. On the basis of the precise relative molecular mass data and multistage mass spectrum fragment ions, combined with control articles, databases and literature reports, 64 chemical components in TZQ were identified, among which 42 chemical components were identified in negative ion mode and 22 chemical components were identified in positive ion mode. Among them, 14 components originated from mulberry leaves, 21 components originated from red paeony roots, 16 from danshen root , 14 from hawthorn leaves, and 11 from lotus leaves.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Characterization of the chemical constituents of TZQ by UPLC-Q-TOF-MS.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"926\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003et\u003csub\u003eR\u003c/sub\u003e/min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eCompound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eFormula\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003eIon mode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003ePrecursor ion（m/z）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003eMS/MS（m/z）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.78\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e2-O-\u0026alpha;-D-galactosyranosid-1-deoxynopyrimycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eNO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e326.1420\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e266.12117、248.10992、230.09875、205.06603、104.10495\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.80\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003ePaeonol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e167.0709\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e138.05245、118.08362、109.02500\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.80\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eEthyl 2-hydroxybenzoate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e167.0709\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e148.05807、138.05245、122.05709\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.83\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eVerbascose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e52\u003c/sub\u003eO\u003csub\u003e26\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e873.2719\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e665.21580、503.1617、377.08501、341.10803、211.06540、179.05462\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.83\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eSorbitol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e,[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e181.0702\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e135.06628、146.05846、149.04555、165.07685\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.88\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e(-)-Catechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e289.0690\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e125.02252、132.02887、219.04418、245.04255\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.89\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eQuinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e191.0547\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e101.02299、127.03774、149.04381、173.04277\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.90\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eDehydroroemerine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e278.1202\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e260.10931、203.04914、174.07348\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e1.18\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e8-Debenzoylpaeoniflorin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e421.1352\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e345.11965、150.04100、128.03377\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e3.13\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eDanshensu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e197.0439\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e135.0435\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e3.13\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eEthyl gallate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e197.0439\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e135.0435\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e3.13\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eAnisic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e197.0439\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e135.0435\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e4.73\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eNeochlorogenic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e353.0869\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e179.03421、135.04334\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e5.86\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eDemethyl-Coclaurine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e270.1130\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e162.0542\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e6.87\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eChlorogenic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e353.0872\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e191.05441、133.02824\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e7.48\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eScopolin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e353.0865\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e223.05997、191.05387、179.03270、173.04415\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e8.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eQuercetin 3, 7-o-beta-d-dipyranoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e17\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e625.1407\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e462.08101、301.03342、299.01916\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e9.57\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003ePaeonol glucoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e505.1565\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e447.13117、445.13390、285.07496、165.05419、121.02814\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e10.13\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eAlbiflorin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e481.1669\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e319.11483、197.07893、133.06243、105.03120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e10.15\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eAlbiflorinR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e, [M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e525.1607\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e357.11805、283.08201、146.96483、121.02795\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e11.27\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eL-Asparagine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e133.0625\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e115.05179、103.05176\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e11.28\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003epaeoniflorin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e, [M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e525.1617\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e449.14490、431.13458、327.10776、283.08152、165.05421、121.02812\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e11.28\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eOxypaeoniflora\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e495.1503\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e449.14490、431.13458、327.10776、283.08152、165.05421、121.02812\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e12.18\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eArmepavine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e314.1718\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e283.13001、268.10534、252.11160、189.07843、107.04914\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e12.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eQuercetin 3-sambubioside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e595.1313\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e300.02585、271.02455、243.02887、146.96484\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e12.70\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eN-Methylcoclaurine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e300.1563\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e283.12993、237.08776、189.08789、174.06452、107.04683\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e13.16\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e611.1628\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e303.0499\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e14.14\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eIsoquercitrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e465.0993\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e303.04681、229.04758、115.05193、105.03101\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e14.20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003ePaeoniolide C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e319.1160\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e133.00989、105.03101\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e14.20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003ePaeoniflorigenone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e319.1160\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e133.00989、105.03101\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e14.43\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e2-O-Rhamnosylvitexin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e577.1562\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e457.11388、413.08698、301.03406、293.04416、271.02405、151.00192\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e14.43\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eHawthorn A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e413.0874\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e301.03406、293.04416、271.02405、151.00192\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e14.74\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eSalvianolic acid D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e463.0880\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e300.02629、271.02356、255.02835、151.00202\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e14.91\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eO-Nornuciferine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e282.1457\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e251.10369、219.07753、208.08535、189.06710、165.06714\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e16.65\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eSalvianolic acid C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e537.1045\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e339.04999、295.05872、146.96452、109.02801\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e19.31\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eRosmarinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e359.0764\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e255.02848、179.03348、161.02275、133.02787、121.02786\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e19.31\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e7-Hydroxycoumarin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e161.0229\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e151.03795、135.04361、133.02787、121.02786\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e20.74\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eSalvianolic acid A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e493.1132\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e311.05425、293.04487、146.96438、109.02784\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e22.08\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eNuciferin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e296.1615\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e265.11951、235.07248、219.07729、191.15789、178.07505、165.06722、122.01285\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e22.18\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eSalvianolic acid G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e339.0498\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e321.03913、293.04405、185.02270、109.02798\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e22.20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eMudanpiosideC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e599.1751\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e519.09301、339.04981、321.03913、293.04405、185.02270、109.02798\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e22.20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eBenzoyloxypaeoniflorin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e599.1751\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e519.09301、339.04981、321.03913、293.04405、185.02270、109.02798\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e22.64\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eLuteolin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e285.0392\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e133.02771、271.98653\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e22.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eSalvianolic acid B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e36\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e717.1457\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e599.17763、519.09260、473.08708、399.04933、321.03929、293.04397、185.02285、109.02793\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e23.21\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eIsorhamnetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e317.0633\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e250.09586、237.08798、207.07754、189.06716、178.07511\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e23.21\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eIsorhamnetin3-O-glucoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e477.1033\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e315.04952、313.03569、300.02815、271.02483、165.01766、146.96444\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e23.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eVitexin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e477.1033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e315.04952、300.02815、271.02482、165.01766、146.96444\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e23.29\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eMonomethyl lithospermate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e551.1198\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e519.09359、353.06552、321.03942、293.04469、231.02809、146.96444\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e23.67\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003ePrzewaquinone E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e313.1039\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e299.10886、189.06848、178.07455\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e24.36\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e285.0394\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e211.07459、146.96431\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e24.55\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eMudanpioside J\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e31\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e629.1877\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e583.18139、553.17095、431.13560、284.03089、146.96436、121.02785\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e24.55\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eBenzoyl paeoniflorin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e, [M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e629.1877\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e583.18139、553.17095、431.13560、284.03089、146.96436、121.02785\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e28.01\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eMiltionone Ⅰ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e313.1423\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e300.28683、212.23423、141.06794、129.06746\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e28.43\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eMoracin C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e309.1112\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e147.9642\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e28.89\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003ePaeonenolide F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e46\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e555.3317\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e487.34280、437.30452、423.32549、405.31648、393.31590\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e28.98\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eNeocryptotanshinone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e313.1434\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e269.15325、226.10103、213.12609\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e28.98\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003e4-Prenylresveratrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e297.1457\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e179.08266、165.06818、152.05940\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e28.98\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eIsocryptotanshinone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e297.1457\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e211.14450、195.11873、189.06818、141.06762\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e31.21\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eTanshinone I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e277.0832\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e249.08887、196.10914、152.05954\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e31.27\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eTanshinoneIIB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e297.1456\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e254.09120、178.07516、115.05180\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e31.53\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eMaslinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e, [M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e471.3479\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e453.33668、299.20013、227.10574\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e32.08\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eHederagenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e, [M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e517.3529\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e423.32552、393.31559\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e32.35\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eOplopanaxogenin C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e48\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e, [M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e517.3535\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e295.22646、277.21606、227.07053、177.08926\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e35.05\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 191px;\"\u003e\n \u003cp\u003eMicrostegiol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 144px;\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e297.1856\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 265px;\"\u003e\n \u003cp\u003e281.19109、205.12340\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003eMolecular docking results\u003c/h2\u003e\n\u003cp\u003eThe ability of TZQ active ingredients to bind to sucrase was investigated through molecular docking studies. The representative docking information of the TZQ active ingredients and sucrase is shown in Figure 9. The sucrase residues GLN-232, LEU-233, LYS-509, ARG-555, and SER-631 formed hydrogen bonds with paeoniflorin, while the surrounding important residues TRP-327, TRP-435, and PHE-604 were hydrophobic with paeoniflorin, in which TRP-327 also formed P-type \u0026pi; stacking with the ligands (Figure 9A). The same docking revealed hydrogen bonds between the sucrase residues ASP-231, SER-631 and the vitexin ligand (Figure 9B). Hydrophobic interactions occur between residues TRP-327 and PHE-479, in which T-type \u0026pi; stacking is formed between the residue TRP-435 ligands (Figure 9B). There are also residues LYS-509, ASP-571, and HIS-629 that form hydrogen bonds with chlorogenic acid ligands, while the hydrophobic interactions are residues LEU-233, TRP-327, TRP-435, and a salt bridge is also formed between LYS-509 (Figure 9C). Salvianolic acid A residues ASP-231, ASP-355, ASP-472, LYS-509, and ASP-632 formed hydrogen bonds with the ligand (Figure 9D). Hydrophobic interactions with the sucrase residues LEU-233, TRP-327, TRP-435, meanwhile TRP-327 also formed P type \u0026pi; stacking. The remaining docking results were shown in Supplementary Material S1. Table 5 summarizes the binding energies of these TZQ active ingredients and sucrase. The docking data revealed that the interaction between the TZQ compounds and sucrase was relatively stable.. Among them, paeoniflorin, vitexin, chlorogenic acid, and salvianolic acid A may have a greater effects on the structure of sucrase. The sucrase structure is affected by \u0026pi; accumulation or salt bridge formation, thus inhibiting its activity and playing a role in lowering glucose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e. The binding energy of sucrase and small-molecule compounds\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"542\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eChemical compound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003eBinding energy（kcal/mol）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eSalvianolic acid A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eSalvianolic acid C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-8.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eDNJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eChlorogenic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eNuciferine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eMaslinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eVitexin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-6.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-7.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 254px;\"\u003e\n \u003cp\u003ePaeoniflorin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 289px;\"\u003e\n \u003cp\u003e-7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eTZQ has a certain inhibitory effect on glycosidases[4], but the specific type and mechanism of inhibition are still unclear, which is not conducive to its rational application and clinical promotion. Therefore, the inhibitory ability of TZQ to invertase activity was investigated in the present study. The inhibitory effect of TZQ on sucrase activity (IC\u003csub\u003e50\u003c/sub\u003e = 1.49 \u0026plusmn; 0.07 \u0026mu;g/mL) was superior to that of acarbose (IC\u003csub\u003e50\u003c/sub\u003e = 3.94 \u0026plusmn; 0.07 \u0026mu;g/mL). As a disaccharide, sucrose is a common carbohydrate eaten by the human body and consists of an \u0026alpha;-glucose molecule and a \u0026beta;-fructose molecule connected by \u0026alpha;-(1,2)-\u0026beta;-glycosidic bonds\u0026nbsp;[14].\u0026nbsp;When encountering either alpha-glucosidase or a beta-fructofuronidase, sucrose can be hydrolyzed into an alpha-D-glucose molecule and a beta-D-fructose molecule\u0026nbsp;[15, 16]. The hydrolysis reactions of sucrose are catalyzed by these two enzymes.\u0026nbsp;These results indicate that the effect of TZQ on \u0026alpha; - (1,2) - \u0026beta; - glycosidic bonds is similar to that of acarbose\u0026nbsp;[17, 18].In terms of enzyme kinetics, there are four types of inhibition, namely, competitive, noncompetitive, anti-competitive, and mixed.\u0026nbsp;Noncompetitive inhibitors present the same affinity for enzymes and enzyme‒substrate complexes. Anti-competitive inhibitors bind only to enzyme‒substrate complexes, whereas competitive inhibitors compete with substrates for the same active site of the enzyme.\u0026nbsp;Regardless of reversibility, the inhibitory effects of TZQ and acarbose on sucrase are the same, with a straight line passing through the origin\u0026nbsp;(Figure\u0026nbsp;5).\u0026nbsp;The slope of the straight line with TZQ is smaller than that without TZQ, and the slope gradually decreases with the\u0026nbsp;increasing of concentration, which is a typical feature of reversible inhibition\u0026nbsp;[19].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe kinetics of enzymes can be used to determine enzyme modification pathways and provide a reference for clinical treatment. According to the results of the double reciprocal kinetic curve of the sucrase reaction rate, TZQ was determined to have a noncompetitive inhibitory effect, similar to white tea extract[20], whereas acarbose was determined to have a competitive inhibitory effect (Figure 6). These results suggested that TZQ and acarbose bind to different targets of sucrase. An open-system study on enzyme inhibition revealed that anti-competitive inhibitors (expected to produce permanent inhibitory effects) may have more effective hypoglycemic effects than competitive inhibitors (expected to have only temporary inhibitory effects) [21]. These findings may provide some reference for explaining the mechanism by which TZQ inhibits sucrase activity more effectively than acarbose does.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCircular dichroism (CD) spectroscopy is an effective method for analyzing structural changes after interactions between proteins and active molecules. An analysis of the CD spectrum changes in the wavelength range of 190\u0026ndash;250 nm revealed that the molecular conformation of sucrase changed after the addition of TZQ, and the \u0026alpha;-helix structure gradually decreased, whereas the \u0026beta;-sheet gradually increased. As the concentration increases, the ratio of the two contents gradually approaches one another. For acarbose, the \u0026alpha;-helix structure and \u0026beta;-sheet ratio changed similarly with increasing reaction time, but its degree was not as significant as that of TZQ. Moreover, the \u0026beta;-turn content is similar to the changes in the \u0026alpha;-helix content. The change in the CD spectrum of the protein secondary structure of the enzyme suggested that some active components of TZQ may combine with sucrase, which promotes the unfolding of the protein polypeptide chain and destroys the \u0026alpha;-(1,2)-\u0026beta;-glycosidic bond hydrogen bond network structure of sucrase. The secondary structure of the enzyme becomes loose, which affects enzyme activity and prevents the substrate from binding to the active site of the enzyme[22, 23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate the pharmacological basis of the inhibitory effect of TZQ on sucrase, we employed high-resolution mass spectrometry to identify the main components of TZQ. A total of 64 chemical ingredients were identified from TZQ via the UPLC‒Q-TOF‒MS method (Table 3). On the basis of previous reports in the literature 24-30, potential compounds with a certain degree of sucrose enzyme inhibition from the 64 compounds were selected. Salvianolic acid C and salvianolic acid A have been reported to have potent \u0026alpha;-glucosidase inhibitory activities, with IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalues of 4.31 and 19.29 \u0026mu;M, respectively. Additionally, salvianolic acid C exhibited mixed-competitive inhibition when it was bound to \u0026alpha;-glucosidase [24]. DNJ and fagomine significantly inhibited the activity of sucrase-mediated intestinal acetone powder in rats, with IC\u003csub\u003e50\u003c/sub\u003e values of 0.14 and 35\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u0026mu;g/mL, respectively [25]. Chlorogenic acid has also been reported to inhibit \u0026alpha;-glycosidase activity, and the IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalues of chlorogenic acid and its isomer were found to be 2.99 and 3.12 mM, respectively[26]. Nuciferine and Paeoniflorin have been reported to have hypoglycemic effects and beneficial effects on the treatment of type 2 diabetes[27, 28]. Vitexin also has an inhibitory effect on a-glycosidase activity，with an IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalue of 4.1 \u0026mu;g/mL [29]. Meanwhile animal experiments have confirmed that maslinic acid has a good hypoglycemic effect[30]. Therefore, 9 of the potential compounds were subjected to molecular docking with sucrase (Table 4). The compound ligands and sucrase interact mostly through hydrophobic interactions, hydrogen bonds, \u0026pi; stacking and salt bridges, thereby altering the structure of sucrase and inhibiting its activity. Further research on the composition and hypoglycaemic mechanism of TZQ compounds is instructive. Compounds with specific hypoglycemic compounds were screened for molecular docking, and the molecular structure changes between the remaining residual compounds and sucrase need to be explored.\u003c/p\u003e\n\u003cp\u003eIn this study, the structural changes in and molecular mechanism underlying the inhibitory activity of TZQ against sucrase were investigated via multiple spectroscopic methods. TZQ notably inhibited sucrase in a reversible and uncompetitive manner. Additionally, the inhibitory activity of TZQ against sucrase was considerable compared with that of the positive control acarbose. Circular dichroism revealed that the binding of TZQ to sucrase causes a rearrangement of the secondary structure of sucrase, with an increase in the content of \u0026alpha;-helix components and a decrease in the content of \u0026beta;-sheet components, therefore inhibiting enzyme activity. The structural relationships between the compounds and sucrase were initially explored via molecular docking. This study provides a scientific basis for the mechanism of interaction between TZQ and sucrase, which may be useful for the potential application of TZQ as an effective \u0026alpha;-glycosidase inhibitor to treat type 2 diabetes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, TZQ showed notable inhibitory activity against sucrase in a reversible and uncompetitive manner. Circular dichroism results revealed that the binding of TZQ to sucrase causes a rearrangement of the secondary structure of sucrase, with an increase in the content of \u0026alpha;-helix components and a decrease in the content of \u0026beta;-sheet components, therefore inhibiting enzyme activity. A total of 64 chemical ingredients were identified from TZQ via UPLC-Q-TOF/MS. In addition to having a binding energy of less than -5 kcal/mol, the potential active ingredients of TZQ, including paeoniflorin, vitexin, chlorogenic acid, and salvianolic acid A, strongly affect the structure of sucrase through \u0026pi; accumulation or salt bridge formation, thus inhibiting its activity and lowering glucose. These findings provide new insight into the inhibitory mechanism of TZQ on sucrase, which may be beneficial for the reasonable use of component-based Chinese medicine to prevent type 2 diabetes mellitus as a novel \u0026alpha;-glycosidase inhibitor.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTZQ Tangzhiqing\u003c/p\u003e\n\u003cp\u003eUPLC-Q-TOF/MS Ultraperformance liquid chromatography-quadrupole-time \u003c/p\u003e\n\u003cp\u003eof flight-mass spectrometry\u003c/p\u003e\n\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e The half-maximal inhibitory concentration\u003c/p\u003e\n\u003cp\u003eDM Diabetes mellitus\u003c/p\u003e\n\u003cp\u003eT2DM Type 2 diabetes mellitus\u003c/p\u003e\n\u003cp\u003eNMPA National Medical Products Administration\u003c/p\u003e\n\u003cp\u003ePBS Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003ePE Polyethylene\u003c/p\u003e\n\u003cp\u003eCD Circular dichroism\u003c/p\u003e\n\u003cp\u003eUV Ultraviolet\u003c/p\u003e\n\u003cp\u003eUPLC Ultra performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eSDF Spatial Data Format\u003c/p\u003e\n\u003cp\u003ePDB Protein Data Bank\u003c/p\u003e\n\u003cp\u003eDNJ 1-Deoxynojirimycin\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (No. 81573741) and the Tianjin Municipal Education Commission Research Project (No. 2021KJ159).\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eYL and ZL designed the experiments. YL, MZ, RW, and ZL wrote the main manuscript text. XX, TJ, FW, and XG conducted the experiments. ST and ZL prepared the figures and analyzed the data. SZ and YH supervised the experiments. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; information\u003c/h2\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eSecond Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300250, China.\u0026nbsp;\u003csup\u003e2\u003c/sup\u003eTianjin University of Traditional Chinese Medicine, Tianjin 301617, China. \u003csup\u003e3\u003c/sup\u003eSchool of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDou ZL, Xia Y, Zhang JW, Li YZ, Zhang YN, Zhao L,et al .Syndrome Differentiation and Treatment Regularity in Traditional Chinese Medicine for Type 2 Diabetes: A Text Mining Analysis. 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European Journal of Pharmacology . 2022; 919-174769\u003c/li\u003e\n \u003cli\u003eChoo CY, Sulong NY, Man F,\u0026nbsp;Wong\u0026nbsp;TW.Vitexin and isovitexin from the Leaves of Ficus deltoidea with in-vivo a-glucosidase inhibition. Journal of Ethnopharmacology. 2012; 142,776\u0026ndash;781\u003c/li\u003e\n \u003cli\u003eMkhwanazi BN., Serumula MR, Myburg RB, Van Heerden FR, Musabayane CT. Antioxidant effects of maslinic acid in livers, hearts and kidneys of streptozotocin-induced diabetic rats: effects on kidney function. Ren Fail.2014; 36(3): 419\u0026ndash;431\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tangzhiqing (TZQ), Sucrase, α-Glucosidase, Inhibitory kinetics, Molecular conformation, Circular dichroism, UPLC-Q-TOF/MS, Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-5255841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5255841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u0026nbsp;\u003c/strong\u003eTangzhiqing (TZQ), a component-based herbal medicine, has been reported to have postprandial hypoglycemic effects on sucrose in humans. Sucrase, a type of α-glucosidase enzyme, breaks down sucrose into its monomers, glucose and fructose. This study investigated the inhibitory activity of TZQ against sucrase and the conformational changes in sucrase induced by interactions between sucrase and TZQ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u0026nbsp;Sucrose was used as an indicator substrate. After confirming the suitable reaction concentrations and time, an assay of the inhibitory activity of TZQ against sucrase was performed with a series of TZQ concentrations ranging from 0.7 to 22 μg/mL. The inhibition kinetics were analyzed in the presence and absence of TZQ or acarbose. Acarbose was used as a positive control drug. The changes in the secondary structure changes of sucrase in the presence and absence of TZQ were determined by circular dichroism spectroscopy. To investigate the pharmacological basis of the inhibitory effect of TZQ, we used a UPLC-Q-TOF/MS method to identify TZQ components. Molecular docking studies were carried out to explore the binding ability of sucrase and potential active ingredients in TZQ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u0026nbsp;TZQ showed a notable inhibitory activity against sucrase in a reversible and uncompetitive manner. The half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values of TZQ and acarbose were 1.49 ± 0.07 and 3.94 ± 0.07 μg/mL, respectively, indicating that TZQ possesses has a considerable inhibitory effect similar to that of acarbose. Circular dichroism results revealed that the binding of TZQ to sucrase causes a rearrangement of the secondary structure of sucrase , with an increase in the content of α-helix components and a decrease in the content of β-sheet components, therefore inhibiting enzyme activity. A total of 64 chemical ingredients were identified from TZQ via UPLC-Q-TOF/MS. In addition to having a binding energy of less than -5 kcal/mol, the potential active ingredients of TZQ, including paeoniflorin, vitexin, chlorogenic acid, and salvianolic acid A, strongly affect the structure of sucrase through π accumulation or salt bridge formation, thus inhibiting its activity and lowering glucose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u0026nbsp;These findings provide a new insight into the inhibitory mechanism of TZQ on sucrase, which may be beneficial for the reasonable use of component-based Chinese medicine to prevent type 2 diabetes mellitus as a novel α-glycosidase inhibitor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number\u0026nbsp;\u003c/strong\u003e：Not applicable.\u003c/p\u003e","manuscriptTitle":"Inhibitory Mechanism of Tangzhiqing on Sucrase: Changed inhibitory kinetics and secondary molecular conformation of sucrase and chemical components from Tangzhiqing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-25 01:10:35","doi":"10.21203/rs.3.rs-5255841/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-27T16:18:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-20T18:04:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-20T13:46:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52897362097445834398157247486316118139","date":"2025-01-16T06:46:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220434402746475346443187950889092910154","date":"2025-01-07T20:19:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105820578624287138682442339880681844954","date":"2025-01-01T07:10:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184775939239737103333538447376594563670","date":"2025-01-01T02:55:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-31T00:48:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168992996973184200613590854743178506126","date":"2024-12-30T21:53:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-30T07:09:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-18T10:18:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-25T08:54:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-24T11:25:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2024-10-24T11:24:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2db86446-cbe2-43f9-8c56-32d2fda15b14","owner":[],"postedDate":"December 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T18:24:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-25 01:10:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5255841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5255841","identity":"rs-5255841","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}