Penthorum chinense pursh compound for resisting non-alcoholic fatty liver and preparation method of penthorum chinense pursh compound

Abstract

The invention relates to the technical field of traditional Chinese medicine extracts, and discloses a penthorum chinense pursh compound for resisting non-alcoholic fatty liver and a preparation method thereof, the penthorum chinense pursh compound comprises 55-80 parts of penthorum chinense pursh stem, 10-20 parts of purple glutinous rice bran and 10-25 parts of phlorizin extract. The preparation method comprises the following steps: (1) mixing the penthorum chinense pursh stem raw material with the purple waxy wheat bran according to the component proportion, pre-cooling, and carrying out low-temperature superfine grinding; and (2) adding a compound enzyme into the crushed raw materials for enzymolysis. The compound enzyme is formed by compounding cellulase, beta-glucosidase and alpha-rhamnosidase; and (3) taking the raw materials after enzyme deactivation, carrying out downstream three-stage extraction, and collecting filtrate. And (4) adding the phlorizin extract into the filtrate obtained in the step (3), and uniformly stirring and mixing. And (5) filtering. According to the prepared penthorum chinense pursh compound, the content of total flavonoids and key components is remarkably increased, the raw and green taste of the medicine is reduced, and the effect of resisting the non-alcoholic fatty liver is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine extracts, and in particular to a Herba Lysimachiae anti-nonalcoholic fatty liver disease-resistant Herba Lysimachiae complex and a preparation method thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Penthorum chinense Pursh is a traditional Miao medicine in Gulin. With the deepening of research on the chemical composition and pharmacological effects of Penthorum chinense Pursh, it has been proven that Penthorum chinense Pursh has the effect of resisting non-alcoholic fatty liver disease. Based on previous studies, quercetin, pine pine, kaempferol, catechin, tannic acid, tagetin-7-O-[6``-(2-methylbutyryl)]-glucoside, puerarin apiglycoside, delphinidin-3,5-O-diglucoside chloride, and acetic acid are considered to be the key components of Penthorum chinense Pursh in its anti-non-alcoholic fatty liver disease activity. Therefore, it is very important to extract and enrich these key components to give full play to their anti-non-alcoholic fatty liver disease effects.

[0004] However, flavonoids and tannins in the herb are mostly present in various forms of glycosides. Compared with flavonoids / tannin aglycones, these glycosides are relatively poorly water-soluble. Therefore, the traditional extraction method cannot fully extract these glycosides, and the directional selectivity is poor. In addition, the type, quantity and position of glycosides will affect their bioavailability. The herb stem extract obtained by the traditional extraction method has a strong green taste, and the extract obtained by the hot extraction method is also accompanied by a pungent smell. The stability is poor during long-term storage, and precipitation is easy to occur, making it difficult to apply to food research and development; and the traditional extraction method has low extraction efficiency, poor directional selectivity, and cannot fully enrich potential active ingredients. Therefore, there is an urgent need for a herb complex with anti-nonalcoholic fatty liver activity, a high content of effective ingredients, and improved flavor and stability of the herb extract. Summary of the invention

[0005] The purpose of the present invention is to provide a Herba Lycopodiellae anti-nonalcoholic fatty liver disease compound and a preparation method thereof in view of the limitations of the current extract preparation method, solve the problems of low extraction efficiency, low content of effective ingredients, strong raw green taste, instability and easy precipitation in the current extraction method, significantly increase the content of flavonoids / tannin aglycones in the compound, and improve the absorption rate of key ingredients.

[0006] The technical solution of the present invention is as follows:

[0007] On one hand, the present invention provides a Herba Lysimachiae complex for resisting non-alcoholic fatty liver disease, comprising 55-80 parts of Herba Lysimachiae stems, 10-30 parts of purple glutinous wheat bran, and 8-25 parts of phlorizin extract; the Herba Lysimachiae stems and purple glutinous wheat bran are hydrolyzed by a composite enzyme and then mixed with the phlorizin extract.

[0008] According to a preferred embodiment, the complex enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 1:0:1 to 3:3:2.

[0009] Another aspect of the present invention provides a method for preparing a Herba Lysimachiae anti-nonalcoholic fatty liver disease compound, comprising the following steps:

[0010] Step (1): mixing the stem of the yellow grass raw material and the purple glutinous wheat bran according to the aforementioned component ratio, precooling, and low-temperature ultrafine grinding;

[0011] Step (2): adding a complex enzyme to the crushed raw material for enzymolysis and enzyme inactivation.

[0012] The complex enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 1:0:1 to 3:3:2;

[0013] Step (3): Divide the raw material of the yellow grass stem after the enzyme is inactivated into three equal parts, perform two downstream three-stage extractions, and collect the filtrate.

[0014] Step (4): Add the phlorizin extract to the filtrate of step (3) and stir to mix.

[0015] Step (5): further filtering using a ceramic membrane.

[0016] According to a preferred embodiment, the precooling temperature in step (1) is -40 to 0°C, and the precooling time is 1 to 5 hours.

[0017] According to a preferred embodiment, in step (1), the temperature of the low-temperature ultrafine grinding chamber is 8 to 20° C., and the grinding time is 25 to 60 minutes.

[0018] According to a preferred embodiment, in step (2), the enzymatic hydrolysis material-liquid ratio is 1g:2-5ml, the enzyme addition amount is 0.5%-2%, the enzymatic hydrolysis pH is 3-6, the enzymatic hydrolysis time is 1-3h, the enzymatic hydrolysis temperature is 30-60°C; the enzyme inactivation temperature is 90°C and the time is 15min.

[0019] According to a preferred embodiment, in step (3), the material-liquid ratio is 1 g:10-50 ml, the extraction temperature is 30-60° C., and the extraction time is 0.5-2.5 h.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. A compound of Herba Lycopodii for preventing non-alcoholic fatty liver disease and a preparation method thereof. The compound enzyme (cellulase, β-glucosidase, α-rhamnosidase) is used to enzymolyze Herba Lycopodii stems and purple glutinous wheat bran. The combined use of the three enzymes has a more synergistic effect, significantly increasing the content of flavonoids / tannin aglycones, thereby improving the bioavailability of flavonoids / tannin glycosides;

[0022] 2. A Herba Lysimachiae complex for resisting non-alcoholic fatty liver disease and a preparation method thereof. The complex provided by the present invention is based on Herba Lysimachiae stems, supplemented with purple glutinous wheat bran and phloridzin. Purple glutinous wheat bran has the effects of removing heat and nourishing liver qi; and it is rich in anthocyanins such as delphinidin and dietary fiber, which can resist oxidation, enhance the body's immunity, and regulate intestinal flora and serum cholesterol. Phloridzin belongs to the dihydrochalcone component in flavonoids, and has multiple biological activities such as anti-oxidation, treatment of diabetes, and improvement of memory. The three are compounded under certain conditions, not only without the need to add additional sweeteners, but also with a synergistic effect, which significantly enhances the anti-non-alcoholic fatty liver activity of the complex. And it has good stability during long-term storage.

[0023] 3. A compound of Herba Chrysanthemi anti-nonalcoholic fatty liver and a preparation method thereof, wherein the Herba Chrysanthemi stems and purple glutinous wheat bran are pre-treated by low-temperature ultrafine grinding, so that the two can be fully mixed, and the particle size and composition distribution are uniform after grinding. At the same time, the heat generation during the grinding process is reduced, and the loss of heat-sensitive active ingredients and volatile components is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of the turbidity of a Herba Chrysanthemi Indici composite for resisting non-alcoholic fatty liver disease (from left to right are Examples 1 to 7);

[0025] Figure 2 A schematic diagram of the turbidity of a Herba Chrysanthemi Indici compound for resisting non-alcoholic fatty liver disease (from left to right are comparative examples 1 to 6);

[0026] Figure 3 A schematic diagram showing the changes in turbidity and centrifugal sedimentation rate of the Herba Chrysanthemi Indici composites for resisting non-alcoholic fatty liver disease prepared in Examples 1 to 7 and Comparative Examples 1 to 6;

[0027] Figure 4 HPLC chromatograms of gallic acid, quercetin, apigenin, kaempferol, and pyrocatechol standards;

[0028] Figure 5 The GC-MS total ion flow diagram of the Herba Chrysanthemi Indici composites with alcohol sobering and liver protecting effects prepared in Examples 1 to 7 and Comparative Examples 1 to 6, corresponding to the order in the upper right corner from top to bottom;

[0029] Figure 6 HE staining images of liver tissues in the animal experiments of Examples 1 to 7 and Comparative Examples 1 to 6. DETAILED DESCRIPTION

[0030] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0032] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0033] Example 1 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0034] The steps include:

[0035] Step (1): 66 parts of the raw material of the yellow grass stem and 18 parts of the purple glutinous wheat bran are respectively mixed, precooled, and low-temperature ultrafine pulverized. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine pulverization chamber temperature is 10°C, and the pulverization time is 25 minutes.

[0036] Step (2): Adding compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 1:1:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0037] Step (3): Divide the raw material of the yellow grass stem after the enzyme is inactivated into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The extraction material-liquid ratio is 1g:30ml, the extraction temperature is 60°C, and the extraction time is 1.5h.

[0038] Step (4): Add 16 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0039] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Example 1.

[0040] The various parameters of the prepared Herba Lysimachiae complex for resisting non-alcoholic fatty liver disease were measured according to the following measurement methods:

[0041] 1. Stability test

[0042] Step (1): placing the prepared anti-non-alcoholic fatty liver disease compound in a 37°C incubator for an accelerated storage test, and taking samples at 0d, 2d, 4d, 8d, 16d, 32d, 64d, and 128d. After taking samples, measuring the OD value at a wavelength of 620nm;

[0043] Step (2): Place the prepared anti-non-alcoholic fatty liver disease compound in a 37°C incubator for accelerated storage test, and take samples at 0d, 2d, 4d, 8d, 16d, 32d, 64d, and 128d. Centrifuge the taken samples at a speed of 4000r / min for 10min, and discard the supernatant. Dry the precipitate at 80°C to constant weight, and record the weight of the precipitate.

[0044] 2. Determination of total flavonoid content

[0045] Determination of total flavonoids content: NaNO2-Al(NO3)3-NaOH colorimetric method was used. Pipette 1 mL of the prepared anti-nonalcoholic fatty liver disease rutin complex into a 25 mL colorimetric tube, first add 1 mL of 5% sodium nitrite solution, shake well and let stand for 5 min, add 1 mL of 10% aluminum nitrate solution, shake well and let stand for 5 min, then add 10 mL of 4% sodium hydroxide solution, and finally add 70% ethanol, make up to 25 mL, mix well and let stand for 15 min, perform color reaction, and measure the absorbance at a wavelength of 510 nm. Linear regression was performed on the absorbance (Y) of the rutin working solution and its concentration (X), and the regression equation was: Y = 11.909X + 0.0011, R 2 =0.998. The measured absorbance value was substituted into the regression equation and the total flavonoid content was calculated.

[0046] 3. Determination of key ingredient content

[0047] High performance liquid chromatography (HPLC) was used to determine the contents of quercetin, pine pine, kaempferol, apigenin and gallic acid. 1 mL of the prepared anti-nonalcoholic fatty liver compound was respectively transferred through a 0.22 μm filter membrane and placed in a brown injection bottle. The chromatographic column was InertSustain C18 (5 μm×4.6×250 mm); mobile phase: 0.1% phosphoric acid (A) and acetonitrile (B); flow rate: 1 mL / min; elution gradient: 0-30 min: 95% A-75% A; 30-55 min: 75% A-55% A; 55-58 min: 55% A-95% A; column temperature: 35°C; injection volume: 20 μL; the measurement was carried out at 220 nm and 254 nm wavelengths, and the peak area was recorded. The absorbance (Y) of the five key component working solutions was linearly regressed with their concentrations (X) to obtain the regression equation. Substitute the measured peak areas into the regression equation and calculate their respective contents.

[0048] 4. Determination of volatile components

[0049] Headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) was used to determine the composition and relative content differences of the volatile components in the prepared Herba Lycopodiellae complex for anti-nonalcoholic fatty liver disease.

[0050] HS-SPME conditions: Accurately measure 5 mL of the test solution into a 20 mL headspace bottle, add 10 μL of 2-octanol solution with a mass concentration of 6.3 μg / mL, seal it and put it in a constant temperature water bath at 60 °C, balance for 5 min, extract and adsorb for 30 min (65-UM), and analyze at 250 °C for 5 min. Repeat the experiment three times.

[0051] GC conditions: chromatographic column is DB-5MS quartz capillary column (30m×0.25mm×0.25μm); injection port temperature: 250℃; carrier gas is high-purity helium (purity ≥99.999%); column flow rate: 1mL / min (constant flow mode), non-split injection. Heating program: initial temperature 50℃, hold for 5min; increase the temperature to 150℃ at 4℃ / min, hold for 5min; then increase the temperature to 250℃ at 10℃ / min, hold for 3min, total running time: 48min.

[0052] MS conditions: electron impact ion source (EI); transfer line temperature: 280°C, ion source: 230°C; ionization: 70 eV; quadrupole: 150°C; full scan; scan range: 40-600 m / z; interface: 250°C, column temperature: 40°C, injection port: 250°C.

[0053] 5. Animal Experiments

[0054] Step (1): Experimental grouping: 150 SPF-grade C57BL / 6 mice were randomly divided into a blank group, a model group, an embodiment group (seven groups), and a comparative example group (six groups), with 10 mice in each group.

[0055] Step (2): Modeling: After 5 days of adaptive feeding, the model group, the example group and the comparative example group were fed a high-fat diet for modeling, and the blank group was given a normal feed. The mice were allowed to eat and drink freely during the modeling period.

[0056] Step (3): Administration: In the fifth week, the Example group and the Comparative Example group were given the corresponding test substance at 3 ml / kg BW, and the Blank group and the Model group were gavaged with an equal volume of pure water. The administration lasted for 5 weeks in total.

[0057] Step (4): Testing indicators:

[0058] Organ index: The livers of mice in each group were taken, weighed, and the organ index was calculated. The calculation formula is as follows:

[0059]

[0060] Hepatic lipid levels: The livers of mice in each group were taken to prepare 10% liver homogenate, and the levels of total cholesterol (TC) and triglyceride (TG) in the liver homogenate were determined using a biochemical analyzer.

[0061] Pathological tissue observation: The livers of mice in each group were fixed with 4% paraformaldehyde, dehydrated, embedded, sliced, and stained with HE after fixation, and the tissue conditions were observed under a microscope.

[0062] Example 2 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0063] The steps include:

[0064] Step (1): 66 parts of the raw material of the yellow grass stem and 18 parts of the purple glutinous wheat bran are mixed, precooled, and low-temperature ultrafine ground. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine ground chamber temperature is 10°C, and the grounding time is 35 minutes.

[0065] Step (2): Adding compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 1:1:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0066] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0067] Step (4): Add 16 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0068] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Example 2.

[0069] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0070] Example 3 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0071] The steps include:

[0072] Step (1): 66 parts of the raw material of the yellow grass stem and 18 parts of the purple glutinous wheat bran are respectively mixed, precooled, and low-temperature ultrafine pulverized. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine pulverization chamber temperature is 10°C, and the pulverization time is 25 minutes.

[0073] Step (2): Adding compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase, and α-rhamnosidase in a ratio of 3:0:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0074] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0075] Step (4): Add 16 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0076] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Example 3.

[0077] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0078] Example 4 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0079] The steps include:

[0080] Step (1): 66 parts of the raw material of the yellow grass stem and 18 parts of the purple glutinous wheat bran are respectively mixed, precooled, and low-temperature ultrafine pulverized. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine pulverization chamber temperature is 10°C, and the pulverization time is 25 minutes.

[0081] Step (2): Add compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 3:2:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0082] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0083] Step (4): Add 16 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0084] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Example 4.

[0085] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0086] Example 5 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0087] The steps include:

[0088] Step (1): 60 parts of the raw material of the yellow grass stem and 27 parts of the purple glutinous wheat bran are mixed, precooled, and low-temperature ultrafine pulverized. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine pulverization chamber temperature is 10°C, and the pulverization time is 25 minutes.

[0089] Step (2): Add compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 3:2:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0090] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0091] Step (4): Add 13 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0092] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Example 5.

[0093] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0094] Example 6 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0095] The steps include:

[0096] Step (1): 55 parts of the raw material of the yellow grass stem and 20 parts of the purple glutinous wheat bran are mixed, precooled, and low-temperature ultrafine pulverized. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine pulverization chamber temperature is 10°C, and the pulverization time is 25 minutes.

[0097] Step (2): Add compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 3:2:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0098] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0099] Step (4): Add 25 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0100] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Example 6.

[0101] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0102] Example 7 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0103] The steps include:

[0104] Step (1): 80 parts of the raw material of the yellow grass stem and 10 parts of the purple glutinous wheat bran are mixed, precooled, and low-temperature ultrafine ground. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine ground chamber temperature is 10°C, and the grounding time is 25 minutes.

[0105] Step (2): Add compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 3:2:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0106] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0107] Step (4): Add 10 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0108] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Example 7.

[0109] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0110] Comparative Example 1 Preparation of a Herba Lysimachiae Extract for Anti-nonalcoholic Fatty Liver Disease

[0111] This comparative example is a further improvement on Example 4. The difference from Example 4 is that purple glutinous wheat bran and phlorizin extract are not added in this comparative example, and the other conditions are consistent with Example 4.

[0112] The specific steps include:

[0113] Step (1): 66 portions of the raw material of the stem of the yellow grass were taken respectively, and precooled and ultrafinely crushed at low temperature. The precooling temperature was -20°C, and the precooling time was 2 hours. The temperature of the ultrafinely crushing chamber was 10°C, and the crushing time was 25 minutes.

[0114] Step (2): Add compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 3:2:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0115] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0116] Step (4): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Comparative Example 1.

[0117] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0118] Comparative Example 2 Preparation of a Herba Lysimachiae Extract for Anti-nonalcoholic Fatty Liver Disease

[0119] This comparative example is a further improvement on Example 4. The difference from Example 4 is that purple glutinous wheat bran is not added in this comparative example, and the other conditions are consistent with Example 4.

[0120] The specific steps include:

[0121] Step (1): 66 portions of the raw material of the stem of the yellow grass were precooled and ultrafinely crushed at low temperature. The precooling temperature was -20°C, the precooling time was 2 hours, the temperature of the ultrafinely crushing chamber was 10°C, and the crushing time was 25 minutes.

[0122] Step (2): Add compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 3:2:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0123] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0124] Step (4): Add 16 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0125] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Comparative Example 2.

[0126] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0127] Comparative Example 3 Preparation of a Herba Lysimachiae Extract for Anti-nonalcoholic Fatty Liver Disease

[0128] This comparative example is a further improvement of Example 4. The difference from Example 4 is that the phlorizin extract is not added in this comparative example, and the other conditions are consistent with those of Example 4.

[0129] The specific steps include:

[0130] Step (1): 66 parts of the raw material of the yellow grass stem and 18 parts of the purple glutinous wheat bran are respectively mixed, precooled, and low-temperature ultrafine pulverized. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine pulverization chamber temperature is 10°C, and the pulverization time is 25 minutes.

[0131] Step (2): Add compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 3:2:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0132] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0133] Step (4): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Comparative Example 3.

[0134] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0135] Comparative Example 4 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0136] This comparative example is a further improvement on Example 4. Different from Example 4, this comparative example does not perform the pre-cooling low-temperature crushing and enzymatic hydrolysis steps.

[0137] The specific steps include:

[0138] Step (1): 66 parts of yellow grass stem raw material and 18 parts of purple glutinous wheat bran are respectively mixed.

[0139] Step (2): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0140] Step (3): Add 16 parts of phlorizin extract to the filtrate of step (2) and stir to mix.

[0141] Step (4): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Comparative Example 4.

[0142] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0143] Comparative Example 5 Preparation of a Herba Lysimachiae Compound for Anti-nonalcoholic Fatty Liver Disease

[0144] This comparative example is a further improvement on Example 4. Unlike Example 4, this comparative example does not perform a pre-cooling low-temperature pulverization step.

[0145] The specific steps include:

[0146] Step (1): 66 parts of yellow grass stem raw material and 18 parts of purple glutinous wheat bran are respectively mixed.

[0147] Step (2): Add compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 3:2:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 1.2%, the enzymolysis pH is 4.5, the enzymolysis time is 2 hours, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15 minutes.

[0148] Step (3): Divide the enzyme-inactivated stem of the Herba Lysimachiae into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60° C., and the extraction time is 1.5 h.

[0149] Step (4): Add 16 parts of phlorizin extract to the filtrate of step (3) and stir to mix.

[0150] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Comparative Example 5.

[0151] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0152] Comparative Example 6 Preparation of a Herba Lysimachiae Extract for Anti-nonalcoholic Fatty Liver Disease

[0153] This comparative example is a further improvement of comparative example 3. Different from comparative example 3, this comparative example does not perform an enzymatic hydrolysis step.

[0154] The specific steps include:

[0155] Step (1): 66 parts of the raw material of the yellow grass stem and 18 parts of the purple glutinous wheat bran are respectively mixed, precooled, and low-temperature ultrafine pulverized. The precooling temperature is -20°C, the precooling time is 2 hours, the low-temperature ultrafine pulverization chamber temperature is 10°C, and the pulverization time is 25 minutes.

[0156] Step (2): Divide the crushed raw material into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60°C, and the extraction time is 1.5h.

[0157] Step (3): further filtering with a ceramic membrane to obtain the Herba Lycopodii complex for resisting non-alcoholic fatty liver disease prepared in Comparative Example 6.

[0158] The measurement methods of various parameters of the prepared Herba Lysimachiae anti-nonalcoholic fatty liver disease composite are consistent with those in Example 1.

[0159] The measurement results of various parameters of Examples 1 to 7 and Comparative Examples 1 to 6 are summarized as follows:

[0160] Depend on Figures 1 to 3 It can be seen that in the 128-day accelerated storage period, the turbidity of Examples 1 to 7 and Comparative Examples 1 to 6 showed a trend of first significantly increasing, then the rate of increase gradually slowed down, and finally tending to be stable. Among them, the turbidity of Examples 4 and 5 was significantly lower than that of the others, indicating that they had good clarity.

[0161] like Figure 4 HPLC chromatograms of gallic acid, quercetin, apigenin, kaempferol, and pyrocatechol standards are shown in Figure 1. Figure 5 It is a GC-MS total ion flow chart, which corresponds to the order in the upper right corner from top to bottom. The total flavonoids and key component contents in the complex prepared by Examples 1 to 7 and Comparative Examples 1 to 6 are shown in Tables 1 to 2 below:

[0162] Table 1 Total flavonoids content

[0163]

[0164] Table 2 Key ingredient content

[0165]

[0166] As shown in Table 1-2, the contents of total flavonoids and key components in the Herba Lysimachiae stem composites prepared in Examples 1 to 7 were increased to varying degrees compared with those in Comparative Examples 1 to 6.

[0167] Examples 4 to 7 differ only in the addition ratios of the extracts of the stems of the yellow grass, the bran of the purple glutinous wheat and the phloridzin. By comparing the effects of Examples 4 to 7 in Table 1 and Table 2, it can be seen that the total flavonoid content of Examples 4, 6 and 7 is lower than that of Example 5 by 3.43 mg / mL; and the contents of gallic acid, quercetin and apigenin in Example 5 are better than those of the others, which are 95.45, 41.56 and 82.67 μg / mL respectively; while the contents of kaempferol and pyrocatechol in Example 5 are lower than those in Example 4 by 30.79 and 2.29 μg / mL; and the contents in Examples 4 to 7 are significantly higher than those in Comparative Example 1.

[0168] Comparing the results of Examples 1, 3 and 4 in Table 1, it can be seen that when the amount of enzyme added is the same and only the ratio of the complex enzyme is changed, the content of total flavonoids in the extract changes, among which Example 3 lacks β-glucosidase and its total flavonoid content is lower than that of Example 1; and the total flavonoids of Example 1 are lower than those of Example 4. Comparing the results of Examples 1 and 2 in Table 1, it can be seen that in Example 2, the pulverization time is extended, but the total flavonoid content is reduced.

[0169] Comparing the results of Examples 1, 3 and 4 in Table 2, it can be seen that the content of gallic acid in Example 3 is significantly lower than that in Example 1; and although the gallic acid content in Example 1 is increased, the contents of quercetin, apigenin, kaempferol and pyrrolidone are all lower than those in Example 3; and comparing the data of Example 1 and Example 4, it can be seen that the contents of gallic acid, quercetin, apigenin, kaempferol and pyrrolidone are increased to varying degrees in Example 4, among which the extraction effects of quercetin, pyrrolidone and kaempferol are the best.

[0170] In summary, the ratio of the composite enzyme used in Example 4 is the optimal enzymatic hydrolysis ratio. The three have a synergistic effect under this ratio, which not only significantly increases the content of quercetin, pine cones and kaempferol, but also increases the content of other key ingredients.

[0171] The volatile components in the complexes of the stems of the yellow grass prepared in Examples 1 to 7 and Comparative Examples 1 to 4 are shown in Table 3:

[0172] Table 3 Relative content of volatile components

[0173]

[0174] As shown in Table 3, (E)-2-nonenal, 2-undecanone and geranylacetone were not detected in Examples 1 to 7. Although lauryl aldehyde and 2,4-di-tert-butylphenol were detected, their relative contents were significantly reduced compared with those in Comparative Examples 1 to 4, thereby reducing the odor of the Herba Glehniae extract. Comparing the data of Examples 4 and 5 with Comparative Example 1, it can be seen that in Comparative Example 1, purple glutinous wheat bran and phlorizin extract were not added, and its volatile components were significantly higher than those in Examples 4 and 5.

[0175] The results of the animal experiments are shown in Table 4 and Figure 6 As shown,

[0176] Table 4 Animal experiment detection index table

[0177]

[0178] As shown in Table 4, compared with the blank group, the organ index, TC and TG levels of the mice in the model group were significantly increased. Compared with the model group, Examples 1 to 7 had different effects on the mice, among which Example 5 had the best effect.

[0179] like Figure 6 As shown, compared with the blank group, after HE staining, the tissues of comparative examples 1 to 6 and the model group appeared purple in the visual field, and there were a large number of round lipid droplets of varying sizes. Compared with the model group, examples 1 to 7 can alleviate hepatic fatty degeneration to varying degrees, reduce the number of lipid droplets, and make cells evenly arranged.

[0180] In summary, after adding purple glutinous wheat bran and phlorizin extract in Examples 4 and 5, the total flavonoids and key component contents were significantly increased, and the volatile components were reduced compared with the control example 1 without adding. At the same time, the extracts were subjected to animal experiments and had significant effects on anti-nonalcoholic fatty liver disease. It can be seen that the three synergistically produced many excellent effects.

[0181] In order to give full play to the synergistic effect of the compound of the stem of the Chinese yew, the conditions involved in Example 5 are the optimal conditions, wherein the compound includes 60 parts of the stem of the Chinese yew raw material, 27 parts of the purple glutinous wheat bran and 13 parts of the phloridzin extract. The temperature of the low-temperature ultrafine grinding chamber is 10°C, and the grinding time is 25 minutes. Cellulase, β-glucosidase, and α-rhamnosidase are compounded in a ratio of 3:2:1.

[0182] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A Herba Lysimachiae complex for preventing non-alcoholic fatty liver disease, characterized in that: The invention comprises 55-80 parts of scutellaria baicalensis stems, 10-20 parts of purple glutinous wheat bran and 10-25 parts of phlorizin extract. The scutellaria baicalensis stems and purple glutinous wheat bran are hydrolyzed by composite enzyme and then mixed with the phlorizin extract.

2. The Herba Lysimachiae complex for preventing non-alcoholic fatty liver disease according to claim 1, characterized in that: The complex enzyme is composed of cellulase, beta-glucosidase and alpha-rhamnosidase in a ratio of 1:0:1 to 3:3:

2.

3. The method for preparing a Herba Lysimachiae complex for preventing non-alcoholic fatty liver disease according to claim 1 or 2, characterized in that: The steps include: Step (1): mixing the stem of the yellow grass raw material and the purple glutinous wheat bran according to the aforementioned component ratio, precooling, and low-temperature ultrafine grinding; Step (2): adding a complex enzyme to the crushed raw material for enzymolysis and enzyme inactivation; The complex enzyme is composed of cellulase, β-glucosidase and α-rhamnosidase in a ratio of 1:0:1 to 3:3:2; Step (3): Divide the enzyme-inactivated stem of the Herba Lycopodii into three equal parts, perform two downstream three-stage extractions, and collect the filtrate; Step (4): adding the phlorizin extract to the filtrate of step (3) and stirring to mix; Step (5): Filter.

4. The method for preparing a Herba Lycopodiellae anti-nonalcoholic fatty liver disease compound according to claim 3, characterized in that: In the step (1), the precooling temperature is -40 to 0°C, and the precooling time is 1 to 5 hours.

5. The method for preparing a Herba Lycopodiellae anti-nonalcoholic fatty liver disease compound according to claim 3, characterized in that: In the step (1), the temperature of the low-temperature ultrafine grinding chamber is 8-20° C., and the grinding time is 25-60 min.

6. The method for preparing a Herba Lycopodiellae anti-nonalcoholic fatty liver disease compound according to claim 3, characterized in that: In the step (2), the enzymatic hydrolysis material-liquid ratio is 1g:2-5 / ml, the enzyme addition amount is 0.5%-2%, the enzymatic hydrolysis pH is 3-6, the enzymatic hydrolysis time is 1-3h, the enzymatic hydrolysis temperature is 30-60°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

7. The method for preparing a Herba Lycopodiellae anti-nonalcoholic fatty liver disease compound according to claim 3, characterized in that: In the step (3), the material-liquid ratio is 1 g: 10-50 ml, the extraction temperature is 30-60° C., and the extraction time is 0.5-2.5 h.

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