CN112353764A

CN112353764A - Bozuri hot granules and extraction method and application thereof

Info

Publication number: CN112353764A
Application number: CN202010541020.5A
Authority: CN
Inventors: 尹强; 尹海龙; 周琴; 穆丹丹; 刘秋云; 刘子甲; 蒋子鸿; 朱姝; 邓新新; 姜大军; 张鹏
Original assignee: Xinjiang Uygur Pharmaceutical Co ltd
Current assignee: Xinjiang Uygur Pharmaceutical Co ltd
Priority date: 2020-06-15
Filing date: 2020-06-15
Publication date: 2021-02-12

Abstract

The invention discloses a budesonide hot granule and an extraction method and application thereof. The extraction method of the Bozu hot granules comprises the following steps: extracting semen cuscutae, celery root and chicory root in raw materials of the Buzure granules by using 60-75% of ethanol by mass fraction, and filtering to obtain an extract A; and (2) combining the extract A with an extract B of celery seed, chicory seed, fennel root bark and fennel in the raw materials of the bouzura hot granules. The Bozuri granules obtained by the extraction method of the invention have better curative effect on treating liver injury, such as immunological liver injury.

Description

Bozuri hot granules and extraction method and application thereof

Technical Field

The invention relates to a budesonide hot granule, an extraction method and application thereof.

Background

The liver is an important organ of substance metabolism and has various physiological functions such as secretion, excretion, synthesis, biotransformation, immunity and the like. When the intestinal mucosa barrier is destroyed and exogenous toxin invades, the liver biotransformation function is weakened, and the liver is seriously damaged. Immune Liver Injury (ILI) is a group of chronic immune Liver diseases mediated by abnormal immunity and with similar clinical manifestations, is commonly seen in diseases such as viral hepatitis and autoimmune hepatitis, and is an important cause of Liver failure and Liver cirrhosis. In recent years, the detection rate of immunological liver injury gradually increases, accounting for 10-20% of chronic hepatitis worldwide, and seriously harms human health.

The main pathogenesis of immunological liver injury is that an organism is induced by environmental factors, medicines or infection factors under the action of susceptibility genes, and the liver injury is caused by the generation of a liver cell membrane self-target antigen, the immune function disorder of the organism and the reduction of the immunological tolerance of the liver. At present, the drugs for treating immunological liver injury mainly comprise interferon, thymosin, Th cell factor and vaccine, wherein the vaccine is most widely applied, but the drugs have certain problems in treating immunological liver injury. Specifically, some populations do not produce antibodies after vaccination, resulting in being at risk of disease infection soon after vaccination and not self-known. Interferon, thymosin and nucleoside medicaments all belong to antiviral medicaments, and the medicaments have high-efficiency and convenient induction, but have the problems of easy generation of virus resistance after long-term administration, easy relapse after drug withdrawal and the like.

The liver-protecting Buzure granules are traditional vitamins, and the celery seeds in the formula are dry and hot, so that cold-dampness obstruction can be eliminated, stones can be eliminated, pain can be relieved, and a plurality of medicines can be blended; chicory is cold in nature, has the effects of clearing liver obstruction, clearing heat, diminishing inflammation, eliminating jaundice, promoting urination and relieving swelling, and is mainly used for treating liver obstruction, damp-heat hepatitis, icteric hepatitis, systemic edema and the like; heating semen Cuscutae; the celery root is dry and hot; the fennel root bark is dry and hot in nature, and can dissipate cold, warm the kidney and the stomach, circulate water and promote diuresis, and relieve swelling and pain; chicory root has the characteristics of dampness and cold, generates dampness and cold in function, regulates abnormal bile quality, reduces liver fire, clears stomach heat, removes jaundice, promotes urination and reduces edema, and is mainly used for treating dry heat or bile liquid diseases; fennel fruit can dispel cold and alleviate pain. The whole formula is precise in compatibility and small in liver burden.

The heat-clearing particles of the prior art adopt a water extraction mode, and have poor curative effect on immunological liver injury. In the new extraction process of the liver-protecting Buzure granules reported by Wutao et al and the research on the protection effect of the liver-protecting Buzure granules on the liver injury of mice, the new extraction process is adopted to improve the curative effect of the Buzure granules on the treatment of the liver injury, but the new extraction process is a preparation process of an inclusion compound added on the basis of the extraction process. Therefore, an extraction process of the Bozuri granules is lacked at present, and the curative effect on liver injury can be improved on the basis of the original extraction process.

Disclosure of Invention

The invention aims to overcome the defect that the extract obtained by adopting the conventional extraction process of the bushy fever granules in the prior art has poor curative effect on treating liver injury, and provides the bushy fever granules, the extraction method and the application thereof. The Bozuri granules obtained by the extraction method of the invention have better curative effect on treating liver injury, such as immunological liver injury.

The invention solves the technical problems through the following technical scheme.

The invention provides an extraction method of a Bozu hot particle, which comprises the following steps: the raw materials of the bushy damp-fever granules comprise semen cuscutae, celery root, chicory root, celery seed, chicory seed, fennel root bark and fennel, and the extraction method of the bushy damp-fever granules comprises the following steps:

extracting semen cuscutae, celery root and chicory root in raw materials of the bushy-giraldii root granules by using 60-75% of ethanol in mass fraction, and filtering to obtain an extract A;

and (2) combining the extract A with an extract B of celery seeds, chicory seeds, fennel root barks and fennel fruits in the raw materials of the bouzura hot granules.

In the present invention, those skilled in the art know that "dodder seed, celery root and chicory root" described in the step (1) and "celery seed, chicory seed, fennel root bark and fennel" described in the step (2) are selected in proportion to the raw material formulation of the bouzura thermal granule. The raw materials of the Bozushi granules generally comprise the following components in parts by mass: 50-150 parts of celery seeds, 150-250 parts of chicory seeds, 20-100 parts of semen cuscutae, 150-250 parts of celery roots, 150-250 parts of fennel root skins, 50-150 parts of chicory roots and 50-150 parts of fennel. In a preferred embodiment of the invention, the formula of the raw materials of the boughs hot granules comprises the following components in parts by mass: 106 parts of celery seed, 212 parts of chicory seed, 53 parts of semen cuscutae, 212 parts of celery root, 212 parts of fennel root bark, 106 parts of chicory root and 106 parts of fennel.

In the step (1), the mass ratio of the mass of the 60-75% ethanol to the total mass of the dodder, the celery root and the chicory root can be (3-6): 1, e.g. 4: 1.

in the step (1), the temperature of the extraction may be, for example, 80 to 100 ℃.

In step (1), the number of times of extraction may be a number of times of extraction conventional in the art, for example, 2 or more, specifically, for example, 3 times.

In step (1), the total time for extraction is preferably 3 to 6 hours, more preferably 4 to 5 hours, for example 4.5 hours.

In the step (1), the time for each extraction can be, for example, 1-2.5 h, for example, 1.5h or 2 h.

When the number of the extractions in the step (1) is 3, it is known to those skilled in the art that the extract a may be a total of filtrates obtained by filtering after each of the extractions.

In the step (1), when the number of times of the extraction is 3, the mass ratio of the first extraction may be, for example, 6: the mass ratio of the second extraction may be, for example, 4: the mass ratio of the third extraction may be, for example, 3:1, wherein the mass ratio refers to the mass ratio of the mass of the 60-75% ethanol to the total mass of the semen cuscutae, the celery root and the chicory root.

In the step (1), when the number of times of the extraction is 3, the time for the first extraction may be, for example, 2h, the time for the second extraction may be, for example, 1.5h, and the time for the third extraction may be, for example, 1 h.

In the present invention, the extract a may be a concentrate according to the extraction method.

In the step (2), the preparation process of the celery seed, the chicory seed, the fennel root bark and the fennel extract can be conventional in the field, and the preparation process can comprise the steps of sequentially extracting a mixture of the celery seed, the chicory seed, the fennel root bark and the fennel in the raw materials of the budesonant granules by water and filtering.

In step (2), the water extraction may be generally a decoction method.

In the step (2), the temperature of the water extraction can be the extraction temperature conventional in the art, and for example, can be 80-100 ℃.

In step (2), the number of times of water extraction may be conventional in the art, and may be generally 2 or more, for example 3 times.

In the step (2), the mass ratio of the mass of the water in the water extraction to the total mass of the celery seed, the chicory seed, the fennel root bark and the fennel can be a conventional mass ratio in the field, and generally can be (3-6): 1, e.g. 4: 1.

in step (2), the total time of the water extraction can be the total time of water extraction conventional in the art, and generally can be 4-5 h, for example 4.5 h.

In step (2), the time for each water extraction may be the time for water extraction conventional in the art, and generally may be 1-2.5 h, for example 1.5h or 2 h.

In step (2), when the number of times of the water extraction is 3, it is known to those skilled in the art that the extract B may be the sum of filtrates obtained by filtering after each of the water extractions. The extract B refers to the extracts of celery seed, chicory seed, fennel root bark and fennel in the raw materials of the budesonide granules.

In the step (2), when the number of times of the water extraction is 3, the mass ratio of the water extracted for the first time is, for example, 6:1, the mass multiple of the water extracted for the second time is, for example, 4:1, and the mass ratio of the water extracted for the third time is, for example, 3:1, wherein the mass ratio is the mass ratio of the water in the water extraction to the total mass of the celery seeds, the chicory seeds, the fennel root bark and the fennel.

In the step (2), when the number of times of the water extractions is 3, the time for the first water extraction is, for example, 2h, the time for the second water extraction is, for example, 1.5h, and the time for the third water extraction is, for example, 1 h.

In the step (2), the extracts of celery seed, chicory seed, fennel root bark and fennel can be concentrates. The concentrate can be prepared as is conventional in the art by distilling the filtrate from the filtration under reduced pressure to remove the solvent.

The operations after combining said extract a and said "celery seed, chicory seed, fennel root bark and fennel" extract B in step (2) may be conventional in the art, for example by also performing a concentration operation.

Wherein the concentration can be realized by concentrating the mixed extract of the extract A and the extract B of the celery seed, the chicory seed, the fennel root bark and the fennel to a medicine extract with the relative density of 1.32-1.35 at 50 ℃.

Wherein the operation after the concentration is carried out according to the reasonable selection of the required dosage form of the bushy granules. The dosage form can be a dosage form which is conventional in the field, and can be granular dosage forms, injections, tablets, syrups, capsules, pills, oral liquids, aerosols or sprays. Thus, different dosage forms can be adopted conveniently according to different administration objects. For example, tablets, granules, syrups, capsules and oral liquids or pills can be used for facilitating administration, and the dosage form of the drug can be adjusted according to the absorption part of the drug and the release requirement of the drug, so that the bioavailability of the drug is improved, and the release time of the drug is prolonged. For severe patients, injection can be adopted, so that the requirement of large-dose administration is met, and the influence of gastrointestinal circulation on the effective ingredients of the medicine is avoided.

When it is desired to prepare a granular dosage form, the concentration is generally followed by granulation and drying. Wherein, the granulation can be prepared by mixing the concentrated medicine extract with auxiliary materials. The auxiliary materials can be sucrose and dextrin, and can also be dextrin, lactose and xylitol. When the auxiliary materials are sucrose and dextrin, the mass ratio of the medicine extract to the sucrose to the dextrin is 1:4: 1. When the auxiliary materials are dextrin, lactose and xylitol, the mass ratio of the medicine extract to the dextrin to the lactose to the xylitol is 1:3:0.5: 0.5.

Through a plurality of experiments, the inventor of the application finds that the new brezopter particles obtained by extracting specific components in the raw materials of the brezopter particles by 60-75% of ethanol and then mixing the extracted components with the rest raw materials by a water extraction process can realize better curative effect on treating liver injury compared with the original process, for example, liver injury induced by Con A. In the experimental process, the applicant tries to extract one, two or three raw material formulas different from the formula of the application by adopting ethanol, so that the treatment effect of the obtained budesonide hot granules is poor.

The invention provides a bushy fever granule prepared by the extraction method.

The invention also provides application of the prepared Buzure granules in preparation of medicines for treating liver injury.

In the present invention, the liver injury is preferably immunological liver injury.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the Bozuri granules obtained by the extraction method of the invention have better curative effect on treating liver injury, such as immunological liver injury. For example, the survival rate of the brazotherm granules extracted by the invention on the lung injury of mice induced by Con A is higher than that of the prior conventional extraction process; can obviously reduce the content of ALT (alanine aminotransferase), AST (aspartate aminotransferase) and TBIL (total bilirubin) in serum; can obviously reduce the mRNA expression level of IL-1 beta, TNF-alpha, IFN-gamma and IL-17A in the liver and increase the IL-4 and TGF-beta levels; and simultaneously, the relative expression intensity of the ROR-gamma t protein can be reduced.

Drawings

FIG. 1 is a graph of survival rates of five groups of mice following 30mg/mL Con A injection.

FIG. 2 shows the serum concentrations of ALT, AST and TBIL in five groups of mice. In fig. 2B, AST concentration, TBIL concentration, and ALT concentration are shown in fig. 2C and TBIL concentration are shown in fig. 2D.

FIG. 3 is a light-microscopic image (magnification X200) of hematoxylin-eosin staining of liver tissues of five groups of mice.

FIG. 4 shows the qPCR method for detecting the expression levels of mRNA of IL-1 beta, TNF-alpha, IFN-gamma, IL-4, IL-17A and TGF-beta in five groups of mice. Wherein, FIG. 4A is the mRNA expression level of IL-1 β; FIG. 4B is the mRNA expression level of TNF- α; FIG. 4C shows IFN-. gamma.mRNA expression levels; FIG. 4D is the mRNA expression level of IL-4; FIG. 4E is the mRNA expression level of IL-17A; FIG. 4F is the mRNA expression level of TGF-. beta.s.

FIG. 5 shows immunohistochemical staining (magnification X200) to detect expression of T-beta, GATA3 and Foxp3 in liver tissue from five groups of mice obtained at 4 h. Wherein, FIG. 5G is the expression of T-beta; FIG. 5H is the expression of GATA 3; FIG. 5I is the expression of Foxp 3.

FIG. 6 shows the expression level of ROR-gamma t protein detected by Western blotting in liver tissues of five groups of mice obtained from 4 h. Wherein, FIG. 6J is the expression level of ROR-gamma t protein in mouse liver tissue; FIG. 6K shows the relative expression intensity of ROR-gamma t protein in mouse liver tissue.

FIG. 7 is a bar graph comparing the expression levels of IL-6 and IFN- β mRNA in five groups of mice measured by qPCR. Wherein, FIG. 7A is a histogram of mRNA expression levels of IL-6; FIG. 7B is a bar graph comparing IFN- β mRNA expression levels.

FIG. 8 shows the Western blotting method to detect the expression level of JAK1 and p-JAK1 proteins in the livers of five groups of mice. Wherein, FIG. 8C shows the expression level of JAK1, p-JAK1 protein in mouse liver; FIG. 8D shows the relative expression intensity of JAK1, p-JAK1 protein in mouse liver.

FIG. 9 shows the expression of p-STAT1, p-STAT3 and IRF1 in liver tissues of five groups of mice obtained at 4h by immunohistochemical staining detection. Wherein FIG. 9E is expression of p-STAT 1; FIG. 9F is expression of p-STAT 3; FIG. 9G is the expression of IRF 1.

FIG. 10 shows the concentrations of MDA and SOD in the livers of five groups of mice at different time points. Wherein, fig. 10A is the concentration of MDA in mouse liver; FIG. 10B shows the concentration of SOD in mouse liver.

FIG. 11 shows the levels of ROS in the livers of five groups of mice at each time point for ROS fluorescence staining.

FIG. 12 shows the inhibition of ROS-mediated JNK activation by HBG and the induction of hepatocyte apoptosis by HBG detected by different detection methods. Wherein, FIG. 12D shows immunohistochemical staining detection of p-JNK expression in liver tissue of mice obtained 4 h; FIG. 12E shows the level of expression of p-ERK1/2, p-p38, and clean-caspase 3 protein in mouse liver detected by Western blotting; FIG. 12F shows TUNEL staining (magnification X200) showing apoptosis of liver cells of five groups of mice 4h after Con A injection.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The main material sources used in the following examples are as follows: male BALB/C mice 180 were purchased from Hubei province disease prevention and control center only; prednisolone is available from Shanghai-derived leaf Biotech, Inc.; canavarin A was purchased from Sigma-Aldrich; ALT, AST, TBIL, MDA, SOD and other kits are purchased from Nanjing institute of bioengineering.

Example 1

1. The formula of the Buzu hot particle raw material comprises the following components in parts by mass: 106 parts of celery seed, 212 parts of chicory seed, 53 parts of semen cuscutae, 212 parts of celery root, 212 parts of fennel root bark, 106 parts of chicory root and 106 parts of fennel.

2. The extraction process comprises

Step (1): weighing the celery seeds, the chicory seeds, the fennel root barks and the fennel according to the formula, and putting the celery seeds, the chicory seeds, the fennel root barks and the fennel into an extraction tank to obtain a mixture B. Adding 6 times of water of the total mass of the mixture B for decoction for 1 time, wherein the temperature for the decoction for 1 time is 80-100 ℃, the time for the decoction for 1 time is 2 hours, filtering after the decoction for 1 time to obtain 1 time of filtrate and 1 time of filter residue, then adding 4 times of water of the total mass of the mixture B into the filter residue for 1 time for decoction for 2 times, wherein the temperature for the decoction for 2 times is 80-100 ℃, the time for the decoction for 2 times is 1.5 hours, filtering after the decoction for 2 times to obtain 2 times of filtrate and 2 times of filter residue, then adding 3 times of water of the total mass of the mixture B into the filter residue for 2 times for decoction for 3 times, the temperature for 3 times for shearing is 80-100 ℃, the time for 3 times for boiling is 1 hour, and filtering after the decoction for 3 times to obtain 3 times of filtrate and 3 times of.

Step (2): weighing semen Cuscutae, Apii radix and Cichorium intybus root according to the above formula, and placing into an extraction tank to obtain mixture A. Adding 70% ethanol for decocting for 3 times, wherein the decocting time for 3 times is 2h, 1.5h and 1h, the adding amount of 70% ethanol for 3 times is 6 times, 4 times and 3 times of the total weight of semen Cuscutae, Apii radix and Cichorium intybus root, and the rest conditions are the same as in step (1).

And (3): and (3) combining the filtrates obtained in the step (1) for 1-3 times to obtain a mixed filtrate, and concentrating the mixed filtrate under reduced pressure to a proper amount to obtain a concentrated solution B. And (3) combining the filtrates obtained in the step (2) for 1-3 times to obtain a mixed filtrate, concentrating the mixed filtrate under reduced pressure to a proper amount to obtain a concentrated solution A, combining the concentrated solutions A and B, and continuously concentrating under reduced pressure to obtain a medicine extract with the relative density of 1.32-1.35 at the temperature of 50 ℃. And then taking 1 part of the extract, 4 parts of sucrose and 1 part of dextrin, granulating and drying to obtain the sugar-free starch.

Comparative example 1 is the extraction process of Bozuri granule disclosed in the "division of drug Standard Uygur medicine of Ministry of health of the people's republic of China". The method comprises the following specific steps:

step one, preparing materials: weighing the raw materials according to the formula.

And step two, extraction: putting the celery seeds, the chicory seeds, the dodder seeds, the celery roots, the fennel root barks, the chicory roots and the fennel into an extraction tank. Adding 6 times of water for decoction for 1 time, wherein the temperature for decoction for 1 time is 80-100 ℃, the time for decoction for 1 time is 2h, filtering after decoction for 1 time to obtain filtrate for 1 time and filter residue for 1 time, then adding 4 times of water for decoction for 2 times, the temperature for decoction for 2 times is 80-100 ℃, the time for decoction for 2 times is 1.5h, filtering after decoction for 2 times to obtain filtrate for 2 times and filter residue for 2 times, then adding 3 times of water for decoction for 3 times, the temperature for shearing for 3 times is 80-100 ℃, the time for shearing for 3 times is 1h, and filtering after decoction for 3 times to obtain filtrate for 3 times and filter residue for 3 times.

Step three, concentration: and mixing the filtrates for 1-3 times to obtain a mixed filtrate, and concentrating the mixed filtrate under reduced pressure to obtain a drug extract with the density of 1.32-1.35 at 50 ℃. And then taking 1 part of the medicinal extract, 4 parts of sucrose and 1 part of dextrin, granulating and drying to obtain the medicine.

Effect example 1

1. Purpose of experiment

The influence of hepatoprotective Buzure particles (HBG) obtained in example 1 and comparative example 1 on phaseolin A (Con A) -induced immunological liver injury was investigated and the protective mechanism was investigated.

2. Animal modeling and grouping

180 male BALB/C mice were purchased from the disease prevention and control center in Hubei province. After one week of acclimatizing feeding, the animals were divided into five groups of 24 animals each. The control group, the model group, the comparative example 1 group (1.6g/kg HBG), the example 1 group (1.6g/kg HBG), and the (6mg/kg prednisolone) prednisolone positive control group (hereinafter referred to as the prednisolone group), respectively. Then the gavage was performed for 10 days. The blank and model groups were gazed with purified water during dosing. The example 1 group and the comparative example 1 group were each used for intragastric gavage of mice with the prepared HBG. The prednisolone group was administered 6mg/kg prednisolone for intragastric administration.

All groups except the control group were administered Con A tail vein injection at a dose of 20mg/kg on day 11. Liver and blood were collected for subsequent testing at 2h, 4h, and 6h after Con A injection, respectively. The same drug treatment method was then used for survival determination. The death was closely observed within 12 hours after injection of Con A at a dose of 30mg/kg and the time of death was recorded for each mouse.

The data in each table below are expressed as mean ± standard deviation, compared with the control group. p <0.05 is statistically significant for the differences. In each figure, # p <0.01, # p <0.05, # p < 0.01.

3. Detection method

Measuring the amount of the relevant cytokine in the blood and liver of the mouse by using a biochemical kit; the liver was analyzed for mRNA expression levels of IL-1 β, TNF- α, IFN- γ, IL-17A, IL-4, TGF- β, IL-6 and IFN- β by real-time fluorescent quantitative PCR (qPCR). H & E stained sections were used to observe pathological changes in the liver. The expression level of the relevant protein was determined by immunohistochemistry and western blotting.

4. Analysis of detection results

(1) After injection of Con a, the survival of each group of mice was observed for 12 hours. The test results are shown in fig. 1, and the control mice are shown to be all alive in fig. 1. After Con a injection, mice began to die within 3 hours. By 6h, the survival rate of the model group decreased to 16.7% and was maintained for 12 h. The survival rate of the mice in the group of example 1 was high. Specifically, the survival rate of the comparative example 1 group decreased to 58.3% at 4h, to 41.7% at 8h and maintained up to 12 h; the survival rate of the group of example 1 decreased to 70.6% at 4h, to 50.4% at 6h and was maintained up to 12 h; the 4h survival rate of the prednisolone group was reduced to 9.1% and maintained until 12 h. Therefore, the survival rate of the mice injected with Con A can be obviously improved by the Bourriella pyrenoidosa granules prepared by the extraction method.

(2) The serum contents of ALT (glutamic-pyruvic transaminase), AST (aspartate aminotransferase) and TBIL (total bilirubin) were measured, and the test results are shown in fig. 2, and the data of the test results are shown in table 1 below. As can be seen from fig. 2 and table 1, the serum ALT, AST and TBIL levels were significantly elevated at each time point after Con a injection, with a peak at 4h (p <0.01), whereas the HBG pretreatment reversed the elevation of ALT, AST and TBIL. The level of serum ALT, AST and TBIL reduction in the mice of the comparative example 1, example 1 and prednisolone groups was statistically significant (p <0.01 or p <0.05) compared to the model group. And the group of the example 1 has obviously better effect than the group of the comparative example 1.

TABLE 1

Note that the number of the holes is equal to or less than the number of the holes,^##：p<0.01；^*：p<0.05；^**：p<0.01。

to further determine the protective effect of these drugs on liver injury, we performed hematoxylin-eosin staining (H & E staining) on liver tissue, and the results are shown in fig. 3. Light microscopy showed that Con a treated mouse liver showed massive evidence of hepatocyte death, inflammatory cell infiltration, and structural liver injury. However, the comparative example 1 group and the example 1 group were less damaged. Especially, the group of example 1 significantly reduced hepatic necrosis. The result shows that HBG prepared in example 1 of the invention can more effectively reduce mouse immune liver injury induced by ConA compared with comparative example 1.

(3) The qPCR method is used for detecting the expression of the inflammatory factors in the liver of the mouse, the test results are shown in figures 4-6, and the data of the test results are shown in the following table 2.

As shown in FIG. 4, mRNA expression levels of IL-1 beta, TNF-alpha, IFN-gamma, IL-4, IL-17A and TGF-beta in five groups of mice were measured by the qPCR method. Wherein, FIG. 4A is the mRNA expression level of IL-1 β; FIG. 4B is the mRNA expression level of TNF- α; FIG. 4C shows IFN-. gamma.mRNA expression levels; FIG. 4D is the mRNA expression level of IL-4; FIG. 4E is the mRNA expression level of IL-17A; FIG. 4F is the mRNA expression level of TGF-. beta.s. As can be seen, the model group liver IL-1 beta, TNF-alpha, IFN-gamma and IL-17A are obviously increased in each time point compared with the control group, and peak value is reached in 4h (p < 0.01). While the expression of IL-4 and TGF-beta decreased significantly at each time point, reaching a trough at 4h (p < 0.01). Pretreatment of the comparative example 1 group, the example 1 group and the prednisolone group significantly reduced the hepatic IL-1 β, TNF- α, IFN- γ and IL-17A mRNA expression levels (p <0.01) in the model group (fig. 4A-C, fig. 4E), increased the hepatic IL-4 and TGF- β levels (p <0.05 or p <0.01) (fig. 4D, fig. 4F), while the effect of example 1 was particularly significant. The contents of the two groups of immune cells, namely Th1/Th2 and Th17/Treg, are usually in dynamic balance in normal cells, and once the two groups of immune cells are unbalanced, a series of immune and inflammation-related reactions are caused. IFN-gamma, IL-4, IL-17A and TGF-beta are cytokines secreted by four immune cells, namely Th1, Th2, Th17 and Treg. The above results indicate that the protective effect of the brezotherm granules on the mouse liver injury caused by Con a may be partly due to the inhibition of inflammatory factors and the regulation of cytokine secretion by Th1, Th2, Th17 and Treg cells.

Meanwhile, T-beta, GATA3, ROR-gamma T and Foxp3 are specific transcription factors of Th1, Th2, Th17 and Treg respectively, and the four transcription factors are researched by an immunohistochemical method, and the expression of T-beta, GATA3 and Foxp3 in liver tissues of five groups of mice obtained for 4h is detected by immunohistochemical staining (magnification times are multiplied by 200) as shown in FIG. 5. Wherein, FIG. 5G is the expression of T-beta; FIG. 5H is the expression of GATA 3; FIG. 5I is the expression of Foxp 3. FIG. 5G shows that the T-beta positive expression in the model group is significantly increased compared to the control group. T-beta expression was significantly reduced in liver cells of the comparative example 1 group, the example 1 group and the prednisolone group, compared to the model group. Figure 5H shows a significant reduction in positive expression of GATA3 following Con a injection. While the HBG of comparative example 1 and example 1 significantly increased the positive expression of GATA 3. As shown in fig. 5I, the expression level of Foxp3 in the model group was significantly reduced compared to the control group. And the Foxp3 expression levels were higher in the comparative example 1 and example 1 pretreatment groups. Compared with the control group, the ROR-gamma t protein expression level of the model group is obviously increased (p <0.01) (FIG. 6J-K). Wherein, FIG. 6J is the expression level of ROR-gamma t protein in mouse liver tissue; FIG. 6K shows the relative expression intensity of ROR-gamma t protein in mouse liver tissue. The group of comparative example 1 and the group of example 1 can both significantly reduce the expression of ROR-gamma t (p <0.01), while the effect of example 1 is more significant. These results show that cytokines and related transcription factors secreted by four immune cells in the cells are disturbed after Con A injection, but pre-administration of HBG can effectively improve the disturbance, suggesting that HBG can protect the liver from being damaged by regulating the production of inflammatory factors and the balance of immune cells.

TABLE 2

(4) HBG inhibits IL-6/IFN-beta induced STATs pathway

IL-6 can regulate immune response, acute phase response and hematopoietic function, and plays an important role in body anti-infection immune response. FIG. 7 is a bar graph comparing the expression levels of IL-6 and IFN- β mRNA in five groups of mice measured by qPCR. Wherein, FIG. 7A is a histogram of the mRNA expression level of IL-6, and FIG. 7B is a histogram of the mRNA expression level of IFN- β. The expression levels of IL-6 and IFN-beta in liver tissues of the mice in the model group are obviously higher than those in the blank group at each time point and reach a peak value (p is less than 0.01) at 4 h. The expression levels of IL-6 and IFN- β mRNA in liver tissues of mice in HBG of comparative example 1 and HBG-treated group of example 1 were significantly lower than those in model group (P <0.01), while the expression level of example 1 was significantly lower than that in comparative example 1, and the data of the test results are shown in Table 3 below.

The Western blot method shown in FIG. 8 detects the expression levels of JAK1 and p-JAK1 proteins in the livers of five groups of mice. Wherein, FIG. 8C is the expression level of JAK1, p-JAK1 protein in mouse liver, and FIG. 8D is the relative expression strength of JAK1, p-JAK1 protein in mouse liver. The groups of comparative example 1 and example 1 reduced the expression of p-JAK1 in mice with liver injury, and the reduction in the group of example 1 was more significant. But there were no significant differences in total JAK expression between groups.

Next, we examined the phosphorylation of STAT1 and STAT3 in mouse liver tissue, and showed that immunohistochemical staining examined the expression of p-STAT1, p-STAT3, and IRF1 in five groups of mouse liver tissue obtained at 4 h. FIG. 9E is expression of p-STAT1, FIG. 9F is expression of p-STAT3, and FIG. 9G is expression of IRF 1. The results showed that the positive expression of p-STAT1 and p-STAT3 was significantly increased in the model group compared to the control group. However, the comparative example 1 group and the example 1 group were effective in down-regulating the positive expression of p-STAT1 and p-STAT3, as compared with the model group. From immunohistochemistry results, IRF1 (interferon regulatory factor 1 recombinant protein) was highly positively expressed in model group mice. The IRF1 positive expression was reduced to different extents in the comparative example 1, example 1 and prednisolone groups compared to the model group (fig. 9G).

TABLE 3

(5) in this study, we observed significant oxidative stress in hepatocytes from liver-injured mice. We first measured the levels of superoxide dismutase (SOD) and Malondialdehyde (MDA) in liver tissue of mice, as shown in figure 10 as the concentrations of MDA, SOD in the livers of five groups of mice at different time points. The results show that the MDA content in the liver tissue of the mice in the model group is increased at each time point after model building and reaches a peak value (p is less than 0.01) after Con A4 h is injected. As shown in fig. 10A, the concentration of MDA in the liver of the mouse is shown, and the control group and the example 1 group can effectively inhibit the increase of MDA concentration (p <0.05 or p <0.01) at each time point, and especially the inhibition effect of the example 1 group is more obvious. As shown in FIG. 10B, the SOD concentration in mouse liver was significantly decreased in the model group at each time point, and was decreased to the minimum value (p <0.01) 4 hours after Con A injection. Interestingly, the SOD activity was significantly increased at each time point in the HBG-treated group compared to the model group (p <0.01), and the test results are shown in Table 4 below.

Subsequently, liver Reactive Oxygen Species (ROS) levels were detected using DCFH-DA staining of frozen sections of liver. FIG. 11 shows ROS levels at three time points. (DAPI and Cy3 in FIG. 11 represent 4', 6-diamidino-2-phenylindole and immunofluorescence kit-anti-mouse staining, respectively, and Merged represents the fluorescence pattern after fusion of the DAPI and Cy3 fluorescence patterns.) compared to the control group, a significant increase in ROS levels occurred in the model mice, peaking 4h after Con A injection. The ROS level of the HBG administration group is obviously reduced at each time point, and the increase of the ROS level induced by Con A can be obviously reduced by HBG. In order to determine whether p-JNK participates in Con A-induced liver injury, an immunohistochemical method is adopted to detect the expression of p-JNK, and as shown in FIG. 12, HBG detected by different detection methods inhibits ROS-mediated JNK activation and further induces hepatocyte apoptosis. FIG. 12D shows that p-JNK expression in liver tissue of mice obtained from 4h was detected by immunohistochemical staining, which indicates that p-JNK expression is significantly increased in mice of the model group compared to the control group, and that p-JNK expression can be effectively reduced by HBG treatment. The expression levels of p-ERK1/2, p-p38 and cleaned-caspase 3 proteins in mouse livers are detected by a western blot method as shown in FIG. 12E, which shows that the protein expression of the model group mouse p-ERK1/2 and p-p38 is higher than that of the blank group (p < 0.01). HBG significantly down-regulated p-ERK1/2 and p-p38 protein expression (p <0.05 or p < 0.01). At the same time, we investigated the effect of HBG on ConA-induced apoptosis of mouse hepatocytes. First, apoptosis of mouse hepatocytes was detected by TUNEL method, and TUNEL staining (magnification × 200) showed apoptosis of five groups of mouse hepatocytes 4h after Con a injection as shown in fig. 12F. Figure 12F shows the presence of a large number of TUNEL positive hepatocytes in model mice compared to control. Interestingly, TUNEL staining also showed a significant improvement in apoptosis in HBG treated mice. To further investigate the role of HBG in ConA-induced hepatocyte apoptosis, we investigated the expression of clear-caspase 3 protein in mouse liver. The results show that the protein expression of the model mouse Cleaved-caspase 3 is significantly increased (p <0.01) compared with the control group (FIG. 12E), and the Bourricum particles extracted in example 1 can significantly reduce the protein expression of Cleaved-caspase 3.

TABLE 4

5. conclusion

This study shows that the group of example 1 can prevent immune liver injury induced by Con A. Example 1 preparationToThe protective effect of the liver-protecting Buzure granules is to regulate immune balance and inhibit the activation of STATs induced by IL-6/IFN-beta, thereby reducing ROS-mediated JNK activation and further reducing cell apoptosis induced by the JNK activation.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. The extraction method of the bushy giraldii nitsche granules is characterized in that the raw materials of the bushy giraldii nitsche granules comprise semen cuscutae, celery root, chicory root, celery seed, chicory seed, fennel root bark and fennel, and the extraction method of the bushy giraldii nitsche granules comprises the following steps:

2. The extraction method of claim 1, wherein the raw materials of the boughs thermal granules comprise the following components in parts by mass: 50-150 parts of celery seeds, 150-250 parts of chicory seeds, 20-100 parts of semen cuscutae, 150-250 parts of celery roots, 150-250 parts of fennel root skins, 50-150 parts of chicory roots and 50-150 parts of fennel;

the raw materials of the Bozu hot particle are preferably the following components in parts by mass: 106 parts of celery seed, 212 parts of chicory seed, 53 parts of semen cuscutae, 212 parts of celery root, 212 parts of fennel root bark, 106 parts of chicory root and 106 parts of fennel.

3. The extraction method according to claim 1, wherein in the step (1), the mass ratio of the mass of the 60-75% ethanol to the total mass of the dodder, celery root and chicory root is (3-6): 1, e.g. 4: 1;

and/or in the step (1), the extraction temperature is 80-100 ℃;

and/or, in step (1), the number of times of extraction is 2 or more, for example 3 times;

and/or, in the step (1), the total time of the extraction is 3-6 h, preferably 4-5 h, for example 4.5 h;

and/or in the step (1), the time for each extraction is 1-2.5 h, such as 1.5h or 2 h.

4. The extraction method according to claim 3, wherein in the step (1), when the number of times of the extraction is 3, the extract A is the sum of filtrates obtained by filtration after each extraction;

and/or in the step (1), when the extraction times are 3, the mass ratio of the first extraction is 6:1, the mass ratio of the second extraction is 4:1, the mass ratio of the third extraction is 3:1, wherein the mass ratio is the mass ratio of the mass of the 60-75% ethanol to the total mass of the semen cuscutae, the celery root and the chicory root;

and/or in the step (1), when the number of times of extraction is 3, the time for the first extraction is 2 hours, the time for the second extraction is 1.5 hours, and the time for the third extraction is 1 hour.

5. The extraction method according to any one of claims 1 to 4, wherein in the step (2), the preparation process of the extracts of celery seed, chicory seed, fennel root bark and fennel comprises the steps of sequentially extracting the mixture of celery seed, chicory seed, fennel root bark and fennel in the raw materials of the budesonant granules with water and filtering.

6. The extraction method according to claim 5, wherein in the step (2), the water extraction is performed by a decoction method;

and/or in the step (2), the temperature of water extraction is 80-100 ℃;

and/or, in the step (2), the number of times of water extraction is more than 2, such as 3;

and/or in the step (2), the mass ratio of the mass of the water in the water extraction to the total mass of the celery seeds, the chicory seeds, the fennel root bark and the fennel is (3-6): 1, e.g. 4: 1;

and/or in the step (2), the total time of water extraction is 4-5 h;

and/or in the step (2), the time for each water extraction is 1-2.5 h.

7. The extraction method as claimed in claim 6, wherein in the step (2), when the number of times of the water extraction is 3, the mass ratio of the first water extraction is 6: 1; the mass ratio of the water extraction for the second time is 4: 1; the mass ratio of the water extraction for the third time is 3:1, wherein the mass ratio is the mass ratio of the mass of water in the water extraction to the total mass of the celery seed, the chicory seed, the fennel root bark and the fennel;

and/or, in the step (2), when the number of times of the water extraction is 3, the time of the first water extraction is 2h, the time of the second water extraction is 1.5h, and the time of the third water extraction is 1 h;

and/or, concentrating after combining the extract A and the extract B;

wherein the concentration is preferably to concentrate the mixed extract of the extract A and the extract B of the celery seed, the chicory seed, the fennel root bark and the fennel to a medicine extract with the relative density of 1.32-1.35 at 50 ℃;

when the desired form of the bushy granules is a granular form, the concentration is preferably followed by granulation and drying; the granulation is preferably to mix the concentrated medicine extract with auxiliary materials; the auxiliary materials are preferably sucrose and dextrin; wherein the mass ratio of the medicine extract to the sucrose to the dextrin is 1:4: 1.

8. a bouzoite produced by the extraction process of any one of claims 1 to 7.

9. Use of the budesonide particles of claim 8 in the preparation of a medicament for the treatment of liver injury.

10. The use of claim 9, wherein the liver injury is an immunological liver injury.