Semen cannabis hangover alleviating and liver protecting peptide chewable tablet and preparation method thereof
By isolating and purifying ADH from the hemp protein, the strongest polypeptide component, Hanmasin peptide DSF2, and combining the medicinal and food homologous components, Hanmasin sanitary liver protection peptide chewable tablets were prepared, which solved the problems of complex ingredients and low purification of existing polypeptide products, and achieved efficient and stable liver protection effects.
Patent Information
- Application Number
- CN202510263589.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-07-11
AI Technical Summary
The existing polypeptide products have complex ingredients, low purification degree, unstable functional activity, and lack clear polypeptide sequences, making it difficult to effectively prevent or treat alcoholic liver damage.
Using hanma selengenin protein as raw material, separated and purified by biological enzymatic chromatography technology, screened out the strongest peptide component hanma selengenin peptide DSF2, and combined with the homologous components of medicine and food, Hanma selengenin halting and liver protection peptide chewable tablets were prepared, and the peptide sequence was measured to ensure the activity and stability of the product.
The chewable tablets of Hanma Ren ulcet alcoholic liver protection peptide have a single component, strong ADH activation and vitality, good taste, fast absorption, suitable for modern life, have significant liver protection effects, are suitable for children, the elderly and patients with dysphagia, and show significant liver protection effects in mouse models.
Smart Images
Figure CN120285145A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a chewable tablet of hemp seed hangover and liver protection peptide and a preparation method thereof. Background Art
[0002] The history of human alcohol consumption is long. However, excessive drinking can not only lead to alcohol dependence but also increase the risk of people getting sick, such as alcoholic fatty liver, liver cirrhosis, alcoholic hepatitis, digestive system diseases, etc. Alcohol has become the second leading cause of liver damage after viruses. The liver protection drugs on the market are expensive and have little effect, and the drugs will further cause harm after being metabolized by the liver. Therefore, developing food-derived natural liver protection products to regulate human metabolism, prevent and relieve alcoholic liver injury through daily dietary supplements has great value for our health and life.
[0003] Bioactive peptides exhibit various biological activities due to different amino acid compositions and sequences, and are considered natural functional factors for regulating body metabolism and promoting human health. Bioactive peptides have advantages such as small molecular weight, easy absorption, wide sources of food proteins, and good safety, and are widely favored. Therefore, finding a safe and effective liver protection active peptide to prevent or treat ALD has become a research and attention hotspot.
[0004] Alcohol dehydrogenase (ADH) is the rate-limiting enzyme for normal human metabolism of alcohol, catalyzing the dehydrogenation of alcohol to produce acetaldehyde. Acetaldehyde is further catalyzed by aldehyde dehydrogenase (ALDH) to produce acetic acid harmless to humans, and finally decomposed into water and carbon dioxide and excreted from the body. Activating the activity of ADH can not only accelerate alcohol metabolism and the excretion of alcohol and metabolic intermediates from the body, but also reduce its adverse effects on the liver.
[0005] At present, the development of polypeptide products is still in its infancy, mostly staying in the mixture after protease hydrolysis, with relatively complex components and low extraction and purification levels. It is difficult to guarantee the functional activity of the developed products, and there is no product development that measures the sequence of the product and clarifies the polypeptide sequence. Summary of the Invention
[0006] The purpose of the present invention is to solve the problems of complex components, low purification level, and unstable functional activity of existing polypeptide products, and to provide a chewable tablet of hemp seed hangover and liver protection peptide and a preparation method thereof.
[0007] Hemp seeds contain 20% - 25% protein and are high-quality raw materials for preparing bioactive peptides. In this invention, hemp seed protein is used as the raw material, and the activation of alcohol dehydrogenase is taken as the target to prepare hemp seed anti-alcohol and liver-protecting peptides. Chromatography technology is used to separate and purify hemp seed polypeptides, and the component with the strongest ADH activation polypeptide is screened out. The sequence of hemp seed peptides is determined. The strongest polypeptide component is combined with food and drug homologous components, and modern nutritional theory is used to prepare hemp seed anti-alcohol and liver-protecting peptide chewable tablets. The in vivo liver-protecting effect of hemp seed anti-alcohol and liver-protecting peptide chewable tablets is verified using a mouse model of alcoholic liver injury.
[0008] The hemp seed anti-alcohol and liver-protecting peptide chewable tablets are developed based on hemp seed peptides, and are compounded with a variety of food and drug homologous functional active substances and their excipients. The product does not require a disintegration process, has rapid drug dissolution, fast absorption, and high bioavailability; it is convenient to carry, has stable quality, is sweet and sour, and is suitable for children, the elderly, and patients with swallowing difficulties. It is convenient to take and carry, does not need to be taken with water, does not require disintegration, has a fast absorption rate, and fast drug dissolution. In the whole technological process, the raw materials do not come into contact with water and are not easily contaminated by microorganisms, meeting the needs of modern high-paced urban life.
[0009] To achieve the above objectives, the technical solutions adopted in this invention are as follows:
[0010] A kind of hemp seed anti-alcohol and liver-protecting peptide chewable tablet, the formula of the hemp seed anti-alcohol and liver-protecting peptide chewable tablet is 225 - 35 parts of hemp seed peptide DSF2, 8 - 18 parts of ultrafine powder of Astragalus membranaceus, 10 - 15 parts of powder of Hovenia dulcis, 8 - 17 parts of powder of Pueraria thomsonii, 6 - 20 parts of powder of Lycium barbarum, 10 - 15 parts of powder of Lonicera japonica, 5 - 8 parts of powder of Rosa roxburghii tratt, 6 - 12 parts of maltodextrin, 0.1 - 1.2 parts of microcrystalline cellulose, 1 - 8 parts of xylitol, 1 - 5 parts of magnesium stearate, 0.5 - 1.5 parts of malic acid; the amino acid sequence of the hemp seed peptide DSF2 is Ile-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg.
[0011] A preparation method of the above-mentioned hemp seed anti-alcohol and liver-protecting peptide chewable tablet, the method is as follows:
[0012] Step 1: Airflow pulverize the hemp seed peptide DSF2 and weigh the polypeptide powder;
[0013] Step 2: Add other raw and auxiliary materials according to the parts of the raw materials, mix evenly, and make tablets.
[0014] Further, in Step 1, the preparation method of the hemp seed peptide DSF2 is as follows:
[0015] (1) Preparation of hemp seed liver-protecting peptide
[0016] Weigh hemp kernel protein powder, add appropriate amount of deionized water, fully dissolve, adjust the pH of the enzymatic solution to 6.2, add neutral protease, enzymatic hydrolysis at 44°C, enzymatic hydrolysis time 2-3h, inactivate enzyme at high temperature after enzymatic hydrolysis, centrifuge, take the supernatant and vacuum freeze-dry for later use;
[0017] (2) Isolation of Hemp Seed Liver Protective Peptide
[0018] Place anhydrous ethanol in a refrigerator overnight, and add it dropwise to the prepared 40 mg / mL hemp peptide solution until the ethanol volume fraction reaches 20%; then let the solution stand at 4°C for 1 hour, and then centrifuge at 7500r / min for 10 minutes, and the precipitate is recorded as E20; add the supernatant to cold anhydrous ethanol until the ethanol volume fraction reaches 40%, and let it stand at 4°C for 1 hour, and then centrifuge to obtain the precipitate, which is recorded as E40; add the supernatant to cold anhydrous ethanol until the ethanol volume fraction reaches 60%, and let it stand at 4°C for 1 hour, and then centrifuge to obtain the precipitate, which is recorded as E60, and the supernatant is recorded as SPT; all components are concentrated under reduced pressure to remove ethanol, and then freeze-dried, and the alcohol dehydrogenase activation rate of each component is determined to screen the most active component;
[0019] (3) Purification of hemp seed liver-protecting peptide
[0020] DEAE Sepharose Fast Flow chromatography resin was used to further separate and purify the hemp peptide powder of the component SPT. The chromatography column specification was (diameter) 2.6*35cm (column height), and the strongest alcohol dehydrogenase activation component was screened; the SPF component was dissolved in a buffer solution of 20mM Tris-HCl, pH 8.0, and a 100mg / mL polypeptide component was prepared. The loading volume was 6mL, and the gradient elution method was used for separation and purification. Elution Buffer A: 20mM Tris-HCl, 0.15M NaCl, pH 8.0; Elution Buffer B: 20mM Tris-HCl, 0.25M NaCl, pH 8.0; Elution Buffer C: 20mM Tris-HCl, 0.35M NaCl, pH 8.0 was used for elution for 8 column volumes, and the elution flow rate was 2.5 mL / min. The most active component was collected, and the eluted component was DSF2. The hemp peptide component DSF2 was obtained by vacuum freeze-drying. The most active component DSF2 isolated and purified was sequenced and analyzed by LC-MS / MS mass spectrometry. It was mainly Ile-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg. The most active component was vacuum freeze-dried.
[0021] Further, in (2), the ADH activity was determined by the Warburg-Hoch method. First, 1.5 mL (0.1 M, pH 8.8) of sodium pyrophosphate buffer, 1.0 mL of 0.01 M NAD + , 0.5 mL (11.5%, v / v) of ethanol, and 0.1 mL of the hydrolyzate of hemp seed protein were mixed at 25 °C and reacted for 5 min. Then, 0.1 mL of 0.25 U / mL ADH was added to the reaction system. The absorbance was immediately measured at 340 nm, and the measurement time was 10 min. The initial linear part of the reaction was plotted, and the slope was denoted as Ka. For the control group, 0.2 mL of distilled water was used instead of the sample, and the slope was denoted as Kc. It was calculated according to formula (1)
[0022]
[0023] The beneficial effects of the present invention compared with the prior art are as follows:
[0024] 1. The hemp seed peptide component DSF2 used in the present invention is a product purified by ethanol fractional extraction and DEAE Sepharose Fast Flow column chromatography. It has strong ADH activation activity, and the obtained product components are relatively single. The molecular weight and sequence have been determined. The main component in the product is a peptide segment with a molecular weight of 989 Da, and the polypeptide sequence is Ile-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg.
[0025] 2. The hemp seed peptide powder used in the present invention is mixed with the hepatoprotective substances that are both medicine and food to act synergistically, which not only improves the taste and color of the hemp seed peptide chewable tablets, but also enhances the efficacy of the product.
[0026] 3. The hemp seed peptide anti-alcoholism and hepatoprotective chewable tablets of the present invention have the characteristics
[0027]
[0028] Description of the Drawings
[0029] Figure 1 It is a comparison chart of the activation rate of ethanol dehydrogenase by various components of hemp seed peptides.
[0030] Figure 2 It is a schematic diagram of the effect of hemp seed peptide chewable tablets on mouse inflammatory factors.
[0031] Figure 3 It is a schematic diagram of the effect of hemp seed peptide chewable tablets on mouse serum physiological and biochemical indexes.
[0032] Figure 4 It is a schematic diagram of the effect of hemp seed peptide chewable tablets on mouse SOD and MDA indexes.
[0033] Figure 5 Schematic diagram of the effects of hemp seed peptide chewable tablets on GSH and GSH-Px indices in mice.
[0034] Figure 6 Pathological analysis diagram of the liver of mice by hemp seed peptide chewable tablets. Specific implementation manners
[0035] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, shall be covered by the protection scope of the present invention.
[0036] The present invention uses hemp seed protein as a raw material, and uses biological enzymolysis technology, chromatography technology, and modern nutrition technology to prepare hemp seed hangover and liver protection peptides, and conducts sequence analysis to obtain a hemp seed peptide product with clear functional components. And compounding medicine and food homologous components to develop hemp seed peptide hangover and liver protection chewable tablets, with multi-component synergistic effect, which can be applied to the fields of biopharmaceuticals and functional foods.
[0037] Example 1:
[0038] 1. Preparation of hemp seed liver protection peptide
[0039] Weigh 10 g of hemp seed protein powder, add 50 mL of deionized water, fully dissolve and stir evenly, adjust the pH of the enzymolysis solution to 6.2, add 3.36% neutral protease, enzymolysis at 44 °C for 2.75 h, inactivate the enzyme at 95 °C for 15 min after enzymolysis, centrifuge at 10000 r / min at 4 °C for 15 min, and take the supernatant for vacuum freeze-drying for standby.
[0040] 2. Separation of hemp seed liver protection peptide
[0041] Put anhydrous ethanol in a 4 °C refrigerator overnight, and add it dropwise to the prepared 40 mg / mL hemp seed peptide solution until the ethanol volume fraction reaches 20%. Then let the solution stand at 4 °C for 1 h, and then centrifuge at 7500 r / min for 10 min. The precipitate part is denoted as E20. Add cold anhydrous ethanol to the supernatant part until the ethanol volume fraction reaches 40%, and the precipitate part obtained after standing at 4 °C for 1 h and centrifuging is denoted as E40. Add cold anhydrous ethanol to the supernatant part until the ethanol volume fraction reaches 60%, and the precipitate part obtained after standing at 4 °C for 1 h and centrifuging is denoted as E60, and the supernatant is denoted as SPT. All components are concentrated under reduced pressure at 55 °C to remove ethanol, and then freeze-dried, and the ethanol dehydrogenase activation rate of each component is measured to screen the strongest active component ( Figure 1 ).
[0042] The ADH activity was determined by the Warburg-Hoch method. First, 1.5 mL of sodium pyrophosphate buffer (0.1 M, pH 8.8), 1.0 mL of 0.01 M NAD + , 0.5 mL of ethanol (11.5%, v / v), and 0.1 mL of hydrolyzed hemp seed protein were mixed at 25 °C and reacted for 5 min. Then, 0.1 mL of 0.25 U / mL ADH was added to the reaction system. The absorbance was immediately measured at 340 nm for 10 min. The initial linear part of the reaction was plotted, and the slope was denoted as Ka. For the control group, 0.2 mL of distilled water was used instead of the sample, and the slope was denoted as Kc, which was calculated according to formula (1).
[0043]
[0044] 3. Purification of hemp seed liver-protecting peptide
[0045] The hemp seed peptide powder of component SPT was further separated and purified using DEAE Sepharose Fast Flow chromatography resin. The chromatography column had a specification of (diameter) 2.6 * 35 cm (column height) to screen the component with the strongest ethanol dehydrogenase activation. The SPF component was dissolved in a buffer solution of 20 mM Tris-HCl, pH 8.0 to prepare a polypeptide component of 100 mg / mL. The sample loading volume was 6 mL, and gradient elution was used for separation and purification. Elution Buffer A: 20 mM Tris-HCl, 0.15 M NaCl, pH 8.0; Elution Buffer B: 20 mM Tris-HCl, 0.25 M NaCl, pH 8.0; Elution Buffer C: 20 mM Tris-HCl, 0.35 M NaCl, pH 8.0 were used to elute for 8 column volumes respectively at an elution flow rate of 2.5 mL / min. The component with the strongest activity was collected, and the eluted component was DSF2. It was vacuum freeze-dried to obtain the hemp seed peptide component DSF2. For the component with the strongest activity DSF2 obtained by separation and purification ( Figure 1 ), LC-MS / MS mass spectrometry was used for polypeptide sequence determination, analysis, and identification, which was mainly Ile-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg. The component with the strongest activity was vacuum freeze-dried for the preparation of the next polypeptide chewable tablets.
[0046] 4. Process flow of hemp seed anti-alcoholism and liver-protecting peptide chewable tablets
[0047] Hemp seed peptide DSF2 → Airflow pulverization → Precision weighing of polypeptide powder → Adding other excipients in a certain ratio → Mixing → Tablet pressing.
[0048] The tablet weight of the Hemp Seed Peptide Hangover and Liver Protecting Chewable Tablets is 2.0 ± 0.05 g. The basic composition formula of the Hemp Seed Peptide for hangover and liver protection is as follows: 25 - 35 parts of Hemp Seed Peptide DSF2 powder, 8 - 18 parts of Astragalus membranaceus ultrafine powder, 10 - 15 parts of Hovenia dulcis powder, 8 - 17 parts of Pueraria lobata pollen, 6 - 20 parts of Lycium barbarum powder, 10 - 15 parts of Lonicera japonica powder, 5 - 8 parts of Rosa roxburghii powder, 6 - 12 parts of maltodextrin, 0.1 - 1.2 parts of microcrystalline cellulose, 1 - 8 parts of xylitol, 1 - 5 parts of magnesium stearate, and 0.5 - 1.5 parts of malic acid.
[0049] 5. Research on the Liver Protecting Effect of Hemp Seed Peptide Chewable Tablets
[0050] Forty-eight mice were randomly divided into 4 groups, with 12 mice in each group, namely: Control group, Acute Alcohol Poisoning Model group, Positive Control group (RU21), and Hemp Seed Peptide Chewable Tablet group (HEPD). Before each experiment, the mice were weighed to determine the actual gavage sample volume. The mice in the Hemp Seed Peptide Chewable Tablet group were gavaged with a sample of 800 mg / kg body weight, the Positive Control group was gavaged with RU21 (300 mg / kg body weight), while the mice in the Control group and the Acute Alcohol Poisoning Model group were gavaged with the same volume of normal saline instead. After 30 min, except for the Control group, the mice in other groups were gavaged with 56-degree white liquor at 8.4 g / kg body weight to cause acute alcohol poisoning in the mice. The mice in the Control group were gavaged with the same volume of normal saline instead. The sample and the white liquor were gavaged once every 24 h for a total of three times to induce acute liver injury in the mice. After the first gavage of the white liquor, the time of appearance and disappearance of the righting reflex in the mice was recorded. After the last gavage of the white liquor, the blood was taken from the mouse eyeballs at an interval of 2 h, the mice were killed, the liver was weighed and stored for later use. Eight samples were randomly selected from each group for the detection of blood and liver biochemical indexes. The physiological and biochemical indexes of the blood and liver and the results of the liver pathological sections showed that the Hemp Seed Peptide Chewable Tablets had the effects of relieving hangover and protecting the liver (Table 1, Figures 2 - 6 ).
[0051] Table 1 Effects of Hemp Seed Peptide Chewable Tablets on the Disappearance of the Righting Reflex in Mice
[0052] Group Quantity Latent time (min) Duration (min) Model 12 <![CDATA[18.89±6.75 b > <![CDATA[754.23±95.23 a > RU21 12 <![CDATA[28.363±23.45 a,b > <![CDATA[586.32±89.29 b > Hemp seed peptide chewable tablets 12 <![CDATA[46.23±32.21 a > <![CDATA[389.56±156.32 c >
Claims
1. A chewing tablet of hemp seed anti-alcoholic and liver-protecting peptide, characterized in that: The formula of the hemp seed peptide hangover and liver protection chewable tablets is as follows: 225-35 parts of hemp seed peptide DSF2, 8-18 parts of ultrafine powder of astragalus membranaceus, 10-15 parts of powder ofhovenia dulcis, 8-17 parts of kudzu pollen, 6-20 parts of wolfberry powder, 10-15 parts of honeysuckle flower powder, 5-8 parts of rosa roxbunghii powder, 6-12 parts of maltodextrin, 0.1-1.2 parts of microcrystalline cellulose, 1-8 parts of xylitol, 1-5 parts of magnesium stearate, 0.5-1.5 parts of malic acid; the amino acid sequence of the hemp seed peptide DSF2 is Ile-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg.
2. A preparation method of the hemp seed hangover and liver-protecting peptide chewable tablets according to claim 1, characterized in that: The method is as follows: Step 1: Airflow pulverize the hemp seed peptide DSF2 and weigh the polypeptide powder. Step 2: Add other raw and auxiliary materials according to the parts by weight of the raw materials, mix well, and tablet.
3. The preparation method of a hemp seed hangover and liver-protecting peptide chewable tablet according to claim 2, characterized in that: In Step 1, the preparation method of the hemp seed peptide DSF2 is as follows: (1) Preparation of hemp seed liver protection peptide Weigh hemp seed protein powder, add an appropriate amount of deionized water, fully dissolve it, adjust the pH of the enzymatic hydrolysis solution to 6.2, add neutral protease, carry out enzymatic hydrolysis at an enzymatic hydrolysis temperature of 44 °C and an enzymatic hydrolysis time of 2-3 h, inactivate the enzyme at high temperature after enzymatic hydrolysis is completed, centrifuge, and take the supernatant for vacuum freeze-drying for standby. (2) Separation of hemp seed liver protection peptide Put anhydrous ethanol in the refrigerator for overnight treatment, and add it dropwise to the prepared 40 mg / mL hemp seed peptide solution until the ethanol volume fraction reaches 20%; then let the solution stand at 4 °C for 1 h, and then centrifuge at 7500 r / min for 10 min, and record the precipitate part as E20; add the supernatant part to cold anhydrous ethanol until the ethanol volume fraction reaches 40%, and centrifuge after standing at 4 °C for 1 h to obtain the precipitate part recorded as E40; add the supernatant part to cold anhydrous ethanol until the ethanol volume fraction reaches 60%, and centrifuge after standing at 4 °C for 1 h to obtain the precipitate part recorded as E60, and record the supernatant as SPT; Concentrate all components under reduced pressure to remove ethanol, and then freeze-dry, measure the alcohol dehydrogenase activation rate of each component, and screen the strongest active component. (3) Purification of hemp seed liver protection peptide The hemp seed peptide powder of component SPT was further separated and purified using DEAE Sepharose Fast Flow chromatography resin. The chromatography column had a specification of (diameter) 2.6 * 35 cm (column height), and the component with the strongest ethanol dehydrogenase activation was screened. The SPF component was dissolved in a buffer solution of 20 mM Tris-HCl, pH 8.0 to prepare a polypeptide component at 100 mg / mL. The sample loading volume was 6 mL, and gradient elution was used for separation and purification. Elution Buffer A: 20 mM Tris-HCl, 0.15 M NaCl, pH 8.0; Elution Buffer B: 20 mM Tris-HCl, 0.25 M NaCl, pH 8.0; Elution Buffer C: 20 mM Tris-HCl, 0.35 M NaCl, pH 8.0 were used to elute for 8 column volumes respectively. The elution flow rate was 2.5 mL / min, and the component with the strongest activity was collected. The eluted component was DSF2, and the hemp seed peptide component DSF2 was obtained by vacuum freeze-drying. For the component DSF2 with the strongest activity after separation and purification, LC-MS / MS mass spectrometry was used for polypeptide sequence determination, analysis and identification, which was mainly Ile-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg, and the component with the strongest activity was vacuum freeze-dried.
4. The preparation method of a hemp seed hangover and liver-protecting peptide chewable tablet according to claim 3, characterized in that: (2) In the case of, the Waler-Hoch method was used to determine the ADH activity. First, 1.5 mL (0.1 M, pH 8.8) of sodium pyrophosphate buffer solution, 1.0 mL of 0.01 M NAD + , 0.5 mL (11.5%, v / v) of ethanol, and 0.1 mL of the hydrolyzed hemp seed protein solution were mixed at 25 °C and reacted for 5 min. Then, 0.1 mL of 0.25 U / mL ADH was added to the reaction system; the absorbance was immediately measured at 340 nm, and the measurement time was 10 min; the initial linear part of the reaction was plotted, and the slope was denoted as Ka; for the control group, 0.2 mL of distilled water was used to replace the sample, and the slope was denoted as Kc, and calculated according to formula (1)