Preparation method and application of abelmoschus manihot extract

Abstract

The invention relates to a preparation method and application of an abelmoschus manihot extract, and belongs to the technical field of natural product preparation. The preparation method of the abelmoschus manihot extract comprises the steps of abelmoschus manihot pretreatment, extraction after enzymolysis, macroporous resin separation, silica gel column separation and the like. The abelmoschus manihot extract prepared by the method has relatively high physiological activity, can effectively prevent and treat diarrhea, and can be used for the breeding industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product preparation, and relates to a preparation method of abelmoschus manihot extract and application thereof. BACKGROUND

[0002] Abelmoschus manihot, also known as wild hibiscus, is a plant of Malvaceae and is a plant on the verge of extinction. Abelmoschus manihot contains rich active ingredients, and the whole plant can be used as medicine. The main components include flavonoids, unsaturated fatty acids, polysaccharides, trace elements and alkaloids, etc. Abelmoschus manihot has the effects of antioxidant, lipid-lowering, anti-inflammatory, antipyretic analgesic, and immune enhancement. In the flower, the content of the above components is in the top position, especially the content of total flavonoids is significantly rich compared with the content in the root, stem, pericarp and other parts, and the flower is an ideal medicine part.

[0003] Abelmoschus manihot is suitable for people with hypertension, hyperlipidemia, diabetes, and soreness of waist and legs, but it is not suitable for people with spleen and stomach deficiency, infants, and people with abdominal pain and diarrhea.

[0004] It has been disclosed in the prior art that Abelmoschus manihot stem is used for treating high fever, constipation, difficulty in urination, boils and sores, and scalding. Abelmoschus manihot and Abelmoschus manihot are of the same genus, and it has been reported that Abelmoschus manihot stem powder has a special effect on preventing and treating three highs and constipation.

[0005] However, there is no report or literature about the treatment of diarrhea by using Abelmoschus manihot. SUMMARY

[0006] The main purpose of the present application is to provide an Abelmoschus manihot extract which can effectively treat or improve diarrhea and be used in the breeding industry.

[0007] The present application adopts the following technical scheme to achieve the above purpose: A preparation method of an Abelmoschus manihot extract mainly includes the following steps: Step 1, removing the roots and leaves of Abelmoschus manihot, only leaving the stem part, cleaning, soaking in HCl solution for 4-5 h, then washing with deionized water, drying to a water content of 10-15%, crushing to obtain Abelmoschus manihot stem powder, and reserving; Step 2, mixing the Abelmoschus manihot stem powder with water, adjusting the pH to 5.0-5.5, adding cellulase, and incubating at 50℃ for 2-2.5 h, then adding anhydrous ethanol, adjusting the alcohol content to 10%, and refluxing at 90℃ for 1-1.5 h, filtering, and reserving the filter residue and the extract I; Step 3, mixing the filter residue obtained in step 2 with ethanol solution, adding citric acid, refluxing at 70℃ for 1-1.5 h, filtering, and reserving the extract II; Step 4: Combine extract I and extract II, concentrate under reduced pressure to remove ethanol, and obtain a concentrated solution; add the concentrated solution and extract with petroleum ether and ethyl acetate in sequence, concentrate the lower layer solution after extraction, dry it, and redissolve it with a 30% ethanol solution to obtain extract I solution for later use. Step 5: Load the macroporous resin with the extract I solution obtained in step 4, elute with ethanol solution, collect the eluent, concentrate, dry, and redissolve with water-methanol at a volume ratio of 3:10 to obtain extract II solution for later use. Step 6: Load the Extract II solution obtained in Step 5 onto a silica gel column, elute with dichloromethane-methanol-formic acid solution, collect the target eluent, remove the organic solvent, and dry to obtain the golden sunflower extract.

[0008] Preferably, the HCl solution in step 1 has a mass fraction of 10%.

[0009] Preferably, in step 2, the ratio of sunflower stem powder to water is 1:(2-3) in kg / L; the amount of cellulase added is 3-4% of the mass of sunflower stem powder.

[0010] Preferably, in step 3, the ratio of filter residue to ethanol solution is 1:(5-6) in kg / L; the mass fraction of the ethanol solution is 50%; and the amount of citric acid added is 50-60 g / L.

[0011] Preferably, the elution procedure in step 5 is as follows: elution is performed sequentially using a 30% ethanol solution and a 50% ethanol solution.

[0012] More preferably, in step 5, the 30% ethanol solution contains 3% choline chloride and the 50% ethanol solution contains 5% choline chloride.

[0013] Preferably, the elution procedure of the dichloromethane-methanol-formic acid solution in step 6 is as follows: elution is performed sequentially using dichloromethane-methanol-formic acid solutions with volume ratios of 50:1:0.05, 20:1:0.02, 10:1:0.01, 5:1:0.01, and 2:1:0.01.

[0014] Preferably, the target eluent in step 6 is a dichloromethane-methanol-formic acid solution eluent with a volume ratio of 20:1:0.02, 10:1:0.01, or 5:1:0.01.

[0015] This invention provides an extract of golden hibiscus obtained by the above preparation method.

[0016] The present invention also provides specific uses for the above-mentioned golden hibiscus extract, namely, the golden hibiscus extract can be used to prepare products for the prevention and treatment of diarrhea; the products include, but are not limited to, food, medicine or feed.

[0017] Specifically, the golden sunflower extract can be used to prepare medicines for the prevention and treatment of diarrhea in poultry and livestock; it can also be used as a feed additive for the prevention and treatment of diarrhea in poultry and livestock.

[0018] The present invention has the following beneficial effects: Compared to existing technologies, the golden hibiscus extract prepared in this invention has a better effect on preventing and treating diarrhea. The golden hibiscus stem extract disclosed in the prior art has a constipation-treating effect, which is the opposite of the diarrhea-treating pharmacological activity of the golden hibiscus extract in this invention. This invention discovers a new use for the golden hibiscus extract. In the preparation process of the golden hibiscus extract in this invention, the components are extracted through enzymatic hydrolysis, acidic environment, and ethanol extraction. After extraction to remove impurities, the target fraction is obtained by elution with macroporous resin, and then further separated using a silica gel column. Finally, the golden hibiscus extract with anti-diarrheal effect is obtained. The elution solvent in the macroporous resin and silica gel column separation was modified, and the resulting elution product has good pharmacological activity. It has a good preventive and therapeutic effect on diarrhea in mice caused by different reasons. When used in feeding weaned piglets, it can prevent diarrhea in piglets, thereby promoting piglet growth. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope of protection of the claims of this application.

[0020] Example 1 Step 1: Remove the roots and leaves of the golden sunflower, keeping only the stem. After cleaning, soak it in a 10% HCl solution for 5 hours, then rinse it with deionized water, dry it in a 120℃ constant temperature oven until the moisture content is 15%, pulverize it, and pass it through a 100-mesh sieve to obtain golden sunflower stem powder for later use. Step 2: Take 1 kg of golden sunflower stem powder, add 3 L of water and mix well. Adjust the pH to 5.0, add 40 g of cellulase (commercial product, 50 U / mg, CAS No. 9012-54-8), and incubate at 50℃ for 2 h for enzymatic hydrolysis. Then add anhydrous ethanol and adjust the alcohol content to 10%. Reflux at 90℃ for 1.5 h, filter, and keep the filter residue and extract I for later use. Step 3: Add the above-obtained filter residue to a 50% ethanol solution at a material-to-liquid ratio of 1:6 (kg / L), mix well, add 60 g / L citric acid, reflux at 70°C for 1 h, filter, and keep extract II for later use. Step 4: Combine extract I and extract II, concentrate under reduced pressure to remove ethanol, and obtain a concentrated solution; add the concentrated solution and extract with petroleum ether and ethyl acetate in sequence, concentrate the lower layer solution after extraction, dry it, and redissolve it with a 30% ethanol solution to obtain extract I solution for later use. Step 5: After fully swelling the D101 macroporous resin in a 30% ethanol solution, pack it into a column using a wet method; load the extract I solution onto the column and elute at a rate of 100 mL / h with a 30% ethanol solution (containing 3% choline chloride, w / v) and a 50% ethanol solution (containing 5% choline chloride, w / v). Collect the eluent, concentrate it, dry it, and redissolve it in water-methanol at a volume ratio of 3:10 to obtain extract II solution for later use. Step 6: Take 200-300 mesh silica gel and mix thoroughly with a dichloromethane-methanol mixed solution at a volume ratio of 50:1. Pack the mixture into a glass column and allow the silica gel to settle freely to obtain a silica gel column. Mix the obtained extract II solution with 100-200 mesh coarse silica gel, dry it, and obtain a dry powder for later use. Sprinkle the dry powder into the packed silica gel column, let it stand, and allow the sample to settle completely. Then elute with dichloromethane-methanol-formic acid solutions at volume ratios of 50:1:0.05, 20:1:0.02, 10:1:0.01, 5:1:0.01, and 2:1:0.01. Combine the eluents from the dichloromethane-methanol-formic acid (20:1:0.02, 10:1:0.01, and 5:1:0.01) segments, remove the organic solvent, and dry to obtain the golden sunflower extract.

[0021] Example 2 Step 1: Remove the roots and leaves of the golden sunflower, keeping only the stem. After cleaning, soak it in a 10% HCl solution for 4 hours, then rinse it with deionized water, dry it in a 120℃ constant temperature oven until the moisture content is 10%, pulverize it, and pass it through a 100-mesh sieve to obtain golden sunflower stem powder for later use. Step 2: Take 1 kg of golden sunflower stem powder, add 2 L of water and mix well. Adjust the pH to 5.5, add 30 g of cellulase (commercial product, 50 U / mg, CAS No. 9012-54-8), and incubate at 50℃ for 2.5 h for enzymatic hydrolysis. Then add anhydrous ethanol and adjust the alcohol content to 10%. Reflux at 90℃ for 1.5 h, filter, and keep the filter residue and extract I for later use. Step 3: Add the above-obtained filter residue to a 50% ethanol solution at a material-to-liquid ratio of 1:5 (kg / L), mix well, add 50 g / L citric acid, reflux at 70°C for 1.5 h, filter, and keep extract II for later use. Step 4: Combine extract I and extract II, concentrate under reduced pressure to remove ethanol, and obtain a concentrated solution; add the concentrated solution and extract with petroleum ether and ethyl acetate in sequence, concentrate the lower layer solution after extraction, dry it, and redissolve it with a 30% ethanol solution to obtain extract I solution for later use. Step 5: After fully swelling the D101 macroporous resin in a 30% ethanol solution, pack it into a column using a wet method; load the extract I solution onto the column and elute at a rate of 100 mL / h with a 30% ethanol solution (containing 3% choline chloride, w / v) and a 50% ethanol solution (containing 5% choline chloride, w / v). Collect the eluent, concentrate it, dry it, and redissolve it in water-methanol at a volume ratio of 3:10 to obtain extract II solution for later use. Step 6: Take 200-300 mesh silica gel and mix thoroughly with a dichloromethane-methanol mixed solution at a volume ratio of 50:1. Pack the mixture into a glass column and allow the silica gel to settle freely to obtain a silica gel column. Mix the obtained extract II solution with 100-200 mesh coarse silica gel, dry it, and obtain a dry powder for later use. Sprinkle the dry powder into the packed silica gel column, let it stand, and allow the sample to settle completely. Then elute with dichloromethane-methanol-formic acid solutions at volume ratios of 50:1:0.05, 20:1:0.02, 10:1:0.01, 5:1:0.01, and 2:1:0.01. Combine the eluents from the dichloromethane-methanol-formic acid (20:1:0.02, 10:1:0.01, and 5:1:0.01) segments, remove the organic solvent, and dry to obtain the golden sunflower extract.

[0022] Example 3 Step 1: Remove the roots and leaves of the golden sunflower, keeping only the stem. After cleaning, soak it in a 10% HCl solution for 5 hours, then rinse it with deionized water, dry it in a 120℃ constant temperature oven until the moisture content is 13%, pulverize it, and pass it through a 100-mesh sieve to obtain golden sunflower stem powder for later use. Step 2: Take 1 kg of golden sunflower stem powder, add 3 L of water and mix well. Adjust the pH to 5.5, add 35 g of cellulase (commercial product, 50 U / mg, CAS No. 9012-54-8), and incubate at 50℃ for 2.5 h for enzymatic hydrolysis. Then add anhydrous ethanol and adjust the alcohol content to 10%. Reflux at 90℃ for 1.5 h, filter, and keep the filter residue and extract I for later use. Step 3: Add the above-obtained filter residue to a 50% ethanol solution at a material-to-liquid ratio of 1:6 (kg / L), mix well, add 55 g / L citric acid, reflux at 70°C for 1.5 h, filter, and keep extract II for later use. Step 4: Combine extract I and extract II, concentrate under reduced pressure to remove ethanol, and obtain a concentrated solution; add the concentrated solution and extract with petroleum ether and ethyl acetate in sequence, concentrate the lower layer solution after extraction, dry it, and redissolve it with a 30% ethanol solution to obtain extract I solution for later use. Step 5: After fully swelling the D101 macroporous resin in a 30% ethanol solution, pack it into a column using a wet method; load the extract I solution onto the column and elute at a rate of 100 mL / h with a 30% ethanol solution (containing 3% choline chloride, w / v) and a 50% ethanol solution (containing 5% choline chloride, w / v). Collect the eluent, concentrate it, dry it, and redissolve it in water-methanol at a volume ratio of 3:10 to obtain extract II solution for later use. Step 6: Take 200-300 mesh silica gel and mix thoroughly with a dichloromethane-methanol mixed solution at a volume ratio of 50:1. Pack the mixture into a glass column and allow the silica gel to settle freely to obtain a silica gel column. Mix the obtained extract II solution with 100-200 mesh coarse silica gel, dry it, and obtain a dry powder for later use. Sprinkle the dry powder into the packed silica gel column, let it stand, and allow the sample to settle completely. Then elute with dichloromethane-methanol-formic acid solutions at volume ratios of 50:1:0.05, 20:1:0.02, 10:1:0.01, 5:1:0.01, and 2:1:0.01. Combine the eluents from the dichloromethane-methanol-formic acid (20:1:0.02, 10:1:0.01, and 5:1:0.01) segments, remove the organic solvent, and dry to obtain the golden sunflower extract.

[0023] Comparative Example 1 Step 1: Remove the roots and leaves of the golden sunflower, keeping only the stem. After cleaning, soak it in a 10% HCl solution for 5 hours, then rinse it with deionized water, dry it in a 120℃ constant temperature oven until the moisture content is 13%, pulverize it, and pass it through a 100-mesh sieve to obtain golden sunflower stem powder for later use. Step 2: Take 1 kg of golden sunflower stem powder, add 3 L of water and mix well. Adjust the pH to 5.5, add 35 g of cellulase (commercial product, 50 U / mg, CAS No. 9012-54-8), and incubate at 50℃ for 2.5 h for enzymatic hydrolysis. Then add anhydrous ethanol and adjust the alcohol content to 10%. Reflux at 90℃ for 1.5 h, filter, and keep the filter residue and extract I for later use. Step 3: Add the above-obtained filter residue to a 50% ethanol solution at a material-to-liquid ratio of 1:6 (kg / L), mix well, add 55 g / L citric acid, reflux at 70°C for 1.5 h, filter, and keep extract II for later use. Step 4: Combine extract I and extract II, concentrate under reduced pressure to remove ethanol, and obtain a concentrated solution; add the concentrated solution and extract with petroleum ether and ethyl acetate in sequence, concentrate the lower layer solution after extraction, dry it, and redissolve it with a 30% ethanol solution to obtain extract I solution for later use. Step 5: After fully swelling the D101 macroporous resin in a 30% ethanol solution, pack it into a column using a wet method; load the extract I solution onto the column and elute at a rate of 100 mL / h with a 30% ethanol solution (containing 3% choline chloride, w / v) and a 50% ethanol solution (containing 5% choline chloride, w / v). Collect the eluent, concentrate it, dry it, and redissolve it in water-methanol at a volume ratio of 3:10 to obtain extract II solution for later use. Step 6: Take 200-300 mesh silica gel and mix thoroughly with a dichloromethane-methanol mixed solution at a volume ratio of 50:1. Pack the mixture into a glass column and allow the silica gel to settle freely to obtain a silica gel column. Mix the obtained extract II solution with 100-200 mesh coarse silica gel, dry it, and obtain a dry powder for later use. Sprinkle the dry powder into the packed silica gel column, let it stand, and allow the sample to settle completely. Then elute with dichloromethane-methanol-formic acid solutions at volume ratios of 50:1:0.05, 20:1:0.02, 10:1:0.01, 5:1:0.01, and 2:1:0.01 in sequence. Combine the eluents of the dichloromethane-methanol-formic acid (50:1:0.05, 2:1:0.01) segments, remove the organic solvent, and dry to obtain the golden sunflower extract.

[0024] Comparative Example 2 Step 1: Remove the roots and leaves of the golden sunflower, keeping only the stem. After cleaning, soak it in a 10% HCl solution for 5 hours, then rinse it with deionized water, dry it in a 120℃ constant temperature oven until the moisture content is 13%, pulverize it, and pass it through a 100-mesh sieve to obtain golden sunflower stem powder for later use. Step 2: Take 1 kg of golden sunflower stem powder, add 3 L of water and mix well. Adjust the pH to 5.5, add 35 g of cellulase (commercial product, 50 U / mg, CAS No. 9012-54-8), and incubate at 50℃ for 2.5 h for enzymatic hydrolysis. Then add anhydrous ethanol and adjust the alcohol content to 10%. Reflux at 90℃ for 1.5 h, filter, and keep the filter residue and extract I for later use. Step 3: Add the above-obtained filter residue to a 50% ethanol solution at a material-to-liquid ratio of 1:6 (kg / L), mix well, add 55 g / L citric acid, reflux at 70°C for 1.5 h, filter, and keep extract II for later use. Step 4: Combine extract I and extract II, concentrate under reduced pressure to remove ethanol, and obtain concentrated solution; add petroleum ether and ethyl acetate to extract the concentrated solution in sequence, concentrate the lower layer solution after extraction, and dry to obtain golden sunflower extract.

[0025] Comparative Example 3 Step 1: Remove the roots and leaves from the golden sunflower, keeping only the stems. After cleaning, dry them in a 120℃ constant temperature oven until the moisture content is 13%. Crush them and pass them through a 100-mesh sieve to obtain golden sunflower stem powder for later use. Step 2: Take 1 kg of golden sunflower stem powder, add 3 L of water and mix well. Adjust the pH to 5.5, add 35 g of cellulase (commercial product, 50 U / mg, CAS No. 9012-54-8), and incubate at 50℃ for 2.5 h for enzymatic hydrolysis. Then add anhydrous ethanol and adjust the alcohol content to 10%. Reflux at 90℃ for 1.5 h, filter, and keep the filter residue and extract I for later use. Step 3: Add the above-obtained filter residue to a 50% ethanol solution at a material-to-liquid ratio of 1:6 (kg / L), mix well, reflux at 70°C for 1.5 h, filter, and keep extract II for later use. Step 4: Combine extract I and extract II, concentrate under reduced pressure to remove ethanol, and obtain a concentrated solution. Add the concentrated solution to the solution and extract with petroleum ether and ethyl acetate in sequence. Concentrate the lower layer solution after extraction, dry it, and redissolve it with a 30% ethanol solution to obtain an extract solution for later use. Step 5: Thoroughly mix 200-300 mesh silica gel with a dichloromethane-methanol mixed solution at a volume ratio of 50:1, and pack the mixture into a glass column, allowing the silica gel to settle freely to obtain a silica gel column. Mix the obtained extract solution with 100-200 mesh coarse silica gel, dry it, and obtain a dry powder for later use. Sprinkle the dry powder into the packed silica gel column, let it stand, and allow the sample to settle completely. Then elute with dichloromethane-methanol-formic acid solutions at volume ratios of 50:1:0.05, 20:1:0.02, 10:1:0.01, 5:1:0.01, and 2:1:0.01, respectively. Combine the eluates from the dichloromethane-methanol-formic acid (20:1:0.02, 10:1:0.01, and 5:1:0.01) segments, remove the organic solvent, and dry to obtain the golden sunflower extract.

[0026] Comparative Example 4 Step 1: Remove the roots and leaves of the golden sunflower, keeping only the stem. After cleaning, soak it in a 10% HCl solution for 5 hours, then rinse it with deionized water, dry it in a 120℃ constant temperature oven until the moisture content is 13%, pulverize it, and pass it through a 100-mesh sieve to obtain golden sunflower stem powder for later use. Step 2: Take 1 kg of golden sunflower stem powder, add 5 L of 10% ethanol solution, mix well, reflux at 90℃ for 1.5 h, filter, and keep the residue and extract I for later use; Step 3: Add the obtained filter residue above into an ethanol solution with a mass fraction of 50% at a solid-liquid ratio of 1:6 (kg / L). After mixing evenly, add citric acid at 55 g / L, reflux and extract at 70 °C for 1.5 h, then filter. Reserve the extract II. Step 4: After combining Extract I and Extract II, concentrate under reduced pressure to remove ethanol to obtain a concentrated solution. Add the concentrated solution and extract successively with petroleum ether and ethyl acetate. Concentrate the lower layer solution after extraction, dry it, and redissolve it with an ethanol solution with a mass fraction of 30% to obtain the Extract I solution for standby. Step 5: Place the D101 macroporous resin in an ethanol solution with a mass fraction of 30% and fully swell it, then load the column by the wet method. Load the Extract I solution, and elute successively with an ethanol solution with a mass fraction of 30% and an ethanol solution with a mass fraction of 50% at an elution rate of 100 mL / h. Collect the eluate, concentrate it, dry it, and redissolve it with a water-methanol mixture with a volume ratio of 3:10 to obtain the Extract II solution for standby. Step 6: Thoroughly mix 200 - 300 mesh silica gel with a dichloromethane-methanol mixed solution with a volume ratio of 50:1, and then load it into a glass column to allow the silica gel to settle freely to obtain a silica gel column. Mix the obtained Extract II solution with 100 - 200 mesh coarse silica gel, dry it to obtain a dry powder for standby. Sprinkle the dry powder into the loaded silica gel column, let it stand still to allow the sample to fully settle, and then elute successively with dichloromethane-methanol solutions with volume ratios of 50:1, 20:1, 10:1, 5:1, and 2:1. Combine the eluates of the dichloromethane-methanol (20:1, 10:1, 5:1) segments, remove the organic solvent, dry it to obtain the Abelmoschus manihot extract.

[0027] Performance Test I. Study on the effect of Abelmoschus manihot extract on mice with diarrhea induced by Senna leaf 1. Experimental design SPF-grade KM mice, license number: SCXK(Shandong) 2022 0006, weighing 18 - 22 g, were adaptively fed for 10 days before the experiment. Relevant experiments were carried out by referring to existing literature.

[0028] The mice were randomly divided into 10 groups, namely the blank group, the model group, the positive drug group, and experimental groups 1 - 7, with 10 mice in each group. The positive drug group and experimental groups 1 - 7 were given corresponding drugs by gavage, the blank group and the model group were given an equal amount of normal saline by gavage, once a day for 5 consecutive days. Starting from the evening of the 6th day of drug administration, food was withheld but water was not restricted. One hour after drug administration on the 7th day, the mice in the model group, the positive drug group, and experimental groups 1 - 7 were given 0.5 mL of senna extract by gavage (the senna was boiled with water for extraction to make a senna extract with 1 g of crude drug content per mL), and the blank group was given an equal amount of normal saline by gavage. After modeling, the mice were fed individually in single cages with filter paper placed in the cages, and the diarrhea situation of the mice was observed. The filter paper was changed every 30 minutes, and the observation continued for 5 hours. The filter paper was collected to record the time of the first loose stool and the overall defecation situation of the mice, and the loose stool rate and diarrhea index were calculated.

[0029] Loose stool grade: Graded according to the contaminated diameter of the filter paper, diameter ≤ 1 cm is grade 1; 1 cm < diameter ≤ 2 cm is grade 2; 2 cm < diameter ≤ 3 cm is grade 3; diameter > 3 cm is grade 4.

[0030] 2. Experimental results and analysis As can be seen from the results in Table 1, the extracts of Abelmoschus manihot prepared in Examples 1 - 3 of the present invention have a high improvement effect on diarrhea in mice caused by senna, can significantly prolong the time of the first loose stool, reduce the loose stool rate, and the diarrhea index decreases significantly; it is worth noting that some mice gavaged with the extract of Comparative Example 1 had loose stools before being given senna, and after being given senna, the diarrhea situation worsened, and their loose stool rate and diarrhea index were higher than those of the model group; although the diarrhea situation decreased after gavaging with the extracts of Abelmoschus manihot in Comparative Examples 2 - 4, the data had no significant difference (P > 0.05), but the time of the first loose stool was prolonged. There are differences in the components of the extracts of Abelmoschus manihot obtained by different preparation methods, and the pharmacological activities between the components are quite different. The extracts of Abelmoschus manihot in the present invention have a good effect on preventing and treating diarrhea.

[0031] Table 1 Comparison of defecation situations of mice in different groups Note: Indicates P < 0.05 compared with the model group. <000012 / / 7>II. Study on the effect of different extracts of Abelmoschus manihot on bacterial diarrhea in mice 1. Experimental design SPF - level KM mice, license number: SCXK(Shandong) 2022 0006, weighing 18 - 22 g, were adaptively fed for 10 days before the experiment. Relevant experiments were carried out referring to existing literature.

[0033] Mice were randomly divided into 10 groups: a control group, a model group, a positive control group, and experimental groups 1-7, with 10 mice in each group. The positive control group and experimental groups 1-7 were administered the corresponding drugs via gavage, while the control group and model group were administered an equal volume of physiological saline via gavage. The administration was repeated once daily for 5 consecutive days. On the evening of the 6th day of administration, mice were kept on a fasting policy but allowed free access to water. One hour after administration on the 7th day, mice in the model group, positive control group, and experimental groups 1-7 were intraperitoneally injected with 5 × 10⁻⁵ mg / L of the drug. 8 0.4 mL of CFU / mL Escherichia coli solution was used. After modeling, mice were fed individually in cages with filter paper placed in the cages to observe diarrhea. The filter paper was changed every 30 minutes, and observations were conducted continuously for 5 hours. The diarrhea rate was recorded, filter paper was collected to record mouse defecation, and the loose stool rate and diarrhea index were calculated.

[0034] Stool looseness grade: graded according to the diameter of the filter paper contamination: grade 1 is ≤1cm; grade 2 is 1cm < diameter ≤2cm; grade 3 is 2cm < diameter ≤3cm; grade 4 is diameter >3cm.

[0035] 2. Experimental Results and Analysis As shown in Table 2, the extracts of *Hippophae rhamnoides* prepared in Examples 1-3 of this invention have a high ameliorative effect on bacterial diarrhea in mice, and can significantly reduce the rate of loose stools and the diarrhea index. Similar to the results in Experiment 1, some mice that were given the extract of Comparative Example 1 by gavage already had loose stools before the injection of bacteria. After the injection of bacteria, the diarrhea worsened, and their rate of loose stools and the diarrhea index were higher than those of the model group. Although the rates of loose stools were reduced by gavage of *Hippophae rhamnoides* extract in Comparative Examples 2-4, there was no significant difference in the data (P>0.05). The rate of loose stools in Comparative Example 3 was lower, but the diarrhea index was not significantly different.

[0036] Table 2 Comparison of defecation patterns in different groups of mice Note: This indicates that, compared to the model group, P < 0.05.

[0037] III. Effects of Golden Sunflower Extract on Diarrhea in Weaned Piglets 1. Experimental Design Weaned piglets aged 30±2 days and weighing 7±0.2 kg were randomly divided into three groups: a control group, experimental group 1, and experimental group 2, with 10 piglets in each group. The control group was fed a basal diet, experimental group 1 was fed a basal diet plus 0.2 g / kg of *Hippophae rhamnoides* extract from Example 1, and experimental group 2 was fed a basal diet plus 0.2 g / kg of *Hippophae rhamnoides* extract from Comparative Example 2. The piglets were observed for 15 consecutive days, and the diarrhea rate was analyzed and calculated.

[0038] Diarrhea in piglets was scored based on existing diagnostic criteria in the literature.

[0039] Normal: Stool moisture content <70%, stool shape or granules, score 0; Mild: Stool moisture content 70-75%, soft stool, formed, 1 point; Moderate: Stool moisture content 75-80%, pasty, unformed, scored 2 points; Severe: Moisture content > 80%, liquid, feces and water separated, score 3 points.

[0040] Piglets scoring ≥1 point are classified as having diarrhea.

[0041] 2. Results and Analysis As can be seen from the results in Table 3, the extract of *Hymenochloa crus-galli* obtained in Example 1 of this invention has a good effect on improving diarrhea in weaned piglets and can significantly reduce the diarrhea rate. The extract of *Hymenochloa crus-galli* in Comparative Example 2 also showed a good effect on improving diarrhea in piglets.

[0042] Table 3 Comparison of diarrhea in different groups of piglets Note: This indicates that compared with the control group, P < 0.05.

Claims

1. A method for preparing an extract of *Hippophae rhamnoides*, characterized in that, Includes the following steps: Step 1: Remove the roots and leaves from the golden sunflower, keeping only the stem. After cleaning, soak it in HCl solution for 4-5 hours, then rinse with deionized water, dry it until the moisture content is 10-15%, and crush it to obtain golden sunflower stem powder for later use. Step 2: After mixing the golden sunflower stem powder with water, adjust the pH to 5.0-5.5, add cellulase, and incubate at 50℃ for 2-2.5h for enzymatic hydrolysis. Then add anhydrous ethanol, adjust the alcohol content to 10%, and reflux at 90℃ for 1-1.5h. Filter and keep the residue and extract I for later use. Step 3: Add the filter residue obtained in Step 2 to the ethanol solution and mix well. Then add citric acid and reflux at 70°C for 1-1.5 hours. Filter and keep extract II for later use. Step 4: Combine extract I and extract II, concentrate under reduced pressure to remove ethanol, and obtain a concentrated solution; add the concentrated solution and extract with petroleum ether and ethyl acetate in sequence, concentrate the lower layer solution after extraction, dry it, and redissolve it with a 30% ethanol solution to obtain extract I solution for later use. Step 5: Load the macroporous resin with the extract I solution obtained in step 4, elute with ethanol solution, collect the eluent, concentrate, dry, and redissolve with water-methanol at a volume ratio of 3:10 to obtain extract II solution for later use. Step 6: Load the Extract II solution obtained in Step 5 onto a silica gel column, elute with dichloromethane-methanol-formic acid solution, collect the target eluent, remove the organic solvent, and dry to obtain the golden sunflower extract.

2. The preparation method according to claim 1, characterized in that, The HCl solution in step 1 has a mass fraction of 10%.

3. The preparation method according to claim 1, characterized in that, In step 2, the ratio of sunflower stem powder to water is 1:(2-3) in kg / L; the amount of cellulase added is 3-4% of the mass of sunflower stem powder.

4. The preparation method according to claim 1, characterized in that, In step 3, the ratio of filter residue to ethanol solution is 1:(5-6) kg / L; the mass fraction of ethanol solution is 50%; and the amount of citric acid added is 50-60 g / L.

5. The preparation method according to claim 1, characterized in that, The elution procedure in step 5 is as follows: elution is performed sequentially using a 30% ethanol solution and a 50% ethanol solution.

6. The preparation method according to claim 5, characterized in that, In step 5, the 30% ethanol solution contains 3% choline chloride, and the 50% ethanol solution contains 5% choline chloride.

7. The preparation method according to claim 1, characterized in that, The elution procedure for the dichloromethane-methanol-formic acid solution in step 6 is as follows: elution is performed sequentially using dichloromethane-methanol-formic acid solutions with volume ratios of 50:1:0.05, 20:1:0.02, 10:1:0.01, 5:1:0.01, and 2:1:0.

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8. The preparation method according to claim 1, characterized in that, In step 6, the target eluent is a dichloromethane-methanol-formic acid solution with a volume ratio of 20:1:0.02, 10:1:0.01, or 5:1:0.

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9. The use of a golden hibiscus extract prepared by any one of claims 1-8 in the preparation of a product for preventing and treating diarrhea.

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