Meconopsis quintuplingensis extract as well as preparation method and application thereof

Abstract

The invention relates to the field of traditional Chinese medicine extracts, and discloses a meconopsis quintuplingensis extract as well as a preparation method and application thereof. According to the application, the meconopsis quintuplingensis ethyl acetate extract or the total flavone ethanol extract is obtained through a specific extraction process, and the meconopsis quintuplingensis ethyl acetate extract or the total flavone ethanol extract has remarkable protection and improvement effects on a mouse acute alcoholic liver injury model; wherein the improvement effect of the ethyl acetate extract on a plurality of indexes such as liver index, ALT, MDA, TG and liver tissue pathological state is superior to that of a clinical medicine silibinin, and the improvement effect of the meconopsis quintuplingensis water extract and the improvement effect of the trichloromethane extract are superior to that of the clinical medicine silibinin; the total flavonoids with higher purity and yield can be obtained by a total flavone ethanol extract process; in addition, the capsule based on the meconopsis quintuplingensis extract is simple in preparation process and stable and controllable in quality, and reference is provided for further development and utilization of traditional Chinese medicines such as meconopsis quintuplingensis and the like.

Description

Technical Field

[0001] The present application relates to the field of Chinese medicine extracts, and in particular to a Meconopsis quinqueneri extract and a preparation method and application thereof. Background Art

[0002] Alcoholic liver injury refers to a series of lesions such as liver damage caused by excessive alcohol intake, which is divided into acute alcoholic liver injury and chronic alcoholic liver injury.

[0003] Excessive drinking and alcoholism are increasingly becoming serious problems that affect human health. The liver is the main place to metabolize alcohol and drugs. Clinically, drugs for the treatment of alcoholic liver damage will increase the burden on the liver. Therefore, the development of safe drugs with few side effects is of great guiding significance in the treatment of liver damage.

[0004] Traditional Chinese medicine has its unique advantages in the treatment of chemical liver damage, and Chinese herbal medicine plays an irreplaceable role in the early treatment of alcoholic liver damage. However, although there are many Chinese herbal preparations for the treatment of alcoholic liver damage in clinical practice, there are still many types of Chinese medicine used. Frequent use still has side effects, and the effect is slow and the efficacy is low. Summary of the invention

[0005] In view of this, the purpose of the present application is to provide a Meconopsis quinqueneri (MQR) extract and a preparation method thereof, so that the prepared Meconopsis quinqueneri extract can have a significant improvement effect on alcoholic liver damage alone, and the improvement effect on multiple indicators such as liver index, ALT, MDA, TG, and liver tissue pathological state is better than that of the clinical drug silybin;

[0006] Another object of the present application is to provide a Meconopsis quinqueneri (MQR) extract and a preparation method thereof, so that the preparation method can improve the purity and yield of total flavonoids in the Meconopsis quinqueneri extract, reduce the loss of total flavonoids, and have a higher enrichment and purification efficiency;

[0007] Another object of the present application is to provide a use of the above-mentioned Meconopsis quinquenervais extract in the preparation of a drug for improving alcoholic liver damage, and to provide a product for improving alcoholic liver damage.

[0008] In order to solve the above technical problems / achieve the above objectives or at least partially solve the above technical problems / achieve the above objectives, as a first aspect of the present application, a method for preparing a Meconopsis quinquenervata extract is provided, comprising:

[0009] S1. Extracting the medicinal material of Meconopsis quinquenervata with ethanol to obtain an ethanol extract;

[0010] S2, extracting the alcohol extract with petroleum ether, separating the aqueous layer and extracting with ethyl acetate, and removing the ethyl acetate to obtain the Meconopsis quinqueneri extract; or

[0011] The alcohol extract is loaded onto D101 macroporous resin for adsorption, left to stand for elution and impurities are removed, and then eluted with ethanol solution, the eluate is collected, and concentrated until there is no alcohol taste, so as to obtain the Meconopsis quinquenervata extract.

[0012] Optionally, step S1 includes:

[0013] Weigh the medicinal material of Meconopsis quinquenervais, add ethanol for reflux extraction, concentrate until there is no ethanol, and dilute with water to obtain an alcohol extract.

[0014] Further optionally, the amount of ethanol used is 5-9 times the amount of the Meconopsis quinquenervata medicinal material; and the concentration of the ethanol is 50-95%.

[0015] Optionally, the loading amount of the alcohol extract is 2BV based on the crude drug concentration of 0.5 g / mL, and the adsorption flow rate is 1 BV / h.

[0016] Optionally, the elution is performed with a 50% ethanol solution, the elution flow rate is 1-2 BV / h, and at least 8 BV of the eluate is collected. As a second aspect of the present application, a Meconopsis quinquenervais extract prepared by the preparation method is provided.

[0017] As a third aspect of the present application, provided is the use of the Meconopsis quinquenervais extract in the preparation of a product for improving alcoholic liver damage.

[0018] As a fourth aspect of the present application, a product for improving alcoholic liver damage comprises the Meconopsis quinquenervata extract described in the present application.

[0019] Optionally, the product is a capsule.

[0020] Further optionally, the product also includes auxiliary materials, and the auxiliary materials include one or more of fillers and lubricants; the fillers include corn starch, dextrin and microcrystalline cellulose, and the lubricants include magnesium stearate.

[0021] The present application obtains the ethyl acetate extract or total flavonoid ethanol extract of Meconopsis quinqueneri through a specific extraction process, which has a significant protective and improving effect on the mouse acute alcoholic liver injury model; among them, the ethyl acetate extract is better than the clinical drug silybin, as well as the water extract, ethanol extract and chloroform extract of Meconopsis quinqueneri in improving multiple indicators such as liver index, ALT, MDA, TG, and liver tissue pathological state; and the total flavonoid ethanol extraction process can obtain total flavonoids with higher purity and yield, and at the same time, the improvement effect on multiple indicators such as liver index, ALT, MDA, TG, and liver tissue pathological state is better than the clinical drug silybin; in addition, the capsule preparation process based on Meconopsis quinqueneri extract provided in the present application is simple, and the quality is stable and controllable, which provides a reference for the further development and utilization of Chinese medicines such as Meconopsis quinqueneri. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown is the flow chart of animal experiments;

[0023] Figure 2 The results show the effects of different extraction parts of MQR on serum transaminase levels in mice with acute alcoholic liver injury; A is the result of ALT, B is the result of AST; ###p<0.001, compared with the blank group; *p<0.05, **p<0.01, ***p<0.001, compared with the model group (x±s, n=6);

[0024] Figure 3 The figure shows the effect of different extraction parts of MQR on the body weight and liver index of mice with acute alcoholic liver injury; A is the body weight of mice in each group before alcohol modeling; B is the change of body weight of mice in each group after alcohol modeling; C is the liver index of mice in each group; ###p<0.001, compared with the blank group; ***p<0.001, compared with the model group (x±s, n=6);

[0025] Figure 4 The results show the effects of different extraction parts of MQR on liver oxidative damage in mice with acute alcoholic liver injury; A is the result of MDA, B is the result of SOD, and C is the result of GSH-PX; ###p<0.001, compared with the blank group; *p<0.05, **p<0.01, ***p<0.001, compared with the model group (x±s, n=6);

[0026] Figure 5 The results show the effects of different extraction parts of MQR on lipid levels in liver tissue of mice with acute alcoholic liver injury; A is the TG result, B is the TC result; ###p<0.001, compared with the blank group; *p<0.05, **p<0.01, ***p<0.001, compared with the model group (x±s, n=6);

[0027] Figure 6 Shown are the effects of different extraction parts of MQR on liver pathology in mice with acute alcoholic liver injury (n=6); from top to bottom are liver appearance, H&E staining (×200 and ×400 magnification);

[0028] Figure 7 The different macroporous resins shown are the results of dynamic elution investigation;

[0029] Figure 8 Shown are the results of the investigation of elution concentration of D101 macroporous resin;

[0030] Fig. 9 Shown are the results of the investigation of the concentration of the sample solution loaded on the D101 macroporous resin;

[0031] Fig.10The figure shows the curve of the sample loading amount of D101 macroporous resin;

[0032] Fig.11 Shown is the result of the D101 macroporous resin adsorption flow rate investigation;

[0033] Fig.12 Shown is the result of investigation on elution rate of D101 macroporous resin;

[0034] Fig.13 Shown is the elution curve of D101 macroporous resin;

[0035] Fig.14 The figure shows the effect of the total flavonoids extract of Meconopsis quinqueneri on the pathology of liver tissue; A is the effect on body weight, B is the effect on liver index, and C is the HE staining result; Normal is the blank group, EtOH is the model group, and EtOH+MQRF is the experimental group; ### p<0.001, compared with the blank group; ** p<0.01, compared with the model group; (x±s, n=6);

[0036] Fig.15 The figure shows the effect of the total flavonoids extract of Meconopsis quinqueneri on acute alcoholic liver injury in mice; A is the results of ALT and AST, B is the results of TG and TC; Normal is the blank group, EtOH is the model group, and EtOH+MQRF is the experimental group; #p<0.05, ### p<0.001, compared with the blank group; * p<0.05, ** p<0.01, *** p<0.001, compared with the model group; (x±s, n=6);

[0037] Fig.16 The figure shows the effect of the total flavonoids extract of Meconopsis quinqueneri on the body weight and liver index of mice with chronic alcoholic liver injury; A is the effect on body weight, B is the effect on liver index; Normal is the blank group, EtOH is the model group, EtOH+MQRF-L / M / H are the experimental low, medium and high dose groups, and EtOH+Silymarin is the positive control group; ### p<0.001, compared with the blank group; ** p<0.01, *** p<0.001, compared with the model group; (x±s, n=8);

[0038] Fig.17 The figure shows the effect of total flavonoids extract of Meconopsis quinqueneri on serum biochemical indexes in mice with chronic alcoholic liver injury; EtOH is the model group, EtOH+MQRF-L / M / H is the experimental low, medium and high dose groups, and EtOH+Silymarin is the positive control group;### p<0.001, compared with the blank group; * p<0.05, ** p<0.01, *** p<0.001, compared with the model group; (x±s, n=8);

[0039] Fig.18 The figure shows the effect of the total flavonoids extract of Meconopsis quinqueneri on the lipid level in liver tissue of mice with chronic alcoholic liver injury; EtOH is the model group, EtOH+MQRF-L / M / H is the low, medium and high dose groups of the experiment, and EtOH+Silymarin is the positive control group; ### p<0.001, compared with the blank group; * p<0.05, ** p<0.01, *** p<0.001, compared with the model group; (x±s, n=8);

[0040] Fig.19 The figure shows the effect of total flavonoids extract of Meconopsis quinqueneri on oxidative damage in mice with chronic alcoholic liver injury; EtOH is the model group, EtOH+MQRF-L / M / H is the experimental low, medium and high dose groups, and EtOH+Silymarin is the positive control group; ### p<0.001, compared with the blank group; * p<0.05, ** p<0.01, *** p<0.001, compared with the model group; (x±s, n=8);

[0041] Fig. 20 The figure shows the effect of the total flavonoids extract of Meconopsis quinqueneri on inflammatory cytokines and mediators of alcoholic liver injury in mice; EtOH is the model group, EtOH+MQRF-L / M / H is the experimental low, medium and high dose groups, and EtOH+Silymarin is the positive control group; ### p<0.001, compared with the blank group; * p<0.05, ** p<0.01, *** p<0.001, compared with the model group; (x±s, n=8);

[0042] Fig.21 The figure shows the effect of the total flavonoids extract of Meconopsis quinqueneri on the liver tissue pathology of mice with alcoholic liver injury; Normal is the blank group, EtOH is the model group, EtOH+MQRF-L / M / H are the experimental low, medium and high dose groups, and EtOH+Silymarin is the positive control group; (n=6). DETAILED DESCRIPTION

[0043] The present application discloses a Meconopsis quinqueneri extract and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0044] It should be noted that, in this article, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of further restrictions, the elements defined by the sentence "comprise one..." do not exclude the presence of other identical elements in the process, method, article or equipment including the elements. At the same time, the embodiments in this application and the features in the embodiments may be combined with each other without conflict.

[0045] In a first aspect of the present application, a method for preparing a Meconopsis quinqueneri extract is provided, comprising:

[0046] S1. Extracting the medicinal material of Meconopsis quinquenervata with ethanol to obtain an ethanol extract;

[0047] S2, extracting the alcohol extract with petroleum ether, separating the aqueous layer and extracting with ethyl acetate, and removing the ethyl acetate to obtain the Meconopsis quinqueneri extract; or

[0048] The alcohol extract is loaded onto D101 macroporous resin for adsorption, left to stand for elution and impurities are removed, and then eluted with ethanol solution, the eluate is collected, and concentrated until there is no alcohol taste, so as to obtain the Meconopsis quinquenervata extract.

[0049] In certain embodiments of the present application, step S1 includes:

[0050] Weigh the medicinal material of Meconopsis quinquenervais, add ethanol for reflux extraction, concentrate until there is no ethanol, and dilute with water to obtain an alcohol extract.

[0051] In some other embodiments of the present application, the amount of ethanol used is 5-9 times the amount of the Meconopsis quinqueneri medicinal material; in some other embodiments of the present application, the number of reflux extractions is 1-3 times, and the time is 1-2h; when multiple extractions are performed, the first amount of ethanol used is 7-9 times the amount of the Meconopsis quinqueneri medicinal material, and the amount of ethanol used in the remaining reflux extractions is 5-6 times the amount of the Meconopsis quinqueneri medicinal material.

[0052] In certain embodiments of the present application, the concentration of ethanol is 50-95%, for example, 50%, 60%, 70%, 80%, 90% or 95%.

[0053] In certain embodiments of the present application, step S1 includes:

[0054] Weigh an appropriate amount of Meconopsis quinqueneri medicinal material, place it in a round-bottom flask, add 5-6 times the amount of 50-95% ethanol for reflux extraction 3 times (7-9 times the amount for the first time), each time for 1-2 hours, filter, combine the filtrate, concentrate until there is no alcohol taste, dilute the residue with an appropriate amount of hot distilled water to obtain an alcohol extract.

[0055] In some embodiments of the present application, petroleum ether and ethyl acetate can be extracted multiple times in step S2, for example, 10 times, and each extraction liquid is combined after extraction. In some other embodiments of the present application, step S2 includes:

[0056] The alcohol extract is extracted with petroleum ether for 10 times, the petroleum ether layer is discarded, the water layer is extracted with ethyl acetate for 10 times, the ethyl acetate layers are combined and evaporated to dryness to obtain the ethyl acetate extract of Meconopsis quinqueneri, or the residue after evaporation is heated with distilled water to be dissolved and diluted to a required concentration to obtain the ethyl acetate extract of Meconopsis quinqueneri.

[0057] In certain embodiments of the present application, the loading amount of the alcohol extract in step S2 is 2BV based on the crude drug concentration of 0.5 g / mL, and the adsorption flow rate is 1 BV / h.

[0058] In certain embodiments of the present application, the sample is allowed to stand for adsorption for 12 hours after loading, and then 1BV of water is used to wash and remove impurities.

[0059] In some embodiments of the present application, elution is performed with a 50% ethanol solution, the elution flow rate is 1-2 BV / h, and the eluate is collected to at least 8 BV. In other embodiments of the present application, 50% ethanol solution is used for standing desorption for more than 12 hours, elution is performed at a flow rate of 1-2 BV / h, and the eluate is collected to 8 BV.

[0060] In some other embodiments of the present application, the concentration of the Meconopsis quinqueneri extract is equivalent to 0.017 g / mL, 0.234 g / mL, 0.468 g / mL, 1.17 g / kg, 2.34 g / kg or 4.68 g / kg of the raw medicinal material, which can be achieved by dilution with water or concentration.

[0061] In a second aspect of the present application, a Meconopsis quinquenervais extract prepared by the preparation method is provided.

[0062] In the third aspect of the present application, an acute ALD mouse model was established by gavage with edible wine to evaluate the anti-ALD effects of different extraction parts of MQR. The results showed that the four extracts of MQR (water extract, alcohol extract, ethyl acetate extract, chloroform extract) and silybin all improved acute ALD to varying degrees, among which the ethyl acetate extract (MQR-E) had the best effect. MQR-E can significantly reduce the liver index, serum transaminase levels (AST, ALT), and malondialdehyde (MDA) content of acute ALD mice, increase the activity of total superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and reduce the histopathological changes caused by alcohol.

[0063] In addition, the present application uses the alcohol extract to separate and purify the total flavonoids extract of Meconopsis quinqueneri (MQRF) through D101 macroporous resin. Then, a mouse model of acute alcoholic liver injury was established to verify whether MQRF has a protective and preventive effect on ALD. The results showed that compared with the model group, the liver index, ALT, AST, TG and TC levels in the MQRF group were significantly reduced. Histopathological observations found that the edema of hepatocytes, enlarged vacuoles, and inflammatory cell infiltration in the MQRF group were significantly improved. This shows that MQRF has a certain protective effect on acute alcoholic liver injury in mice. At the same time, MQRF also has a significant protective effect on various indicators of chronic alcoholic liver injury in mice.

[0064] Based on this, the present application proposes the use of the Meconopsis quinqueneri extract in the preparation of products for improving alcoholic liver damage.

[0065] In the fourth aspect of the present application, based on the above application, a product for improving alcoholic liver damage is provided, comprising the Meconopsis quinquenervata extract described in the present application, the dosage of which is determined according to the actual efficacy, i.e., the effective dose.

[0066] In certain embodiments of the present application, the product is a medicine, and its dosage form includes but is not limited to capsules, tablets, granules, and any form of liquid preparations.

[0067] In some other embodiments of the present application, the product further includes auxiliary materials, and the auxiliary materials include one or more of fillers and lubricants; wherein the filler includes corn starch, dextrin and microcrystalline cellulose, and the lubricant includes magnesium stearate.

[0068] In each group of comparative experiments provided in this application, unless otherwise specified, except for the differences indicated in each group, other experimental conditions, materials, etc. are kept consistent to ensure comparability.

[0069] The following is a further description of a Meconopsis quinqueneri extract provided in the present application and its preparation method and application.

[0070] Example 1: Preparation of different Meconopsis quinqueneri extracts

[0071] (1) Water extract of Meconopsis quinquenervais (MQR-W): Weigh an appropriate amount of Meconopsis quinquenervais medicinal material, add 5 times the amount of distilled water, reflux and extract 3 times (add 7 times the amount for the first time), each time for 1 hour, filter, combine the filtrate, and concentrate to a solution equivalent to 0.234 g / mL of the raw medicinal material.

[0072] (2) Alcohol extract of Meconopsis quinquenervais (MQR-A): Weigh an appropriate amount of Meconopsis quinquenervais medicinal material, add 5 times the amount of 95% ethanol for reflux extraction 3 times (7 times the amount for the first time), each time for 1 hour, filter, combine the filtrate, concentrate until there is no alcohol taste, and dilute with hot distilled water to a solution equivalent to 0.234 g / mL of the raw medicinal material.

[0073] (3) Ethyl acetate extract of Meconopsis quinqueneri (MQR-E): The ethanol was recovered from the Meconopsis quinqueneri alcohol extract until there was no alcohol taste. The residue was diluted with an appropriate amount of hot distilled water and extracted with petroleum ether for 10 times. The petroleum ether layer was discarded and the water layer was extracted with ethyl acetate for 10 times. The ethyl acetate layers were combined and evaporated to dryness. The residue was dissolved in distilled water and diluted to a solution equivalent to 0.234 g / mL of the raw medicinal material.

[0074] (4) Chloroform extract of Meconopsis quinqueneri (MQR-C): The ethanol extract of Meconopsis quinqueneri was recovered until there was no alcohol smell. The residue was shaken with petroleum ether to defat it, and then 100 mL of 0.01 mol / L hydrochloric acid solution was added to dissolve it and the pH value was adjusted to 2-3. The filtrate was filtered and concentrated ammonia was added to adjust the pH value to 9-10. The filtrate was extracted with chloroform 10 times. The chloroform layers were combined and the solvent was recovered. The residue was heated with distilled water to dissolve and dilute to a solution equivalent to 0.234 g / mL of the raw medicinal material.

[0075] Example 2: Validation of the mouse acute alcoholic liver injury model

[0076] 1. Experimental Animals

[0077] Ninety male C57BL / 6J mice (weighing 18 - 22 g), (SPF, SYXK(Yu) 2017 - 0003), were adaptively fed for one week.

[0078] 2. Experimental grouping and dosage

[0079] All mice were randomly divided into seven groups, with 12 mice in each group (18 mice in the model group). The administration groups were divided into the water extraction group, ethanol extraction group, ethyl acetate extraction group, and chloroform extraction group (each extract in Example 1), and the administration dose was 2.34 g (raw drug amount) / kg·BW·d -1 ; the positive control group was given silybin (0.1 g / kg·BW·d -1 ); the blank group and the acute alcohol model group (EtOH) were given distilled water according to body weight. The recommended clinical dosage of MQR recorded in the third volume of "Chinese Tibetan Medicine" is 6 - 9 grams. According to the previous research and the dosage of the positive drug, the dosage selected in this experiment was 18 grams. Calculated based on the body surface area ratio of mice to humans (1:0.0026), the dosage of MQR in mice was 2.34 g / kg.

[0080] 3. Experimental procedure

[0081] According to the above administration dose, the drug was administered continuously for 7 days. From the 8th day, except for the blank group which was given distilled water, other groups were given commercially available liquor with an alcohol content of 52% for gavage (12 mL / kg·BW), once a day, and then acute alcoholic liver injury was induced for 14 consecutive days. During the morning of the experiment, the blank group and the model group were given an equal amount of distilled water instead of the drug, and the other groups were continuously administered (for a total of 3 weeks).

[0082] After the last administration, the mice were fasted for 12 hours, and serum and liver were collected. The detailed modeling process is as Figure 1 shown. Two to three mice were randomly selected from the model group and the blank group to detect whether liver injury occurred. The judgment indexes were as follows: (1) The levels of serum transaminases (AST, ALT) and liver tissue TG, TC in the model group mice were significantly increased, with statistical differences; (2) Pathological changes occurred in liver tissue examination, such as hepatocyte edema, fat accumulation, and infiltration of inflammatory factors.

[0083] 4. Experimental methods

[0084] (1) Sampling

[0085] Each group of mice was fasted for 12 hours, weighed, and blood was collected by removing the eyeballs. After standing at room temperature for 1 hour, the mice were centrifuged for 15 minutes (4°C, 3000rpm), and the serum was separated and stored at -20°C for testing. The abdomen of the experimental animals was cut open, and then the livers of the mice in each group were completely removed, photographed, and the color, size, weight, etc. were compared to observe whether there were signs of swelling or atrophy; tissue blocks from the fixed part of the right lobe of the mouse liver were taken and fixed in 5-10 times the amount of 4% paraformaldehyde solution for HE staining; after flushing the blood with physiological saline, the remaining livers were marked one by one, put into tin foil, and inserted into an ice box for temporary low-temperature storage, and then transferred to liquid nitrogen or stored at -80°C for use.

[0086] (2) Determination of serum biochemical indicators

[0087] The collected serum samples were used to measure the activity values ​​of ALT and AST in the serum according to the instructions of the kit.

[0088] (3) Determination of oxidative stress indicators in liver tissue

[0089] Weigh an appropriate amount of liver tissue and place it in a grinding tube. Add physiological saline equal to 9 times the volume of the tissue weight for grinding. After sufficient homogenization, centrifuge for 10 minutes (2500rpm, 4°C) and take the supernatant for detection. The specific experimental steps are as required by the instructions, and the content of MDA, SOD, and GSH-PX in the tissue is calculated.

[0090] MDA, SOD and GSH-PX were measured according to the instructions of commercially available kits.

[0091] (4) Determination of lipid levels in liver tissue

[0092] Prepare liver tissue supernatant, perform the experiment according to the instruction manual, and calculate the TG and TC contents in the tissue after detection by ELISA.

[0093] (5) HE staining

[0094] The liver samples of mice in each group were fixed and embedded in paraffin, sliced, stained with hematoxylin and eosin in sequence, dehydrated, sealed, and examined under a microscope.

[0095] 5. Results

[0096] (1) Effects of different MQR extraction parts on the physiological state of mice with acute alcoholic liver injury

[0097] After alcohol was administered orally, the mice's eating and drinking activities were severely interfered with, and they showed varying degrees of drunkenness or even poisoning reactions. A large number of mice died in the early stage of alcohol administration, especially in the model group; the weight of mice in each group dropped severely, and the reason for this was analyzed to be the large initial oral dose. As the experiment progressed, the mortality rate of mice in each group decreased. During the experiment, the mice in the experimental group sobered up faster than those in the model group, and the glossiness of their fur was closer to that of the blank group.

[0098] (2) Effects of different MQR extraction parts on body weight and liver index in mice with acute alcoholic liver injury

[0099] During the preventive medication period, no mice died. After the experiment, there were no deaths in the blank group; 5 mice died in the Meconopsis quinquenervii water extract group, alcohol extract group, and ethyl acetate group; 6 mice died in the positive control group and the Meconopsis quinquenervii chloroform group; 10 mice died in the model group, and a total of 53 survived. Except for the blank group, the mice in the experimental groups showed drowsiness, decreased fur gloss, slow reaction, and reduced daily food intake. Compared with the mice in the model group, the MQR extraction part groups were in a shorter state of drunkenness, had a lower mortality rate, ate more, and were more active than the model group. After the liver was weighed, the liver index was calculated and the results are as follows: Figure 3 .

[0100] Depend on Figure 3 A and B show that before alcohol modeling, there was no significant difference in the weight of mice in each group. After alcohol modeling, the weight of mice in the experimental group decreased compared with the blank control group. And under alcohol intervention, the weight of mice in the group decreased significantly compared with the initial stage of the experiment ( Figure 3 B). This indicates that the alcohol dose used in the study was more harmful to mice in the early stage of the experiment. Compared with the blank group, the liver index of the mice in the model group increased significantly (p<0.001). After intervention with different extraction parts of MQR, the liver index of mice decreased to a certain extent, among which MQR-E had a significant effect, and the difference was statistically significant (p<0.001). Figure 3 As shown in C.

[0101] (3) Effects of different MQR extraction parts on serum transaminases in mice with acute alcoholic liver injury

[0102] After acute drinking, liver cells will be damaged, which is manifested by increased serum transaminase concentrations. Figure 2 In A, the serum ALT level of mice in the model group was significantly higher than that in the blank control group (p<0.05). After administration of MQR, the serum ALT level of mice in each group decreased to a certain extent (p<0.05). The serum ALT level of mice in the MQR-E group was close to that of the blank control group (p>0.05).

[0103] Compared with the blank group, the serum AST levels of mice in the model group and the MQR alcohol extraction group and water extraction group were significantly increased. Compared with the model group, the AST levels of the positive control group and the MQR ethyl acetate group and chloroform group were significantly decreased (p<0.05). Figure 2 B. This indicates that different extraction parts of MQR have a certain degree of protective effect on acute alcoholic liver injury.

[0104] (4) Effects of different MQR extraction parts on liver oxidative damage index in mice with acute alcoholic liver injury

[0105] To reflect the lipid peroxidation and anti-oxidation status of liver tissue, the levels of MDA, SOD, and GSH-PX in the liver tissue of each group of experimental animals were detected. Figure 4 . The results showed that compared with the blank group, the SOD and GSH-PX enzyme activities in the liver tissue of the model group mice decreased, and the MDA content increased, and the difference was statistically significant (p<0.05). After intervention with different extraction parts of MQR, the MDA level in the liver tissue of mice with acute alcoholic liver injury was significantly lower than that in the model group (p<0.05); the SOD level increased to a certain extent (p<0.05); the GSH-PX enzyme activity showed significant statistical differences between the other groups and the model group except the MQR water extraction group (p<0.05).

[0106] (5) Effects of different MQR extraction parts on lipid levels in liver tissue of mice with acute alcoholic liver injury

[0107] from Figure 5 It can be seen that the TG and TC contents in the liver tissue of mice in the model group were significantly higher than those in the blank control group (p<0.05). After intervention with different extraction parts of MQR, the TG and TC levels of mice in each group decreased significantly compared with the model group (p<0.05).

[0108] (6) Effects of different MQR extraction parts on liver pathology in mice with acute alcoholic liver injury

[0109] Depend on Figure 6 As can be seen in the figure, the liver of mice in the normal group had a smooth surface, soft texture and clear edges. In the model group, the liver tissue of mice was swollen, rough and discolored. After administration of different extracts of silybin or MQR, the liver surface was smooth and the swelling was reduced. Moreover, the liver morphology of the ethyl acetate group of Meconopsis quinqueneri (MQR-E) was close to that of the blank control group.

[0110] HE staining results showed that the hepatocytes of the blank control group were intact, with large round nuclei, red cytoplasm, and no lesions. Compared with the blank group, the model group had enlarged hepatocytes, fatty degeneration, and liver fibrosis, as shown by hepatocyte edema, ballooning, and lymphocyte infiltration in the central vein and edge of the liver lobule. Compared with the model group, the hepatocyte structure and morphology of the mice in the MQR different extraction parts groups were improved, especially the liver tissue of the ethyl acetate group (MQR-E) was close to the blank control group.

[0111] Example 3: Process Optimization of Meconopsis quinqueneri Total Flavonoids Extract (MQRF)

[0112] 1. Liquid absorption rate inspection

[0113] Take 100 g of Meconopsis quinquenervata (batch number: 20220108), accurately weigh it, put it in a 2000 mL beaker, measure 900 mL of ethanol, 50% ethanol, and aqueous solution respectively, seal it with plastic wrap for more than 12 hours, filter it, measure the remaining amount, and calculate the liquid absorption rate, see Table 1.

[0114] Table 1 Liquid absorption rate investigation

[0115]

[0116] The results showed that within the scope of investigation, the liquid absorption rate was above 300%. In order to improve the extraction efficiency, the present application added 3 times more solvent in the first extraction when ethanol reflux was used to extract the alcohol extract for multiple times.

[0117] 2. Investigation of extraction solvent

[0118] Take 20 g of Meconopsis quinquefolia medicinal material, a total of 12 portions, accurately weighed, add water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 90% ethanol 140 mL (the first 200 mL), heat to reflux, extract 3 times, each time for 1 hour, filter, and dilute the combined filtrate to about 400 mL.

[0119] Take the test solution and determine the amount of total flavonoids and luteolin as well as the cream yield according to the proposed method. Calculate the percentages (%) of total flavonoids, luteolin and cream yield, and the comprehensive score (%) = (percentage of total flavonoids + percentage of luteolin + percentage of cream yield) / 3.

[0120] Table 2 Extraction solvent investigation

[0121]

[0122] As shown in Table 2, the difference in the extract yield of different solvents is not significant, but the amount of total flavonoids and luteolin is quite different. 50-90% ethanol is the best, and 60-90% ethanol can be further selected. The best extraction solvent is 70% ethanol.

[0123] 3. Determination of reflux extraction process

[0124] Take 20g (9 portions) of Meconopsis quinquenervae medicinal material, add a certain amount of 70% ethanol in sequence according to Table 3, reflux extraction, filter, and combine the filtrate to 400mL. Determine the amount of total flavonoids, the amount of luteolin and the extract yield in the Meconopsis quinquenervae extract, and the results are shown in Tables 3 to 5.

[0125] Table 3 Orthogonal test factor level table

[0126]

[0127] Table 4 Results and analysis of orthogonal test

[0128]

[0129]

[0130] Table 5 Orthogonal test results and variance analysis

[0131]

[0132] Note: **P<0.01, significant difference

[0133] It can be seen from the orthogonal intuitive analysis table that the order of factors affecting the extraction process is: number of extractions > solid-liquid ratio > extraction time. Through variance analysis, the number of extractions has a significant effect on the extraction amount of total flavonoids. Based on the principles of high extraction efficiency, economy and time saving, A1B1C3 was determined as the extraction process for total flavonoids in Meconopsis quinqueneri.

[0134] 4. Reflux extraction process verification

[0135] Since the liquid absorption rate of the medicinal material is large during the extraction process, the extraction efficiency of 4 times the solvent is low, so the amount of extraction solvent is appropriately increased. Take 20g of the five-veined Meconopsis medicinal material, a total of 3 portions, add 6 times the amount of 70% ethanol (9 times the first amount), reflux extraction 3 times, each time for 1 hour, filter, and combine the filtrate to 400mL. The total flavonoids, luteolin and paste yield in the five-veined Meconopsis extract were determined, and the results are shown in Table 6.

[0136] Table 6 Verification test results

[0137]

[0138] As shown in Tables 4 and 6, the extraction efficiency of this process is higher than that of A1B1C3, and the differences in total flavonoid content and paste recovery rate are not large, indicating that the extraction process is stable and feasible. Combined with the solvent investigation and liquid absorption rate, the optimal extraction process is determined as follows: weigh the medicinal material of Meconopsis quinquenervais, add 6 times the amount of 70% ethanol (add 9 times the amount of 70% ethanol for the first time), and reflux extraction 3 times, each time for 1 hour.

[0139] 5. Research on macroporous resin purification process

[0140] (1) Preparation of crude total flavonoids extract (ethanol extract) from Meconopsis quinquefolia

[0141] Weigh 500 g of Meconopsis quinquenervais medicinal material, add 6 times the amount of 70% ethanol (9 times the amount for the first time), reflux extraction 3 times, 1 hour each time, filter, combine the filtrate, recover the ethanol, and concentrate until there is no alcohol taste to obtain the crude total flavonoids extract of Meconopsis quinquenervais.

[0142] (2) Macroporous resin pretreatment

[0143] Weigh appropriate amounts of NKA-9, D101, HP-20, DM-301, HPD100, SP207, and HP2MGL resins respectively. Before wet column loading, soak them in 95% ethanol for more than 24 hours, then rinse the resin with 95% ethanol, mix the effluent with deionized water (1:5), and when no white turbidity is produced, wash with a large amount of deionized water until there is no alcohol smell. Then soak them in 2BV 5% HCl overnight, and rinse them with a large amount of deionized water until they are neutral; soak them in 2BV 5% NaOH overnight, and rinse them with a large amount of deionized water until they are neutral, and set aside.

[0144] (3) Determination methods of adsorption amount and adsorption rate, desorption amount and desorption rate

[0145] Adsorption amount and adsorption rate:

[0146] Adsorption amount = (C0V0-C1V1) / M

[0147] Adsorption rate = (C0V0-C1V1) / C0V0×100%

[0148] Where: C1 is the mass concentration of total flavonoids in the adsorption solution (mg / mL), C0 is the mass concentration of total flavonoids in the loading solution (mg / mL), V0 is the volume of the loading solution (mL), V1 is the volume of the adsorption solution (mL), and M is the mass of the resin (g).

[0149] Desorption amount and desorption rate:

[0150] Desorption amount = C2V2 / M

[0151] Desorption rate = C2V2 / (C0V0-C1V1)×100%

[0152] Wherein: C0, V0, C1, V1, M are the same as above, C2 is the mass concentration of total flavonoids in the ethanol eluent (mg / mL), and V2 is the volume of the elution concentration.

[0153] (4) Investigation of static adsorption and desorption of resin

[0154] Take 5g of different types of macroporous resins, accurately add 50mL of the test solution, statically adsorb for 24h at 37℃ and 150rmp, filter, determine the amount of total flavonoids in the adsorbed solution, and calculate the adsorption amount and adsorption rate of total flavonoids of Meconopsis quinqueneri on each resin.

[0155] Accurately pipette 50 mL of 70% ethanol and add it to the adsorbed resin. Perform static analysis at 37°C and 150 rpm for 24 h. Filter and make the filtrate volume up to 50 mL. Determine the amount of total flavonoids of Meconopsis quinqueneri in the filtrate, and calculate the desorption amount and desorption rate of total flavonoids of Meconopsis quinqueneri by each resin.

[0156] Table 7 Resin static saturation adsorption and analytical determination results (n = 2)

[0157]

[0158] As shown in Table 7, the desorption amounts of HP2MGL, NKA-9 and DM-301 resins were all relatively low. Therefore, the remaining four resins D101, HPD100, HP-20 and SP207 were selected for further dynamic elution investigation.

[0159] (5) Dynamic elution study

[0160] Accurately pipette an appropriate amount of the total flavonoids extract of Meconopsis quinquefolia (equivalent to 0.2 g of the medicinal material) and add it to the treated HP-20, SP207, HPD100, and D101 macroporous resin columns (column volume 20 mL). After 24 hours, the adsorption equilibrium is reached. After adsorption at a flow rate of 2 BV / h, elution is performed with 3 BV of water. Then, 70% ethanol is used for 24 hours until complete decomposition is performed, and elution is performed at a flow rate of 2 BV / h. The eluate is collected to 40 mL, and the amount of total flavonoids therein is determined respectively, and the adsorption amount and adsorption rate, desorption amount and desorption rate are calculated. The results are shown in Table 8.

[0161] Table 8 Resin dynamic saturation adsorption and analytical determination results (n = 2)

[0162]

[0163] As shown in Table 8, the adsorption and desorption of HPD100 in dynamic adsorption are relatively low. Therefore, the remaining three resins D101, HP-20, and SP207 were selected to conduct dynamic elution studies of different concentrations of ethanol.

[0164] Accurately pipette an appropriate amount of total flavonoids extract from Meconopsis quinqueneri (equivalent to 0.3 g of medicinal material) and add it to the top of the treated HP-20, SP207, and D101 macroporous resin columns (column volume 20 mL). Then elute with 3 BV of water, 3 BV of 30% ethanol, and 2.5 BV of 50% to 90% ethanol in turn. After collecting the extracts, determine the amount of total flavonoids therein. The results are shown in Table 1. Figure 7 and Table 9.

[0165] Table 9 Dynamic elution investigation (n=2)

[0166]

[0167] Depend on Figure 7 As can be seen from Table 9, the amount of total flavonoids eluted by D101 resin is greater than that of SP207 and HP-20 resins, and the loss of flavonoids in the water eluate (1BV) is less than 5%. Therefore, D101 macroporous resin is the best purification filler and is beneficial to the preparation of Meconopsis quinquefolia extract.

[0168] (6) Elution concentration investigation

[0169] Accurately pipette an appropriate amount of total flavonoids extract of Meconopsis quinquefolia (equivalent to 0.3 g of medicinal material) and add it to the top of the treated D101 macroporous resin column (column volume 20 mL). The adsorption flow rate is 2 BV / h. Desorb it with different volume fractions of ethanol (water, 30%, 50%, 60%, 70%, 80%, 90%) in turn. The elution flow rate is 2 BV / h. Collect the eluents and determine the amount of total flavonoids and the amount of paste collected in the eluents. The results are shown in Figure 8 .

[0170] Table 10 Investigation of water washing amount in dynamic adsorption of resin

[0171]

[0172] Table 11 Verification of elution concentration in dynamic adsorption of resin (n=2)

[0173]

[0174] The results showed that the total flavonoids of MQR were mainly concentrated in the 30%-50% ethanol elution part ( Figure 8 ). In the verification experiment in Table 11, the elution rate of 50% ethanol reached 94.86%, so 50% ethanol was selected as the elution solution.

[0175] It can be seen from Table 10 that 1 BV of water can remove most of the impurities, and the loss of MQR total flavonoids is less than 5%. Therefore, in order to achieve a higher purity of MQR total flavonoids, it is determined that 1 BV of water can be used to remove impurities after adsorption.

[0176] (7) Investigation of sample solution concentration

[0177] Accurately pipette 3mL of total flavonoids extract of Meconopsis quinquefolia (equivalent to 6g of medicinal materials), dilute with water to 2g / mL, 1.5g / mL, 1g / mL, and 0.5g / mL, respectively, and add to the top of the treated D101 macroporous resin column (column volume is 20mL), the adsorption flow rate is 2BV / h, after standing adsorption for 12 hours, 1BV of water is used to remove impurities, and then 10BV of 50% ethanol is used to elute to 200mL, the elution flow rate is 2BV / h, the eluate is collected, and the amount of total flavonoids and the recovery rate of MQR are determined. The results are shown in Fig. 9 .

[0178] Table 12 Verification of elution concentration in dynamic adsorption of resin (n=2)

[0179]

[0180] Depend on Fig. 9 As shown in Table 12, when the concentration of the drug solution is 0.5 g / mL, the elution rate of total flavonoids is the highest, and the loss is within 5%, so the drug solution mass concentration is selected as 0.5 g (raw drug amount) / mL.

[0181] (8) Sample loading amount

[0182] The extract of Meconopsis quinquenervata was accurately pipetted and added to the top of the treated D101 macroporous resin column (column volume was 20 mL). The adsorption was carried out at a flow rate of 2 BV / h. 0.5 BV of the effluent was collected each time. The total flavonoid content in the effluent was determined and the leakage curve was drawn. The results are shown in Fig.10 .

[0183] Table 13 Sample loading amount investigation (n=2)

[0184]

[0185]

[0186] The results show that when the sample volume exceeds 2BV, the total amount of flavonoids leaked increases sharply, and the leakage ratio reaches about 3%. In order to reduce the loss of total flavonoids in production, the sample volume is selected as 2BV, and the weight of medicinal materials (kg): wet resin volume (L) is calculated to be 1:1.

[0187] (9) Investigation of adsorption flow rate

[0188] Accurately pipette 3 portions of 2BV of Meconopsis quinqueneri extract (0.5 g / mL) and add them to the top of the treated D101 macroporous resin column (column volume is 20 mL), respectively, with adsorption flow rates of 1, 2, and 3BV / h, respectively, and elute with 1BV of water to remove impurities, and elute with 10BV of 50% ethanol, with an elution flow rate of 2BV / h, collect the eluate, and determine the amount of total flavonoids in MQR. The results are shown in Fig.11 .

[0189] Depend on Fig.11 It can be seen that when the adsorption rate is 2-3 BV / h, the elution rate decreases significantly, and when the adsorption rate is 1 BV / h, the amount of MQR total flavonoids in the eluate is the highest, so 1 BV / h is selected as the adsorption rate.

[0190] (10) Investigation of elution flow rate

[0191] Three portions of Meconopsis quinquenervata extract (0.5 g / mL) were accurately transferred and added to the top of the treated D101 macroporous resin column (column volume was 20 mL). The adsorption flow rate was 1 BV / h. 1 BV of water was used to remove impurities. The elution flow rates were 1, 2, and 3 BV / h, respectively. 50% ethanol eluate was collected to 10 BV. The amount of total flavonoids in MQR was determined. The results are shown in Fig.12 .

[0192] Depend on Fig.12 It can be seen that when the elution rate is 2-3BV / h, the elution rate decreases too fast and the total flavonoids cannot be completely eluted. The enrichment capacity decreases with the increase of the elution rate, and a too slow elution rate will affect the work efficiency. It can be seen from the figure that the elution rate is not much different at 1BV / h and 2BV / h elution flow rates, so 2BV / h is selected as the optimal elution flow rate.

[0193] (11) Elution endpoint investigation

[0194] Accurately pipette 2BV of MQR extract and add it to the top of the treated D101 macroporous resin column (column volume is 20mL), adsorb at a flow rate of 1BV / h, elute with 1BV of water to remove impurities, elute with 50% ethanol, the elution flow rate is 2BV / h, collect once for each column volume, and determine the amount of total flavonoids in each flow fraction. The results are shown in Fig.13 .

[0195] Depend on Fig.13 It can be seen that when the elution dosage is 3BV, a large amount of flavonoids are eluted; when the elution dosage is 8BV, the total flavonoids have been basically eluted completely, so 8BV is selected as the minimum elution dosage of MQR total flavonoids.

[0196] (12) Verification experiment

[0197] Accurately transfer 3 portions of 2BV of MQR extract (0.5g / mL) and add them to the top of the treated D101 macroporous resin column (column volume is 20mL), adsorb at a flow rate of 1BV / h, remove impurities with 1BV of water, elute with 50% ethanol, the elution flow rate is 2BV / h, collect the eluate to 200mL, determine the amount of total flavonoids and the recovery rate, calculate the purity and yield of total flavonoids, and the results are shown in Table 14.

[0198] Purification of Total Flavonoids from MQR by Macroporous Resin D101 (n = 3)

[0199]

[0200] As can be seen from the table, compared with the extract before column loading, the purity of total flavonoids from Meconopsis quintuplinervia increased from 7.35% to 15.96%, and the recovery rate of total flavonoids reached 93.13%, indicating that this process has a high enrichment and purification efficiency.

[0201] Example 4: Effect of Total Flavonoid Extract from Meconopsis quintuplinervia (MQRF) on Mice with Alcoholic Liver Injury

[0202] 1. Effect on Acute Alcoholic Liver Injury

[0203] Take an appropriate amount of MQR medicinal materials (about 1 cm in length), reflux extract with 6 times the amount of 70% ethanol for 3 times, 1 h each time, filter, combine the filtrates, concentrate until the alcohol smell disappears, and dilute with water to a sample solution of 0.5 g (raw medicinal materials) / mL, that is, obtain the crude total flavonoid extract of MQR.

[0204] Take 2 BV (0.5 g / mL) of the crude total flavonoid extract of MQR, add it to the top of the treated D101 macroporous resin column, adsorb at a flow rate of 1 BV / h, then statically adsorb for 12 hours, wash with 1 BV of water to remove impurities, and then statically desorb with 50% ethanol for more than 12 hours, elute at a flow rate of 2 BV / h, collect the eluate until 8 BV, concentrate until the alcohol smell disappears, and dilute with water to a sample solution of 0.234 g (raw medicinal materials) / mL, that is, obtain the MQRF extract.

[0205] (1) Experimental Grouping and Treatment

[0206] Thirty-six C57BL / 6J mice, weighing 18 g - 22 g, SPF grade, male (SYXK(Yu)2022 - 0006), were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0207] After 7 days of adaptive feeding, the 36 mice were randomly divided into 3 groups: 10 in the blank group, 12 in the MQRF group, and 14 in the model group. The sample group was given the MQRF extract every day (2.34 g / kg×BW×d -1 ); the blank group and the model group were given an equal volume of distilled water.

[0208] According to the above dosage, the administration was continued for 7 days. From the 8th day, except for the blank group which was given distilled water, the other groups were given commercially available liquor with an alcohol content of 52% by gavage (10 mL / kg×BW×d -1 , maintained at this concentration until the model was successfully established), once a day, and then acute alcoholic liver injury was induced continuously for 2 weeks.

[0209] (2) Results

[0210] Natural medicines take effect slowly. In order to better observe the preventive and protective effects of the total flavonoids of Meconopsis quinquefolia on acute alcoholic liver injury, the mice were first given preventive medication for one week. During this period, the weight of the mice did not change significantly (the weight of the first three times). After that, a high dose (10 mL / kg × BW × d -1 ) were intragastrically administered with 52% high-proof liquor to induce acute alcoholic liver injury. During the modeling experiment, it was clearly observed that the weight of the mice in the alcohol group decreased significantly (weighed 3 to 5 times), but there was a trend of slow recovery in the subsequent experiments ( Fig.14 -A). From the liver index chart ( Fig.14 -B) It can be seen that the liver weight of mice in the model group increased significantly compared with the blank group (p<0.001), but after drug administration, it decreased significantly (p<0.01). HE staining showed ( Fig.14 -C), compared with the blank group, the liver cells of the mice in the model group showed edema, enlarged vacuoles, lipid droplets, etc., and the portal vein area was infiltrated by inflammatory cells. After administration of MQRF extract, the pathological conditions were improved, but a small number of inflammatory cells still existed.

[0211] ALT and AST are biochemical indicators that reflect liver damage, while TG and TC can evaluate the lipid level of liver tissue. Fig.15 As shown in the results, compared with the normal control group, the levels of ALT, AST, TG and TC in the model group were significantly different (p<0.05), indicating that the mice had obvious liver damage. Compared with the model group, the serum transaminase levels of mice with acute alcoholic liver injury were significantly reduced after administration of MQRF extract (p<0.01). This shows that MQRF has a certain protective effect on ethanol-induced liver damage. In addition, compared with the EtOH group, the TG and TC levels of liver tissue in the MQRF group were significantly decreased (p<0.05), indicating that MQRF can improve fatty degeneration caused by acute alcoholic liver injury. In summary, MQRF has a certain preventive and protective effect on acute alcoholic liver injury.

[0212] 2. Effects on biochemical indicators and histopathology of chronic alcoholic liver injury

[0213] Take an appropriate amount of MQR medicinal material (about 1 cm in length), add 6 times the amount of 70% ethanol (add 9 times the amount of 70% ethanol for the first time), reflux extraction 3 times, 1 hour each time, filter, combine the filtrate, concentrate until there is no alcohol taste, and dilute with water to obtain the MQR total flavonoids extract with a concentration equivalent to 0.5 g / mL of the original medicinal material.

[0214] Take 2 BV (0.5 g / mL) of MQR extraction solution, add it to the top of the D101 macroporous resin column, let it stand for adsorption for more than 12 hours, adsorb it at a flow rate of 1 BV / h, wash away impurities with 1 BV of water, then desorb it with 50% ethanol by standing for more than 12 hours, elute it at a flow rate of 2 BV / h for 8 BV, collect the eluate, concentrate it until there is no alcohol smell, and dilute it with water to a sample solution with a concentration equivalent to 0.468 g / mL of the crude drug, thus obtaining the MQRF extraction solution.

[0215] (1) Experimental grouping and treatment

[0216] Ninety C57BL / 6J mice, weighing 18 g - 22 g, SPF grade, male (SYXK(Yu)2022 - 0006), provided by the Experimental Animal Center of Chongqing Academy of Chinese Materia Medica and purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. The breeding environment temperature is 25°C ± 2°C, and the relative humidity is 60% ± 10%. They are fed freely with food and water. After 7 days of adaptive feeding, the 90 mice are randomly divided into six groups, with 15 mice in each group (20 mice in the model group).

[0217] The sample groups are divided into low, medium, and high-dose groups of total flavonoids from Meconopsis quintuplinervia, and different concentrations of MQRF extraction solution with the same dose are given every day; the positive control group is given silybin (0.1 g / kg×BW×d-1); the blank group and the model group are given the same volume of distilled water. The third volume of "Tibetan Medicine in China" records that the recommended clinical dosage of MQR is 6 - 9 grams. Calculated based on the body surface area ratio of mice to humans (1∶0.0026), the dosage of mice in the total flavonoids from Meconopsis quintuplinervia dose groups is 1.17 g / kg, 2.34 g / kg, and 4.68 g / kg respectively.

[0218] According to the above dosage, administer the drugs continuously for 4 weeks. At the same time, in the afternoon during the experiment, except for the control group given distilled water, other groups are given commercially available liquor with an alcohol content of 52% (2 - 6 mL / kg×BW in the first week, increasing by 2 mL / kg every week thereafter, and finally maintaining at 10 mL / kg×BW until the model is successfully established), once a day, and then induce chronic alcoholic liver injury for 4 consecutive weeks. After the last drug administration, fast for 12 hours, draw blood from the eyeballs, and take the liver.

[0219] (2) Results

[0220] During the experiment, compared with the blank group, the body weight of the alcohol-fed group first decreased significantly, and then gradually recovered. In the end, there was no significant difference between the final body weight and the initial body weight of the six groups, which indicated that the total energy intake was equivalent. Body weight was measured 5 times in the first two weeks, and except for the blank group, the weight loss of the other groups was more obvious. This shows that alcohol intervention has a certain impact on the daily food intake of mice. However, in the later stage of the experiment, the weight of the mice gradually recovered, and there was no significant difference between the groups. This also shows that the way of alcohol intake is relatively reasonable, and it does not affect the energy intake and metabolism of mice overall ( Fig.16 -A). In addition, the liver index of the model group was significantly different from that of the blank group (p<0.001). However, after drug treatment, there was a downward trend, and the difference was statistically significant (p<0.01). Among them, the effects of the medium and high doses of total flavonoid extracts of Meconopsis quinquefolia were particularly obvious (p<0.001). Fig.16 -B.

[0221] In order to evaluate the effects of total flavonoids from Meconopsis quinqueneri on serum biochemical parameters of alcoholic liver injury, the levels of ALT, AST, and ADH were measured. Fig.17 As shown in the results, the serum transaminase levels after 52% alcohol treatment were significantly increased, which was significantly alleviated after Silymarin and MQRF administration (p<0.05). In particular, the serum transaminase levels of the MQRF-H and MQRF-M treatment groups were very close to those of the blank group. In addition, compared with the model group, the ADH level showed a significant downward trend after MQRF administration, and the differences between the other two groups were statistically significant except for the MQRF-L group (p<0.05). Moreover, the serum ADH level of MQRF-H mice has fallen back to be close to that of the positive control group. In summary, the levels of ALT, AST and ADH in the low, medium and high doses of Meconopsis quinqueneri total flavonoids extract groups (MQRF-L, MQRF-M, MQRF-H) were dose-dependently reduced compared with the EtOH group. The results showed that MQRF can improve ethanol-induced liver damage in mice, and the effects of the MQRF-M and MQRF-H treatment groups were particularly obvious.

[0222] Excessive intake of ethanol will hinder the tricarboxylic acid cycle and fatty acid oxidation, affect lipid metabolism, and cause a large amount of triglyceride (TG) and cholesterol (TC) to accumulate in the liver. Acetyl-CoA carboxylase (ACC) is a rate-limiting enzyme that catalyzes the first step of fatty acid synthesis and metabolism and plays an important role in lipid metabolism. Fig.18As shown in the results, compared with the blank group, the TG and TC levels in the alcoholic liver injury model group (EtOH) were significantly increased (p<0.01). However, after treatment with low, medium, and high doses of MQRF, the levels of TG and TC were reduced in a dose-dependent manner. In addition, compared with the model group, the ACC level in the treatment group was significantly reduced (p<0.001) and was lower than that in the blank group. In summary, MQRF can improve hepatocyte fatty degeneration and lipid metabolism disorders caused by alcoholic liver injury.

[0223] To explore the intervention mechanism of Meconopsis quinqueneri total flavonoids extract (MQRF) on ethanol-induced liver injury, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA) were used as indicators to evaluate the oxidative stress level in the liver. Fig.19 As shown in the results, there were significant differences in SOD, MDA, and GSH-PX values ​​between the model group and the blank group (p<0.001). Among them, the SOD activity of liver tissue in the low, medium, and high dose groups of MQRF was significantly increased compared with the model group (p<0.05). Compared with the model group, the GSH-PX activity of liver tissue increased after MQRF administration. Except for the MQRF-L group, the GSH-PX activity of the medium and high dose groups showed a significant or extremely significant increase (p<0.05 or p<0.01). In addition, compared with the blank group, the MDA level of the model group of alcoholic liver injury was significantly increased (p<0.001), but these changes were reversed after MQRF treatment and decreased in a dose-dependent manner. These results show that MQRF has a protective effect on oxidative damage of liver tissue, among which MQRF-M and MQRF-H have obvious effects.

[0224] Oxidative stress is closely related to the occurrence and development of inflammation. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) in mice were detected to analyze whether MQRF could improve the inflammatory response of the liver. Enzyme-linked immunosorbent assay (ELISA) showed that the levels of TNF-α and IL-6 in the liver tissue of the model group were significantly increased compared with the blank group (p<0.001)( Fig. 20 -A, B). Compared with the model group, the levels of TNF-α and IL-6 in the drug-treated group were significantly decreased (p<0.001). Fig. 20 -C shows that compared with the blank group, the NO content in the model group was significantly increased (p<0.05). However, the difference in NO content in the liver of mice in the low, medium and high doses of MQRF groups and the model group was also statistically significant (p<0.05). This shows that MQRF can improve the inflammatory response of alcoholic liver injury in mice.

[0225] like Fig.21-A, the liver in the blank group was soft, bright red, and had sharp edges. In contrast, the liver in the model group was fragile, swollen, rough, dull, and light red. After administration of MQRF or silybin, the swelling was reduced; however, fibrous fissures were still visible on the surface of the liver tissue in the MQRF-L group. After treatment with MQRF-M or MQRF-H, the degree of liver lesions was significantly improved compared with the model group, almost approaching that of the blank group.

[0226] H&E analysis showed ( Fig.21 -B), the blank group had clear liver lobule structure, hepatic cords radiated outward from the central vein and were arranged neatly, the hepatocytes were normal in shape, polygonal, and the nuclei were round. The liver tissue was evenly stained, and no obvious abnormalities were found in the tissue. However, the hepatocytes in the model group showed typical liver injury feathers, with disordered cell arrangement, edema, enlarged vacuoles, and infiltration of inflammatory cells in the portal vein area. After treatment with silybin or MQRF, the pathological damage of liver tissue caused by alcohol was significantly improved. Among them, the MQRF-L group had improved pathological conditions such as blurred intercellular space, edema and necrosis, and a small number of inflammatory cells were seen in the whole liver; the edema, ballooning and other pathological changes of hepatocytes in the MQRF-M group were significantly improved, but there were still a small number of inflammatory cells infiltrating; H&E staining in the MQRF-H group showed no obvious pathological changes in hepatocytes, which was close to the blank group.

[0227] In addition, Oil Red O staining showed that compared with the blank group, ethanol induced the formation of intrahepatic lipid droplets; however, compared with the model group, the lipid droplet content in the MQRF group and silybin group was less, close to that in the blank group, indicating that MQRF can alleviate alcohol-induced hepatic fatty degeneration. Fig.21 -C).

[0228] Example 5: Preparation of Meconopsis quinquefolia extract capsules

[0229] Investigation of filler excipients

[0230] According to the selection of commonly used auxiliary materials such as dextrin, corn starch and microcrystalline cellulose as fillers, 23g of Meconopsis quinquenervais extract powder was weighed respectively, a total of 4 parts, 4g of different types of auxiliary materials were added, water was added as a wetting agent, granulated through a 24-mesh sieve, dried, sieved through a 24-mesh sieve, 0.3% of magnesium stearate was added for lubrication, and mixed to obtain the product.

[0231] Table 15 Results of samples prepared with different excipients

[0232]

[0233] The results show that when no auxiliary materials are added, the material itself is very sticky and difficult to granulate; the angle of repose of the particles made by the other auxiliary materials is: corn starch < dextrin < microcrystalline cellulose, and the angle of repose of the particles made by each auxiliary material is less than 30°, with corn starch being the best, and the fluidity is improved; bulk density: microcrystalline cellulose < dextrin < corn starch. The bulk density of the particles made by corn starch is higher and more economical, so corn starch is selected as the filler auxiliary material.

[0234] Capsule preparation

[0235] Weigh 23 g of Meconopsis quinqueneri extract powder, add 2 g of corn starch and 1 mL of water, prepare a soft material according to the standard of "forming a ball when held in the hand and falling apart when touched", sieve through a 24-mesh sieve for granulation, dry, sieve through a 24-mesh sieve, add magnesium stearate to obtain 0.3% of the total weight, mix well, and obtain the product.

[0236] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for preparing a Meconopsis quinqueneri extract, characterized in that: include: S1. Extracting the medicinal material of Meconopsis quinquenervata with ethanol to obtain an ethanol extract; S2, extracting the alcohol extract with petroleum ether, separating the aqueous layer and extracting with ethyl acetate, and removing the ethyl acetate to obtain the Meconopsis quinqueneri extract; or The alcohol extract is loaded onto D101 macroporous resin for adsorption, left to stand for elution and impurities are removed, and then eluted with ethanol solution, the eluate is collected, and concentrated until there is no alcohol taste, so as to obtain the Meconopsis quinquenervata extract.

2. The preparation method according to claim 1, characterized in that: Step S1 includes: Weigh the medicinal material of Meconopsis quinquenervais, add ethanol for reflux extraction, concentrate until there is no ethanol, and dilute with water to obtain an alcohol extract.

3. The preparation method according to claim 2, characterized in that: The amount of ethanol used is 5-9 times the amount of the Meconopsis quinquenervata medicinal material.

4. The preparation method according to claim 2 or 3, characterized in that: The concentration of the ethanol is 50-95%.

5. The preparation method according to claim 1, characterized in that: The loading amount of the alcohol extract is 2 BV based on the crude drug concentration of 0.5 g / mL, and the adsorption flow rate is 1 BV / h.

6. The preparation method according to claim 1, characterized in that: The elution was performed with 50% ethanol solution at a flow rate of 1-2 BV / h, and at least 8 BV of the eluate was collected.

7. The Meconopsis quinquenervais extract prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the Meconopsis quinquenervais extract according to claim 7 in the preparation of a product for improving alcoholic liver damage.

9. A product for improving alcoholic liver damage, characterized in that: The invention comprises the Meconopsis quinqueneri extract as claimed in claim 7.

10. The product according to claim 9, characterized in that The product also includes auxiliary materials, which include one or more of fillers and lubricants.

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