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

Abstract

The invention relates to the technical field of traditional Chinese medicine extracts, and discloses a penthorum chinense pursh extract for resisting non-alcoholic fatty liver and a preparation method thereof, and the method comprises the following steps: (1) pre-cooling penthorum chinense pursh stem raw materials, and carrying out low-temperature superfine grinding; (2) adding compound enzymes (cellulase, beta-glucosidase and alpha-rhamnosidase) into the raw materials subjected to superfine grinding, and carrying out enzymolysis treatment; and (3) carrying out two times of down-flow three-stage extraction on the raw materials in the step (2). And (4) further filtering by adopting a ceramic membrane to obtain the penthorum chinense pursh extract. The extraction method provided by the invention can greatly retain and enrich active components, and improves the bioavailability of glycoside substances. In addition, the original pungent smell and raw and green taste of penthorum chinense pursh are also weakened; meanwhile, the levels of total cholesterol and triglyceride can be remarkably reduced, and the compound has definite activity of resisting the non-alcoholic fatty liver disease and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine extracts, and in particular to a Herba Lysimachiae extract for resisting non-alcoholic fatty liver disease 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 tin, apigenin, 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, extracting and enriching these key components will help further research.

[0004] The existing extraction processes for Herba Chrysanthemi serrata mainly include the following problems: 1) Existing studies mostly use decoction or reflux methods, using water as a solvent to extract Herba Chrysanthemi serrata. However, flavonoids and tannins in Herba Chrysanthemi serrata are mostly present in various forms of glycosides. Compared with flavonoids / tannin aglycones, these glycosides are relatively poorly water-soluble, and the type, quantity and position of glycosides will affect their bioavailability. 2) The extraction efficiency of existing extraction methods is not high, the directional selectivity is poor, and the potential active ingredients cannot be fully enriched. 3) The Herba Chrysanthemi serrata stem extract obtained by the existing extraction method has a raw green taste, and is accompanied by a pungent smell when extracted at high temperature. 4) The Herba Chrysanthemi serrata extract is prone to precipitation during long-term storage and has poor stability. These problems limit the research and utilization of Herba Chrysanthemi serrata and its key components.

[0005] The stems of the Chinese yew are natural plant resources, and contain a large amount of cellulose, which hinders the dissolution of cell contents. Cellulase is a type of catalyst that plays a biocatalytic role in the decomposition of cellulose. It can decompose cellulose into monosaccharides or oligosaccharides. Its working principle is to catalyze the hydrolysis of β-1,4 glycosidic bonds and destroy long-chain cellulose molecules; and it is difficult for a single cellulase to completely degrade the natural cellulose substrate. The structure of glycosides in the stems of the Chinese yew is complex and varied. The current preparation method of the Chinese yew extract has a low extraction rate of effective ingredients, and the concentration of effective ingredients in the extract is low, making it difficult to fully utilize the Chinese yew resources. Therefore, there is an urgent need for an extraction method with high extraction efficiency, good selectivity, and the ability to effectively enrich components with anti-non-alcoholic fatty liver activity. Summary of the invention

[0006] The purpose of the present invention is to provide a Herba Chrysanthemi Indici extract for resisting non-alcoholic fatty liver disease and a preparation method thereof, in view of the problem that the extraction efficiency of the effective components of the Herba Chrysanthemi Indici extract is low, improve the extraction efficiency, and significantly increase the content of flavonoids / tannin aglycones. At the same time, the absorption rate of key components is improved, thereby being able to reduce the levels of total cholesterol and triglycerides, and having a clear activity of resisting non-alcoholic fatty liver disease.

[0007] The technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a method for preparing a Herba Lysimachiae extract for resisting non-alcoholic fatty liver disease, comprising the following steps:

[0009] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing;

[0010] Step (2): precooling and low-temperature ultrafine grinding of the crude raw material;

[0011] Step (3): 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] Preferably, the cellulase, β-glucosidase and α-rhamnosidase are compounded in a ratio of 1:1:1 to 2:1:1.

[0014] Step (4): Divide the enzyme-inactivated stem of the herb into three equal parts, perform two downstream three-stage extractions, and collect the filtrate;

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

[0016] According to a preferred embodiment, the precooling in step (2) is specifically to precool the crude raw material of the yellow grass stem to a brittle state; more specifically, the precooling temperature in step (2) is -40 to 0°C, and the precooling time is 1 to 5 hours.

[0017] According to a preferred embodiment, the temperature of the low-temperature ultrafine grinding chamber is 8-20° C., and the grinding time is 20-60 min.

[0018] According to a preferred embodiment, in step (3), the enzymatic hydrolysis material-liquid ratio is 1:2-5 (g / ml), the enzyme addition amount is 0.1%-1%, 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 (4), the material-liquid ratio is 1:10-50 (g / ml), the extraction temperature is 30-60° C., and the extraction time is 0.5-2.5 h.

[0020] Another aspect of the present invention provides a Herba Lysimachiae extract for resisting non-alcoholic fatty liver disease, which is prepared by the aforementioned preparation method.

[0021] According to a preferred embodiment, the gallic acid in the Herba Lysimachiae extract is greater than 20 μg / mL, the quercetin is greater than 14 μg / mL, the apigenin is greater than 39 μg / mL, the kaempferol is greater than 6 μg / mL, and the pyrocatechol is greater than 0.63 μg / mL.

[0022] According to a preferred embodiment, the Herba Lysimachiae extract does not contain (E)-2-nonenal.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. A Herba Chrysanthemi extract for preventing non-alcoholic fatty liver disease and a preparation method thereof. Cellulase, β-glucosidase and α-rhamnosidase are compounded in a ratio of 2:1:1, which has a synergistic effect and can significantly improve the hydrolysis efficiency of glycosides. Cellulase combines with cellulose constituting the cell wall, effectively destroys the cell wall, promotes the dissolution of a large number of active ingredients, and effectively improves the extraction efficiency.

[0025] 2. A Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease and a preparation method thereof. The extraction method provided by the present invention uses low-temperature ultrafine grinding to pre-treat the Herba Lysimachiae stems, which can ensure the uniformity of particle size and component distribution after grinding. At the same time, it can reduce the high temperature generated by mechanical action during the grinding process, and better retain the heat-sensitive active ingredients.

[0026] 3. A Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease and a preparation method thereof, which can achieve the purpose of enriching key components and improving extraction efficiency by optimizing various parameters in the low-temperature ultrafine grinding and compound enzyme hydrolysis process and combining low-temperature downstream multi-stage extraction; and can also reduce the relative content of characteristic volatile components in the Herba Lycopodiellae extract, weaken its irritating odor and raw green taste, and improve palatability.

[0027] 4. A Herba Lysimachiae extract for resisting non-alcoholic fatty liver disease and a preparation method thereof, which can significantly reduce the levels of total cholesterol and triglycerides and have clear activity against non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the clarity of the Herba Lycopodii stem extracts prepared in the examples of the present application (from left to right are examples 1 to 8);

[0029] Figure 2 This is a schematic diagram of the clarity of the Herba Lycopodii stem extracts prepared in the comparative examples of the present application (Comparative Examples 1 to 2 from left to right);

[0030] Figure 3 This is a schematic diagram of the turbidity change of the stem extract of the present invention's examples and comparative examples during storage;

[0031] Figure 4 HPLC chromatograms of gallic acid, quercetin, apigenin, kaempferol and pyrocatechol standards of this application;

[0032] Figure 5 The GC-MS total ion current diagram of the stem extract of the herbaceous chrysanthemum in the examples and comparative examples of the present application;

[0033] Figure 6 This is the HE staining image of liver tissue for animal experiments in this application. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0037] Example 1 Preparation method of a Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease

[0038] The steps include:

[0039] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0040] Step (2): The crude raw material is precooled and subjected to low-temperature ultrafine grinding. The precooling temperature is -20°C and the precooling time is 2 hours. The temperature of the low-temperature ultrafine grinding chamber is 10°C and the grinding time is 20 minutes.

[0041] Step (3): 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 0.8%, the enzymolysis pH is 3.0, the enzymolysis time is 2h, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

[0042] Step (4): Divide the enzyme-inactivated stem of the herb 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.

[0043] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii extract for resisting non-alcoholic fatty liver disease.

[0044] Measurement method:

[0045] 1. Stability test

[0046] (1) The prepared Herba Chrysanthemi anti-nonalcoholic fatty liver extract was placed in a 37°C incubator for an accelerated storage test, and samples were taken at 0d, 2d, 4d, 8d, 16d, 32d, 64d, and 128d. After sampling, the OD value was measured at a wavelength of 620nm.

[0047] (2) The prepared Herba Chrysanthemi anti-nonalcoholic fatty liver extract was placed in a 37°C incubator for accelerated storage test, and samples were taken at 0d, 2d, 4d, 8d, 16d, 32d, 64d, and 128d. The samples were centrifuged at a speed of 4000r / min for 10min, and the supernatant was discarded. The precipitate was dried at 80°C to constant weight, and the weight of the precipitate was recorded.

[0048] 2. Determination of total flavonoid content

[0049] Determination of total flavonoids content: NaNO2-Al(NO3)3-NaOH colorimetric method was used. 1 mL of the prepared anti-nonalcoholic fatty liver extract was transferred to a 25 mL colorimetric tube, and 1 mL of 5% sodium nitrite solution was added first, and the mixture was shaken and allowed to stand for 5 min. 1 mL of 10% aluminum nitrate solution was added, and the mixture was shaken and allowed to stand for 5 min. 10 mL of 4% sodium hydroxide solution was added, and finally 70% ethanol was added, and the volume was adjusted to 25 mL. The mixture was mixed and allowed to stand for 15 min, and a color reaction was performed. The absorbance value was determined at a wavelength of 510 nm. A linear regression was performed between 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.

[0050] 3. Determination of key ingredient content

[0051] 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 extract of Herba Chrysanthemi chinensis for resisting non-alcoholic fatty liver disease 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 a wavelength of 220 nm and 254 nm, and the peak area was recorded. The absorbance (Y) of the working solution of the five key components was linearly regressed against its concentration (X) to obtain the regression equation. Substitute the measured peak areas into the regression equation and calculate their respective contents.

[0052] 4. Determination of volatile components

[0053] The composition and relative content differences of the volatile components in the examples and comparative examples were determined by headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 5. Animal Experiments

[0058] 1) Experimental grouping: 120 SPF-grade C57BL / 6 mice were randomly divided into a blank group, a model group, an embodiment group (eight groups), and a comparative example group (two groups), with 10 mice in each group.

[0059] 2) Modeling: After 5 days of adaptive feeding, the mice in the model group, embodiment group and control group were fed a high-fat diet for modeling, and the blank group was given a normal feed. During the modeling period, the mice were allowed to eat and drink freely.

[0060] 3) Administration: In the fifth week, the Example group and the Comparative Example group were given the corresponding test substances 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.

[0061] 4) Detection indicators:

[0062] Organ index: The livers of mice in each group were taken, weighed, and the organ index was calculated. The calculation formula is as follows:

[0063]

[0064] 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.

[0065] 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.

[0066] Example 2 Preparation method of a Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease

[0067] The steps include:

[0068] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0069] Step (2): The crude raw material is precooled and subjected to low-temperature ultrafine grinding. The precooling temperature is -20°C and the precooling time is 2 hours. The temperature of the low-temperature ultrafine grinding chamber is 10°C and the grinding time is 20 minutes.

[0070] Step (3): 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 0.8%, the enzymolysis pH is 4.5, the enzymolysis time is 2h, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

[0071] Step (4): Divide the enzyme-inactivated stem of the herb 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.

[0072] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii extract for resisting non-alcoholic fatty liver disease.

[0073] Example 3 Preparation method of a Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease

[0074] The steps include:

[0075] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0076] Step (2): The crude raw material is precooled and subjected to low-temperature ultrafine grinding. The precooling temperature is -20°C and the precooling time is 2 hours. The temperature of the low-temperature ultrafine grinding chamber is 10°C and the grinding time is 20 minutes.

[0077] Step (3): 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 0.8%, the enzymolysis pH is 6.0, the enzymolysis time is 2h, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

[0078] Step (4): Divide the enzyme-inactivated stem of the herb 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.

[0079] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease prepared in Example 3.

[0080] Example 4 Preparation method of a Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease

[0081] The steps include:

[0082] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0083] Step (2): The crude raw material is precooled and subjected to low-temperature ultrafine grinding. The precooling temperature is -20°C and the precooling time is 2 hours. The temperature of the low-temperature ultrafine grinding chamber is 10°C and the grinding time is 20 minutes.

[0084] Step (3): 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 0.8%, the enzymolysis pH is 4.5, the enzymolysis time is 2h, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

[0085] Step (4): Divide the enzyme-inactivated stem of the herb 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.

[0086] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract for preventing and treating non-alcoholic fatty liver disease prepared in Example 4.

[0087] Measurement method

[0088] The same as Example 1.

[0089] Example 5 Preparation method of a Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease

[0090] The steps include:

[0091] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0092] Step (2): The crude raw material is precooled and subjected to low-temperature ultrafine grinding. The precooling temperature is -20°C and the precooling time is 2 hours. The temperature of the low-temperature ultrafine grinding chamber is 10°C and the grinding time is 20 minutes.

[0093] Step (3): 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 2:0:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 0.8%, the enzymolysis pH is 4.5, the enzymolysis time is 2h, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

[0094] Step (4): 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.

[0095] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease prepared in Example 5.

[0096] Measurement method

[0097] The same as Example 1.

[0098] Example 6 Preparation method of a Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease

[0099] The steps include:

[0100] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0101] Step (2): The crude raw material is precooled and subjected to low-temperature ultrafine grinding. The precooling temperature is -20°C and the precooling time is 2 hours. The temperature of the low-temperature ultrafine grinding chamber is 10°C and the grinding time is 20 minutes.

[0102] Step (3): 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 2:1:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.8%, the enzymolysis pH is 4.5, the enzymolysis time is 2h, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

[0103] Step (4): 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.

[0104] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii extract for preventing non-alcoholic fatty liver disease prepared in Example 6.

[0105] Measurement method

[0106] The same as Example 1.

[0107] Example 7 Preparation method of a Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease

[0108] The steps include:

[0109] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0110] Step (2): The crude raw material is precooled and subjected to low-temperature ultrafine grinding. The precooling temperature is -20°C and the precooling time is 2 hours. The temperature of the low-temperature ultrafine grinding chamber is 10°C and the grinding time is 20 minutes.

[0111] Step (3): 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 2:1:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 0.8%, the enzymolysis pH is 4.5, the enzymolysis time is 2h, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

[0112] Step (4): Divide the enzyme-inactivated stem of the herb 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 30° C., and the extraction time is 1.5 h.

[0113] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease prepared in Example 7.

[0114] Measurement method

[0115] The same as Example 1.

[0116] Example 8 Preparation method of a Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease

[0117] The steps include:

[0118] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0119] Step (2): The crude raw material is precooled and subjected to low-temperature ultrafine grinding. The precooling temperature is -20°C and the precooling time is 2 hours. The temperature of the low-temperature ultrafine grinding chamber is 10°C and the grinding time is 20 minutes.

[0120] Step (3): 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 2:1:1; the enzymolysis material-liquid ratio is 1:5 (g / ml), the enzyme addition amount is 0.8%, the enzymolysis pH is 4.5, the enzymolysis time is 2h, the enzymolysis temperature is 50°C; the enzyme inactivation temperature is 90°C, and the time is 15min.

[0121] Step (4): Divide the enzyme-inactivated stem of the herb 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.

[0122] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodii extract for preventing non-alcoholic fatty liver disease prepared in Example 8.

[0123] Measurement method

[0124] The same as Example 1.

[0125] Comparative Example 1: A method for preparing a Herba Lysimachiae extract for resisting non-alcoholic fatty liver disease

[0126] Compared with Example 1, this comparative example does not include the precooling and low-temperature ultrafine grinding steps in step (2) and the enzymatic hydrolysis step in step (3).

[0127] The steps include:

[0128] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0129] Step (2): Divide the raw material of the yellow grass stem treated in step (1) into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The extraction material-liquid ratio is 1:30 (g / ml), the extraction temperature is 60°C, and the extraction time is 1.5h.

[0130] Step (3): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract for preventing and treating non-alcoholic fatty liver disease prepared in Comparative Example 1.

[0131] Measurement method

[0132] The same as Example 1.

[0133] Comparative Example 2: A method for preparing a Herba Lysimachiae extract for resisting non-alcoholic fatty liver disease

[0134] Compared with Example 1, this comparative example does not include the precooling and low-temperature ultrafine grinding steps of step (2) and the enzymatic hydrolysis step of step (3). Compared with Comparative Example 1, the extraction temperature of this application is higher than that of Comparative Example 1.

[0135] The steps include:

[0136] Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0137] Step (2): Divide the raw material of the yellow grass stem treated in step (1) into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The extraction material-liquid ratio is 1:30 (g / ml), the extraction temperature is 95° C., and the extraction time is 1.5 h.

[0138] (3) Further filtering was performed using a ceramic membrane to obtain the Herba Lysimachiae extract for resisting non-alcoholic fatty liver disease prepared in Comparative Example 2.

[0139] Measurement method

[0140] The same as Example 1.

[0141] The measurement data of Examples 1-8 and Comparative Examples 1-2 are as follows:

[0142] Depend on Figures 1 to 3 It can be seen that the turbidity of Examples 1 to 8 and Comparative Examples 1 to 2 showed a trend of first increasing rapidly, then slowing down, and finally gradually stabilizing during the 128-day accelerated storage period. The turbidity of Example 8 was significantly lower than that of the others, indicating that it had good clarity.

[0143] like Figure 4 and Figure 5 The standard curve and measurement curve of the total flavonoids and key component contents are shown in Table 1-2 below. The total flavonoids and key component contents in the stem extracts of Example 1-8 and Comparative Example 1-2 are shown in Table 1-2 below:

[0144] Table 1 Total flavonoids content

[0145]

[0146] Table 2 Key ingredient content

[0147]

[0148] As shown in Table 1-2, the total flavonoids and key components in the extracts of the stem of the Chinese yew prepared in Examples 1 to 8 are increased to varying degrees compared with those in Comparative Examples 1-2; among them, the total flavonoids content in Example 8 is the highest, which is 2.19 mg / mL. The contents of gallic acid, quercetin, apigenin, kaempferol, and pyracantha are better than those in the others, which are 50.24, 17.79, 51.94, 8.69, and 1.04 μg / mL, respectively.

[0149] The relative contents of volatile components in the stem extracts of the Herba Lycopodii of Examples 1-8 and Comparative Examples 1-2 are shown in Table 3:

[0150] Table 3 Relative content of volatile components

[0151]

[0152] Note: “ / ” means not detected.

[0153] As shown in Table 3, (E)-2-nonenal was not detected in Examples 1 to 8, and 2-undecanone and geranyl acetone were not detected in Examples 6, 7, and 8. The relative contents of lauryl aldehyde and 2,4-di-tert-butylphenol in Examples 1 to 8 were reduced.

[0154] The effects of the stem extracts of Example 1-8 and Comparative Example 1-2 on mouse organs are shown in Table 4:

[0155] Table 4 Animal experiment detection index table

[0156]

[0157] 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 8 had different degrees of effect on the mice. Among them, the effect of Example 8 was the most significant. Comparative Examples 1 to 2 had some improvement, but the improvement was far less than that of Examples 1 to 8.

[0158] like Figure 6 It can be seen that compared with the blank group, after HE staining, the tissues of comparative examples 1 to 2 and the model group appeared purple in the visual field, and there were a large number of white fat droplets of varying sizes. Compared with the model group, Examples 1 to 8 were able to reduce the number of white fat droplets and improve hepatic fatty degeneration.

[0159] In summary, the conditions involved in Example 8 are optimal conditions, wherein the enzymatic hydrolysis pH is 4.5, the enzymatic hydrolysis liquid ratio is 1:5 (g / ml); the low-temperature ultrafine grinding chamber temperature is 10°C, and the grinding time is 20min; cellulase, β-glucosidase and α-rhamnosidase are compounded in a ratio of 2:1:1; and the extraction temperature of the downstream three-stage extraction is 60°C.

[0160] 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 method for preparing a Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease, characterized in that: The steps include: Step (1): picking fresh yellow grass stems of uniform maturity and good quality, removing impurities, washing, drying and crushing; Step (2): precooling and low-temperature ultrafine grinding of the crude raw material; Step (3): 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 (4): Divide the enzyme-inactivated stem of the herb into three equal parts, perform two downstream three-stage extractions, and collect the filtrate; Step (5): further filtering using a ceramic membrane.

2. The method for preparing a Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease according to claim 1, characterized in that: The precooling in step (2) specifically involves precooling the crude raw material of the herbaceous stalks to a brittle state.

3. The method for preparing a Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease according to claim 1, characterized in that: The pulverizing time in the low-temperature ultrafine pulverizing chamber in the step (2) is 20 to 60 minutes.

4. The method for preparing a Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease according to claim 1, characterized in that: In step (3), the enzymatic hydrolysis material-liquid ratio is 1:2-5 (g / ml), the enzymatic hydrolysis pH is 3-6, the enzymatic hydrolysis temperature is 30-60° C., and the enzyme inactivation temperature is 90° C. for 15 min.

5. The method for preparing a Herba Lycopodiellae extract for preventing non-alcoholic fatty liver disease according to claim 1, characterized in that: The extraction temperature in step (4) is 30-60°C.

6. A Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease, characterized in that: The preparation is carried out by the preparation method according to any one of claims 1 to 5.

7. The Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease according to claim 6, characterized in that: The extract of the herb contains more than 20 μg / mL of gallic acid, more than 14 μg / mL of quercetin, more than 39 μg / mL of apigenin, more than 6 μg / mL of kaempferol, and more than 0.63 μg / mL of pyrocatechol.

8. The Herba Lysimachiae extract for preventing non-alcoholic fatty liver disease according to claim 6, characterized in that: The Herba Lysimachiae extract does not contain (E)-2-nonenal.

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