A Kunlun chrysanthemum extract complex with intestinal probiotic and hypoglycemic activities and preparation method thereof

Abstract

The present invention discloses a Kunlun fetus chrysanthemum extract complex with intestinal prebiotic and hypoglycemic activity and a preparation method thereof, belonging to the technical field of natural compound extraction. The present invention first uses Lactobacillus acidophilus to ferment Kunlun fetus chrysanthemum powder, then uses gradient ethanol solvent for repeated extraction, and uses water as a solvent to prepare an extract complex, which significantly improves the stability of the organic solvent extract in aqueous solution; further studies have shown that when the anhydrous ethanol extract, water extract and extract complex of Kunlun fetus chrysanthemum are used alone as prebiotics, they can all change intestinal microorganisms; when used as an α-glucosidase inhibitor, they also show in vitro hypoglycemic activity, however, when the complex prepared by using anhydrous ethanol and water extract can better play the above role, and the complex shows a synergistic effect when playing the above role, providing a theoretical basis for its application in the field of prebiotics and hypoglycemic.

Description

Technical Field

[0001] The present invention relates to the technical field of natural compound extraction, in particular to a Kunlun chrysanthemum extract complex with intestinal probiotic and blood sugar-lowering activities and a preparation method thereof. Background Art

[0002] Kunlun chrysanthemum (Coreopsis tinctoria buds) is a processed variety of the Coreopsis genus (Coreopsis L.) that is steam-cured and then dried after incompletely opening its inflorescence. Kunlun chrysanthemum grows primarily in the high-altitude areas of my country's Xinjiang Uyghur Autonomous Region, particularly near the Kunlun Mountains. This unique growing environment endows Kunlun chrysanthemum with a rich array of medicinal compounds and biological activities.

[0003] With the advancement of modern science and technology, research on Kunlun Chrysanthemum (Chrysanthemum chinense) continues to deepen, and the pharmacological effects and functional components of its extracts have been scientifically verified and understood. Studies have shown that Kunlun Chrysanthemum (Chrysanthemum chinense) extracts have multiple benefits, including lowering blood pressure, blood lipids, and blood sugar, as well as anti-tumor and antioxidant properties. They also have potential as prebiotics, demonstrating broad application prospects in pharmaceuticals, health supplements, and cosmetics.

[0004] There are various methods for preparing Kunlun chrysanthemum extract, including water extraction, organic solvent extraction, supercritical extraction, etc. Different preparation methods have a significant impact on the components and yield of the extract. The extract obtained by organic solvent extraction contains a variety of small molecules and has good biological activity, but due to its poor water solubility, its utilization rate is low. The large molecules extracted by water as a solvent have good water solubility and high utilization rate, but the product composition is complex and the purity is not high. Therefore, it is urgent to develop a new method for preparing Kunlun chrysanthemum extract so that the obtained extract has high stability while increasing its utilization as much as possible to achieve better regulation of intestinal microorganisms and in vitro hypoglycemic effects. Summary of the Invention

[0005] The present invention aims to provide a Kunlun chrysanthemum extract complex with intestinal prebiotic and hypoglycemic activities and a preparation method thereof to address the aforementioned problems of the prior art. The Kunlun chrysanthemum extract complex prepared by the present invention significantly improves the stability of the organic solvent extract in aqueous solution while exhibiting enhanced prebiotic and hypoglycemic activities, providing a theoretical basis for its application in the prebiotic and hypoglycemic fields.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a Kunlun chrysanthemum extract complex, comprising the following steps:

[0008] Kunlun fetal chrysanthemum powder was first fermented with Lactobacillus acidophilus, then extracted with anhydrous ethanol reflux, centrifuged and filtered to obtain a residue, and the filtrate was freeze-dried to obtain extract A;

[0009] The obtained filter residue is sequentially subjected to gradient reflux extraction with ethanol solutions having volume fractions of 80%, 50% and 20% to obtain a filter residue;

[0010] The obtained filter residue is subjected to pure water reflux extraction to obtain a water extract;

[0011] The obtained water extract is ultrafiltered through a filter membrane, and the filtrate is freeze-dried to obtain macromolecular substance B;

[0012] The extract A and the macromolecular substance B are mixed, homogenized under high pressure with water as a solvent, and then ground with a ball mill. The obtained mixed solution is dried to obtain the Kunlun fetal chrysanthemum extract complex C.

[0013] Furthermore, the fermentation treatment is to inoculate Lactobacillus acidophilus in the aqueous solution of Kunlun fetal chrysanthemum powder for shaking fermentation, the inoculation amount of Lactobacillus acidophilus is 4%; the shaking fermentation temperature is 37°C, the fermentation time is 48h, and the rotation speed is 200r / min.

[0014] Furthermore, the temperature of the anhydrous ethanol reflux extraction is 60°C and the time is 2 hours; the temperature of the ethanol solution gradient reflux extraction is 60°C and the extraction time is 2 hours; the temperature of the pure water reflux extraction is 90°C and the extraction time is 2 hours.

[0015] Furthermore, the molecular weight cut-off of the filter membrane is 5000Da, and the number of ultrafiltration times is 6 times.

[0016] Furthermore, when mixed, the mass ratio of the extract A to the macromolecular substance B is 1:2.

[0017] Furthermore, the components of the extract A include 49.37wt% xanthophylloside, 6.24wt% kaempferol, 8.42wt% scutellariae and 5.47wt% marimoside; the molecular weight of the macromolecular substance B is 2297.841kDa, and the components include 27.64wt% arabinose, 28.31wt% galactose, 15.30wt% glucose and 15.05wt% galacturonic acid.

[0018] Furthermore, the particle size of the Kunlun chrysanthemum extract complex C is 469±5.57 nm, the dispersion coefficient is 0.21±0.01, and the potential is -50.13±1.12 mV.

[0019] The present invention also provides a Kunlun chrysanthemum extract complex obtained by the preparation method.

[0020] The present invention also provides the use of the Kunlun chrysanthemum extract complex in preparing products with intestinal beneficial properties.

[0021] The present invention also provides the use of the Kunlun chrysanthemum extract complex in preparing a product with hypoglycemic activity.

[0022] The present invention discloses the following technical effects:

[0023] The present invention firstly ferments Kunlun chrysanthemum powder with Lactobacillus acidophilus, then repeatedly extracts with gradient ethanol solvents, and uses water as a solvent to prepare an extract complex, thereby significantly improving the stability of the organic solvent extract in aqueous solution; further studies have shown that when the anhydrous ethanol extract, water extract and extract complex of Kunlun chrysanthemum are used alone as prebiotics, they can all change intestinal microorganisms; when used as α-glucosidase inhibitors, they also exhibit in vitro hypoglycemic activity; however, when the complex is prepared using anhydrous ethanol and water extracts, the above-mentioned effects can be better exerted, and the complex exhibits a synergistic effect when exerting the above-mentioned effects, providing a theoretical basis for its application in the fields of prebiotics and hypoglycemics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 is the ion chromatogram of extract A;

[0026] Figure 2 The stability test results of the substances obtained in Examples 1 to 3 are as follows;

[0027] Figure 3 Chao 1 index of intestinal flora detected in samples of each treatment group;

[0028] Figure 4 Simpson index of intestinal flora detected for samples of each treatment group;

[0029] Figure 5 Shannon index of intestinal flora detected for samples of each treatment group;

[0030] Figure 6 is the effect of each treatment group on the level of intestinal microbial phyla;

[0031] Figure 7 is the effect of each treatment group on the level of intestinal microbial genus;

[0032] Figure 8 The results of starch load in each treatment group are shown in Figure 2. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1 Preparation of Kunlun Fetal Chrysanthemum Anhydrous Ethanol Extract

[0039] (1) Soaking Kunlun chrysanthemum in water to remove excess impurities, then drying it in an oven, crushing it with an ultra-high pressure cell crusher (pressure 1200 bar, flow rate 15 L / H, machine stroke number 140 times / min, crushing temperature 4°C), and passing it through a 60-mesh sieve to obtain Kunlun chrysanthemum powder;

[0040] (2) Place the conical flask containing Kunlun chrysanthemum powder in an autoclave at 121°C for 30 minutes, and then cool to room temperature;

[0041] (3) Lactobacillus acidophilus was inoculated into MRS broth, cultured at 37°C and shaken at 200 rpm for 48 h for activation;

[0042] (4) Inoculate 4% activated Lactobacillus acidophilus into the sterilized Kunlun fetal chrysanthemum powder aqueous solution in a clean bench, culture at 37°C and 200 rpm for 48 h to ferment the Kunlun fetal chrysanthemum powder;

[0043] (5) The fermented Kunlun chrysanthemum powder aqueous solution was centrifuged at 4°C and 10,000 rpm for 15 min, and the precipitate was collected and dried in an oven at 60°C for later use;

[0044] (6) 100 g of fermented Kunlun Fetal Chrysanthemum powder was mixed with 100% ethanol at a solid-liquid ratio of 1 g:15 mL, and reflux extracted at 60°C for 2 h. After cooling, the mixture was centrifuged at 10,000 rpm and 4°C for 15 min, filtered through gauze, and the filtrate was concentrated to 1 / 4 of its original volume at 60°C and freeze-dried to obtain anhydrous ethanol extract (A). The filter residue was oven-dried for subsequent extraction.

[0045] Example 2 Preparation of Kunlun Fetal Chrysanthemum Macromolecules

[0046] The filter residue finally obtained in Example 1 was extracted with 80%, 50%, and 20% ethanol in a gradient reflux manner (the extraction conditions were the same as in Example 1), and filtered to obtain a filter residue;

[0047] The extraction solvent was replaced with water, and the filter residue was subjected to reflux extraction again at 90°C. The remaining steps were the same as in Example 1 to obtain an aqueous extract. The aqueous extract was repeatedly ultrafiltered six times using a 5000Da ultrafiltration membrane, concentrated at 60°C, and freeze-dried to obtain the macromolecular substance (B).

[0048] Example 3 Preparation of composites of A and B

[0049] 150 mg of A prepared in Example 1 and 300 mg of B prepared in Example 2 were dissolved in 600 mL of water, homogenized five times in a high-pressure homogenizer at a pressure of 100 MPa, and then ball milled at a speed of 250 r / min for 4 h. Finally, the mixture was spray-dried to obtain a composite (C).

[0050] Test Example 1 Physicochemical properties of the substances obtained in each example

[0051] 1. The total sugar, total phenol, and protein contents of A, B, and C were determined using the phenol-sulfuric acid method, the folin-phenol method, and the Coomassie brilliant blue method, respectively. The particle size and potential of A, B, and C were measured using a particle size potential tester. The results are shown in Tables 1 and 2.

[0052] Table 1 Analysis of the content of total sugar, total phenol and protein in the extract

[0053]

[0054] Table 2 Particle size potential analysis of extracts and complexes

[0055]

[0056] As can be seen from Table 1, the total phenol content in A is 88.81±2.10%, and the total sugar content in B is 86.05±2.08%. The total phenol content in the prepared complex is significantly higher than that in B, indicating that the preparation of complex C is successful; from the particle size potential of the extract and the complex in Table 2, it can be seen that the dispersion coefficient of C is reduced, and the absolute value of the potential is higher, indicating that its solution can exist in a more stable state.

[0057] 2. Composition of the substances obtained in each embodiment

[0058] Detection method:

[0059] Composition analysis of A: Accurately weigh 1 mg of A and dissolve it in methanol by ultrasonication. After centrifugation, remove the supernatant, filter it through a 0.22 μm filter, dilute it 10-fold with methanol, and store it in a refrigerator at 4°C for subsequent UPLC / Q-TOF-MS analysis. The mobile phases were (A) an aqueous solution containing 0.1% formic acid and (B) an acetonitrile solution. The flow rate of the mobile phase was 0.2 mL / min, the column temperature was 30°C, and the injection volume was 2 μL. Equilibrate each column for 5 minutes before injection. The mobile phase examples are shown in Table 3.

[0060] Table 3 Mobile phase composition (volume ratio)

[0061]

[0062] Mass spectrometry was performed using an electrospray ionization (ESI) source in negative ion mode. Detection conditions were capillary voltage: 2.5 kV; cone voltage: 30 V; source temperature: 120°C; desolvation temperature: 500°C; primary mass spectrometry collision energy: 6 eV; secondary mass spectrometry collision energy: 30 eV; and the mass-to-charge ratio (m / z) acquisition range was 50–750 Da. Each sample was tested in triplicate, and a methanol blank was injected to minimize sample cross-contamination.

[0063] B. Monosaccharide Composition Analysis: Monosaccharide composition was determined using the PMP derivatization method. First, hydrolyze the sample. Take 1 mL of a 10 mg / mL sample solution and place it in an ampoule. Add 1 mL of trifluoroacetic acid (4 M), mix thoroughly, seal the ampoule, and react at 110°C for 8 h. Complete the hydrolysis by rotary evaporation to dryness. Add an appropriate amount of methanol and repeat the rotary evaporation six times to ensure complete removal of the trifluoroacetic acid. Then, add 100 μL of 0.6 M NaOH solution and 100 μL of 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution. Mix thoroughly, transfer to a stoppered tube, seal the ampoule, and derivatize in a 70°C waterbath for 100 min. Add 100 μL of 0.3 M HCl to a final volume of 2 mL. Extract with 2 mL of chloroform to remove unreacted PMP. Repeat six times. The aqueous phase is filtered and ready for HPLC analysis. Prepare 10 mg / mL standard solutions of each monosaccharide (mannose, glucose, arabinose, galactose, rhamnose, galacturonic acid, glucuronic acid, and xylose). Pipette equal volumes of these solutions and mix to create a mixed standard. Place 50 μL of each monosaccharide standard solution or mixed standard solution in a stoppered test tube. Add 50 μL of 0.6 M NaOH solution and 100 μL of PMP solution for derivatization. Subsequent procedures are the same as for the above samples. HPLC injection analysis is performed, and a standard curve is constructed. The HPLC operating parameters are shown in Table 4.

[0064] Table 4 HPLC operating parameters

[0065]

[0066] B. Molecular weight determination: A 5 mg / mL solution was prepared using the mobile phase. After passing through the membrane, the molecular weight of the polysaccharide was determined using size exclusion gel chromatography coupled with an 18-angle laser light scattering instrument and a differential detector. Data analysis was performed using ASTRA 6.1 software. The relevant parameters are shown in Table 5.

[0067] Table 5 Relevant working parameters for average molecular weight determination

[0068]

[0069] result: Figure 1The ion chromatogram of A is shown. A is composed of 24 compounds, including (xanthoside, kaempferol, scutellarin, mariside, taxifolin-3'-O-galactoside, plantagoside, 2-isopropylmalic acid, chlorogenic acid, caffeic acid, naringenin-7-O-glucoside, myricetin-3-O-β-D-galactoside, eriodictyol, luteolin, isoquercetin, isochlorogenic acid C, dihydrobaicalein, luteolin-7-O-acetyl-β-D-glucoside, coreopsis glycoside, methyl 4-hydroxycinnamate, genistein, oxalin , quercetin, luteolin and butein), among which xanthophylloside, kaempferol, calciphyllin and mariside are the main components with the highest content. The mass percentages of the main components xanthophylloside, kaempferol, calciphyllin and mariside in A are 49.37%, 6.24%, 8.42% and 5.47%, respectively; B has a molecular weight of 2297.841kDa and is composed of arabinose, galactose, glucose and galacturonic acid, with mass percentages of 27.64%, 28.31%, 15.30% and 15.05%, respectively.

[0070] 3. The extracts and complexes prepared in Examples 1 to 3 were subjected to stability tests. The substances obtained in each example were prepared into 1 mg / mL aqueous solutions, mixed and placed at room temperature for 60 minutes. The state of the solution was observed and the state of the solution after 0 minute and 60 minutes was recorded. The results are shown in Tables 6 and Figure 2 shown.

[0071] Table 6 Stability results of the materials obtained in each example

[0072]

[0073] Depend on Figure 2 As shown in Table 6, the composite C prepared from A and B improves the stability of A in aqueous solution.

[0074] Test Example 2 Prebiotic activity of the substances obtained in each example

[0075] 1. Preparation of basal culture medium and fecal homogenate

[0076] Basal nutrient medium (1.0 L, pH 7.0), containing yeast extract (2.0 g), peptone (2.0 g), NaCl (0.1 g), K2HPO4 (0.04 g), KH2PO4 (0.01 g), CaCl2·2H2O (0.01 g), MgSO4·7H2O (0.01 g), NaHCO3 (2.0 g), heme (0.02 g), L-cysteine ​​(0.5 g), bile salts (0.5 g), Tween 80 (2.0 mL), resazurin solution (1.0 mL, 1%, w / v), vitamin K (10.0 μL) and ultrapure water, was sterilized at 121°C for 20 min.

[0077] Fecal samples were collected from fresh feces of four healthy volunteers (two men and two women aged 22-30 years, with no history of gastrointestinal diseases and no antibiotics taken within 3 months). The feces were diluted with 0.9% sterile saline (w / v) to prepare a fecal suspension (10%, w / v). The fecal suspension was centrifuged at 500 r / min for 5 min, and the supernatant was collected to prepare a fecal homogenate.

[0078] 2. Grouping

[0079] Test Group 1: 1.0 mL of fecal homogenate was mixed with 9.0 mL of basal nutrient medium containing 100.0 mg of the anhydrous ethanol extract (A) prepared in Example 1 and added to a vial;

[0080] Test Group 2: 1.0 mL of fecal homogenate was mixed with 9.0 mL of basal nutrient medium containing 100.0 mg of the aqueous extract (B) prepared in Example 2 and added to a vial;

[0081] Test group 3: 1.0 mL of fecal homogenate was mixed with 9.0 mL of basal medium containing 100.0 mg of the complex (C) prepared in Example 3 and added to a vial;

[0082] Blank group: 1.0 mL of fecal homogenate was mixed with 9.0 mL of basal nutrient medium and added to a vial.

[0083] The three experimental groups and the blank group were incubated in a carbon dioxide anaerobic incubator at 37°C. The fermentation broth was taken out at 0 and 48 hours to analyze the changes in intestinal flora composition and short-chain fatty acid content.

[0084] 3. Composition of intestinal flora

[0085] After 48 h of fermentation, the intestinal microbial DNA of each experimental group was extracted using the NucleoSpin96So kit.

[0086] The bacterial 16S rRNA gene in the V3-V4 region was amplified by PCR using forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3', SEQ ID NO. 1) and reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO. 2) and tested on a microarray. The results were as follows: Figures 3 to 7 shown.

[0087] from Figures 3 to 7It can be seen that the complex (C) prepared in Example 3 has a significant regulatory effect on intestinal microorganisms after in vitro fermentation, significantly changing the abundance of the flora, among which the levels of Firmicutes and Lactobacillus are significantly higher than those in other groups. It can be seen that the complex prepared in Example 3 can better regulate intestinal microorganisms as a prebiotic.

[0088] 4. Detection of short-chain fatty acids after in vitro fermentation

[0089] Take 1 mL of fermentation broth after 0 h and 48 h of fermentation, add 4 times the volume of methanol, let it stand at 4°C overnight, centrifuge at 12000r for 15 min, transfer the supernatant to a 2 mL EP tube containing 0.25 g of anhydrous Na2SO4, and then centrifuge under the same conditions. Take the upper methanol phase and add it to a gas phase sample bottle, and use GC-MS instrument to analyze the content of short-chain fatty acids (acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid and isovaleric acid) in the sample.

[0090] Preparation of short-chain fatty acid standards:

[0091] Take 50 μL (52.46 μg) of acetic acid, 30 μL (29.7 μg) of propionic acid, 50 μL (47.9 μg) of n-butyric acid, 25 μL (23.75 μg) of isobutyric acid, 25 μL (23.48 μg) of n-valeric acid and 25 μL (23.25 μg) of isovaleric acid in a 10 mL volumetric flask and add methanol to make up to volume as a stock solution.

[0092] 1 mL, 0.5 mL, 0.25 mL, 0.125 mL, 0.05 mL, and 0.025 mL of the stock solution were taken, methanol was added to 2 mL, and the samples were injected and analyzed to draw a standard curve. The test results are shown in Table 7.

[0093] Table 7 Changes in short-chain fatty acid content after 48 h of fermentation

[0094]

[0095] From the short-chain fatty acid detection results in Table 7, it can be seen that the complex (C) prepared in Example 3 has a better effect of promoting the production of short-chain fatty acids.

[0096] Test Example 3 Hypoglycemic activity of the substances obtained in each example

[0097] 1. In vitro hypoglycemic activity

[0098] The in vitro hypoglycemic activity test was determined by detecting the inhibition rate of α-glucosidase.

[0099] A, B and C were prepared into 1 mg / mL solutions, respectively, and diluted into 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL and 0.0625 mg / mL test solutions, respectively. 30 μL of each concentration of test solution was added to 30 μL of α-glucosidase solution, and incubated at 37°C for 10 min. Then 30 μL of p-NPG solution was added to react for 15 min, and 100 μL of 0.1 mol / L Na2CO3 was added to terminate the reaction. The absorbance was measured at 405 nm. Acarbose was used as a positive control in the test. By calculating the IC values ​​of A, B and C 50 The hypoglycemic activity of the ingredients was determined by calculating the DRI values, and the synergistic effect of the ingredients was analyzed by calculating the DRI values. The results are shown in Tables 8 and 9.

[0100] Table 8 Half inhibition concentration of each group of samples on α-glucosidase

[0101]

[0102] Table 9 DRI values ​​of each group of samples at half inhibition rate

[0103]

[0104] IC 50 The value refers to the sample concentration when the enzyme activity inhibition rate is 50%. It can be seen from Table 8 that the IC 50 The IC values ​​of A and B were 0.343 mg / mL and 0.777 mg / mL, respectively. 50 The DRI values ​​decreased by 53.06% and 46.59%, respectively, indicating that the α-glucosidase inhibition rate of the complex C prepared by A and B was significantly improved. The larger the DRI value, the better the synergistic effect. As can be seen in Table 9, B has the best synergistic effect on the complex C prepared by A and B.

[0105] 2. Starch loading test of the materials obtained in each example:

[0106] Test preparation:

[0107] Forty SD rats were fed normally for 7 days and then fasted for 12 hours. The blood glucose levels of each rat were measured and divided into a blank group, test group 1, test group 2, and test group 3 based on the blood glucose levels (ensuring no significant differences between the groups). All rats were gavaged with 4 g / kg of starch and then immediately gavaged with 400 mg / kg of the substance obtained in Examples 1 to 3 or an equivalent amount of normal saline (blank group). Specific treatments are shown in Table 10.

[0108] Table 10 Treatment methods of SD rats in each group

[0109]

[0110] Blood glucose levels of all rats were measured at 15, 30, 60, and 90 min by taking blood from the tip of the tail. Figure 8 shown.

[0111] Depend on Figure 8 It can be seen that 15 minutes after gavage with 4 g / kg starch, Test Group 3 exhibited a better starch loading capacity than Test Groups 1 and 2, indicating that the complex C prepared in Example 3 used in Test Group 3 has better hypoglycemic activity. In summary, the complex C prepared by the present invention has good stability in water, can better regulate intestinal microorganisms, and promote the production of short-chain fatty acids. In vivo tests have confirmed that it has polar hypoglycemic activity, and in vitro tests have confirmed that it can inhibit the activity of α-glucosidase as an antagonist. Compared with A and B, it has a synergistic and synergistic hypoglycemic effect, providing a theoretical basis for its application in the fields of prebiotics and hypoglycemics.

[0112] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a Kunlun chrysanthemum extract complex, characterized in that: The following steps are involved: Kunlun fetal chrysanthemum powder was first fermented with Lactobacillus acidophilus, then extracted with anhydrous ethanol reflux, centrifuged and filtered to obtain a residue, and the filtrate was freeze-dried to obtain extract A; The obtained filter residue is sequentially subjected to gradient reflux extraction with ethanol solutions having volume fractions of 80%, 50% and 20% to obtain a filter residue; The obtained filter residue is subjected to pure water reflux extraction to obtain a water extract; The obtained water extract is ultrafiltered through a filter membrane, and the filtrate is freeze-dried to obtain macromolecular substance B; Extract A and macromolecular substance B are mixed, homogenized under high pressure using water as a solvent, and then ground using a ball mill. The resulting mixed solution is dried to obtain the Kunlun fetal chrysanthemum extract complex C; during the mixing, the mass ratio of the extract A to the macromolecular substance B is 1:2; The fermentation treatment is to inoculate the bacterial liquid of Lactobacillus acidophilus into the aqueous solution of Kunlun fetal chrysanthemum powder for shaking fermentation, and the inoculation amount of Lactobacillus acidophilus is 4%; The molecular weight cut-off of the filter membrane is 5000 Da.

2. The preparation method according to claim 1, characterized in that The shaking fermentation temperature was 37° C., the fermentation time was 48 h, and the rotation speed was 200 r / min.

3. The preparation method according to claim 1, characterized in that The temperature of the anhydrous ethanol reflux extraction is 60°C and the time is 2 hours; the temperature of the ethanol solution gradient reflux extraction is 60°C and the extraction time is 2 hours; the temperature of the pure water reflux extraction is 90°C and the extraction time is 2 hours.

4. The preparation method according to claim 1, characterized in that The number of ultrafiltrations was 6 times.

5. The preparation method according to claim 1, characterized in that The components of the extract A include 49.37wt% xanthophylloside, 6.24wt% kaempferol, 8.42wt% scutellariae and 5.47wt% marimoside; the molecular weight of the macromolecular substance B is 2297.841kDa, and the components include 27.64wt% arabinose, 28.31wt% galactose, 15.30wt% glucose and 15.05wt% galacturonic acid.

6. The preparation method according to claim 1, characterized in that The particle size of the Kunlun chrysanthemum extract complex C is 469±5.57 nm, the dispersion coefficient is 0.21±0.01, and the potential is -50.13±1.12 mV.

7. The Kunlun chrysanthemum extract complex obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the Kunlun chrysanthemum extract complex according to claim 7 in the preparation of products for regulating intestinal microorganisms.

9. Use of the Kunlun chrysanthemum extract complex according to claim 7 in the preparation of a product having hypoglycemic activity.

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