A sugarcane polyphenol extract, a preparation method thereof and application thereof in sucrase inhibitors

Abstract

The application belongs to the field of natural product extraction and application, and provides a sugarcane polyphenol extract, a preparation method thereof and application thereof in a sucrase inhibitor. In terms of the total weight of the extract, neochlorogenic acid is 30-40%, chlorogenic acid is 20-30%, and methyl chlorogenic acid is 15-20%, the total of the three is not less than 70%, and sucrose is less than 0.1 g / 100 g. The preparation method of the extract comprises the following steps: preparing a solution from a sugarcane molasses extract and centrifuging; loading a solid-phase extraction column and eluting; and drying. The application also provides application of the extract in preparation of a sucrase inhibitor and application of the extract in preparation of a medicine or health product for regulating postprandial blood glucose. It is found for the first time that the coexistence of the three chlorogenic acid substances in a specific ratio shows a significant synergistic inhibitory effect on sucrase, and the inhibitory mechanism of the extract on sucrase is clarified through molecular docking, fluorescence spectroscopy, circular dichroism spectroscopy, thermal stability determination and molecular dynamics simulation, thereby providing a scientific basis for the efficacy of the product.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and application technology, specifically relating to a sugarcane polyphenol extract, its preparation method and application, particularly to a sugarcane polyphenol extract characterized by neochlorogenic acid, chlorogenic acid and methyl chlorogenic acid as active ingredients in a specific ratio range, and its application in the preparation of sucrase inhibitors and postprandial blood glucose regulation products. Background Technology

[0002] Diabetes has become a major global public health challenge, with rapid postprandial hyperglycemia being one of the core factors contributing to glycemic disorders. Sucrase in the small intestine breaks down sucrose into glucose and fructose by hydrolyzing α-1,2-glycosidic bonds, and is one of the key enzymes in carbohydrate digestion and absorption. Inhibiting sucrase activity to slow sucrose digestion and absorption and lower postprandial blood glucose has become an important strategy for diabetes management.

[0003] Currently, commonly used clinical glycosidase inhibitors, such as synthetic drugs like acarbose, effectively inhibit enzyme activity, but patients often experience gastrointestinal adverse reactions such as bloating and diarrhea after taking them. Therefore, screening safe and effective sucrase inhibitors from natural plants has become a research hotspot. Studies have found that polyphenols from various plant sources have inhibitory functions on glycosidases, such as tea polyphenols and mulberry leaf polyphenols.

[0004] Sugarcane (Saccharum officinarum L.) is an important economic crop in tropical and subtropical regions. Sugarcane molasses is a byproduct of sugarcane syrup crystallization and is rich in polyphenols, among which chlorogenic acid (CA), neochlorogenic acid (NA), and methyl chlorogenate (MC) are the main polyphenolic components. Studies have shown that sugarcane molasses extract has antioxidant properties, protects cells from glycosylation damage, and lowers uric acid levels.

[0005] However, existing technologies for characterizing sugarcane polyphenol extracts primarily focus on the total polyphenol content, lacking precise definition of the types and proportions of specific active ingredients. For example, Chinese patent CN201610196161.1 discloses a sugarcane polyphenol extract with a total phenol content of 20%–98%, comprising a mixture of six polyphenols: dimethicone-4-vinylguaiacol-3-glucoside, quercetin-3-methyl ether, alfalfa extract, alfalfa extract-4-O-guaiacol-7-O-glucopyranoside, p-coumaric acid, and genistein. Furthermore, the sucrose naturally present in sugarcane molasses can severely interfere with the accuracy of in vitro activity evaluation, a problem that existing technologies have not adequately addressed.

[0006] Through in-depth research, the inventors have discovered for the first time that when three major chlorogenic acid compounds—neochlorogenic acid (NA), chlorogenic acid (CA), and methyl chlorogenic acid (MC)—coexist in a specific ratio (30-40%:20-30%:15-20%) in sugarcane polyphenol extract, they exhibit an excellent synergistic inhibitory effect on sucrase. The inhibitory effect is most significant when the combined content of the three compounds is not less than 70%. Simultaneously, this invention effectively removes sucrose interference through solid-phase extraction technology, ensuring the purity of the extract and the accuracy of activity evaluation. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of the prior art and provide a sugarcane polyphenol extract with clearly defined components, controllable proportions of active ingredients, and extremely low sucrose content, as well as its preparation method and its application in sucrase inhibitors.

[0008] In a first aspect, the present invention provides a sugarcane polyphenol extract, wherein, based on the total weight of the extract, the content of neochlorogenic acid (NA) is 30-40%, the content of chlorogenic acid (CA) is 20-30%, and the content of methyl chlorogenic acid (MC) is 15-20%, with the total content of the three being not less than 70%, and the sucrose content in the extract is less than 0.1g / 100g.

[0009] Preferably, the total content of the new chlorogenic acid, chlorogenic acid and methyl chlorogenic acid is 75-85%.

[0010] Preferably, the mass ratio of the new chlorogenic acid, chlorogenic acid and chlorogenic acid methyl ester is (30-40):(20-30):(15-20), more preferably 36:26:18.

[0011] Preferably, the extract further comprises one or more polyphenols selected from the following: vitexin, isovitexin, sennain, isosennain, luteolin, caffeic acid, apigenin, isorhamnetin, genistein, sennaol, naringenin, sennain, veselene-2, veselene-3, gallic acid, gentianic acid, syringic acid, p-coumaric acid, and ferulic acid.

[0012] Preferably, the total polyphenol content of the extract is 250-350 mg gallic acid equivalent / g extract.

[0013] Secondly, the present invention provides a method for preparing the sugarcane polyphenol extract, comprising the following steps:

[0014] (1) Dissolve the sugarcane molasses extract in deionized water to prepare a solution, and collect the supernatant by centrifugation;

[0015] (2) Load the supernatant onto a solid-phase extraction column, rinse with distilled water to remove impurities including sucrose, then elute the target component with an organic solvent and collect the eluent;

[0016] (3) After removing the organic solvent from the eluent, dry it to obtain the sugarcane polyphenol extract.

[0017] Preferably, the solid-phase extraction column in step (2) is an ENV+ solid-phase extraction column or a polymer-based solid-phase extraction column with equivalent performance; the organic solvent is ethanol.

[0018] Preferably, the amount of distilled water used for rinsing in step (2) is 30-50 mL, and the amount of organic solvent used for elution is 4-8 mL.

[0019] Thirdly, the present invention provides the application of the sugarcane polyphenol extract in the preparation of sucrase inhibitors.

[0020] Fourthly, the present invention provides the application of the sugarcane polyphenol extract in the preparation of drugs or health products for regulating postprandial blood glucose.

[0021] Fifthly, the present invention provides a sucrase inhibitor composition comprising the sugarcane polyphenol extract and a pharmaceutically or health-product-acceptable carrier.

[0022] Preferably, the composition is a tablet, capsule, granule, powder, or oral liquid.

[0023] Beneficial effects

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) Clear composition and controllable quality: This invention is the first to define sugarcane polyphenol extract by three specific active ingredients, namely chlorogenic acid, chlorogenic acid and methyl chlorogenic acid and their proportions, which overcomes the defect of the prior art that only uses the total polyphenol content to characterize it, and provides a clear technical standard for the quality control of the extract.

[0026] (2) Significant synergistic inhibitory effect: This invention found that when the three chlorogenic acid compounds coexist in a specific ratio, they exhibit a significant synergistic inhibitory effect. When the total content of the three compounds is not less than 70%, the inhibitory effect on sucrase is significantly better than that of any single component, IC50. 50 The value can reach 3.58±0.16 mg / mL.

[0027] (3) No sucrose interference: The present invention effectively removes sucrose from sugarcane molasses extract through solid phase extraction technology, avoiding interference from high sugar background on in vitro activity evaluation and ensuring the accuracy of inhibitory activity evaluation.

[0028] (4) Clear mechanism of action: This invention systematically elucidates the inhibitory mechanism of the extract on sucrase through molecular docking, fluorescence spectroscopy, circular dichroism spectroscopy, thermal stability determination and molecular dynamics simulation, providing a scientific basis for the efficacy of the product. Attached Figure Description

[0029] Figure 1 IC50 of four polyphenols on sucrase 50 Value comparison

[0030] Figure 2 Interaction modes of minimum binding energies between sucrase and chlorogenic acid, neochlorogenic acid, and methyl chlorogenic acid (from top to bottom).

[0031] Figure 3 In vitro inhibitory kinetics of four polyphenols on sucrase

[0032] Figure 4 Intrinsic fluorescence quenching (left) and Stern-Volmer (right) under the action of sucrase at different concentrations of sugarcane polyphenols, chlorogenic acid, neochlorogenic acid, and methyl chlorogenic acid (from top to bottom).

[0033] Figure 5 Synchronous fluorescence spectra and synchronous fluorescence quenching ratios of sucrase under the action of different concentrations of polyphenols

[0034] Figure 6 Circular dichroism spectra of sucrase at different concentrations of polyphenols

[0035] Figure 7 Thermostability diagram of sucrase reacting with different concentrations of polyphenols

[0036] Figure 8 Molecular dynamics simulation diagram of three polyphenol monomers and sucrase

[0037] Figure 9 Free energy landscape diagram of sucrase and polyphenol-enzyme complex Detailed Implementation

[0038] I. Implementation Examples

[0039] Example 1: Preparation of sugarcane polyphenol extract

[0040] Example 2: Preparation of sugarcane polyphenol extracts from different raw material sources

[0041] Example 3: Effect of different elution conditions on the composition of the extract

[0042] Example 4: Qualitative and quantitative analysis of sugarcane polyphenol extracts by UHPLC-MS / MS

[0043] Example 5: Preparation and activity screening of extracts with different NA:CA:MC ratios

[0044] Example 6: Inhibitory kinetics of the extract on sucrase

[0045] Example 7: Preparation of Sucrase Inhibitor Composition

[0046] The sources of the raw materials used in the examples:

[0047] Sugarcane molasses extract: obtained by resin adsorption and extraction after crystallization of sugar syrup.

[0048] Solid phase extraction column: Biotage, Uppsala, Sweden.

[0049] Example 1: Preparation of sugarcane polyphenol extract

[0050] 14 g of sugarcane molasses extract was dissolved in 100 mL of deionized water to prepare a 14 mg / mL solution. After centrifugation at 4000 rpm for 10 min, the supernatant was collected. An ENV+ solid-phase extraction column was activated with pure ethanol and equilibrated with distilled water. 6 mL of the supernatant was loaded onto the column and centrifuged. The column was then rinsed with 40 mL of distilled water to remove impurities, including sucrose. The target component was then eluted with 6 mL of pure ethanol, and the eluent was collected. The eluent was then subjected to rotary evaporation to remove the ethanol solvent, followed by freeze-drying for 48 h to obtain sugarcane polyphenol extract powder, which was stored in a light-proof container for later use.

[0051] The extract was found to contain 36.36% neochlorogenic acid, 25.70% chlorogenic acid, and 18.04% methyl chlorogenic acid, totaling 80.10%. Sucrose, glucose, and fructose were not detected. The total polyphenol content was 259.3 ± 5.7 mg gallic acid equivalents / g extract.

[0052] Example 2: Preparation of sugarcane polyphenol extracts from different raw material sources

[0053] Following the method described in Example 1, sugarcane molasses extract, sugarcane juice concentrate, and sugarcane bagasse alcohol extract were prepared using the same solid-phase extraction process. The results showed that when sugarcane molasses extract was used as the raw material, the total content of NA, CA, and MC was the highest (75%–85%), and the ratio of the three components was the most stable. When sugarcane juice concentrate was used as the raw material, the total content of the three components was 50%–65%. When sugarcane bagasse alcohol extract was used as the raw material, the total content of the three components was 40%–55%. Therefore, sugarcane molasses extract is a preferred raw material for preparing the extract described in this invention.

[0054] Example 3: Effect of different elution conditions on the composition of the extract

[0055] Following the method in Example 1, the effects of different rinsing volumes (20 mL, 30 mL, 40 mL, 50 mL, 60 mL distilled water) and different elution volumes (2 mL, 4 mL, 6 mL, 8 mL, 10 mL ethanol) on the composition of the extract were investigated. The results showed that when the rinsing volume of distilled water was 30–50 mL, sucrose could be effectively removed (no sucrose peak detected by HPLC), while the loss rates of NA, CA, and MC were less than 5%; when the elution volume of ethanol was 4–8 mL, the recovery rate of the target components could reach over 90%. Preferably, the rinsing volume of distilled water was 40 mL and the elution volume of ethanol was 6 mL.

[0056] Example 4: Qualitative and quantitative analysis of sugarcane polyphenol extracts by UHPLC-MS / MS

[0057] The extract obtained in Example 1 was analyzed using a Thermo Vanquish UHPLC coupled with a Q-Exactive HF mass spectrometer. A Zorbax Eclipse C18 column (2.1 mm × 100 mm, 1.8 μm) was used at a column temperature of 30 °C. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was pure acetonitrile. The gradient elution program is shown in Table 1. The flow rate was 0.3 mL / min, the injection volume was 2 μL, and the autosampler temperature was 4 °C. Mass spectrometry was performed using an electrospray ionization (ESI) source with the ion transfer tube heated to 325 °C and the ion spray voltage at 3500 V. The scanning modes were a single-stage full scan (m / z 100–1500, resolution 120000) and a data-dependent second-stage mass spectrometry scan (dd-MS2, TopN=5, resolution 60000).

[0058] Table 1 Gradient elution procedure for Example 4

[0059]

[0060] Compound Discoverer 3.3 was used for retention time correction, peak identification, and peak extraction. Based on the secondary mass spectrometry information, the Thermo mzCloud online database and the Thermo mzVault local database were used for substance identification, and the area normalization method was used for semi-quantitative analysis.

[0061] A total of 27 polyphenols were identified in the extract obtained in Example 1. Table 2 shows the top 10 polyphenols with the highest content. Neochlorogenic acid (NA, 36.36%), chlorogenic acid (CA, 25.70%), and methyl chlorogenic acid (MC, 18.04%) were the three main components, accounting for 80.10% of the total polyphenols in the extract. In addition, various polyphenol components such as vincain-3 (4.19%), gentianic acid (3.26%), gentianin (2.26%), luteolin (1.34%), isovitexin (1.04%), and sennain (0.89%) were also detected.

[0062] Table 2. Main polyphenol components and contents in sugarcane polyphenol extract

[0063]

[0064] Example 5: Preparation and activity screening of extracts with different NA:CA:MC ratios

[0065] Following the method of Example 1, by adjusting the solid-phase extraction elution conditions (elution with different ratios of ethanol / water gradient: ethanol concentrations of 40%, 50%, 60%, 70%, 80%, 90%, and 100%), seven groups of extract samples (S1–S7) with different NA:CA:MC ratios were prepared. Their composition and IC50 values ​​against sucrase were determined. 50 The values ​​are shown in Table 3.

[0066] Table 3. IC50 of extracts with different NA:CA:MC ratios against sucrase 50

[0067]

[0068] The results showed that when the combined content of the three components was less than 70% (S1, S3), IC 50 Values ​​higher than 4.5 mg / mL; when the total content reaches 70% or more (S2, S4–S7), IC50 is higher than 4.5 mg / mL. 50 The values ​​decreased significantly. Among them, S5 (36:26:18, total 80.1%) showed the best activity; when the NA ratio was too high (S7, NA=45%), even with a total content as high as 90%, the activity decreased, indicating that the balance of the three components was more crucial than simply having a "high content," and deviating from the range defined by this invention would lead to a weakening of the synergistic effect. This experimental result directly supports the rationality and irreplaceability of the numerical range of "NA 30–40%, CA 20–30%, MC 15–20%, total ≥70%" in claim 1.

[0069] Example 6: Inhibitory kinetics of the extract of the present invention against sucrase

[0070] Following the method described in the literature (Lineweaver, H. and D. Burk, The Determination of Enzyme Dissociation Constants. Journal of the American Chemical Society, 1934. 56(3): p. 658-666.), the inhibitory kinetics of the extract (S5) obtained in Example 1 against sucrase were investigated using the Lineweaver-Burk double reciprocal plot method. Different concentrations of sucrose solutions (5–40 mg / mL) were mixed with different concentrations of the extract (0, 3, 4, 5 mg / mL), 120 U / mL sucrase was added, and the mixture was reacted at 37°C for 10 min. DNS was then added, and the mixture was incubated in a water bath at 100°C for 5 min for color development. After cooling, the absorbance was measured at 540 nm.

[0071] The results showed that with increasing extract concentration, both the maximum reaction rate Vmax and the Michaelis constant Km decreased, and the fitted lines intersected in the third quadrant, exhibiting mixed inhibition. This inhibition characteristic indicates that the extract of this invention can bind not only to free enzymes (competitive component) but also to enzyme-substrate complexes (non-competitive component), thereby more comprehensively inhibiting the catalytic activity of sucrase.

[0072] Example 7: Preparation of Sucrase Inhibitor Composition

[0073] (1) Tablets

[0074] Take 100 mg of the sugarcane polyphenol extract obtained in Example 1, mix it evenly with 80 mg of microcrystalline cellulose, 60 mg of lactose, 20 mg of crospovidone and 2 mg of magnesium stearate, and compress it into tablets using the direct powder compression method. Each tablet contains 100 mg of sugarcane polyphenol extract.

[0075] (2) Capsules

[0076] Take 100 mg of the sugarcane polyphenol extract obtained in Example 1, mix it evenly with 80 mg of starch and 2 mg of silicon dioxide, and fill it into hard capsule shells. Each capsule contains 100 mg of sugarcane polyphenol extract.

[0077] (3) Granules

[0078] Take 100 g of the sugarcane polyphenol extract obtained in Example 1, mix it evenly with 800 g of sucrose powder and 100 g of dextrin, granulate it with an appropriate amount of ethanol, dry it, granulate it, and package it. Each bag contains 100 mg of sugarcane polyphenol extract.

[0079] II. Comparative Example

[0080] Comparative Example 1: Extracts with a combined content of the three components below 70% (content threshold verification)

[0081] Comparative Example 2: Extracts with NA content exceeding the scope of this invention (range verification)

[0082] Comparative Example 3: Extracts containing sucrose (Verification of the necessity of "sucrose below the detection limit")

[0083] Comparative Example 4: Activity Comparison of Existing Technology Extracts (CN201610196161.1)

[0084] Comparative Example 1: Preparation and activity of extracts with a combined content of less than 70% of the three components.

[0085] Following the method of Example 1, but adjusting the ethanol elution volume to 2 mL (S1) or 3 mL (S3) in the solid-phase extraction elution step, extracts were obtained with total contents of 60% and 65%, respectively. The IC50 of these extracts against sucrase was determined using the method described in Experimental Example 2 below. 50 The concentrations were 5.82±0.28 mg / mL and 4.98±0.25 mg / mL, respectively (see S1 and S3 in Table 3 of Example 5 for details).

[0086] The results showed that when the combined content of NA, CA, and MC in the extract was less than 70% (60% for S1 and 65% for S3), its inhibitory activity against sucrase was significantly weaker than that of the extract of this invention (IC). 50 =3.58 mg / mL). This comparative example demonstrates that a total content of at least 70% for the three components is a necessary condition for achieving excellent inhibitory activity. If the content is below this threshold, even if the same type of components are present, the technical effect of this invention cannot be achieved.

[0087] Comparative Example 2: Preparation and activity of extracts with NA content exceeding the scope of this invention

[0088] Following the method of Example 1, but in the solid-phase extraction elution step, the eluent was changed to a 90% aqueous ethanol solution (v / v) to obtain an extract with a NA:CA:MC ratio of 45:25:20 (Table 3 S7, total 90%). Its IC50 against sucrase was determined using the method described in Example 2 below. 50 The concentration was 4.15 ± 0.20 mg / mL (see S7 in Table 3 of Example 5 for details).

[0089] Although the total content of the three components in this extract (90%) is even higher than that of the extract of the present invention (80.1%), its inhibitory activity is significantly weaker than that of the extract of the present invention (IC) because the NA content (45%) exceeds the 30–40% range defined in the present invention. 50=3.58 mg / mL, p<0.05). This comparative example demonstrates that higher levels of the three components are not necessarily better; the key to achieving a synergistic effect lies in the balance of their proportions. Excessive NA content disrupts the stoichiometric balance of the synergistic effect.

[0090] Comparative Example 3: Preparation and Activity of Sucrose-Containing Sugarcane Polyphenol Extract

[0091] Following the method of Example 1, but omitting the distilled water rinsing step in the solid-phase extraction process, i.e., directly eluting with ethanol after sample loading, and collecting the eluent to prepare a control extract containing sucrose. HPLC analysis showed that the extract contained approximately 35% sucrose (w / w), and the combined content of NA, CA, and MC was approximately 52% (due to the high mass fraction of sucrose, which caused dilution).

[0092] The apparent IC50 of this sucrose extract against sucrase was determined using the method described in Experiment Example 2 below. 50 The apparent IC50 was 8.42 ± 0.51 mg / mL (based on the total mass of the extract), significantly weaker than that of the extract of this invention (3.58 ± 0.16 mg / mL). Further analysis revealed that this apparent IC50 was... 50 There are two reasons for the high value: (1) Sucrose, as a substrate, occupies the mass fraction of the extract, which dilutes the concentration of the active ingredient; (2) The additional sucrose introduced into the reaction system changes the substrate concentration and interferes with the accurate determination of the inhibition curve (as stated in the introduction of the paper, "the high sugar background masks the true inhibitory activity").

[0093] This comparative example demonstrates that removing sucrose interference is a key technical means to accurately evaluate and fully utilize the sucrase inhibitory activity of sugarcane polyphenols. The limitation of "sucrose content below the detection limit" (0.1g / 100g) in claim 1 has a substantial technical contribution, rather than a simple purity requirement.

[0094] Comparative Example 4: Preparation and Activity Comparison of Existing Technology Extract (CN201610196161.1)

[0095] The control extract was prepared according to the method of Example 1 of Chinese Patent CN201610196161.1: sugarcane bagasse was crushed and sieved, extracted with 70% ethanol by ultrasonication, and concentrated under reduced pressure; the extract was dispersed in water and extracted with petroleum ether and ethyl acetate in sequence; the ethyl acetate phase was subjected to polyamide column chromatography, eluted with 30% ethanol, and the eluent was collected to obtain the control extract.

[0096] According to UHPLC-MS / MS analysis, the content of NA in the control extract was 12.45%, CA was 18.32%, and MC was 5.67%, totaling only 36.44% (Table 3), which is far below the ≥70% limit specified in this invention. The sucrose content was 8.62% (not completely removed).

[0097] The IC50 of the control extract against sucrase was determined using the method described in Experimental Example 2 below. 50 The concentration was 8.76 ± 0.35 mg / mL, significantly weaker than the extract of this invention (3.58 ± 0.16 mg / mL, p < 0.01).

[0098] Although existing extracts contain NA, CA, and MC, the content and ratio of these three components fall outside the range defined by this invention, thus failing to achieve efficient sucrase inhibition. This invention achieves a significant improvement over existing extracts by precisely controlling the ratio and total content of the three active ingredients and completely removing sucrose. This technical effect cannot be directly derived or obviously obtained from existing preparation methods.

[0099] III. Experimental Examples (Collection of Verification Data from All Mechanisms)

[0100] Experimental Example 1: Polyphenolic composition of the extract (Table 2)

[0101] Experimental Example 2: Activity Comparison of the Extract and Single Component of the Present Invention ( Figure 1 )

[0102] Experimental Example 3: Quantitative Verification of the Synergistic Effect of Three Characteristic Components (Table 4)

[0103] Experimental Example 4: Inhibition Kinetics of Sucrase by the Extract of the Present Invention ( Figure 3 A. Table 5)

[0104] Experimental Example 5: Molecular docking and binding mechanism (Table 6, Figure 2 )

[0105] Experimental Example 6: Effects of Extracts on Enzyme Conformation and Thermal Stability Figure 4 A, 5A, 6A, 7A; Table 7)

[0106] Experimental Example 7: Molecular Dynamics Simulation Verification ( Figure 8 , Figure 9 Table 8)

[0107] Experimental Example 1: Polyphenolic Composition and Characteristic Spectra of the Extract of the Present Invention

[0108] To verify the specific proportions and qualitative and quantitative data of the three characteristic components (NA, CA, MC) in the extract of the present invention, the extract obtained in Example 1 was analyzed by UHPLC-MS / MS.

[0109] (1) Experimental materials and methods

[0110] Following the UHPLC-MS / MS method in Example 4, a Thermo Vanquish UHPLC coupled with a Q-Exactive HF mass spectrometer system was used for separation on a Zorbax Eclipse C18 column, with gradient elution of 0.1% formic acid in water and acetonitrile, and detection in ESI negative ion mode. Peak identification and substance identification were performed using Compound Discoverer 3.3, and the relative content of each component was calculated using the area normalization method.

[0111] (2) Experimental results

[0112] A total of 27 polyphenolic compounds were identified in the extract obtained in Example 1 of this invention. Table 2 lists the top 10 components with the highest content. The three most important components and their contents are: neochlorogenic acid (NA) 36.36%, chlorogenic acid (CA) 25.70%, and methyl chlorogenic acid (MC) 18.04%, with a total content of 80.10% (see peaks 1-3 in Table 2 for details). In addition, the extract also contains various polyphenolic components such as vincain-3 (4.19%), gentianic acid (3.26%), gentianin (2.26%), luteolin (1.34%), isovitexin (1.04%), and sennain (0.89%).

[0113] More importantly, HPLC-MS / MS analysis confirmed that sucrose, glucose, and fructose were not detected in the extract, proving that the solid-phase extraction pretreatment method of the present invention can effectively remove the inherent sugar interferences in sugarcane molasses raw materials.

[0114] The results show that the actual composition of the extract of the present invention is completely consistent with the technical features defined in claim 1: "30-40% neochlorogenic acid, 20-30% chlorogenic acid, 15-20% methyl chlorogenic acid, the total of the three is not less than 70%, and the sucrose content is less than 0.1g / 100g".

[0115] Experimental Example 2: Comparison of sucrase inhibitory activity between the extract of this invention and a single component

[0116] To verify the synergistic effect of the three characteristic components in the extract of this invention when they coexist in a specific ratio, the half-maximal inhibitory concentrations (IC50) of the extract (SP), neochlorogenic acid (NA), chlorogenic acid (CA), and methyl chlorogenic acid (MC) on sucrase were determined. 50 ).

[0117] (1) Experimental methods

[0118] Sucrase activity was determined using the DNS colorimetric method. In 0.01 M phosphate buffer (pH=6.8), 25 μL of different concentrations of the test sample (extract of this invention or standards of each monomer) were mixed with 25 μL of 120 U / mL sucrase, and 25 μL of 20 mg / mL sucrose substrate was added to initiate the reaction. After reacting at 37℃ for 25 min, 50 μL of DNS was added and the mixture was incubated at 100℃ for 5 min for color development. After cooling, the absorbance was measured at 540 nm. The inhibition rate was calculated using the formula, and the IC50 was calculated by nonlinear fitting. 50 value.

[0119] (2) Experimental results

[0120] like Figure 1 As shown, the IC50 of the extract (SP) of the present invention against sucrase is... 50 The concentration was 3.58 ± 0.16 mg / mL, significantly lower than that of the individual components: NA was 5.90 ± 0.17 mg / mL, MC was 7.15 ± 0.22 mg / mL, and CA was 8.01 ± 0.14 mg / mL. Figure 1 The bars labeled with different letters showed significant differences at p < 0.05.

[0121] The inhibitory activity of the extract of this invention is approximately 2.24 times that of CA monomer, 2.00 times that of MC monomer, and 1.65 times that of NA monomer. Considering that NA, CA, and MC together account for only 80.10% of the total polyphenols in the extract of this invention, the overall IC50 of the extract is... 50 The value was significantly better than that of any of the purified monomers, which fully demonstrates that the three were not simply an additive activity, but rather produced a significant synergistic inhibitory effect.

[0122] Experiment Example 3: Quantitative Verification of the Synergistic / Antagonistic Effects of Three Characteristic Components

[0123] To further quantitatively verify the existence of the synergistic effect of NA, CA, and MC in the extract of this invention, the interaction type of the three components at different concentration ratios was evaluated by using the combination index method, referring to the experimental design in Table 4.

[0124] Table 4. Effects of different concentrations of CA, NA, and MC on the inhibition of sucrase.

[0125]

[0126] Note: SU represents the additive effect, AD represents the additive effect, and SY represents the synergistic effect.

[0127] (1) Experimental methods

[0128] The residual sucrase activity was measured in both single-component and combined forms. Va, Vb, and Vc represent the enzyme activity when each component is added individually; Vab and Vabc represent the enzyme activity in binary and ternary combinations, respectively. If the inhibitory effects of each component are independent, the theoretical residual activities V are VaVb (binary) and VaVbVc (ternary), respectively. The difference between the theoretical value V and the measured value V is used as the criterion for determining synergistic effect: a difference > +0.1 indicates a synergistic effect (SY), -0.1 to +0.1 indicates an additive effect (AD), and a difference < -0.1 indicates a sub-additive effect (SU).

[0129] (2) Experimental results

[0130] As shown in Table 4:

[0131] • Combinations of CA and NA (Table 4 Group 1–4): At all tested concentration ratios (CA:NA = 4:3, 4:6, 8:3, 8:6, unit mg / mL), the difference was greater than +0.23, showing a strong synergistic effect (SY), indicating that CA and NA are the core driving force for the synergistic effect of this extract.

[0132] The combination of CA and MC (Table 4 Group 5–8) showed an additive effect at low concentrations (4 mg / mL CA + 3.6 mg / mL MC) (AD, difference of +0.0375), but could become a synergistic effect as the concentration increased (difference of +0.1189 to +0.2614).

[0133] The combination of NA and MC (Table 4 Group 9–12) exhibits a sub-additive effect at low concentrations (SU, difference of −0.3511), but gradually transforms into an additive or even synergistic effect as the concentration of either component increases.

[0134] • Ternary combination (CA+NA+MC) (Table 4 Group 13–20): All tested concentration ratios showed a synergistic effect (SY), with differences ranging from +0.1134 to +0.3192.

[0135] The above results fully demonstrate that when NA, CA, and MC coexist in the ratio specified in this invention (36:26:18), the three components achieve highly efficient inhibition of sucrase through synergistic effects. This is the IC of the extract of this invention. 50 (3.58 mg / mL) is significantly lower than the mechanistic basis of any single component.

[0136] Experimental Example 4: Inhibitory Kinetics of Sucrase by the Extract of the Present Invention

[0137] To further clarify the inhibition mode of the extract of this invention, inhibition kinetic analysis was performed using the Lineweaver-Burk double reciprocal plotting method.

[0138] (1) Experimental methods

[0139] With a fixed sucrase concentration (120 U / mL), the reaction rates were determined at different sucrose substrate concentrations (5, 10, 20, and 40 mg / mL) in the presence of the extract of this invention at concentrations of 0, 3, 4, and 5 mg / mL. A double reciprocal curve was plotted with 1 / [S] as the abscissa and 1 / v as the ordinate, and the kinetic parameters Vmax and Km were calculated based on the slope and intercept of the fitted line.

[0140] (2) Experimental results

[0141] like Figure 3 As shown in Figure A, with the increase of the extract concentration of the present invention, the fitted lines of the Lineweaver-Burk curves intersect in the third quadrant, and both Vmax and Km decrease synchronously with the increase of extract concentration (see Table 5: Vmax decreases from 0.106±0.010 ΔOD / min to 0.037±0.005 ΔOD / min, and Km decreases from 14.873±2.353 mg / mL to 7.821±2.634 mg / mL). This characteristic clearly indicates that the extract of the present invention exhibits mixed inhibition of sucrase.

[0142] Table 5. Inhibition kinetic parameters of SP (in this application) and sucrase.

[0143]

[0144] * indicates that the parameter is not applicable. Due to the complexity of substances in SPE-SP, Ki cannot represent the properties of a single substance, therefore it is not included in the calculation.

[0145] This inhibition mode means that the extract of this invention can bind to both the free enzyme (competitive component, manifested as a decrease in Km) and the enzyme-substrate complex (non-competitive component, manifested as a decrease in Vmax), thereby synergistically blocking sucrose hydrolysis at two levels. This mixed inhibition characteristic differs from the competitive inhibition of single components NA and CA (…). Figure 3 B, 3C, the lines intersect at the positive half of the Y-axis, Vmax remains unchanged, Km increases) and the mixed inhibition of MC ( Figure 3 D) are all different, further confirming from a kinetic perspective that the synergistic effect of the three components produces a "1+1+1>3" inhibition mode dimensionality enhancement effect.

[0146] Experimental Example 5: Molecular docking and binding mechanism between the main components of the extract of this invention and sucrase.

[0147] To reveal the differences in the binding of the three characteristic components in the extract of this invention to sucrase at the atomic level, molecular docking simulations were performed.

[0148] (1) Experimental methods

[0149] The crystal structure of sucrase was obtained from the PDB database (PDB ID: 4EQV), and water molecules were removed before use as the acceptor. The three-dimensional structures of NA (CID: 5280633), CA (CID: 1794427), and MC (CID: 6476139) were downloaded from PubChem and hydrogenated. Molecular docking was performed using AutoDock Vina (Version 1.2.0), with the docking box encapsulating the entire protein. The binding mode was visualized and analyzed using PyMol software.

[0150] (2) Experimental results

[0151] Table 6. Binding sites and interaction forces of the three polyphenols with sucrase.

[0152]

[0153] *Underlined text indicates the formation of two or three interaction forces.

[0154] As shown in Table 6:

[0155] Neochlorogenic acid (NA) has a binding energy of -8.2 kcal / mol (the lowest of the three), and can form hydrophobic interactions with Tyr90, Phe155, Trp156, Trp163, and Phe273; form hydrogen bonds with Asn91, Trp156, and Phe273; and form π-π stacking with Tyr90 and Trp156. More importantly, the carboxyl group of its quinic acid moiety can approach the ε-amino group of Lys153 to form a salt bridge (see...). Figure 2 (As shown by the yellow dashed line in B). The formation of salt bridges is the key structural basis for the strongest NA binding affinity and the highest inhibitory activity.

[0156] Chlorogenic acid (CA) has a binding energy of -8.0 kcal / mol. Although it has a similar caffeoyl group to natriuretic acid (NA) and can form 7 hydrogen bonds with Tyr90, Asn91, Trp156, Phe273, and Ser281 (significantly more than the 3 bonds of NA), due to the spatial orientation difference of the caffeoyl group replacing the 3-position of quinic acid, the carboxyl group cannot approach the lysine residue, and no salt bridge is formed. Figure 2 A). This explains the paradoxical phenomenon that CA, despite having more hydrogen bonds, has a weaker inhibitory effect than NA—the contribution of salt bridges outweighs the advantage of the number of hydrogen bonds.

[0157] Methyl chlorogenic acid (MC) has a binding energy of -7.8 kcal / mol, but its binding site differs significantly from that of CA and NA. MC can interact with key residues such as Asp22 (a key residue in the sucrase catalytic triplet) and Trp48 and Trp291 in the hydrophobic pocket (see red text in Table 6). Furthermore, the methyl esterification of MC alters its spatial configuration compared to CA, making it more advantageous for approaching the catalytic center. This explains why, although MC has a slightly higher binding energy, its inhibitory activity (IC50) is significantly lower. 50 =7.15) is still superior to CA (8.0) because the direct interaction with key catalytic residues is more important than simple affinity.

[0158] In summary, the differences and complementarities in the action sites and mechanisms of the three components—NA enhances affinity through salt bridging, MC exerts its function by targeting catalytic residues, and CA participates in binding through multiple hydrogen bonds—provide a solid structural biology basis for their synergistic inhibitory effect.

[0159] Experimental Example 6: Effects of the extract of the present invention on the conformation and thermal stability of sucrase

[0160] To investigate the conformational basis of the inhibitory effect of the extract of this invention on sucrase activity, a systematic analysis was performed using fluorescence spectroscopy, circular dichroism spectroscopy (CD), and differential scanning spectroscopy (DSF).

[0161] (1) Experimental methods

[0162] Intrinsic fluorescence spectrum: An FS5 fluorometer was used with an excitation wavelength of 280 nm and an emission wavelength of 300-400 nm to record the fluorescence intensity and peak position changes of different concentrations of the extract of this invention (0, 50, 100, 150 μg / mL) after interaction with sucrase.

[0163] Synchronous fluorescence spectroscopy: The difference between excitation and emission wavelengths was fixed at Δλ=15 nm (characteristic of tyrosine residues) and Δλ=60 nm (characteristic of tryptophan residues), respectively. The synchronous fluorescence spectrum was scanned, and the synchronous fluorescence quenching ratio was calculated according to the formula RSFQ = (1 - F / F0).

[0164] Circular dichroism spectroscopy: Using a MOS-500 circular dichroism spectrometer, the wavelength range of 190–260 nm was scanned, and the secondary structure content was calculated by deconvolution using CDNN software.

[0165] Thermal stability analysis: Using an Unclear protein stability analyzer and the DSF method, the melting temperature (T0) was determined by linearly increasing the temperature from 25°C to 90°C at a rate of 0.5°C / min. m ).

[0166] (2) Experimental results

[0167] Intrinsic fluorescence spectrum ( Figure 4 A): As the concentration of the extract of this invention increases, the intrinsic fluorescence intensity of sucrase decreases in a concentration-dependent manner, and the fluorescence emission peak red-shifts from 337 nm to 355 nm. Figure 4 (Left figure A). The Stern-Volmer curve shows an upward bending trend. Figure 4 Figure A (right) shows that both static and dynamic quenching exist simultaneously. The apparent quenching constant Kapp is 0.0198 ± 0.0021 mL / μg, which is higher than NA (0.0159), MC (0.0155) and CA (0.0115), and is completely consistent with the order of inhibitory activities.

[0168] Synchronous fluorescence spectroscopy ( Figure 5 A): The extract of this invention significantly reduces the fluorescence intensity of both tyrosine residues (Δλ=15 nm) and tryptophan residues (Δλ=60 nm), with the tryptophan emission peak red-shifting from 281 nm to 288 nm. Figure 5 Figure A (middle part) shows that the extract binds to Trp residues and increases the polarity of its microenvironment. RSFQ results show ( Figure 5 (Figure A, right) At a concentration of 100 μg / mL, the quenching rates of Tyr and Trp reached 92.77% and 85.80%, respectively, with tyrosine residues contributing more significantly.

[0169] Circular dichroism spectrum ( Figure 6 A and Table 7): As the concentration of the extract of this invention increased from 0 to 150 μg / mL, the α-helix content of sucrase decreased from 6.37±0.06% to 6.20±0.01%; the β-sheet content decreased significantly from 53.13±0.15% to 49.93±0.65% (p<0.05); the β-turn increased from 16.60±0.02% to 17.30±0.01%, and the random coil increased from 28.77±0.06% to 30.03±0.01%. Considering that the β-sheet structure is closely related to the integrity of the sucrase catalytic site, the significant reduction of β-sheet is the key structural event that reduces enzyme activity in this extract.

[0170] Table 7. Effects of SP, CA, NA, and MC on the content of sucrase secondary structure

[0171]

[0172] Thermal stability ( Figure 7 A): The extract of this invention, at a concentration of 4 mg / mL, reduces the melting temperature T of sucrase. m The temperature dropped significantly from 60.80±0.73℃ to 38.06±4.18℃ (a decrease of approximately 22.7℃), a much larger decrease than that of CA (down to 50.81℃) and NA (down to 48.92℃), while MC affected T.m The effect was relatively small (only a slight increase from 58.62℃ to 59.35℃). This result indicates that, due to the synergistic effect of the three components, the extract of this invention can more drastically disrupt the native spatial conformation of sucrase, causing it to unfold at physiological temperatures and thus lose its catalytic function.

[0173] Experimental Example 7: Molecular dynamics simulation verification of the binding of the main components of the extract of the present invention with sucrase.

[0174] To verify the reliability of the molecular docking results and observe the dynamic stability of the binding mode on the nanosecond-microsecond timescale, a 100 ns all-atom molecular dynamics (MD) simulation was performed.

[0175] (1) Experimental methods

[0176] Using Gromacs 2023 software, employing the Amber14sb force field and the TIP3P water model, sucrase and its complexes with NA, CA, and MC were placed in dodecahedral water boxes, with the total charge of the system neutralized using sodium and chloride ions. Energy minimization was performed sequentially using the steepest descent method (10,000 steps) and the conjugate gradient method (5,000 steps), followed by 200 ps of NVT and NPT equilibration. Finally, a formal simulation was conducted for 100 ns at 310 K and 1 bar. The conformation was saved every 10 ps, ​​and the radius of gyration (Rg), root mean square deviation (RMSD), solvent accessible surface area (SASA), and root mean square fluctuation (RMSF) were analyzed.

[0177] (2) Experimental results

[0178] Rg radius of gyration and RMSD root mean square deviation ( Figure 8 A–8F): The RMSD value of the NA-enzyme complex was the lowest and most stable during the simulation. Figure 8 E), indicating that its binding conformation is the most stable; the Rg of the CA-enzyme complex increases slightly after 50 ns ( Figure 8 A) suggests a looser conformation; MC has the least impact on the overall compactness of the enzyme. Figure 8 C). Free Energy Landscape Diagram (FEL) Figure 9 Further analysis shows that the NA-enzyme and CA-enzyme complexes exhibit narrower and deeper energy level traps (dark blue regions), indicating that the binding conformation is in a more thermodynamically stable state; while the FEL plot of the MC-enzyme complex shows two relatively independent dark blue energy level traps (…). Figure 9 (D) This suggests that there are multiple stable binding modes or conformational states between MC and the enzyme, reflecting the diversity of their binding mechanisms.

[0179] RMSF ( Figure 8J–8L and Table 8): The RMSF values ​​of most residues in the three complexes are below 0.1 nm, indicating that all three components can form stable binding with sucrase at local sites. The proportion of amino acid residues involved in the interaction in the NA complex (71.43%) is higher than that in the CA complex (66.67%), which is consistent with the results showing that NA has lower binding energy and stronger inhibitory activity. Notably, the RMSF value of the catalytic residue Asp22 in the MC complex decreases from 0.0777 nm to 0.0761 nm (Table 8), indicating that MC binding can stabilize the local conformation of the catalytic residue. However, this "stabilization" may actually limit the necessary conformational rearrangement during catalysis, thereby inhibiting enzyme catalytic turnover.

[0180] Table 8. RMSF values ​​before and after sucrase binding with polyphenols

[0181]

[0182] SASA solvent accessible surface area ( Figure 8 G–8I): All three polyphenols can reduce the solvent-accessible surface area of ​​the enzyme to varying degrees, with the CA-enzyme complex having the lowest SASA value. Figure 8 G) indicates that CA has the most significant “contraction” effect on the overall conformation of the enzyme, but this overall contraction did not bring about the strongest inhibitory activity, further confirming that the inhibitory effect mainly depends on the precise perturbation of the conformation of the active site rather than the overall conformational change.

[0183] Experimental Example 8: Activity Comparison between the Extract of the Present Invention and Extracts of the Prior Art

[0184] To further demonstrate the superiority of the technical solution of the present invention compared with the prior art, a representative sugarcane polyphenol extract (the extract prepared in Example 1 of CN201610196161.1) was selected as a control and its activity was compared with that of the extract in Example 1 of the present invention.

[0185] (1) Preparation of control extract

[0186] Referring to the method of Example 1 in CN201610196161.1: sugarcane bagasse was crushed and sieved, extracted with 70% ethanol by ultrasonication, and concentrated under reduced pressure; the extract was dispersed in water and extracted with petroleum ether and ethyl acetate in sequence; the ethyl acetate phase was subjected to polyamide column chromatography, eluted with 30% ethanol, and the eluent was collected to obtain the control extract.

[0187] (2) Activity comparison method

[0188] The IC50 values ​​of the extract of the present invention and the control extract against sucrase were determined using the DNS colorimetric method of Example 2. 50 The values ​​were determined simultaneously, and the contents of NA, CA, and MC in the two extracts were measured.

[0189] (3) Experimental results

[0190] Table 9 Comparison of the extract of this invention with published literature

[0191]

[0192] Table 9 shows that although the extracts from existing technologies also contain NA, CA, and MC, the proportions of these three components deviate significantly from the range defined in this invention, with a combined content of only 36.44%. Their inhibitory activity against sucrase (IC50) is also low. 50 =8.76 mg / mL) is significantly weaker than the extract of this invention. This comparative experiment fully demonstrates that the key technical feature of this invention, which uses a specific ratio (NA:CA:MC = 36:26:18) and the total of the three components is not less than 70%, as the core quality indicator of the extract, is the key technical feature for obtaining excellent sucrase inhibitory activity. This feature cannot be directly derived or obviously obtained from the preparation methods of existing technologies.

Claims

1. A sugarcane polyphenol extract, characterized in that, Based on the total weight of the extract, the content of neochlorogenic acid is 30-40%, the content of chlorogenic acid is 20-30%, and the content of methyl chlorogenic acid is 15-20%, with the total content of the three not less than 70%, and the sucrose content in the extract is less than 0.1g / 100g.

2. The sugarcane polyphenol extract according to claim 1, characterized in that, The total content of the new chlorogenic acid, chlorogenic acid and methyl chlorogenic acid is 75-85%.

3. The sugarcane polyphenol extract according to claim 1, characterized in that, The mass ratio of the new chlorogenic acid, chlorogenic acid and chlorogenic acid methyl ester is (30–40):(20–30):(15–20).

4. The sugarcane polyphenol extract according to claim 3, characterized in that, The mass ratio of the new chlorogenic acid, chlorogenic acid and chlorogenic acid methyl ester is 36:26:

18.

5. The sugarcane polyphenol extract according to claim 1, characterized in that, The extract also contains one or more polyphenols selected from the following: vitexin, isovitexin, sennain, isosennain, luteolin, caffeic acid, apigenin, isorhamnetin, genistein, sennaol, naringenin, sennain, veselene-2, veselene-3, gallic acid, gentianic acid, syringic acid, p-coumaric acid, ferulic acid.

6. The sugarcane polyphenol extract according to claim 1, characterized in that, The total polyphenol content of the extract is 250–350 mg gallic acid equivalent / g extract.

7. A method for preparing a sugarcane polyphenol extract according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Dissolve the sugarcane molasses extract in deionized water to prepare a solution, and collect the supernatant by centrifugation; (2) Load the supernatant onto a solid-phase extraction column, rinse with distilled water to remove impurities including sucrose, then elute the target component with an organic solvent and collect the eluent; (3) After removing the organic solvent from the eluent, dry it to obtain the sugarcane polyphenol extract.

8. The preparation method according to claim 7, characterized in that, The solid-phase extraction column mentioned in step (2) is an ENV+ solid-phase extraction column or a polymer-based solid-phase extraction column with equivalent performance; the organic solvent is ethanol.

9. The preparation method according to claim 7, characterized in that, The amount of distilled water used for rinsing in step (2) is 30-50 mL, and the amount of organic solvent used for elution is 4-8 mL.

10. The use of the sugarcane polyphenol extract according to any one of claims 1 to 6 in the preparation of sucrase inhibitors.

11. The use of the sugarcane polyphenol extract according to any one of claims 1 to 6 in the preparation of a medicament or health product for regulating postprandial blood glucose.

12. A sucrase inhibitor composition, characterized in that, It comprises the sugarcane polyphenol extract as described in any one of claims 1 to 6 and a pharmaceutically or health-product-acceptable carrier.

13. The composition according to claim 12, characterized in that, The composition is in the form of tablets, capsules, granules, powders, or oral liquids.

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