Penthorum chinense pursh extract with effects of dispelling effects of alcohol and protecting liver and preparation method of penthorum chinense pursh extract

Abstract

The invention relates to the technical field of biological medicines, and discloses a penthorum chinense pursh extract with effects of dispelling effects of alcohol and protecting liver and a preparation method thereof, the preparation method comprises the following steps: step (1): picking mature and fresh penthorum chinense pursh leaves, removing impurities, cleaning, drying, crushing and coarsely processing; (2) precooling the coarsely prepared raw materials, and carrying out low-temperature superfine grinding; (3) adding a compound enzyme into the crushed raw materials for enzymolysis and enzyme deactivation; the compound enzyme is formed by compounding hemicellulase, beta-glucosidase and beta-galactosidase according to the ratio of 1: 1: 0-3: 3: 1; (4) dividing the penthorum chinense pursh leaf raw material subjected to enzyme deactivation into three equal parts, carrying out downstream three-stage extraction twice, and collecting filtrate; and (5) further filtering by adopting a ceramic membrane. According to the preparation method, the extraction efficiency of flavonoid and tannin substances in the penthorum chinense pursh extract can be greatly improved, the medicine taste and the raw and green taste of the extract are reduced, and the stability of the extract is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a herba scutellariae extract with alcohol sobering and liver protecting effects 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. It is rich in a variety of active ingredients. Among them, flavonoids such as quercetin, pinospermone, pinospermone, apigenin, myricetin, kaempferol, and organic acids such as gallic acid, breviscapine acid, and chebulic acid are considered to be the key material basis for Penthorum chinense Pursh to exert its hepatoprotective activity. Based on these active ingredients, Penthorum chinense Pursh has outstanding performance in hepatoprotective activity and is often used in the clinic to treat liver diseases such as alcoholic fatty liver and hepatitis. Therefore, it is very necessary to extract and enrich these key ingredients for further research and utilization.

[0004] The existing extraction methods for Herba Chrysanthemi serrata mainly include the following problems: 1) Traditional extraction methods are mostly water extraction methods, while 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. In addition, the type, quantity and position of glycosides will affect their bioavailability. 2) The extraction efficiency of traditional extraction methods is not high, the directional selectivity is poor, and the potential active ingredients cannot be fully enriched. 3) The Herba Chrysanthemi serrata leaf extract obtained by traditional extraction methods has obvious medicinal and green tastes; and it is easy to form 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] Hemicellulase is an important biocatalyst, including β-galactosidase, xylosidase, mannosidase, etc., which can degrade different types of hemicellulose, and has the characteristics of high efficiency and specificity. Hemicellulase can destroy plant cell walls and promote the dissolution of intracellular substances. Its principle is to break down the glycosidic bonds in hemicellulose into monosaccharides or oligosaccharides, which are often used in food brewing. There is a synergistic effect between the various enzymes in the hemicellulase system. In addition to being able to effectively degrade hemicellulose, it can also promote the degradation and conversion of other glycosides, so that they can be better utilized. This is of great significance for the research and utilization of Chinese medicine ingredients. However, there is no relevant research and enlightenment in the field of hemicellulase suitable for the special form of glycosides in the yellow grass. Summary of the invention

[0006] The purpose of the present invention is to provide a Herba Chrysanthemi Indici extract with alcohol sobering and liver protecting effects and a preparation method thereof in view of the problem that Herba Chrysanthemi Indici glycosides are poorly soluble in water and the traditional extraction method has low extraction efficiency. The Herba Chrysanthemi Indici extract can effectively convert glycosides into corresponding aglycones, greatly enrich flavonoids and tannins, and significantly improve the extraction efficiency; and the bioavailability of key components is improved, which has a good effect in alcohol sobering and liver protecting.

[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 Lycopodiellae extract having alcohol sobering and liver protecting effects, comprising the following steps:

[0009] Step (1): picking fresh yellow grass leaves 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 composite enzyme to the crushed raw material for enzymolysis and enzyme inactivation; the composite enzyme is composed of hemicellulase, β-glucosidase, and β-galactosidase in a ratio of 1:1:0 to 3:3:1; preferably, the ratio of hemicellulase, β-glucosidase, and β-galactosidase is 1:1:1 to 2:2:1.

[0012] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal portions, perform two downstream three-stage extractions, and collect the filtrate.

[0013] Step (5) is further filtered using a ceramic membrane.

[0014] According to a preferred embodiment, the precooling in step (2) is specifically to precool the crude raw material of the yellow grass leaf to a brittle state; more specifically, the precooling temperature is set to -40 to 0°C, and the precooling time is 1 to 5 hours. In step (2), the temperature of the low-temperature ultrafine grinding chamber is 8 to 20°C, and the grinding time is 15 to 60 minutes.

[0015] 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 10min.

[0016] 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.

[0017] On the other hand, the present invention provides a Herba Lysimachiae extract with alcohol sobering and liver protecting effects, wherein the total flavonoids content is greater than 5.88 mg / mL, the gallic acid content is greater than 115 μg / mL, the quercetin content is greater than 12 μg / mL, the apigenin content is greater than 38 μg / mL, the kaempferol content is greater than 60 μg / mL and the pyracantha content is greater than 4 μg / mL.

[0018] Preferably, it is prepared by the preparation method as described above.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. A Herba Lysimachiae extract with alcohol sobering and liver protection effects and a preparation method thereof. The preparation method provided by the present invention uses low-temperature ultrafine grinding to pre-treat Herba Lysimachiae leaves, thereby ensuring the uniformity of the particle size and composition of the raw materials after grinding. During the grinding process, the heat generated by mechanical action is reduced, and the heat-sensitive active ingredients are better retained.

[0021] 2. A Herba Lycopodii extract with alcohol sobering and liver protecting effects and a preparation method thereof, wherein the preparation method provided by the present invention uses a composite enzyme to enzymatically hydrolyze Herba Lycopodii leaves. Hemicellulase, β-glucosidase and β-galactosidase are used in combination in a certain proportion to achieve a more synergistic effect.

[0022] 3. A Herba Chrysanthemi extract with alcohol sobering and liver protection effects and a preparation method thereof. The present invention adjusts various parameters in the low-temperature ultrafine grinding and compound enzyme hydrolysis process, and further combines low-temperature downstream multi-stage extraction, so that flavonoids, tannins and the like are dissolved in large quantities and effectively enriched, and the extraction efficiency is significantly improved. At the same time, the relative content of characteristic volatile components in the Herba Chrysanthemi leaf extract is reduced, and the medicinal taste and green taste of the Herba Chrysanthemi leaf extract are reduced. It is a preparation method with significant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison chart of the turbidity of the Herba Lysimachiae leaf extracts of Examples 1 to 10 of the present application (from left to right are Examples 1 to 10);

[0024] Figure 2 This is a comparison chart of the turbidity of the Herba Lysimachiae leaf extracts of Comparative Examples 1 to 2 of the present application (Comparative Examples 1 to 2 from left to right);

[0025] Figure 3 Schematic diagram of the changes in turbidity and centrifugal sedimentation rate of the Herba Lysimachiae leaf extracts of Examples 1 to 10 and Comparative Examples 1 to 2 of the present application;

[0026] Figure 4 HPLC chromatograms of gallic acid, quercetin, apigenin, kaempferol, and pyrocatechol standards;

[0027] Figure 5 The figure is a difference diagram of the composition and relative content of the volatile components in the examples and comparative examples determined by GC-MS, from top to bottom: comparative example 2, comparative example 1, example 10, example 9, example 8, example 7, example 6, example 5, example 4, example 3, example 2 and example 1;

[0028] Figure 6 This is a schematic diagram of the test results of the mouse open field test;

[0029] Figure 7 This is a schematic diagram of the test results of mouse liver function indicators;

[0030] Figure 8 This is a schematic diagram of the results of the mouse liver enzyme content test;

[0031] Fig. 9 Schematic diagram of the blood ethanol concentration test results in mice. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0035] Example 1 Preparation method of a Herba Lycopodiellae extract having alcohol sobering and liver protecting effects

[0036] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0037] Step (2): pre-cooling and low-temperature ultrafine grinding of the crude raw materials.

[0038] The precooling temperature is -20℃, the precooling time is 2h, the temperature of the low temperature ultrafine grinding chamber is 12℃, and the grinding time is 15min.

[0039] Step (3): adding a complex enzyme to the crushed raw material for enzymolysis and enzyme inactivation.

[0040] The complex enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 1:1:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.1%, the enzymolysis pH is 4.8, the enzymolysis time is 2h, the enzymolysis temperature is 40°C; the enzyme inactivation temperature is 90°C and the time is 10min.

[0041] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0042] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 1.

[0043] Measurement method

[0044] 1. Stability test

[0045] (1) The extract of Herba Chrysanthemi Indici, which has the effect of sobering up and protecting the liver, was placed in an incubator at 37°C for 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.

[0046] (2) The extract of the Herba Aspergiae chinensis, which has the effect of sobering up and protecting the liver, 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. 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 a constant weight, and the weight of the precipitate was recorded.

[0047] 2. Determination of total flavonoid content

[0048] Determination of total flavonoid content: NaNO2-Al(NO3)3-NaOH colorimetric method was used. 1 mL of Examples 1 to 4 and Comparative Examples 1 to 2 were respectively transferred into 25 mL colorimetric tubes, 1 mL of 5% sodium nitrite solution was first added, shaken and allowed to stand for 5 min, 1 mL of 10% aluminum nitrate solution was added, shaken and allowed to stand for 5 min, 10 mL of 4% sodium hydroxide solution was added, and finally 70% ethanol was added, the volume was fixed to 25 mL, mixed and allowed to stand for 15 min, a color reaction was performed, and the absorbance value was measured at a wavelength of 510 nm. A 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.

[0049] 3. Determination of key ingredient content

[0050] High performance liquid chromatography (HPLC) was used to determine the contents of quercetin, pine pine, kaempferol, apigenin and gallic acid. 1 mL of the extract of the herb with alcohol sobering and liver protecting effects 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.

[0051] 4. Determination of volatile components

[0052] 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).

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 5. Animal Experiments

[0057] 1) Experimental grouping: 143 SPF male balb / c mice were randomly divided into a model group, an embodiment group (ten groups), and a comparative example group (two groups), with 11 mice in each group.

[0058] 2) Administration: The Example Group and the Comparative Example Group were given the corresponding test substance at 3 mL / kg BW for 7 consecutive days, and the Model Group was gavaged with an equal volume of saline. 30 minutes after the last gavage, 15 mL / kg of 56% ethanol was gavaged.

[0059] 3) Detection indicators:

[0060] Observe the activity of mice: 8 mice were selected from each group. Unsteady crawling, dragging of the back abdomen on the ground, and laziness with eyes closed were taken as indicators of drunkenness, and free movement, flexible limbs, and mental recovery were taken as indicators of soberness. The drunkenness and soberness time of each group of mice were recorded, and the duration of drunkenness (min, the time from drunkenness to soberness) was calculated.

[0061] Open field test: 30 minutes after the mice woke up from drunkenness, 8 mice from each group were taken to perform open field test to evaluate the extent of changes in mobility caused by drunkenness.

[0062] Serum liver function indexes: After the open field test, serum was collected from 8 mice in each group, and the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin (T-BIL) were determined by biochemical method.

[0063] Liver enzyme content: The livers of 8 mice in each group were taken and prepared into 10% liver homogenate. The content of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) was determined by Elisa method.

[0064] Blood ethanol: Three mice were selected from each group, and the ethanol content in the blood of mice was detected by biochemical method.

[0065] Example 2 Preparation method of a Herba Lycopodiellae extract having alcohol sobering and liver protecting effects

[0066] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0067] Step (2): pre-cooling and low-temperature ultrafine grinding of the crude raw materials.

[0068] The precooling temperature is -20℃, the precooling time is 2h, the temperature of the low temperature ultrafine grinding chamber is 12℃, and the grinding time is 15min.

[0069] Step (3): adding a complex enzyme to the crushed raw material for enzymolysis and enzyme inactivation.

[0070] The complex enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 1:1:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.5%, the enzymolysis pH is 4.8, the enzymolysis time is 2h, the enzymolysis temperature is 40°C; the enzyme inactivation temperature is 90°C and the time is 10min.

[0071] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0072] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 2.

[0073] Measurement method

[0074] The same as Example 1.

[0075] Example 3 Preparation method of a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0076] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0077] Step (2): pre-cooling and low-temperature ultrafine grinding of the crude raw materials.

[0078] The precooling temperature is -20℃, the precooling time is 2h, the temperature of the low temperature ultrafine grinding chamber is 12℃, and the grinding time is 15min.

[0079] Step (3): adding a complex enzyme to the crushed raw material for enzymolysis and enzyme inactivation.

[0080] The complex enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 1:1:1; the enzymatic hydrolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 1%, the enzymatic hydrolysis pH is 4.8, the enzymatic hydrolysis time is 2h, the enzymatic hydrolysis temperature is 40°C; the enzyme inactivation temperature is 90°C and the time is 10min.

[0081] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0082] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 3.

[0083] Measurement method

[0084] The same as Example 1.

[0085] Example 4 Preparation method of a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0086] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0087] Step (2): pre-cooling and low-temperature ultrafine grinding of the crude raw materials.

[0088] The precooling temperature is -20℃, the precooling time is 2h, the temperature of the low temperature ultrafine grinding chamber is 12℃, and the grinding time is 15min.

[0089] Step (3): adding a complex enzyme to the crushed raw material for enzymolysis and enzyme inactivation.

[0090] The complex enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 1:1:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.5%, the enzymolysis pH is 4.8, the enzymolysis time is 2h, the enzymolysis temperature is 30°C; the enzyme inactivation temperature is 90°C and the time is 10min.

[0091] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0092] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 4.

[0093] Example 5 Preparation method of a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0094] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0095] Step (2): pre-cooling and low-temperature ultrafine grinding of the crude raw materials.

[0096] The precooling temperature is -20℃, the precooling time is 2h, the temperature of the low temperature ultrafine grinding chamber is 12℃, and the grinding time is 15min.

[0097] Step (3): adding a complex enzyme to the crushed raw material for enzymolysis and enzyme inactivation.

[0098] The complex enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 1:1:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.5%, the enzymolysis pH is 4.8, the enzymolysis time is 2h, the enzymolysis temperature is 60°C; the enzyme inactivation temperature is 90°C and the time is 10min.

[0099] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0100] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 5.

[0101] Example 6 Preparation method of a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0102] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0103] Step (2) The crude raw material is precooled and subjected to low temperature ultrafine grinding. The precooling temperature is -20°C, the precooling time is 2 hours, the low temperature ultrafine grinding chamber temperature is 12°C, and the grinding time is 60 minutes.

[0104] Step (3): Adding compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 1:1:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.5%, the enzymolysis pH is 4.8, the enzymolysis time is 2 hours, the enzymolysis temperature is 40°C; the enzyme inactivation temperature is 90°C, and the time is 10 minutes.

[0105] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0106] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 6.

[0107] Measurement method

[0108] The same as Example 1.

[0109] Example 7 Preparation method of a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0110] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0111] 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 12°C and the grinding time is 15 minutes.

[0112] Step (3): Adding compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 1:1:0; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.5%, the enzymolysis pH is 4.8, the enzymolysis time is 2 hours, the enzymolysis temperature is 40°C; the enzyme inactivation temperature is 90°C, and the time is 10 minutes.

[0113] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0114] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 7.

[0115] Measurement method

[0116] The same as Example 1.

[0117] Example 8 Preparation method of a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0118] Step (1): picking fresh yellow grass leaves 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 12°C and the grinding time is 15 minutes.

[0120] Step (3): Adding compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 2:2:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.5%, the enzymolysis pH is 4.8, the enzymolysis time is 2 hours, the enzymolysis temperature is 40°C; the enzyme inactivation temperature is 90°C, and the time is 10 minutes.

[0121] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:10 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0122] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 8.

[0123] Measurement method

[0124] The same as Example 1.

[0125] Example 9 Preparation method of a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0126] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0127] 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 12°C and the grinding time is 15 minutes.

[0128] Step (3): Adding compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 2:2:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.5%, the enzymolysis pH is 4.8, the enzymolysis time is 2 hours, the enzymolysis temperature is 40°C; the enzyme inactivation temperature is 90°C, and the time is 10 minutes.

[0129] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0130] Step (5): further filtering with a ceramic membrane to obtain the Herba Lycopodiellae extract with alcohol sobering and liver protecting effects prepared in Example 9.

[0131] Measurement method

[0132] The same as Example 1.

[0133] Example 10 Preparation method of a Herba Lycopodiellae extract having alcohol sobering and liver protecting effects

[0134] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0135] 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 12°C and the grinding time is 15 minutes.

[0136] Step (3): Adding compound enzyme to the crushed raw material for enzymolysis and enzyme inactivation. The compound enzyme is composed of hemicellulase, β-glucosidase and β-galactosidase in a ratio of 2:2:1; the enzymolysis material-liquid ratio is 1:2 (g / ml), the enzyme addition amount is 0.5%, the enzymolysis pH is 4.8, the enzymolysis time is 2 hours, the enzymolysis temperature is 40°C; the enzyme inactivation temperature is 90°C, and the time is 10 minutes.

[0137] Step (4): Divide the enzyme-inactivated Herba Lysimachiae leaves into three equal parts, perform two downstream three-stage extractions, and collect the filtrate. The material-liquid ratio is 1:50 (g / ml), the extraction temperature is 40° C., and the extraction time is 1.5 h.

[0138] Step (5): further filter using a ceramic membrane to obtain Example 10.

[0139] Measurement method

[0140] The same as Example 1.

[0141] Comparative Example 1: A method for preparing a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0142] The difference between this comparative example and Examples 1-4 is that the low-temperature pulverization in step (2) and the enzymatic hydrolysis in step (3) are not performed.

[0143] The specific steps include:

[0144] Step (1): picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0145] Step (2): Divide the crude raw material of the yellow grass leaf into three equal parts and perform two downstream three-stage extractions, and collect the filtrate. The extraction material-liquid ratio is 1:20 (g / ml), the extraction temperature is 40°C, and the extraction time is 1.5h.

[0146] Step (3): further filtering with a ceramic membrane to obtain Comparative Example 1.

[0147] Measurement method

[0148] The same as Example 1.

[0149] Comparative Example 2: A method for preparing a Herba Chrysanthemi Indici extract having alcohol sobering and liver protecting effects

[0150] The difference between this comparative example and Examples 1-4 is that the low-temperature pulverization in step (2) and the enzymatic hydrolysis in step (3) are not performed, and the extraction temperature is high temperature extraction.

[0151] The specific steps include:

[0152] Step (1) picking fresh yellow grass leaves of uniform maturity and good quality, removing impurities, washing, drying and crushing.

[0153] Step (2) The crude raw material of the yellow grass leaf is divided into three equal parts, and two downstream three-stage extractions are performed, and the filtrate is collected. The extraction material-liquid ratio is 1:20 (g / ml), the extraction temperature is 95° C., and the extraction time is 1.5 h.

[0154] In step (3), a ceramic membrane is used for further filtration to obtain Comparative Example 2.

[0155] Measurement method

[0156] The same as Example 1.

[0157] Statistics:

[0158] Depend on Figures 1 to 3 As shown, the turbidity of Examples 1 to 10 and Comparative Examples 1 to 2 increased to varying degrees during the 128-day accelerated storage period. The overall trend was a rapid increase at first, then a slow increase rate, and finally a gradual stabilization. The overall trend of the centrifugal sedimentation rate of Examples 1 to 10 during the accelerated storage process was also a rapid increase at first, then a stabilization, and the centrifugal sedimentation rate was significantly lower than that of Comparative Examples 1 to 2.

[0159] The contents of total flavonoids and key components in the extract of the herbaceous plant are shown in Table 1-2:

[0160] Table 1 Total flavonoids content

[0161]

[0162] Table 2 Key ingredient content

[0163]

[0164] It can be seen from Tables 1-2 above that the total flavonoids and key components in the extracts of the leaves of the Herba Lysimachiae prepared in Examples 1 to 10 are increased compared with those in Comparative Examples 1-2. Among them, Example 9 has the most significant increase, with a total flavonoid content of 7.71 mg / mL, which is about 5 times that of Comparative Example 1. The contents of gallic acid, quercetin, apigenin, kaempferol, and pyracantha are 144.39, 24.57, 54.17, 69.24, and 5.59 μg / mL, respectively; among them, the content of kaempferol and gallic acid is particularly significantly increased compared with Comparative Examples 1-2, with the content of kaempferol being 70 times that of Comparative Examples 1-2; and the content of gallic acid being 5 times that of Comparative Examples 1-2.

[0165] HPLC chromatograms of gallic acid, quercetin, apigenin, kaempferol, and pyrifos Figure 4 As shown; GC-MS total ion current diagram is as follows Figure 5 As shown, the relative content of volatile components in the extract of the leaves of the present application is shown in Table 3:

[0166] Table 3 Relative content of volatile components

[0167]

[0168] Note: “ / ” means not detected.

[0169] As shown in Table 3, (E)-2-nonenal, lauryl aldehyde and 2-undecanone were not detected in Examples 1 to 10. Although safranal a, geranyl acetone and 2,4-di-tert-butylphenol were detected, their relative contents were significantly lower than those in Comparative Examples 1 to 2. The relative content of volatile components in Comparative Example 2 was significantly higher than that in Comparative Example 1 and Examples 1 to 10.

[0170] Animal experiment - drunkenness duration test results are shown in Table 4:

[0171] Table 4 Animal experiment - drunkenness duration test results

[0172]

[0173] As shown in Table 4, compared with the model group and comparative examples 1 to 2, Examples 1 to 7 can delay the time of intoxication of mice after intragastric administration of ethanol to a certain extent and reduce the duration of intoxication behavior. The effects of Examples 7, 8, and 9 are comparable, and the hangover effect is very significant. After intragastric administration of ethanol, the mice did not show intoxication behavior.

[0174] Depend on Figure 6It can be seen that the total distance, the number of times of crossing between squares, and the number of times of crossing the central square of the mice in Examples 1 to 10 are significantly higher than those in the model group and Comparative Examples 1 to 2, and the time of staying in the central square is significantly lower than that in the model group and Comparative Examples 1 to 2. Among them, Example 9 has the best effect, which can significantly improve the activity ability of mice after being drunk.

[0175] Depend on Figure 7 It can be seen that Examples 1 to 10 can all reduce the levels of AST, ALT and T-BIL in the serum of mice after drinking. Among them, Example 9 has a better effect, with the levels of AST, ALT and T-BIL being 26.134±1.126U / L, 9.743±0.927U / L and 6.324±7.190μmol / L respectively.

[0176] Depend on Figure 8 It can be seen that the contents of ADH and ALDH in the liver of mice in Examples 1 to 10 were significantly higher than those in the model group and Comparative Examples 1 to 2. Among them, the contents of ADH and ALDH in the liver of mice intervened in Example 9 were the highest, which were 1.699±0.082pg / mg and 1.568±0.057ng / mg respectively.

[0177] Depend on Fig. 9 It can be seen that Examples 1 to 10 can all reduce the blood ethanol concentration of mice in a drunken state. The blood ethanol concentration of mice intervened by Example 9 was the lowest, at 24.651±2.176mmol / L.

[0178] In summary, the conditions involved in Example 9 are the optimal conditions, wherein the temperature of the low-temperature ultrafine grinding chamber is 12°C, the grinding time is 15 min, the enzyme addition amount is 0.5%, the enzymatic hydrolysis temperature is 40°C, the hemicellulase, β-glucosidase, and β-galactosidase are compounded in a ratio of 2:2:1, and the extraction material-liquid ratio is 1:20.

[0179] 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 having the effects of sobering up and protecting the liver, characterized in that: The steps include: Step (1): picking fresh yellow grass leaves 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 composite enzyme to the crushed raw material for enzymolysis and enzyme inactivation; the composite enzyme is composed of hemicellulase, β-glucosidase, and β-galactosidase in a ratio of 1:1:0 to 3:3:1; Step (4): Divide the enzyme-inactivated yellow grass leaf material into three equal portions, perform two downstream three-stage extractions, and collect the filtrate; Step (5) is further filtered using a ceramic membrane.

2. The method for preparing the Herba Lycopodiellae extract having the effects of sobering up and protecting the liver according to claim 1, characterized in that: The precooling in step (2) specifically involves precooling the crude raw material of the herb to a brittle state.

3. The method for preparing the Herba Lysimachiae extract having the effects of sobering up and protecting the liver according to claim 1, characterized in that: In the step (2), the temperature of the low-temperature ultrafine grinding chamber is 8 to 20° C., and the grinding time is 15 to 60 minutes.

4. The method for preparing the Herba Lycopodiellae extract having the effects of sobering up and protecting the liver according to claim 1, characterized in that: In step (3), the enzyme addition amount is 0.1% to 1%, the enzymolysis temperature is 30 to 60° C., and the enzyme inactivation temperature is 90° C. for 10 min.

5. The method for preparing the Herba Lysimachiae extract having the effects of sobering up and protecting the liver according to claim 1, characterized in that: In the step (4), the extraction 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.

6. A Herba Lysimachiae extract having the effects of sobering up and protecting the liver, characterized in that: The total flavonoids content is greater than 5.88 mg / mL, the gallic acid content is greater than 115 μg / mL, the quercetin content is greater than 12 μg / mL, the apigenin content is greater than 38 μg / mL, the kaempferol content is greater than 60 μg / mL, and the pyrocatechol content is greater than 4 μg / mL.

7. The Herba Lysimachiae extract with alcohol sobering and liver protecting effects according to claim 6, characterized in that: The method is as described in any one of claims 1 to 5.

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