Efficient active separation and purification method and application of elaeagnus bocknaliana polyphenol

Abstract

The present application relates to a kind of efficient active separation and purification method and purposes of elaeagnus conferta polyphenol, comprising: elaeagnus conferta powder is extracted with ethanol solution, after ultrasonic extraction, centrifugal supernatant is obtained, repeat multiple times after being combined, concentrated by rotary evaporation, and obtain elaeagnus conferta polyphenol concentrated extract;Elaeagnus conferta polyphenol concentrated extract is centrifuged, and the supernatant is used as chromatography sample liquid;Chromatography sample liquid is added to Flash liquid chromatography by liquid sample loading mode, is separated by gradient elution mode to chromatographic column, and each elution component is collected in turn, and concentrated respectively;The concentrated solution of each elution component is added to chromatographic column again to carry out second gradient elution separation, and chromatographic column separation elution is repeated three times, and three kinds of purified elaeagnus conferta polyphenol components are obtained after freeze drying.The present application realizes the high-resolution fractionation elution of polyphenol monomer with high structural similarity by the synergistic regulation of mobile phase polarity gradient and elaeagnus conferta polyphenol molecular hydrophobic interaction.

Description

Technical Field

[0001] This invention relates to the field of natural product separation, purification, and activity evaluation, specifically to a highly efficient method for the separation and purification of Yunnan olive polyphenols and its applications. Background Technology

[0002] Diabetes mellitus (DM) is a metabolic disease caused by insufficient insulin secretion or impaired insulin action. Type 2 diabetes accounts for more than 90% of all diabetes cases. Fasting blood glucose and postprandial blood glucose levels are two key indicators for diagnosing type 2 diabetes. Currently, there is no effective cure for hyperglycemia. Conventional treatment generally involves regularly taking hypoglycemic drugs and controlling diet. However, most of these hypoglycemic drugs are chemically synthesized, such as sulfonylureas, biguanides, and chemically synthesized alpha-glucosidase inhibitors (such as acarbose, voglibose, and miglitol). Clinical practice has shown that long-term use of these drugs can lead to many toxic side effects, such as gastrointestinal discomfort, bloating, and diarrhea. Therefore, the extraction of active substances with hypoglycemic effects from natural plants is gaining increasing acceptance among consumers.

[0003] *Phyllanthus emblica* Linn., a plant belonging to the genus *Phyllanthus* in the family Euphorbiaceae, is also known as Indian gooseberry, Indian olive, olive (Sichuan), and green fruit. It is widely distributed in Yunnan, Fujian, Guizhou, and Guangxi provinces of my country. Its fruit is nearly spherical, with smooth, pale yellow-green flesh. The taste is initially sour, then astringent, and finally sweet. It is a wild plant resource with high edible and medicinal value. *Phyllanthus emblica* is rich in polyphenols, possessing various pharmacological activities such as anti-inflammatory, anti-cancer, hypoglycemic, hypolipidemic, and antioxidant effects. Current research on *Phyllanthus emblica* polyphenols mainly focuses on optimizing the extraction process, while research on the efficient separation of structurally similar monomers from the extracted polyphenol mixture has not been reported. Among them, mucolytic gallate, mucolytic lactone gallate, and galloyl glucose are highly similar in molecular skeleton, polarity, and functional group composition. Conventional single-stage chromatographic separation methods easily lead to elution overlap and insufficient purity, making it difficult to obtain high-purity monomer products suitable for functional studies. Therefore, it is urgent to establish a multi-stage separation and purification method for Yunnan olive polyphenols, which can effectively separate the above-mentioned polyphenol monomers while ensuring the reproducibility and scalability of the operation, and provide a reliable material basis for the study of their hypoglycemic activity.

[0004] Patent CN115645452A describes a method for graded extraction and preparation of polyphenols from Yunnan olives, comprising the following steps: Fresh Yunnan olive pulp is freeze-dried, pulverized, and sieved; extraction is performed using ethanol solution combined with ultrasonication; the extract is placed in a vacuum filtration flask and filtered under reduced pressure; the residue after filtration is hydrolyzed in NaOH solution; the pH is adjusted with HCl solution; the acidic supernatant is collected by centrifugation; the precipitate is washed with ultrapure water and combined with the acidic supernatant; ethyl acetate is added to the supernatant and thoroughly mixed and extracted; the extracts are combined; the filtrate and extract are combined and concentrated to a paste using a rotary evaporator at 35°C; distilled water is added to disperse the concentrate; and the concentrate is freeze-dried to obtain Yunnan olive polyphenols, with a purity exceeding 95%. However, the above patent yields a mixture of Yunnan olive polyphenols without separating the mixture, and it is unknown which polyphenol in the mixture has a hypoglycemic effect. This fails to provide a basis for subsequent analysis, identification, or further utilization, or to meet the needs of subsequent research, production, or application. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one of the defects in the prior art by providing a highly efficient method for the active separation and purification of *Oliveum yunnanense* polyphenols and its applications. This invention solves the problem of effectively separating structurally similar polyphenol monomers through a fractional design of the chromatographic separation process, thereby obtaining high-purity mucolytic gallate, mucolytic lactone gallate, and galloyl glucose. The *Oliveum yunnanense* polyphenols prepared by this invention can significantly inhibit the activity of α-glucosidase and α-amylase, reduce blood glucose in diabetic mice, and improve insulin resistance in diabetic mice.

[0006] Based on existing research on the extraction of polyphenols from Yunnan olives, this invention focuses on the separation of polyphenol mixtures after extraction and constructs a multi-stage reversed-phase chromatography separation strategy. This strategy improves the separation resolution and final purity of structurally similar polyphenol monomers by combining primary fractional separation with secondary fine purification. The specific separation and purification process involves four stages: Stage 1: Extraction and primary purification of Yunnan olive polyphenols. Utilizing the high solubility of Yunnan olive polyphenols in an ethanol-water mixture, ultrasonic extraction is performed at low temperature to avoid hydrolysis or oxidation of heat-sensitive ester bonds and lactone structures. Impurities such as plant residues, proteins, polysaccharides, and microorganisms are removed by centrifugation and microporous membrane filtration to obtain a clear, sterile filtrate, providing suitable samples for subsequent preparative chromatography and protecting the chromatographic column; Stage 2: Primary Flash reversed-phase chromatography coarse separation. A C18 reversed-phase column is used with gradient elution in a water-methanol system containing 0.1% formic acid. Preliminary separation and enrichment are achieved by utilizing the differences in polarity, hydrophobicity, and secondary interactions (such as π-π interactions) of different polyphenol components; Stage 3: Secondary chromatographic purification. The first separation yields enriched fractions, but these may still contain trace impurities or homologues with extremely similar structures. Each enriched fraction obtained from the first separation is concentrated and then subjected to gradient elution using a C18 column in Flash chromatography to further remove structurally similar trace impurities or homologues, ultimately obtaining high-purity oleuropein monomer. Stage Four: Product Acquisition and Preservation. Freeze-drying technology is used to remove the solvent at low temperature (-80℃) and under vacuum. This method avoids hydrolysis, oxidation, or Maillard reactions that may occur with traditional heating and drying, maximizing the preservation of the target compound's structural integrity and biological activity.

[0007] The objective of this invention can be achieved through the following technical solutions: One objective of this invention is to provide a highly efficient method for the active separation and purification of polyphenols from Yunnan olives, comprising the following steps: (1) Extract Yunnan olive powder with ethanol solution, ultrasonically extract and centrifuge to collect the supernatant. Repeat the process several times and combine the supernatants. Concentrate by rotary evaporation to obtain Yunnan olive polyphenol concentrated extract. (2) Centrifuge the concentrated extract of Yunnan olive polyphenols obtained in (1) and take the supernatant as the chromatographic sample solution; (3) The chromatographic loading solution obtained in (2) is added to the Flash liquid chromatograph by liquid loading, and the separation is carried out in the chromatographic column by gradient elution. Each eluted component is collected in sequence and concentrated separately. (4) The concentrated solution of each elution component obtained in (3) is added to the chromatographic column again according to the elution conditions in step (3) for a second gradient elution separation. The chromatographic column separation and elution are repeated three times. The eluent with absorbance value > 0.03 AU is collected and freeze-dried to obtain three purified Yunnan olive polyphenol components.

[0008] Further, in (1), the ratio of the Yunnan olive powder to the ethanol solution is 1g: (15-25)mL, and the ethanol solution refers to an aqueous ethanol solution with a volume fraction of 60%~95%.

[0009] Further, in (1), the ratio of the Yunnan olive powder to the ethanol solution is 1 g: 20 mL.

[0010] Furthermore, in (1), During ultrasonic extraction, the ultrasonic power is (150-250)W and the ultrasonic extraction temperature is 15-25℃. The centrifugation speed is 7000-10000 rpm, and the time is 15-25 min; During the rotary evaporation concentration process, the rotary evaporation temperature is 35-50℃.

[0011] Furthermore, in (1), During the ultrasonic extraction process, the ultrasonic power is 200W; The centrifugation speed was 8000 rpm and the time was 20 min; During the rotary evaporation concentration process, the rotary evaporation temperature is 40℃.

[0012] Further, in (2), the supernatant is filtered through a filter membrane to form a chromatographic loading solution, wherein the filter membrane is a 0.22 μm aqueous filter membrane.

[0013] Furthermore, in (3) and / or (4), the chromatographic conditions are: The chromatographic column is a C18 column; The mobile phase consists of phase A and phase B. Phase A contains an acid with a concentration of 0-0.5%, and phase B is methanol and / or acetonitrile. Flow rate: 25-50 mL / min; Injection volume: 5-15 mL; Detection wavelength: 200-400 nm.

[0014] Preferably, in (3) and / or (4), the chromatographic conditions are: The chromatographic column was an FP ECOFLEX C18; The mobile phase consists of phase A and phase B. Phase A is an aqueous solution containing 0.1% (v / v) formic acid, and phase B is methanol or a solution containing 95% methanol and 5% acetonitrile. Flow rate: 40 mL / min; Injection volume: 10 mL; Detection wavelength: 280 nm.

[0015] Furthermore, the gradient elution conditions for (3) and / or (4) are as follows:

[0016] The second objective of this invention is to utilize the Yunnan olive polyphenols obtained by the highly efficient active separation and purification method described above, for use in hypoglycemic drugs or hypoglycemic supplements.

[0017] Compared with the prior art, the present invention has the following advantages: (1) This invention achieves high-resolution fractional elution of polyphenol monomers with highly similar structures by synergistically regulating the polar gradient of the mobile phase and the hydrophobic interaction of Yunnan olive polyphenol molecules. The process is simple to operate and the separation and purification are fast and efficient, providing a replicable and scalable technical path for the efficient preparation of natural product polyphenol compounds.

[0018] (2) The polyphenols mentioned in this invention have excellent hypoglycemic effects. The half-inhibitory concentration of α-glucosidase is 3.43 ± 0.11 μg / mL, the half-inhibitory concentration of α-amylase is 960.70 ± 23.76 μg / mL, and the half-inhibitory concentration of acarbose of α-glucosidase is 196.31 ± 33.76 μg / mL. Compared with the commercially available hypoglycemic drug acarbose, the Yunnan olive polyphenols in this invention can significantly inhibit the activity of α-glucosidase.

[0019] (3) The Yunnan olive polyphenol (mucosyl gallate) isolated and purified by the present invention can significantly inhibit the fasting blood glucose level of diabetic mice induced by a high-fat diet combined with streptozotocin (STZ) and improve the glucose metabolism disorder in mice. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the extraction, separation, and purification process of the present invention. Figure 2 This is a preparative chromatogram of the separation and purification in an embodiment of the present invention; Figure 3 The LC-MS ion chromatogram of the purified Yunnan olive polyphenols of this invention is shown. Figure 4 This is an HPLC chromatogram of the purified Yunnan olive polyphenols of this invention; Figure 5 This is a schematic diagram illustrating the inhibitory effects of Yunnan olive polyphenols on α-glucosidase and α-amylase according to the present invention; Figure 6 This is a graph showing the results of fasting blood glucose (FBG) measurement in mice in an example of the present invention; Figure 7 This is a graph showing the oral glucose tolerance test (OGTT) and its AUC values ​​in mice in this invention example; Figure 8The graph shows the results of serum insulin (FINS) and insulin resistance index measurements in mice in this invention example; Figure 9 This is a schematic diagram of the peak collection during the secondary chromatographic purification of the single-elution concentrate in Comparative Example 1 of this invention. Figure 10 This is a schematic diagram of the preparation, chromatographic separation, purification, and peak collection in Comparative Example 2 of the present invention; Figure 11 This is a schematic diagram of the peak collection during the preparation, chromatographic separation, purification, and collection in Comparative Example 3 of the present invention; Figure 12 This is a schematic diagram of the preparation, chromatographic separation, purification, and peak collection in Comparative Example 4 of the present invention; Figure 13 This is a schematic diagram of the peak collection during the preparation, chromatographic separation, purification, and collection in Comparative Example 5 of Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments, and are used to explain the present invention, but not to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by other people skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Throughout this specification, unless otherwise specified, the terminology used in this invention should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any conflict, this specification takes precedence. The extraction, separation, and purification process of Yunnan olive polyphenols is attached. Figure 1 As shown.

[0023] Example 1 This embodiment provides a method for the isolation and purification of highly bioactive Yunnan olive polyphenols. The specific process is as follows: Figure 1 It includes the following steps: (1) Remove the pits from fresh Yunnan olives and place them in a vacuum drying oven at a temperature of 40℃ for vacuum drying; (2) After drying, it is put into a pulverizer and pulverized, and then passed through an 80-mesh sieve to obtain Yunnan olive powder; (3) Mix the Yunnan olive powder obtained in step (2) with 70% ethanol solution at a material-to-liquid ratio of 1:20. Extract the mixture by ultrasonication for 20 min at an ultrasonic power of 200 W and an ultrasonic temperature of 15-25 ℃. Centrifuge the mixture at 8000 rpm for 20 min. Take the supernatant and continue to extract the residue by ultrasonication with 70% ethanol solution at a material-to-liquid ratio of 1:20. Repeat the above steps 3 times. Combine the supernatants to obtain the crude extract of Yunnan olive polyphenols. (4) The crude extract of Yunnan olive polyphenols from step (3) was concentrated by rotary evaporation at a temperature of 40 °C to obtain a concentrated crude extract of Yunnan olive polyphenols. (5) The crude extract concentrate of Yunnan olive polyphenols obtained in step (4) is filtered through a 0.22 μm microporous membrane, and the resulting filtrate is used as the loading solution for separation and purification by Flash liquid chromatography (FLC). (6) Using a 10 mL sterile syringe, draw 10 mL of the sample solution from step (5) and inject it into the injection tube of an FLC high-pressure preparative chromatography system equipped with a diode array detector (DAD). Gradient elution is performed using an FP ECOFLEX C18 column packed with 120 g Spherical C18 Monomeric (50 μm, 120 Å) packing material. The elution mobile phases are 0.1% (v / v) formic acid-water solution (mobile phase A) and methanol (mobile phase B). Flow rate: 40 mL / min; injection volume: 10 mL; detection wavelength: 280 nm. Collect the eluent when the absorbance value is >0.03 AU; the gradient elution conditions are shown in the table below: (7) The eluent was collected sequentially. The study found that during gradient elution, when the methanol volume fraction in mobile phase B was between 3% and 30%, the main monomer components of Yunnan olive polyphenols eluted sequentially, forming three eluted components. Thus, three Yunnan olive polyphenol components were separated and purified, such as... Figure 2 As shown, these are designated F1, F2, and F3, respectively. The three collected eluents were then concentrated using a rotary evaporator for secondary reverse-phase chromatography purification. (8) The three elution concentrates from step (7) above were loaded onto the FP ECOFLEX C18 column again according to step (6), and the column was separated and purified twice under gradient elution conditions. The target eluents with different peak times were collected, and the gradient elution was repeated three times. The high-purity eluent collected was concentrated by rotary evaporation. The concentrate was pre-frozen in a -60 ℃ freezer and then freeze-dried to obtain three high-purity and high-biological-activity Yunnan olive polyphenols.

[0024] Example 2 The three highly bioactive Yunnan olive polyphenols prepared in Example 1 were analyzed by LC-MS / MS to determine the active polyphenolic substances contained in the Yunnan olive polyphenols.

[0025] Three polyphenol samples separated and purified were analyzed using a BEH C18 column (2.1 mm × 100 mm × 1.7 μm) packed with octadecyl-bonded silica gel (C18) in a high-performance liquid chromatography-triple quadrupole mass spectrometer. The mobile phases used were: mobile phase A was 0.1% formic acid-water solution, and mobile phase B was 95% methanol + 5% acetonitrile solution. The elution programs were as follows: 0–15 min, 100%–70% A; 15–17 min, 70%–0% A; 17–20 min, 0% A; flow rate: 0.6 mL / min; injection volume: 1 μL; column temperature: 45 ℃.

[0026] Mass spectrometry conditions: Acquisition mode was MSE (low energy / high energy switching scan), ion mode was electrospray scanning of positive and negative ions separately, capillary voltage was 2 kV, nebulizer temperature was 450 ℃, nebulizer flow rate was 900 L / h, cone backflush gas was 50 L / h, ion source temperature was 115 ℃, scan range was m / z 50 ~ 1000, scan rate was 0.2 s, collision energy was 6 eV / 20~45 eV, and flow rate was 10 μL / mL.

[0027] like Figure 3 As shown in Table 1, the purified Yunnan olive polyphenol fractions F1-F3 were identified by LC-MS. F1 was found to be in... RT The peak eluted at 7.632 min. Based on the mass spectrometry information, F1 was identified as mucogallate (MG). F2 mainly consisted of three phenolic compounds, which were observed at [missing information]. RT Peaks eluted at 7.632, 10.159, and 11.334 min. Based on the mass spectrometry information, F2 was identified as mucilage gallate (MG), mucilage digallate (MdG), and mucilage lactone gallate (MLG). F3... RT The peak eluted at 11.832 min. Based on the mass spectrometry information, F3 was identified as galloyl glucose (GG).

[0028] Table 1. Characterization of Yunnan olive polyphenols by high performance liquid chromatography-triple quadrupole mass spectrometry The three highly bioactive Yunnan olive polyphenols prepared in Example 1 were analyzed by HPLC.

[0029] Three polyphenol samples, separated and purified, were analyzed by high-performance liquid chromatography (HPLC) using an Ultimate Plus C18 column (5 μm, 4.6 × 250 mm). The mobile phases used were: mobile phase A was 0.1% formic acid-water solution, and mobile phase B was 95% methanol + 5% acetonitrile solution. The elution programs were as follows: 0–35 min, 100%–70% A; 35–45 min, 70%–0% A; 45–50 min, 0% A; flow rate: 0.6 mL / min; injection volume: 10 μL; column temperature: 25 ℃; detection wavelength: 280 nm. Analysis was performed using Empower software.

[0030] like Figure 4 As shown, based on the HPLC chromatograms of F1-F3, F1 is mucosidic gallate (MG, RT = 0.77 min), with a purity of approximately 99.45%. F2 consists of mucosidic gallate (MG, RT = 0.77 min), mucosidol gallate (MLG, RT = 1.03 min), and mucosidic-methyl ester- O The fractions consisted of gallic acid esters (MMG, RT = 1.30 min) with purities of approximately 26.50%, 71.39%, and 2.11%, respectively. F3 was galloyl glucose (GG, RT = 1.16 min) with a purity of 97.03%.

[0031] The highly bioactive Yunnan olive polyphenols prepared in Example 1 were subjected to in vitro digestive enzyme (α-glucosidase and α-amylase) inhibition experiments, specifically including the following steps: The method for determining the inhibitory activity of α-glucosidase includes: preparing inhibitor solutions of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5 μg / mL using phosphate buffer (0.1 mol / L, pH 6.8). Add 20 μL of α-glucosidase solution (0.04 U / mL) and 60 μL of different concentrations of inhibitor solutions to a 2 mL centrifuge tube, mix well, and incubate in a 37 ℃ water bath for 10 min. Then add 20 μL of 3 mmol / L p-nitrophenyl-α-D-glucopyranoside (pNPG) substrate solution, react in a 37 ℃ water bath for 30 min, and then terminate the reaction by adding 50 μL of 0.2 mol / L Na₂CO₃ stop solution. Shake well and let stand at room temperature for 5 min. Pipette 250 μL of reaction solution from each tube into a 96-well plate. Measure the absorbance at 405 nm using a multi-wavelength microplate reader to calculate the inhibition rate of α-glucosidase by Yunnan olive polyphenols. Calculate the IC50 value using SPSS 24.0 software. Acarbose was used as a positive control. Each experiment was performed in triplicate.

[0032] The formula for calculating the inhibition rate is as follows: In the above formula: A 空白 The absorbance value is the value after the reaction without adding the sample. A 空白背景 The absorbance value after the reaction is without the addition of sample and pNPG; A 样品 This represents the absorbance value of the sample after the reaction.

[0033] The method for determining the inhibitory activity of α-amylase included: Preparing purified olive polyphenols into polyphenol solutions of different concentrations using phosphate buffer (0.1 mol / L, pH 6.8). Adding 50 μL of α-amylase solution and 50 μL of olive polyphenol solutions of different concentrations to 2 mL centrifuge tubes, mixing thoroughly, and incubating in a 37 ℃ water bath for 10 min, then adding 1% soluble starch, mixing well, adding 3 mL of GOPOD reagent, shaking to mix, and incubating in a 50 ℃ water bath for 20 min. After the reaction, aspirating 250 μL from each well into a 96-well plate, and measuring the absorbance at 510 nm using a multi-wavelength microplate reader, calculating the inhibition rate, and calculating the IC50 value using SPSS 24.0 software. Acarbose was used as a positive control. Each experiment was performed in triplicate.

[0034] like Figure 5 As shown, the purified, highly bioactive Yunnan olive polyphenols had a half-inhibitory concentration (WIC) of 3.43 ± 0.11 μg / mL against α-glucosidase and a WIC of 960.70 ± 23.76 μg / mL against α-amylase. Acarbose had a WIC of 196.31 ± 33.76 μg / mL against α-glucosidase. Compared with the commercially available hypoglycemic drug acarbose, the Yunnan olive polyphenols of this invention can significantly inhibit the activity of α-glucosidase.

[0035] Example 3 Animal experiments were conducted on the highly bioactive mucogallate ester prepared in Example 1, specifically including the following steps: Fifty male C57BL / 6 mice (4 weeks old, 14 ± 2 g) were purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. and housed in an animal room at 22 ± 1 ℃, 50 ± 10% humidity, and a 12-hour light-dark cycle, with free access to food and water. After one week of acclimatization, the mice were randomly divided into a normal group (n = 10) and a diabetic group (n = 40). Throughout the experiment, normal mice were fed a normal diet (containing 10% cal fat), while T2DM mice were fed a high-fat diet (containing 60% cal fat). After 8 weeks of housing, the mice were fasted but allowed free access to water for 12 hours. Mice fed the high-fat diet were intraperitoneally injected with 75 mg / kg body weight (BW) of freshly prepared STZ (dissolved in 0.05 mol / L, pH = 4.5 citrate-sodium citrate buffer), while normal mice were injected with an equal volume of citrate-sodium citrate buffer (0.05 mol / L, pH = 4.5). Fasting blood glucose (FBG) was measured in mice on days 3 and 7 after STZ injection. Mice with FBG exceeding 11.1 mmol / L, accompanied by symptoms of polyphagia, polydipsia, polyuria, and significant weight loss, were considered to have successfully modeled T2DM mice. These successfully modeled T2DM mice were used for the next step of the experiment.

[0036] T2DM mice were randomly divided into four groups: high-dose mucogallate group (HMG, 300 mg / kg BW, n=10), low-dose mucogallate group (LMG, 75 mg / kg BW), metformin positive control group (MET, 200 mg / kg BW), and diabetic control group (DM). Mice in the NC and DM groups were administered an equal volume of saline for 10 weeks. Body weight and fasting blood glucose (FBG) were monitored weekly. The changes in fasting blood glucose (FBG) after 10 weeks of gavage are shown in the figure. Figure 6 .

[0037] In the 9th week of continuous gavage, mice underwent an oral glucose tolerance test (OGTT), which included: The day before the experiment, mice were placed in clean cages and fasted for 12 hours with free access to water. Each group of mice was administered glucose solution (1 g / kg BW) by gavage. Tail blood samples were then collected at 0, 30, 60, 90, and 120 minutes. Blood glucose levels were measured using a Roche glucometer and blood glucose test strips, and the area under the glucose curve (AUC) was calculated. The oral glucose tolerance test results and AUC values ​​for mice are shown in the appendix. Figure 7 .

[0038] Serum biochemical indicators and hormone levels were measured using an ELISA kit from Shanghai Tongwei Industrial Co., Ltd., which measured fasting insulin (FINS) levels in mouse serum. The insulin resistance index was calculated based on fasting blood glucose and fasting insulin levels in mice, using the following formula: The HOMA-IR index is calculated as (FBG × FINS) / 22.5.

[0039] Results Analysis The hypoglycemic effect of mucogallate in a diabetic mouse model – fasting blood glucose (FBG) analysis.

[0040] The experimental results of fasting blood glucose (FBG) are as follows: Figure 6 As shown. Fasting blood glucose (FBG) is an important indicator of type 2 diabetes mellitus (T2DM). Fasting blood glucose was monitored weekly in mice after the start of mucogallate (MG) intervention. Figure 6 At week 0, the FBG level in the diabetic model group (DM, FBG > 11.1 mmol / L) was significantly higher than that in the normal control group (NC) (p < 0.05). After 10 weeks of mucogallate (MG) intervention, compared with the DM group (10.59 mmol / L), both metformin and MG intervention significantly reduced fasting blood glucose in T2DM mice. High and low doses of mucogallate achieved the same glycemic regulatory effect as metformin.

[0041] The hypoglycemic effect of mucogallate in a diabetic mouse model: AUC analysis of oral glucose tolerance test (OGTT) and oral glucose tolerance test.

[0042] The oral glucose tolerance test (OGTT) was used to assess the response of test animals to acute hyperglycemia. The experimental results of the OGTT and oral glucose tolerance test (AUC) values ​​are as follows: Figure 7 As shown, blood glucose levels in all mice significantly increased after gavage administration of glucose, peaking at 30 min. At the end of the OGTT experiment (120 min), the blood glucose levels in the metformin (MET) group, high-dose MG intervention (HMG and LMG groups) mice were 15.12, 15.72, and 16.85 mmol / L, respectively, all significantly lower than those in the model DM group (24.03 mmol / L, p < 0.05). Simultaneously, compared with the type 2 diabetes model (DM) group, the area under the curve (AUC) of the OGTT in the NC, MET, HMG, and LMG groups was significantly lower (p < 0.05). These results indicate that MG can significantly improve glucose tolerance in type 2 diabetic mice.

[0043] The hypoglycemic effect of mucogallate in a diabetic mouse model—fasting insulin (FINS) and insulin resistance index (HOMA-IR). Insulin resistance (IR) is a prominent feature of type 2 diabetes mellitus (T2DM). Therefore, to further assess insulin sensitivity, the HOMA-IR value was calculated using fasting blood glucose (FBG) and fasting insulin (FINS) to evaluate the degree of insulin resistance. The experimental results of serum fasting insulin (FINS) and the HOMA-IR index are as follows: Figure 8 As shown in the figure, compared with diabetic model mice (DM), the FINS level in the DM group (35.52 mIU / L) was significantly higher than that in normal (NC) mice (21.61 mIU / L) (p < 0.05), while the FINS levels in the metformin (28.86 mIU / L) and high- and low-dose mucogallate intervention groups (25.66 mIU / L and 27.97 mIU / L, respectively) were significantly lower (p < 0.05). Meanwhile, the HOMA-IR in the DM group was significantly higher than that in the normal NC mouse group and the intervention treatment groups (HMG and LMG groups) (p < 0.05), while the HOMA-IR in T2DM mice treated with MET, HMG, and LMG was significantly lower (p < 0.05). These results indicate that mucogallate can significantly improve insulin sensitivity and insulin resistance in T2DM mice and maintain glycemic homeostasis.

[0044] Comparative Example 1 Comparative Example 1 did not employ secondary reversed-phase chromatography for purification and separation. The Yunnan olive polyphenol extract was pretreated according to steps (1-5) in Example 1, except that only one reversed-phase C18 Flash high-pressure preparative chromatography separation was performed. Using the same gradient elution conditions as in Example 1, the eluted fractions were collected and concentrated after a single gradient elution to obtain concentrated solutions of each fraction.

[0045] Comparative Example 2 Comparative Example 2 shows that the proportion of organic phase in gradient elution exceeds the critical range. The Yunnan olive polyphenol extract was pretreated according to steps (1-5) in Example 1, with the only difference being the modification of the chromatographic gradient elution separation conditions. The specific gradient elution conditions are shown in the table below. Comparative Example 3 Comparative Example 3 shows that the proportion of organic phase in gradient elution exceeds the critical range. The pretreatment methods for the Yunnan olive polyphenol extract and chromatographic sample were the same as in Example 1, with the only difference being the modification of the chromatographic gradient elution separation conditions. The gradient elution conditions are adjusted as shown in the table below. Comparative Example 4 The pretreatment methods for Yunnan olive polyphenol extract and chromatographic sample loading solution are the same as in Example 1, except that the chromatographic gradient elution separation conditions are modified. The gradient elution conditions are adjusted as shown in the table below. Comparative Example 5 The pretreatment methods for Yunnan olive polyphenol extract and chromatographic sample loading solution are the same as in Example 1, except that the chromatographic gradient elution separation conditions are modified. The gradient elution conditions are adjusted as shown in the table below. Comparative analysis of results Analysis of Comparative Example 1 In Comparative Example 1, the same first-stage reversed-phase chromatography gradient elution conditions as in Example 1 were used, but no further secondary reversed-phase chromatography purification was performed after obtaining the initial eluted fraction. Figure 2 and Figure 9 It can be seen that although multiple elution peaks can be formed during the primary separation process, there is still obvious component crossover within each peak. Especially between adjacent elution peaks, the entrainment phenomenon between mucolytic gallate and mucolytic lactone gallate, as well as galloyl glucose, is quite obvious. Figure 9 This result indicates that relying solely on a single reversed-phase chromatographic separation is insufficient to completely eliminate the co-elution problem between structurally similar polyphenol monomers, resulting in insufficient purity of the obtained components, which is difficult to meet the purity requirements for subsequent functional activity evaluation.

[0046] Analysis of results from Comparative Example 2 In Comparative Example 2, the methanol volume fraction during the gradient elution process was set significantly higher than the critical range used in Example 1, causing the main elution process to occur primarily under conditions of a higher organic phase ratio. Figure 10 It can be seen that under these conditions, multiple peaks in the elution curve overlap significantly, the baselines between peaks are difficult to separate, and the collection window is unclear. This result indicates that when the proportion of organic phase is too high, the retention behavior of each monomer in the Yunnan olive polyphenol in the reversed-phase system tends to be consistent, leading to a significant decrease in separation selectivity and hindering effective fractionation elution.

[0047] Analysis of results from Comparative Example 3 In Comparative Example 3, separation was achieved by shortening the gradient elution time and accelerating the rate of change in the methanol ratio. Figure 11 As shown, under these conditions, the elution peaks become narrower overall, and some peaks overlap, making the boundaries between elution regions of adjacent components unclear. Due to the excessively rapid gradient change, the polyphenolic components are eluted before they can establish sufficiently stable retention differences, resulting in insufficient separation resolution. This result indicates that excessively rapid gradient changes are detrimental to the complete separation of structurally similar polyphenols in reversed-phase chromatography.

[0048] Analysis of results from Comparative Example 4 In Comparative Example 4, separation was achieved by extending the gradient elution time or setting a longer elution platform in non-critical regions. Figure 12It can be seen that under these conditions, the elution peaks exhibit significant broadening and tailing, with peaks that are not concentrated and some peaks still overlap. Despite the extended elution time, the separation effect was not substantially improved; in fact, the separation efficiency was reduced. This result indicates that simply extending the gradient time without properly controlling the organic phase ratio range cannot effectively improve the separation and purification effect.

[0049] Analysis of results from Comparative Example 5 In Comparative Example 5, the gradient elution interval setting did not match the elution characteristics of the main monomers of Yunnan olive polyphenols. Figure 13 As shown, the elution peaks of the target polyphenol components are relatively dispersed, and the elution positions of some components shift forward or backward, making it difficult to accurately define the collection interval.

[0050] Comparison of results from different pairs The comparison results of each pair of proportions are shown in the table below: Separation Mechanism Analysis of Examples and Comparative Examples The polyphenols of Yunnan olives, including viscogallate, viscolactone gallate, and galloyl glucose, all contain multiple phenolic hydroxyl groups in their molecular structure. However, their overall polarity and hydrophobicity differ due to the presence or absence of lactone and glycosylation. Among them, galloyl glucose exhibits higher polarity and weaker hydrophobicity due to the introduction of glycosyl groups, while viscogallate and viscolactone gallate, while maintaining the polyphenol backbone, show relatively stronger hydrophobicity with less difference in their properties.

[0051] In the reversed-phase C18 chromatographic system used in this invention, the separation process mainly relies on the difference in the strength of hydrophobic interactions between different polyphenolic components and the stationary phase, which is reflected by the gradual change in the polarity of the mobile phase during gradient elution. As the proportion of organic phase in the mobile phase gradually increases, the polarity of the mobile phase decreases accordingly, thereby weakening the hydrophobic interaction between the solute and the stationary phase. This allows components with weaker hydrophobicity or stronger polarity to elute preferentially, while components with stronger hydrophobicity elute sequentially under conditions of higher organic phase proportions.

[0052] Under the gradient elution conditions used in Example 1, by controlling the methanol volume fraction within the range of 5%–35%, the change in mobile phase polarity was matched with the polarity and hydrophobicity distribution characteristics of the three polyphenol components. This allowed the differences in hydrophobic interactions between different components on the stationary phase to be amplified stepwise, resulting in well-separated elution peaks. Under these conditions, each target polyphenol could elute sequentially and form relatively independent collection zones, providing a stable basis for subsequent secondary reversed-phase chromatographic purification. Compared to the comparative conditions, when the overall organic phase ratio was too high, the rate of change was too rapid, or the gradient zone setting did not match the polarity and hydrophobicity characteristics of the target polyphenols during gradient elution, the differences in hydrophobic interactions between the components on the stationary phase were difficult to fully translate into effective retention time differences, leading to reduced separation or overlap between elution peaks, thus affecting the separation and purification effect.

[0053] This invention, through the rational design of the change range and rate of mobile phase polarity during gradient elution, coordinates the change in mobile phase polarity with the polarity and hydrophobic interaction characteristics of Yunnan olive polyphenol monomers. This is an important technical basis for achieving effective separation of structurally similar polyphenols and obtaining high-purity monomers.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A highly efficient method for the active separation and purification of polyphenols from Yunnan olives, characterized in that, Includes the following steps: (1) Extract Yunnan olive powder with ethanol solution, ultrasonically extract and centrifuge to collect the supernatant. Repeat the process several times and combine the supernatants. Concentrate by rotary evaporation to obtain Yunnan olive polyphenol concentrated extract. (2) Centrifuge the concentrated extract of Yunnan olive polyphenols obtained in (1) and take the supernatant as the chromatographic sample solution; (3) The chromatographic loading solution obtained in (2) is added to the Flash liquid chromatograph by liquid loading, and the separation is carried out in the chromatographic column by gradient elution. Each eluted component is collected in sequence and concentrated separately. (4) The concentrated solution of each elution component obtained in (3) is added to the chromatographic column again according to the elution conditions in step (3) for a second gradient elution separation. The chromatographic column separation and elution are repeated three times. The eluent with absorbance value > 0.03 AU is collected and freeze-dried to obtain three purified Yunnan olive polyphenol components.

2. The method for efficient separation and purification of Yunnan olive polyphenols according to claim 1, characterized in that, In (1), the ratio of the Yunnan olive powder to the ethanol solution is 1g: (15-25)mL, and the ethanol solution refers to an aqueous ethanol solution with a volume fraction of 60% to 95%.

3. A highly efficient method for the active separation and purification of Yunnan olive polyphenols according to claim 1 or 2, characterized in that, In (1), the ratio of the Yunnan olive powder to the ethanol solution is 1 g: 20 mL.

4. The method for efficient separation and purification of Yunnan olive polyphenols according to claim 1, characterized in that, In (1), During ultrasonic extraction, the ultrasonic power is (150-250)W and the ultrasonic extraction temperature is 15-25℃. The centrifugation speed is 7000-10000 rpm, and the time is 15-25 min; During the rotary evaporation concentration process, the rotary evaporation temperature is 35-50℃.

5. A highly efficient method for the active separation and purification of Yunnan olive polyphenols according to claim 1 or 4, characterized in that, In (1), During the ultrasonic extraction process, the ultrasonic power is 200W; The centrifugation speed was 8000 rpm and the time was 20 min; During the rotary evaporation concentration process, the rotary evaporation temperature is 40℃.

6. The method for efficient separation and purification of Yunnan olive polyphenols according to claim 1, characterized in that, (2) The supernatant is filtered through a filter membrane to form a chromatographic sample solution. The filter membrane is a 0.22 μm aqueous filter membrane.

7. The method for efficient separation and purification of Yunnan olive polyphenols according to claim 1, characterized in that, In (3) and / or (4), the chromatographic conditions are: The chromatographic column is a C18 column; The mobile phase consists of phase A and phase B. Phase A contains an acid with a concentration of 0-0.5%, and phase B is methanol and / or acetonitrile. Flow rate: 25-50 mL / min; Injection volume: 5-15 mL; Detection wavelength: 200-400 nm.

8. The method for efficient separation and purification of Yunnan olive polyphenols according to claim 1, characterized in that, In (3) and / or (4), the chromatographic conditions are: The chromatographic column was an FP ECOFLEX C18; The mobile phase consists of phase A and phase B. Phase A is an aqueous solution containing 0.1% (v / v) formic acid, and phase B is methanol or a solution containing 95% methanol and 5% acetonitrile. Flow rate: 40 mL / min; Injection volume: 10 mL; Detection wavelength: 280 nm.

9. The method for efficient separation and purification of Yunnan olive polyphenols according to claim 1, characterized in that, (3) and / or (4) gradient elution conditions are as follows: 。 10. The use of Yunnan olive polyphenols obtained by the highly efficient active separation and purification method according to any one of claims 1-9, characterized in that, Used in hypoglycemic drugs or foods that help lower blood sugar.

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