WO2022143514A1 - Oral preparation containing caffeic acid ester and breviscapine, and preparation method therefor - Google Patents

Abstract

The present invention relates to an oral preparation containing a caffeic acid ester and breviscapine, and a preparation method therefor and an application thereof, comprising common oral dosage forms such as capsules, tablets, oral liquid, dripping pills or granules. The preparation process provided by the present invention fully utilizes caffeic acid ester components in Erigeron breviscapus and reduces the content of other harmful and noneffective substances, and can allow for salt formation of breviscapine and the caffeic acid ester, thereby improving efficacy, having an antitumor effect while protecting liver, and providing a modern traditional Chinese medicine for clinical treatment which is more convenient and effective and more controllable in quality.

Description

Oral preparation containing caffeic acid ester and breviscapine and preparation method thereof technical field

In particular, it relates to a preparation method and application of breviscapine or its salt and caffeic acid ester extract or its salt, especially its application in the preparation of medicines for treating cardiovascular and cerebrovascular diseases and non-alcoholic fatty liver diseases caused by hyperlipidemia , and the application in the preparation of antitumor drugs.

Background technique

In recent years, the spectrum of diseases has undergone significant changes with the acceleration of population aging, deepening urbanization, and gradual changes in lifestyles. Chronic non-communicable diseases such as hypertension, coronary heart disease, and diabetes are widely prevalent, and the incidence of cardiovascular and cerebrovascular diseases and malignant tumors is also accelerating.

Dengzhan Asarum (Dengzhanhua) is a dry whole herb of Erigeron breviscapus (Vant.) Hand-Mazz, a plant of the genus Asteraceae. Removing blood stasis, clearing collaterals and relieving pain, used for colds, headaches, rheumatism pain, paralysis caused by cerebrovascular accidents, etc. ("National Chinese Herbal Medicine Compilation"). Dengzhan Xixin can be made into medicine alone, and the currently listed varieties include Dengzhan Xixin Injection, Dengzhan Xixin Capsules, Dengzhan Xixin Soft Capsules, Dengzhan Xixin Dropping Pills, Yimaikang, Dengzhan Xixin Granules, etc. The active ingredients are flavonoids breviscapine and caffeic acid esters, and the existing products mainly emphasize breviscapine represented by breviscapine, breviscapine products include breviscapine injection, injection Use breviscapine, breviscapine tablets, breviscapine drop pills, etc. Even Dengzhan Asarum capsules, Yimaikang and other Dengzhan Asarum extract products do not regulate the content of caffeic acid esters, let alone enrichment. Dengzhan Xixin injection is the only product that is enriched in caffeic acid esters in terms of technology. However, the ratio of the content of caffeic acid ester to flavonoids is only in the range of 1:1.2-1:5.5. Through the research on caffeic acid esters in scutellaria brevis plant, it is found that caffeic acid esters in scutellaria scutellariae are very rich, including monocaffeic acid ester and dicaffeoate. There are many families and genera containing caffeic acid esters in plants, such as Compositae, Honeysuckle, Eucommia, Chuanxiaceae, Rubiaceae, Convolvulaceae and other plants. At present, it is known that Compositae plants are different from other plants containing caffeoyl quinic acid in that there is also a class of caffeoyl octyl sugar compounds, including monocaffeoyl, dicaffeoyl and tricaffeoyl octyl sugar acids, respectively. . The caffeoyl octulonic acids contained in scutellaria scutellariae are dicaffeoyl octyl saccharides, including scutellaria ester B and scutellaria scutellariae, which are isolated from the genus Asteraceae, and Scutellaria scutellariae is now only obtained from Scutellaria scutellariae. Jianping Zhao, et al. (J. Nat. Prod. 2014, 77, 509-515) reported that six caffeoyl octulonic acids obtained from Syringa spp. have anti-inflammatory and anti-metabolic derangement effects. The content range of these special types of dicaffeoate compounds is listed separately, which can better clearly classify and control the caffeic acid esters obtained from D. The content of the ingredients is easier to control, which also makes the quality of such products more controllable. There is also a class of hepatotoxic substances γ-pyrones in Radix Asarum, the representative compound is pyroconic acid. The method of removing pyroconic acid in the previous patent is to adopt column chromatography, and the properties used are: Pyroconic acid has a high polarity and can be removed by washing with water in column chromatography, but if the extraction solvent must be low-concentration alcohol or water extraction, otherwise impurities such as chlorophyll will easily solidify on the adsorption column, resulting in inability to wash detachment or make column regeneration difficult. This patent removes insoluble macromolecular impurities, including most of chlorophyll, protein, tannin, etc., by adjusting the pH of the acidified supernatant to neutrality, then microfiltration first, and then concentrating it through a nanofiltration membrane. Molecules and water are filtered, and 90% of the pyroconic acid can be removed as pyroconic acid with a molecular weight of only 112 Da, which is both soluble in water. The removed concentrate is applied to the column, so that the applied column can be separated and purified more efficiently. And it can also be extracted with 50-80% ethanol in the first step of extraction to increase the extraction rate. The extracted and refined scutellarin and caffeic acid esters can be directly dried and used as medicines. The pH of the scutellarin and caffeic acid esters can also be adjusted to neutral, and spray-dried after being salted to increase water solubility and increase the water solubility. druggability of the class of ingredients.

SUMMARY OF THE INVENTION

In view of the above-mentioned technical problems, the object of the present invention is to provide an oral preparation containing caffeic acid ester and scutellarin, wherein, the oral preparation contains caffeic acid ester or its salt and scutellarin and its salt and pharmaceutically acceptable excipients, The weight ratio of caffeic acid ester or its salt and breviscapine or its salt is 5:1 to 30:1.

Currently, the extraction method of the breviscapine oral preparation is limited, and the preparation is usually a mixture of caffeic acid ester, breviscapine, and other substances, which cannot be accurately mixed. The present invention adopts a unique preparation method, extracts the main components thereof, especially prepares the salts thereof, and effectively promotes the medicinal effect.

Preferably, the present invention further provides an oral preparation containing caffeic acid ester or its salt and breviscapine or its salt, said caffeic acid ester or its salt comprising dicaffeoate or its salt and monocaffeic acid or Its salt, wherein the weight ratio of dicaffeoate or its salt to breviscapine or its salt is 3.8:1 to 8:1, and the weight ratio of monocaffeate or its salt to dicaffeoate or its salt is 1 :2-1:8.

Preferably, the present invention further provides an oral preparation containing caffeic acid ester or its salt and breviscapine or its salt, said dicaffeoate salt comprising 1,3-O-dicaffeoylquinic acid or its salt, 3,4-O-dicaffeoylquinic acid or its salt, 3,5-O-dicaffeoylquinic acid or its salt, 4,5-O-dicaffeoylquinic acid or its salt, Fibonacci ester B or its salt, scutellarin or its salt; monocaffeic acid ester or its salt including 3-O-caffeoylquinic acid or its salt, 4-O-caffeoylquinic acid or its salt, 5 -O-caffeoylquinic acid or its salt, breviscapine or its salt.

Preferably, the present invention further provides an oral preparation containing caffeic acid ester or its salt and breviscapine or its salt, wherein 1,3-O-dicaffeoylquinic acid in said dicaffeoate or its salt or its salt, 3,4-O-dicaffeoylquinic acid or its salt, 3,5-O-dicaffeoylquinic acid or its salt, 4,5-O-dicaffeoylquinic acid or its Salts, the above-mentioned four are isomers, collectively referred to as simply substituted dicaffeoate or its salts; the described Feponica ester B or its salts and scutellarin or its salts are isomers, which are Dicaffeoyl octanoic acid or its salts, wherein the ratio of the simple substituted dicaffeoate or its salt to dicaffeoyl octanoic acid or its salt is 2:1 to 0.9:1.

The above-mentioned oral preparations are preferably hard capsules, soft capsules, tablets, granules, oral liquids, dripping pills, water pills, powders, and pills.

The scutellarin or its salt of the above-mentioned oral preparation includes scutellarin or its salt, scutellarin or its salt, scutellarin and its salt, etc. scutellarin or its salt-related flavonoid compounds isolated from scutellarin or its salt.

In the above preparation, caffeic acid ester or its salt and breviscapine or its salt account for 80-99% by weight of the preparation, and the balance is auxiliary materials. The oral preparation is preferably an oral preparation such as a capsule or tablet, for example, a hard capsule.

Further, the dicaffeoate salt and the scutellarin salt are both sodium or potassium salts or other salts that can be used medicinally and are soluble in water; the weight ratio thereof is 5:1 to 30:1. Preferably 6:1-15:1, more preferably 6.5:1-12:1, most preferably 7:1-10:1, most preferably around 8:1, that is, other products should also preferably be sodium salts.

The present invention further provides a preparation method of an oral preparation, the preparation method is as follows:

Take Dengzhan Asarum and add 10-90% concentration of aqueous ethanol to extract, and the extract is concentrated under reduced pressure to form an extract; the extract is dissolved in water, adjusted to pH 7-9, filtered, added acid to adjust pH to 1 to 3, placed overnight, and filtered , collect the filtrate and the precipitation, purify the precipitation with water and ethanol, and dry to obtain scutellarin; after the precipitation is refined, add alkali to adjust the pH value to 7-8, and spray dry to obtain scutellarin salt; the acidified filtrate adjusts the pH value to 7 ~9, use ceramic microfiltration membrane for clarification, and the clarified liquid obtained after clarification is then concentrated with organic nanofiltration membrane, the concentrated liquid is passed through a polyamide chromatography column, eluted with water, eluted with 50-80% ethanol, and collected. The ethanol eluent is concentrated and dried to obtain caffeic acid ester; the ethanol eluent is concentrated and adjusted to pH 7-9, filtered, and the filtrate is spray-dried to obtain caffeic acid ester salt. The weight ratio of dicaffeoate or its salt and breviscapine or its salt is 5:1-30:1, preferably 6:1-15:1, more preferably 6.5:1-12:1, most preferably 7:1: 1-10:1, the most preferred embodiment is 8:1, the oral preparation of Dengzhanxixin is obtained, and appropriate auxiliary materials are added to make tablets, capsules, soft capsules, dropping pills, granules and oral liquid preparations .

In the preparation method of the above oral preparation, the pH value of the alkali-adjusted solution is NaOH, Na ₂ CO ₃ , NaHCO ₃ , KOH, K ₂ CO ₃ or KHCO ₃ , or others that can be used to adjust the pH value HCl, H ₂ SO ₄ or H ₃ PO ₄ , or other acid solutions that can be used to adjust pH.

The ethanol concentration of the polyamide chromatographic column eluted with ethanol is 50-95%.

The application of the above-mentioned medicinal composition in the preparation of medicines for treating cardiovascular and cerebrovascular diseases and non-alcoholic fatty liver disease caused by hyperlipidemia; in particular, it has been proved by repeated pharmacological and pharmacodynamic experiments that the above-mentioned composition of the present invention also has Anti-tumor effect, that is, the above-mentioned pharmaceutical composition of the present invention also has application in the preparation of anti-tumor drugs. The above-mentioned tumors include tumors of the respiratory or digestive system such as colorectal cancer, lung cancer, and liver cancer. It is suitable for the occurrence of digestive system tumors in patients with non-alcoholic fatty liver disease, including colorectal cancer, liver cancer, etc.

Currently, the single oral preparation of Asarum brevis on the market does not have the step of removing the hepatotoxic component pyroconic acid, nor the step or process of refining caffeic acid esters or their salts, especially dicaffeoate or their salts. For example, the existing technology of Dengzhan Xixin capsules is to take 2000g of Dengzhan Xixin, and extract it twice with 80% ethanol under reflux for 1.5 hours for the first time and 1 hour for the second time, combine the extracts, add about 640g of activated carbon, boil, filter, and extract the filtrate. Concentrate under reduced pressure to a thick paste, add an appropriate amount of starch, dry under reduced pressure, pulverize, sieve, coat with 3% polyvinyl alcohol solution, add 0.5 g of magnesium stearate, mix well, put into capsules, and make Into 1000 grains, that is.

It has been reported in the literature that caffeoylquinic acid compounds have antioxidant, anti-inflammatory, antibacterial, antiviral, anti-hyperglycemic, hyperlipidemic, and immunomodulatory effects, and are a class of active ingredients. Therefore, refining such active substances and making quantitative regulations are necessary work to make Dengzhan Xixin products better for clinical services. Both breviscapine and caffeic acid esters have poor water solubility and fat solubility, resulting in low bioavailability and weak druggability. The present invention adopts alkali-soluble acid precipitation in the preparation method, firstly precipitating the flavonoid component breviscapine, and then refining; meanwhile, the acidified supernatant liquid part is retained, and the acidified supernatant liquid part is another type of active ingredient caffeic acid esters, Through membrane separation technology, microfiltration is used to remove macromolecular insoluble substances, and then nanofiltration membrane is used to concentrate to remove most of the pyroconic acid, and then column chromatography is performed to enrich the dicaffeoate components, which are again toxic to the liver. of pyroconic acid. After nanofiltration membrane and column chromatography, more than 99% of pyroconic acid was removed.

This result was confirmed by experiments:

Table 1. Changes in composition of the acidified supernatant of Dengzhan Asarum after membrane filtration after pH adjustment

As can be seen from Table 1, the acidified supernatant of Asarum scutellariae was adjusted to pH close to neutral and passed through a clarification membrane and a nanofiltration concentration membrane. 84%. After column chromatography, the content of dicaffeoate was well retained, and pyroconic acid was further removed.

The microfiltration clarification membrane is preferably 100 nm or 200 nm, and the nanofiltration concentration membrane is preferably 300D-400D.

When the flavonoids and caffeic acid esters were salted, the in vitro dissolution experiments in mice proved that the dissolution rate was higher than that of the prototype, so the drugability was better than the prototype. The dissolution profiles of Dengzhanxixin tablets and Dengzhanxixin sodium salt tablets in different dissolution media were evaluated. The research results show that the dissolution rates of the two tablets in 0.1M HCl are both low, reaching saturation in 5 minutes, and the cumulative dissolution percentage is less than 15%; In the dissolution medium of acetate buffer at pH 4.5, the cumulative release of the two tablets in acetate at pH 4.5 basically reached saturation at 30 minutes, but the cumulative dissolution percentage at 30 minutes was higher than that of Dengzhan Xixin sodium salt tablets For the prototype sheet, all are less than 60%. In the dissolution medium of pH 6.8 phosphate buffer, when the rotation speed is 50 rpm, Dengzhan Xixin sodium salt tablets have been completely dissolved in pH 6.8 phosphate buffer for 15 minutes, while Dengzhan Xixin tablets need to accumulate in 45 minutes. The dissolution percentage is greater than 85%; the cumulative dissolution percentage of Dengzhanxixin sodium salt tablet at 30min is greater than Dengzhanxixin tablet.

By feeding C57 mice with MCD (Methionine and Choline Deficient L-Amino Acid Diet) methionine and choline deficient diets, a non-alcoholic steatohepatitis (NASH) model was constructed, and the existing Dengzhanxixin capsules and the invented process Dengzhanxixin were explored. The therapeutic effect of capsules on NASH.

1. Test reagents

MCD feed, item number A02082002BR, manufacturer: Research Diets (USA)

2. Test process

(1) Animal grouping and treatment Twenty C57BL/6J male mice were randomly divided into 4 groups after adaptive feeding for 1 week, with 5 mice in each group. The control group is the normal maintenance feed, the model group is fed with the MCD feed, the existing Dengzhanxixin capsule powder group and the invention process Dengzhanxixin capsule powder group (see Example 6 for the prescription), the oral liquid is administered by gavage (100mg/kg. bw), once a day. The experimental animals had free access to food and water for 4 weeks. (2) Determination of mouse body weight and liver weight The body weight and liver wet weight of each group of mice were weighed, and the liver index was calculated.

3. Test results

(1) Effects on the body weight, liver weight and liver index of mice Compared with the control group, they were significantly reduced, indicating that the test drugs could not effectively improve the reduction of body weight and liver weight induced by MCD diet. In addition, there was no significant difference in body weight, liver weight and liver index between the two tested drugs and the model group, as shown in Table 2.

Table 2. Effects of Dengzhan Xixin capsule powder on body weight, liver weight and liver index of mice

group	Weight (g)	Liver weight (g)	Liver Index (%)
control group	27.08±1.46	1.43±0.15	5.31±0.36
model group	21.94±1.25	1.91±0.24	8.69±0.75
Invented craft Dengzhan Asarum group	22.12±0.56	1.88±0.11	8.49±0.36
Existing Craft Dengzhan Asarum Group	22.68±0.22	1.97±0.35	8.77±0.22

(2) Effects on liver function indexes of mice Compared with the control group, the serum alanine aminotransferase (AST) and aspartate aminotransferase (ALT) levels of the mice in the model group were significantly increased (P<0.001). The serum AST and ALT levels of the mice in the Dengzhanxixin capsule powder group with the invention process were higher than those in the control group, but compared with the model group, the Dengzhanxixin capsule powder with the invention process significantly inhibited the MCD diet-induced increase in serum AST and ALT in mice. High; the existing technology Dengzhanxixin group can significantly reduce the ALT level, and there is a trend of reducing AST, but there is no statistical significance, see Table 3.

Table 3. Effects of Dengzhan Xixin Capsule Powder and Existing Dengzhan Shengmai Capsule Powder on Liver Function Index of Mice

group	Alanine aminotransferase (U/L)	Aspartate aminotransferase (U/L)
control group	37.2±6.57	103.2±22.9
model group	160.8±49.2 ###	181.2±34.1 ###
Invented craft Dengzhan Asarum group	101.6±28.2* #	132.1±22.1*
Existing Craft Dengzhan Asarum Group	100.1±22.4* #	162.2±21.6 ##

^### P<0.005vs control group; ^## P<0.01vs control group; ^# P<0.05vs control group; ^* P<0.05vs model group

4. Analysis and conclusion

(1) Effects on body weight MCD diet-induced weight loss in NASH mice is a disadvantage of this dietary model. This study found that the invented technology Dengzhan Xixin capsule powder and the existing technology Dengzhan Xixin capsule powder could not inhibit the weight loss induced by MCD diet, which may be related to the structure of MCD diet.

(2), the effect on liver function of MCD diet-fed mice serum ALT and AST levels were significantly increased, indicating that the MCD diet-induced NASH model was successfully established. This study found that the invented technology Dengzhan Xixin capsule powder and the existing technology Dengzhan Xixin capsule powder can improve NASH induced by MCD diet, and the invented technology Dengzhan Xixin capsule powder has a down-regulation effect on both ALT and AST.

Existing breviscapine capsule powder is prepared from breviscapine salt and caffeic acid ester in the ratio within the scope of the claims, and the powder prepared with breviscapine or its salt and caffeic acid ester or its salt has similar effects . The difference between the prior art and the present invention is that the production methods are different, and the obtained components and different contents have obvious differences. Although the previous documents have proved that scutellarin or scutellarin have the effect of improving liver factors, but in the scutellarin capsules. It is verified by oral method, and it is found that between different processes, the function of the capsule obtained by the present invention can more comprehensively improve the relevant factors of non-alcoholic fatty liver.

Since non-alcoholic fatty liver disease is usually associated with a high-fat and high-carbohydrate diet, previous literature found that fatty liver is often significantly associated with the incidence of digestive tract tumors, such as gastric cancer and bowel cancer. At the same time of liver disease, whether it has anti-tumor function, the applicant's researchers specially made a multiple combination ratio for Example 6, focusing on research whether it has anti-intestinal cancer function.

Description of drawings:

Fig. 1 Toxicity test of Dengzhanxixin on human intestinal cancer cells HCT116;

Fig. 2 Toxicity test of Dengzhanxixin on human intestinal cancer cells Caco2;

Figure 3. Dengzhanxixin inhibits the proliferation of HCT116 cells;

Figure 4 Dengzhan Asarum 1.25:1 ratio of each concentration inhibits the proliferation of HCT116 cells;

Figure 5 Dengzhan Asarum 2:1 ratio of each concentration inhibits the proliferation of HCT116 cells;

Figure 6 Dengzhan Xixin 4:1 ratio of various concentrations of Xinxin inhibits the proliferation of HCT116 cells;

Figure 7 Dengzhan Asarum 6:1 ratio of each concentration inhibits the proliferation of HCT116 cells;

Figure 8 Dengzhan Asarum 8:1 ratio of each concentration inhibits the proliferation of HCT116 cells;

Figure 9 Controls the effect of different concentrations of breviscapine on inhibiting the proliferation of HCT116 cells;

Figure 10. Brevis asarum inhibits the proliferation of Caco2 cells;

Figure 11. Brevis asarum 1.25:1 ratio group inhibited the proliferation of Caco2 cells by each concentration;

Figure 12. The 2:1 ratio of Dengzhan Asarum inhibits the proliferation of Caco2 cells in each concentration group;

Figure 13. Dengzhanxiu 4:1 ratio group inhibited the proliferation of Caco2 cells by various concentrations of Xinxin;

Fig. 14 The 6:1 ratio of Dengzhan Asarum inhibits the proliferation of Caco2 cells in each concentration group;

Figure 15. The 8:1 ratio of Dengzhan Asarum inhibited the proliferation of Caco2 cells in each concentration group;

Figure 16 Control breviscapine inhibits the proliferation of Caco2 cells;

Figure 17 Body weight change;

Fig. 18 The effect of Dengzhanxixin on the survival rate of tumor-bearing mice;

Figure 19 Colorectal length of mice in each group ( ^* P<0.05, ^** P<0.01, ^*** P<0.005vs sham);

Figure 20 Number of tumors ( ^* P<0.05, ^** P<0.01, ^*** P<0.005vs sham);

Figure 21 Tumor burden ( ^* P<0.05, ^** P<0.01, ^*** P<0.005 vs sham).

Oncology Research Section:

1. Cell test

1. Experimental materials:

Cells: Two types of human colorectal cancer cells in good growth condition: HCT116, CaCo2.

Medium: HCT116: IMDM+10% FBS; CaCo2: MEM+15% FBS.

Culture conditions: 37°C, 5% CO ₂ .

Experimental drugs: experimental Dengzhan Asarum (inventive process) each proportioning drug, control Dengzhan Asarum (existing technology) Capsule contents: drug powder is stored at 4 ° C, and the culture medium is used to prepare a 1 mg/mL mother solution when cells are added. , vortex to dissolve completely, and dilute to the corresponding concentration for use.

2. Cytotoxicity test: (killing effect of drugs on tumor cells)

2.1 Experimental method:

After the cells were digested, single cell suspension was prepared, and HCT116 cells and CaCo2 cells were seeded in a 96-well plate at a density of 4*10 ⁴ cells/well and 1.5*10 ⁴ cells/well, respectively.

Twenty-four hours after inoculation, the 96-well plate was replaced with a drug containing a ratio of drugs (inventive process, dicaffeoate salt: breviscapine salt 1.25:1, 2:1, 4:1, 6:1, 8:1) and The culture medium of the control drug (ds: the content of the existing technology Haobang Dengzhan Xixin Capsules) has a final concentration of 30/100/300 μg/mL. Set 5 duplicate wells for each concentration, and set 5 solvent control wells and 1 cck-8 detection control well at the same time.

The cck-8 assay was performed 24 hours after dosing, and the cytotoxicity curve was drawn according to the assay data.

2.2 Experimental results:

2.2.1 The killing effect of Dengzhanxixin on human rectal cancer cells HCT116

After the cells were treated with different concentrations of the drug for 24 hours, the viable cells were detected by cck-8 method. The results are shown in Figure 1. Using HCT116 cells, the experimental results showed that the two ratios of breviscapine (inventive process) dicaffeoate and breviscapine in 6:1 and 8:1 were effective on intestinal tract when the drug concentration was greater than 100 μg/mL. Cancer cells have a certain killing effect, but there is no significant effect; the three ratios of dicaffeoate and breviscapine 4:1, 6:1, 8:1 have cytotoxic effects when the drug concentration is greater than 300 μg/mL, but not significant.

2.2.2 The cytotoxic effect of Dengzhan Asarum on human rectal cancer cells Caco2

For human colorectal cancer CaCo2 cells, different proportions of Asarum brevis (invention process) all showed a certain killing effect, and the 8:1 ratio of dicaffeoate and breviscapine can produce significant effects at a dose of 30 μg/mL. Cytotoxicity; 4:1, 6:1, 8:1 groups had significant cytotoxicity at the dose of 100 μg/mL; different formulations had significant cytotoxicity at the dose of 300 μg/mL, 4:1, 6: The 1, 8: 1 group is better than the 1.25: 1, 2: 1 group, and even better than the control group (existing technology).

Table 4. Toxicity test of breviscapine on human intestinal cancer cells Caco2

compare\group	ds	1.25:1	2:1	4:1	6:1	8:1
0 vs 30	ns	ns	ns	ns	ns	**
0 vs 100	ns	ns	ns	**	**	**
0 vs 300	*	**	**	***	***	***

The cells were treated with drugs for 24 hours after adherence, and detected by the cck-8 method. (*P<0.05, **P<0.01, ***P<0.001 vs blank control; ns: not significant).

3. Cell proliferation test method: (inhibitory effect of drugs on tumor cell growth)

3.1 Experimental method:

After cell digestion, single cell suspension was prepared, and HCT116 cells and CaCo2 cells were seeded in 6 96-well plates at a density of 5*103 cells/well and 1.5*103 cells/well, respectively. 24 hours after inoculation, a 96-well plate was first taken for cck-8 assay, which was recorded as 0 day; the remaining 96-well plates were added with different ratios of drugs (1.25:1, 2:1, 4:1, 6:1, 8:1). 1) The culture medium of the control drug (ds: the contents of Zhenbang Dengzhan Xixin Capsules), HCT116 cells use the drug doses with final concentrations of 10, 30, and 100 μg/mL, and CaCo2 cells use 3, 10, and 30 μg/mL drugs. Dosage, 5 replicate wells for each concentration, and 5 solvent control wells and 1 cck-8 detection control well. In the middle, the cell medium (containing drugs) was changed once at 3 days. The cck-8 was measured at 24, 48, 72, 96 and 120 hours after dosing, and recorded as 1day, 2day, 3day, 4day, and 5day. The corresponding data were analyzed and cell growth curves were drawn.

3.2 Test results

3.2.1 Inhibitory effect of Dengzhan Xixin on the proliferation of colorectal cancer cells HCT116

The results of the inhibition of HCT116 cell proliferation by different ratios of drugs are shown in Figures 3 to 9, and the detailed statistical differences are shown in Attached Table 2. The results showed that on the 1st and 2nd day of administration, the effect of the drug on inhibiting the proliferation of HCT116 cells was not obvious; /mL), the 8:1 (100ug/mL) ratio group had a significant inhibitory effect on cell proliferation; on the fourth day of administration, 1.25:1 (100ug/mL), 2:1 (30ug/mL, 100ug/mL), 4 : 1 (10ug/mL, 30ug/mL, 100ug/mL), 6:1 (100ug/mL), 8:1 (30ug/mL, 100ug/mL) each group had a significant inhibitory effect on cell proliferation; Day, 6:1 (100ug/mL), 8:1 (30ug/mL, 100ug/mL) ratio group had significant inhibitory effect. Compared with each group, the 8:1 ratio group had the best inhibitory effect on the proliferation of HCT116 cells. (Figure 4-Figure 9)

Table 5 The effect of breviscapine on inhibiting the proliferation of human colorectal cancer cells HCT116

Administration group and concentration\day(s)	0	1	2	3	4	5
0 vs 1.25:1 10ug/mL	ns	ns	ns	ns	ns	**
0 vs 1.25:1 30ug/mL	ns	ns	ns	ns	ns	ns
0 vs 1.25:1 100ug/mL	ns	ns	ns	ns	ns	**
0 vs 2:1 10ug/mL	ns	ns	ns	ns	ns	ns
0 vs 2:1 30ug/mL	ns	ns	ns	ns	**	ns
0 vs 2:1 100ug/mL	ns	ns	ns	ns	**	ns
0 vs 4:1 10ug/mL	ns	ns	ns	ns	*	ns
0 vs 4:1 30ug/mL	ns	ns	ns	ns	*	ns

0 vs 4:1 100ug/mL	ns	ns	ns	ns	**	ns
0 vs 6:1 10ug/mL	ns	ns	ns	ns	ns	ns
0 vs 6:1 30ug/mL	ns	ns	ns	ns	ns	ns
0 vs 6:1 100ug/mL	ns	ns	ns	**	***	***
0 vs 8:1 10ug/mL	ns	ns	ns	ns	ns	ns
0 vs 8:1 30ug/mL	ns	ns	ns	ns	**	**
0 vs 8:1 100ug/mL	ns	ns	ns	**	***	**
0 vs ds 10ug/mL	ns	ns	ns	ns	ns	ns
0 vs ds 30ug/mL	ns	ns	ns	ns	ns	ns

(*P<0.05, **P<0.01, ***P<0.001 vs control; ns, not significant).

3.2.2 Inhibitory effect of Dengzhan Xixin on the proliferation of colorectal cancer cells Caco2

The results of the drug inhibiting the proliferation of Caco2 cells are shown in Figures 10-16, and the detailed statistical differences are shown in Table 6. The results showed that on the first day after administration, the effect of the drug on inhibiting the proliferation of Caco2 cells was not obvious, and on the second day after administration, scutellarin (inventive process) dicaffeoate salt: breviscapine salt 8:1 (30ug/ mL, 100ug/mL) significantly inhibited cell proliferation; on the third day after administration, 6:1 (100ug/mL), 8:1 (30ug/mL, 100ug/mL) significantly inhibited cell proliferation, and on the fourth day after administration and Day 5 1.25:1(30ug/mL), 2:1(30ug/mL), 4:1(30ug/mL), 6:1(10ug/mL, 30ug/mL), 8:1(10ug/mL, 30ug/mL). The control Dengzhan Xixin did not inhibit the proliferation of colorectal cancer cells Caco2. The experimental Dengzhanxixin (the invention process) each proportioning group has better inhibitory effect on the proliferation of Caco2 cells than the control Dengzhanxixin (the existing technology), and the 8:1 proportioning group has the best effect.

Table 6 The effect of breviscapine on inhibiting the proliferation of human colorectal cancer cells HCT116

group\day(s)	0	1	2	3	4	5
0 vs 1.25:1 3	ns	ns	ns	ns	ns	ns
0 vs 1.25:1 10	ns	ns	ns	ns	ns	ns
0 vs 1.25:1 30	ns	ns	ns	ns	**	**
0 vs 2:1 3	ns	ns	ns	ns	ns	ns
0 vs 2:1 10	ns	ns	ns	ns	ns	ns
0 vs 2:1 30	ns	ns	ns	ns	**	**
0 vs 4:1 3	ns	ns	ns	ns	ns	ns
0 vs 4:1 10	ns	ns	ns	ns	ns	ns

0 vs 4:1 30	ns	ns	ns	ns	*	**
0 vs 6:1 3	ns	ns	ns	ns	ns	ns
0 vs 6:1 10	ns	ns	ns	ns	**	**
0 vs 6:1 30	ns	ns	ns	*	**	**
0 vs 8:1 3	ns	ns	ns	ns	ns	*
0 vs 8:1 10	ns	ns	**	**	**	***
0 vs 8:1 30	ns	ns	**	**	***	***
0 vs ds 3	ns	ns	ns	ns	ns	ns
0 vs ds 10	ns	ns	ns	ns	ns	ns
0 vs ds 30	ns	ns	ns	ns	ns	ns

(*P<0.05, **P<0.01, ***P<0.001 vs blank; ns: not significant)

2. Animal testing

1. Experimental materials:

Table 7 Test article information

name	Dengzhan Asarum Proportioning Drugs	control drug
manufacturer	Yunnan Bio Valley Pharmaceutical	Yunnan Haobang Dengzhan Asarum Capsules
batch number
physical state	brown powder	brown powder
Storage conditions	4 degrees refrigerated, airtight	4 degrees refrigerated, airtight

Table 8 Information on experimental animals

species	C57BL/6N mice
level	SPF animal
order age	5 weeks
Age at start of trial	6 weeks
weight range	18-20g
gender	male

supplier	Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
animal identification method	6/cage, identified by ear tag method
Animal order quantity	110
number of animals used	110
Reasons for choosing the number of animals	The animals were divided into 9 groups, with 14 animals in the model group and 12 animals in each other group

Table 9 Feed Information

Feed properties	Control diet (NCD)	medicated feed
feed name	Mice maintenance diet	Rats and mice maintenance diets containing specific drugs
Feed formula	Basic feed	0.4%, drug weight/feed weight (w/w)
supplier	Small millet Yutai	Small millet Yutai
Storage conditions	4℃	4℃

2. Test method:

(1) Test grouping: 5-week-old mice of C57BL/6N strain, whose body weight reached 18-20 grams after 1 week of adaptation to the environment, were divided into sham operation (sham) group, model group and drug treatment group. Among them, the mice in the model and administration groups were injected with AOM (azoxymethane, 10 mg/kg) by intraperitoneal injection for modeling, while the mice in the sham group were injected with an equal volume of normal saline.

(2) Administration: One week after AOM injection, the mice in the treatment group began to be fed medicated feed (0.4%, w/w), while the mice in the sham and model groups continued to be fed the normal rat maintenance feed. At the same time, DSS (Dextran Sulfate Sodium Salt, dextran sulfate sodium) was added to the drinking water of model and treatment group mice to promote tumorigenesis.

(3) The detection indicators are as follows:

Feed intake: weigh once a week and record the feed consumption per cage;

Body weight: Weigh the body weight once a week, and observe the body weight changes of the animals;

Colorectal length and histopathological changes;

The number of tumors was counted, the diameter (mm) of each tumor was measured with a vernier caliper, the diameters of all tumors in the intestinal tract of each mouse were summed, and the total tumor load of the individual was calculated.

3. Test results

3.1 Mice body weight

The body weight of the animals was measured once a week during modeling and administration. The results are shown in Figure 17 and Table 9.

During the modeling period, compared with the sham operation (sham) group, the mice in the model group experienced significant weight loss after the first administration of 1% DSS at the 3rd week and the first administration of 2% DSS at the 8th week (Fig. 2A). . After the first administration of 1% DSS in the 3rd week, the body weight of the mice in each administration group did not show severe weight loss; after the first administration of 2% DSS in the 8th week, each administration group and model group showed significant weight loss. , but relatively speaking, breviscapine (inventive process) has the effect of alleviating the weight loss of experimental animals, among which the dicaffeoate salt: breviscapine salt 8:1 ratio group has the best effect. The control of Asarum brevis (the prior art) did not reduce the weight loss of tumor-bearing animals.

Table 9. Weight change

group\week(s)	1	2	3	4	5	6	7	8	9	10
mode vs control	ns	ns	ns	#	ns	ns	ns	ns	##	##
mode vs ds	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
mode vs 1.25:1	ns	ns	ns	**	ns	ns	ns	ns	ns	ns
mode vs 2:1	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
mode vs 4:1	ns	ns	ns	**	ns	ns	ns	*	ns	ns
mode vs 6:1	ns	ns	ns	*	ns	ns	ns	*	ns	ns
mode vs 8:1	ns	ns	ns	**	ns	ns	ns	**	ns	ns

A: Comparison of the body weight of the mice in the modeling group and the sham operation (sham) group; B: Changes in the body weight of the mice in the administration group. (*P<0.05 vs model,

**P<0.01vs model, ^# P<0.05vs sham, ^## P<0.01vs sham; ns: not significant)

3.2 Mice survival period

At the end of the experiment, the survival of animals in each group is shown in Figure 18 and Table 10. Of these, most mice died after 2% DSS treatment at week 8. The experimental results of the 8:1 and 6:1 ratios of breviscapine asarum (inventive process) dicaffeoate salt: breviscapine salt ratio of mice have higher survival rates, suggesting that these drugs have the effect of improving the survival period of tumor-bearing animals.

Table 10. Effects of Dengzhanxixin on the survival rate of tumor-bearing mice

group	sham	model	ds	1.25:1	2:1	4:1	6:1	8:1
Endpoint Survival (Only)	10	8	5	5	8	7	10	11
Enrolled (only)	10	12	12	12	12	12	12	12
Survival rate (%)	100	66.7	41.7	41.7	66.7	58.3	83.3	91.7

3.3 Mouse colorectal tumor indicators

At the end of the experiment, the mice were sacrificed and the colorectum was taken for measurement. The results are shown in Figure 19. Compared with the sham operation group, the colorectal length of the model group was significantly shorter, and the experimental Dengzhanxixin (inventive process) dicaffeoate Salt: The 8:1 ratio of breviscapine salt can significantly restore the length of the colorectum. There was no statistical difference between the other groups and the model group.

The number and volume of tumors in the colorectum of mice were measured, and the results were shown in Figures 20 and 21, compared with the model group. The experimental breviscapine (inventive process) dicaffeoate salt: breviscapine salt 8:1 ratio group significantly reduced the number of tumors (Figure 20) and tumor burden (Figure 21), and the 6:1 ratio group also reduced tumors number and tumor burden. The effect of other groups was not obvious.

Analysis and Conclusion

(1) The Dengzhan Asarum extract under the new process has a better anti-intestinal cancer effect;

(2) The anti-intestinal cancer effect of breviscapine is related to the ratio of two important components, dicaffeoate and breviscapine;

(3) The control drug breviscapine has no obvious anti-intestinal cancer effect, which may be related to the preparation process, active ingredient content and proportion of the drug.

Detailed ways:

Example 1: Preparation of raw material drug extract

Take 2000 g of Asarum radix and add 50% aqueous ethanol to decoct twice, 2 hours each time, filter, combine the filtrates, and concentrate under reduced pressure to form an extract (relative density 1.15-1.25, 75°C); add 5 times the amount of the extract dissolved in water, add 5% sodium hydroxide solution with stirring, adjust pH to 7.5-8.5, filter, add 10% sulfuric acid solution to adjust pH to 1-3, leave overnight, filter, collect filtrate and precipitate, precipitate with ethanol, and then Add 5% sodium hydroxide to adjust the pH value to 7-8, spray dry to obtain powder 1; adjust the pH value of the acidified filtrate to 7-9, use a 100nm ceramic membrane for clarification, and use a 350D organic membrane for the clarified solution obtained after clarification. Concentrated, the concentrate was passed through a 30-60 mesh polyamide chromatography column (diameter to length ratio 1:4), eluted with water for 3 column volumes, eluted with 65% ethanol for 4 column volumes, and collected the ethanol eluent, After concentration, the pH value is adjusted to 7-9 and spray-dried to obtain powder 2; powder 1 and powder 2 are mixed in proportion to obtain a mixture of scutellarin sodium salt powder and caffeate sodium salt powder 1:4 to obtain the medicine of the present invention API composition ingredients of the composition. Among them, powder 1 contains 36.3% of scutellarin sodium salt, a total of 5.4 g. Powder 2 contains 21.0% of dicaffeic acid ester salt in a total of 30.9 g and monocaffeic acid ester salt 9.1 g. Among them, the simple substituted dicaffeoate salt was 19.0 g, and the dicaffeoyl octanoate was 11.9 g. The obtained total mixed powder of Asarum radix 162g.

Example 2: Preparation of raw drug extracts

Take 2000 g of Asarum radix and add 50% aqueous ethanol to decoct twice, 2 hours each time, filter, combine the filtrates, and concentrate under reduced pressure to form an extract (relative density 1.15-1.25, 75°C); add 5 times the amount of the extract Dissolve in water, add 5% sodium hydroxide solution with stirring, adjust pH to 7.5-8.5, filter, add 10% sulfuric acid solution to adjust pH to 1-3, leave overnight, filter, collect filtrate and precipitate, precipitate with ethanol, reduce Press-dried to obtain powder 1; the acidified filtrate was adjusted to pH 7-9, clarified with a 100nm ceramic membrane, the clarified solution obtained after clarification was concentrated with a 350D organic membrane, and the concentrated solution passed through a 30-60 mesh polyamide chromatography column (diameter to length ratio 1:4), after eluting 3 column volumes with water, eluting 4 column volumes with 65% ethanol, collecting the ethanol eluent, concentrating and drying under reduced pressure to obtain powder 2; 2. Mix according to the proportion to obtain a 1:4 mixture of scutellarin powder and caffeic acid ester powder, and obtain the raw material drug composition components of the pharmaceutical composition of the present invention. Among them, powder 1 contains 35.7% of scutellarin 5.6g in total. Powder 2 contained 21.5% of dicaffeic acid ester in a total of 31.4 g and monocaffeic acid ester 9.1 g. Among them, the simple substituted dicaffeoate was 18.5 g, and the dicaffeoyl octanoic acid was 12.9 g. 161.7 g of the total mixed powder of Asarum scutellariae was obtained.

Example 3: Preparation of raw drug extracts

Take 3000 g of Asarum radix and add 60% aqueous ethanol to decoct twice, 2 hours each time, filter, combine the filtrates, and concentrate under reduced pressure to form an extract (relative density 1.15-1.25, 75°C); add 5 times the amount of extract Dissolve in water, add 5% sodium hydroxide solution under stirring, adjust pH 7~9, filter, add 10% hydrochloric acid solution to adjust pH 1~3, leave overnight, filter, collect filtrate and precipitate, precipitate with ethanol, add Adjust the pH value to 7-8 with 5% sodium hydroxide, and spray dry to obtain powder 1;

The acidified filtrate was adjusted to pH 7-9, clarified with a 200nm ceramic membrane, the clarified solution obtained after clarification was concentrated with a 400D organic membrane, and the concentrated solution passed through a 30-60 mesh polyamide chromatography column (diameter to length ratio of 1). : 4), after eluting with 3 column volumes of water, eluting with 3 column volumes of 70% ethanol, collecting the ethanol eluent, adjusting the pH value to 7-9 after concentration and spray drying to obtain powder 2; 1 about 18.7g, powder 2 about 208.3g, mix powder 1 and powder 2 in proportion to obtain a mixture of scutellarin sodium salt powder and caffeic acid ester sodium salt powder 1:3 to obtain the medicinal composition of the present invention. API composition ingredients.

Among them, powder 1 contains 45% of scutellarin sodium salt, a total of 8.4 g. Powder 2 contained 18.6% of dicaffeoate salt in a total of 38.7 g and 7.3 g of monocaffeate salt. Among them, the simple substituted dicaffeoate salt was 18.3 g, and the dicaffeoyl octanoate was 20.4 g.

Example 4: Preparation of raw drug extracts

Take 3000 g of Asarum radix and add 65% aqueous ethanol to decoct twice, 2 hours each time, filter, combine the filtrates, and concentrate under reduced pressure into extract (relative density 1.15-1.25, 75 ° C); add 5 times the amount of extract Dissolve in water, add 5% sodium hydroxide solution under stirring, adjust pH 7~9, filter, add 10% hydrochloric acid solution to adjust pH 1~3, leave overnight, filter, collect filtrate and precipitate, precipitate with ethanol, reduce Pressed powder 1; the acidified filtrate was adjusted to pH 7-9, clarified with a 200nm ceramic membrane, the clarified solution obtained after clarification was concentrated with a 400D organic membrane, and the concentrated solution passed through a 30-60 mesh polyamide chromatography column ( The ratio of diameter to length is 1:4), after eluting with 3 column volumes of water, eluting with 3 column volumes of 70% ethanol, collecting the ethanol eluent, concentrating and adjusting the pH value to 7-9 for spray drying to obtain Powder 2; obtain about 25.3 g of powder 1 and about 223 g of powder 2, mix powder 1 and powder 2 in proportion to obtain a mixture of breviscapine and dicaffeoate sodium salt powder 1:3.5 to obtain the medicinal combination of the present invention ingredient of the drug substance.

Among them, powder 1 contains 32% scutellarin 8.1g in total. Powder 2 contained 12.7% of dicaffeoate salt in a total of 28.4 g and 7.3 g of monocaffeate salt. Among them, the simple substituted dicaffeoate salt was 13.4 g, and the dicaffeoyl octanoate was 15 g.

Example 5: Preparation of API Extract

Take 2500 g of Asarum sinensis and add 75% concentration of water ethanol to decoct twice, 2 hours each time, filter, combine the filtrates, and concentrate under reduced pressure into extract (relative density 1.15-1.25, 75 ° C); add 5 times the amount of extract Dissolved in water, add 5% sodium hydroxide solution with stirring, adjust pH 7.5~9.0, filter, add 10% sulfuric acid solution to adjust pH 1~3, leave overnight, filter, collect filtrate and precipitate, precipitate with ethanol, add Adjust the pH value to 7-8 with 5% sodium hydroxide, and spray dry to obtain powder 1;

The acidified filtrate was adjusted to pH 7-9, clarified with a 100nm ceramic membrane, and the clarified solution obtained after clarification was then concentrated with a 300D organic membrane, and the concentrated solution passed through a 30-60 mesh polyamide chromatography column (diameter to length ratio 1: 4), after eluting with 3.5 column volumes of water, eluting with 3 column volumes of 75% ethanol, collecting the ethanol eluent, concentrating and adjusting the pH value to 7-9 by spray drying to obtain powder 2; obtain 13.5g of Powder 1 and 164 g of powder 2, the obtained scutellarin sodium salt powder and caffeic acid ester sodium salt powder are mixed according to 1:2 to obtain the raw material drug composition components of the pharmaceutical composition of the present invention. Among them, powder 1 contains 51% of scutellarin sodium salt, totaling 6.9 g. Powder 2 contained 19.5% of dicaffeic acid ester salt, a total of 32.0 g, and 9.7 g of monocaffeic acid ester salt. Among them, the simple substituted dicaffeoate salt was 19.6 g, and the dicaffeoyl octanoate was 12.4 g.

The inventor also used 10%, 30%, 40%, 45%, 50%, 55%, 60%, 75%, 90% of different concentrations of aqueous ethanol to extract Asarum sinensis, and other steps were the same as in Example 1. , after being acidified and concentrated through the membrane, the spray dry powder is obtained. The experiment proves that 10-90% ethanol extraction can get the lampshade extract, but from the yield, membrane passing rate, impurity content, powder bulk density and uniformity, industrial Measured by indicators such as production cost, the extract obtained with more than 40% ethanol is relatively better than the ethanol extract with less than 40%, and the ethanol extract with 40-75% is obviously better and more suitable for the process of the present invention, and 50% is the most preferred. % ethanol to extract Asarum sinensis.

Embodiment 6: the difference of active ingredient content in the inventive process and the existing process

Inventive process: take 2000 g of Asarum radix and add 50% hydrous ethanol to decoct twice, 2 hours each time, filter, combine the filtrates, and concentrate under reduced pressure to form an extract (relative density 1.15-1.25, 75° C.); Dissolve 5 times the amount of water, add 5% sodium hydroxide solution with stirring, adjust pH to 7.5-8.5, filter, add 10% sulfuric acid solution to adjust pH 1-3, leave overnight, filter, collect filtrate and precipitate, and precipitate ethanol After refining, 5% sodium hydroxide was added to adjust the pH to 7-8, and spray-dried to obtain powder 1; the acidified filtrate was adjusted to pH 7-9, clarified with a 100nm ceramic membrane, and the clarified solution obtained after clarification was then clarified with 350D The organic membrane was concentrated, and the concentrated solution was passed through a 30-60 mesh polyamide chromatography column (diameter to length ratio of 1:4), eluted with water for 3 column volumes, eluted with 65% ethanol for 4 column volumes, and collected the ethanol wash. Deliquoring, purifying with ethyl acetate, adjusting the pH value to 7-9 after concentration and spray drying to obtain powder 2; mixing 15 g of powder 1 and 15 g of powder 2 to obtain breviscapine sodium salt powder and caffeate sodium salt powder.

Existing technology: take 2000g of Asarum radix, extract twice with 80% ethanol under reflux, the first time is 1.5 hours, the second time is 1 hour, the extracts are combined, about 640g of activated carbon is added, boiled, filtered, and the filtrate is concentrated under reduced pressure to Thick paste, add appropriate amount of starch, dry under reduced pressure, pulverize and sieve. Obtain Asarum radix extract powder.

Taking the weight of the total powder as the denominator and the weight of each type of ingredients as the numerator to calculate the content Table 11

Table 11. Comparison of the components of the existing process and the inventive process

Breviscapine is the extract specified in the pharmacopoeia, and the main components are scutellarin and other substances, such as scutellarin, apigenin, scutellarin, etc., but the content is expressed as scutellarin.

Example 5: Preparation of capsules

Take 167 g of the extraction mixture prepared in Example 1, add 18 g of starch, mix well, and fill the capsules to obtain the final product.

Example 6: Preparation of capsules

Take 151 g of the extraction mixture prepared in Example 2, add 29 g of starch, mix well, and fill the capsules to obtain the final product.

Example 7: Preparation of capsules

Take 142 g of the extraction mixture prepared in Example 1, add 34 g of starch, and 4 g of magnesium stearate, mix well, and fill the capsules.

Example 8: Preparation of Tablets

Take 151 g of the extraction mixture prepared in Example 2, 100 g of starch, and 10 g of dextrin, pass through a 14-mesh sieve for granulation, ventilate and dry at 60-70° C., and add 3 g of magnesium stearate. Compressed into tablets and coated.

Example 9: Preparation of dripping pills

Take 15 g of the extraction mixture prepared in Example 3, put in 45 g of polyethylene glycol 4000, mix evenly, melt, drop into low-temperature liquid paraffin, select pellets, and remove the liquid paraffin.

Embodiment 10: the preparation of oral liquid

Take 20 g of the extraction mixture prepared in Example 1, mix it with 300 g of honey, 50 g of sucrose, 2 g of sodium benzoate and 300 ml of distilled water, heat to dissolve, and filter for heat preservation.

Example 11: Preparation of Granules

Take 9 g of the extraction mixture prepared in Example 3, mix it with 40 g of microcrystalline cellulose, add 3% povidone ethanol solution to make soft material, pass through an 18-mesh sieve to make granules, dry at 600 ° C for 30-45 minutes, granulate, add 4g talcum powder, mix well, granulate, bag, and that’s it.

Example 12: Preparation of Soft Capsules

Take 120 g of the extraction mixture prepared in Example 3, add 5% glycerol, 1% glycine, add 400 to 400 g of polyethylene glycol, mix well, and press into 1000 soft capsules.

Claims (10)

1. An oral preparation containing caffeic acid ester and scutellarin, characterized in that the oral preparation contains caffeic acid ester or its salt and scutellarin or its salt and pharmaceutically acceptable excipients, caffeic acid ester or its salt and scutellarin The weight ratio of the anthocyanins or their salts is 5:1 to 30:1.
2. The oral preparation according to claim 1, wherein the caffeic acid ester or its salt comprises dicaffeoate or its salt and mono-caffeic acid or its salt, wherein the dicaffeoate or its salt and lampshade The weight ratio of anthocyanin or its salt is 3.8:1 to 8:1, and the weight ratio of monocaffeic acid ester or its salt to dicaffeoate or its salt is 1:2 to 1:8.
3. The oral preparation according to claim 2, wherein the dicaffeoyl ester or its salt comprises 1,3-O-dicaffeoylquinic acid or its salt, 3,4-O-dicaffeoyl Quinic acid or its salts, 3,5-O-dicaffeoylquinic acid or its salts, 4,5-O-dicaffeoylquinic acid or its salts, Fembol ethyl or its salts, scutellarin or its salts; monocaffeic acid ester salts include 3-O-caffeoylquinic acid or its salts, 4-O-caffeoylquinic acid or its salts, 5-O-caffeoylquinic acid or its salts, lampshade Anthoside or its salt.
4. The oral preparation according to claim 3, wherein in the dicaffeoyl quinine, 1,3-O-dicaffeoylquinic acid or its salt, 3,4-O-dicaffeoylquinine Acid or its salt, 3,5-O-dicaffeoylquinic acid or its salt, 4,5-O-dicaffeoylquinic acid or its salt are collectively referred to as simple substituted dicaffeoate or its salt; said The phosphatidyl ester B or its salt and scutellarin or its salt are isomers, which are dicaffeoyl caprylic acid or its salts, wherein simply substituted dicaffeoyl ester or its salt and dicaffeoyl The ratio of caprylic acid or salt is 2:1 to 0.9:1.
5. The oral preparation according to any one of claims 1-4, wherein the oral preparation is a hard capsule, a soft capsule, a tablet, a granule, an oral liquid, a dripping pill, a water pill, a powder, or a pill.
6. The oral preparation of claim 5, wherein the weight ratio of the caffeic acid ester or its salt to breviscapine or its salt is 5:1 to 30:1, and the caffeic acid ester salt and the scutellarin salt are Sodium or potassium salts or other medicinally acceptable and water-soluble salts;
7. The preparation method of the oral preparation as claimed in claim 1, characterized in that: taking Dengzhan Asarum and adding 10-90% concentration of water-containing ethanol to extract, the extract is concentrated under reduced pressure to form an extract; the extract is dissolved in water and adjusted to pH 7-9, Filtration, adding acid to adjust pH 1-3, leaving overnight, filtering, collecting filtrate and precipitation, precipitation refining with water and ethanol, drying to obtain breviscapine; after precipitation refining, adding alkali to adjust pH to 7-8, spray drying to obtain Breviscapine salt; the acidified filtrate is adjusted to pH 7-9, clarified with a ceramic microfiltration membrane, the clarified solution obtained after clarification is then concentrated with an organic nanofiltration membrane, the concentrated solution is passed through a polyamide chromatographic column and washed with water After elution, eluting with 50-80% ethanol, collecting the ethanol eluent, concentrating and drying to obtain caffeic acid ester; after concentrating the ethanol eluent, adjusting the pH to 7-9, filtering, and spray-drying the filtrate to obtain caffeic acid ester salt ; Mix caffeic acid ester or its salt and breviscapine or its salt in a ratio of 5:1 to 30:1 to obtain breviscapine oral preparation, add appropriate auxiliary materials, can be made into tablets, capsules, soft capsules , drop pills, granules and oral liquid preparations.
8. The preparation method according to claim 7, characterized in that: the pH value of the alkali-adjusting solution is NaOH, Na ₂ CO ₃ , NaHCO ₃ , KOH, K ₂ CO ₃ or KHCO ₃ , or others that can be used to adjust The pH value of the alkaline solution; the pH value of the acid adjustment solution is HCl, H ₂ SO ₄ or H ₃ PO ₄ , or other acid solutions that can be used to adjust the pH value.
9. The application of the oral preparation of claim 1 in preparing a medicine for treating cardiovascular and cerebrovascular diseases and non-alcoholic fatty liver diseases caused by hyperlipidemia.
10. The application of the oral preparation of claim 1 in the preparation of antitumor drugs.

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