CN106176843A - Willow herb suppresses HDAC1 enzyme effective site and preparation method and application - Google Patents

Abstract

The active parts of willow orchid for inhibiting HDAC1 enzyme, its preparation method and application belong to the technical field of plant extracts. A mixture of one or two or more selected from the ethyl acetate fraction of Willowberry, the n-butanol fraction of Willowberry and the aqueous phase extraction fraction of Willowberry in any mass ratio. Grind the whole willow orchid, add 65%~95% ethanol aqueous solution to reflux for extraction, concentrate and dry the obtained extract to obtain extract; Ethyl ester and n-butanol are used as solvents to extract the extract dispersion liquid, and after the solvent is evaporated, the ethyl acetate part and the n-butanol part of the willow flower are obtained; the extracted extract dispersion liquid is concentrated and dried to obtain the water phase extraction of the willow flower parts. The application of the effective portion of the willow orchid of the present invention for inhibiting HDAC1 enzyme in the preparation of drugs for inhibiting HDAC1 enzyme can be used for the preparation of antitumor drugs.

Description

The active part, preparation method and application of Willow Orchid for inhibiting HDAC1 enzyme

technical field

The invention relates to an effective part of willow orchid for inhibiting HDAC1 enzyme, a preparation method and an application, and belongs to the technical field of plant extracts.

Background technique

Epilobium angustifolium L. is a perennial stout herbaceous plant of the Onagraceae genus Epilobium angustifolium L., which is a traditional medicinal plant in Tibetan medicine. It is mainly born in forest margins, under forests and wet grasslands of river valleys at an altitude of 2800-4250 meters. Distributed in the southwest, northwest, north and northeast of China. It is widely distributed in the northern temperate zone, and it is also found in North America, Europe to Japan, Asia Minor to the Himalayas and other places. The whole herb of Willow Orchid is bitter in taste, non-toxic, and has the functions of reducing swelling and diuresis, producing breast milk, and moistening the intestines. Indications of insufficient milk, qi deficiency and edema. Geer Gesang Tashi recorded in "Practical Tibetan Medicine Name Library" that it has the functions of clearing away heat and promoting gallbladder, stopping diarrhea and killing insects.

Malignant tumor is one of the major diseases that seriously endanger human health and has become the leading cause of human death. The prevention and treatment of malignant cancer cells has become an important topic concerned by the medical field. However, the onset of malignant tumors is hidden, the etiology is complex, and the pathological changes are diverse, so the treatment is still limited to the diversity and recurrence of cancer diseases. Although the carcinogenic causes of different cancers may be different, their tissue observations all show similar cell cycle disorders and rapid and uncontrollable cell growth and metastasis. Therefore, research on some family proteins involved in cell cycle regulation has become an important factor in promoting cancer therapy. Histone deacetylases (HDACs) are a class of proteases that play an important role in the structural modification of chromosomes and the regulation of gene expression. In general, the acetylation of histones is conducive to the dissociation of DNA and histone octamers, and the relaxation of nucleosome structure, so that various transcription factors and co-transcription factors can specifically bind to DNA binding sites and activate genes transcription. In cancer cells, overexpression of HDAC leads to enhanced deacetylation, which increases the attractive force between DNA and histones by restoring the positive charge of histones, making the relaxed nucleosomes very compact, which is not conducive to specific genes expression of some tumor suppressor genes. Histone deacetylase inhibitors (histone deacetylase inhibitors, HDACi) can regulate the expression and stability of apoptosis and differentiation-related proteins by increasing histone acetylation in specific regions of chromatin, and induce apoptosis and differentiation. Become a new class of anticancer drugs. HDACi not only has a good therapeutic effect on a variety of hematological tumors and solid tumors, but also has the advantages of relatively high selectivity for tumor cells and low toxicity.

The applicant found in the research that there are few raw materials for anti-tumor drugs at present, especially the raw materials of traditional Chinese medicines used to prepare drugs that inhibit HDAC1 enzyme are relatively scarce, and the discovery of raw materials for new drugs is an urgent problem that needs to be solved. Willow Orchid plant contains polyphenols, flavonoids, triterpene acids, tannin compounds and other chemical components. In this field, the main functions of Willow Orchid are clearing heat and promoting gallbladder, reducing swelling and diuresis, breast milk, and moistening intestines. At present, it has not inhibited HDAC1 Research on enzymes.

Contents of the invention

The technical problem to be solved by the present invention is: to overcome the deficiencies of the prior art, to provide the active part of willow orchid for inhibiting HDAC1 enzyme and its preparation method and application. HDAC1 enzyme drug has good anti-tumor effect.

The technical solution adopted by the present invention to solve its technical problems is: the active part of the willow flower inhibiting HDAC1 enzyme, which is characterized in that: it is selected from the parts of the ethyl acetate part of the willow flower, the n-butanol part of the willow flower, and the aqueous phase extraction part of the willow flower A mixture of one or more than two in any mass ratio.

The HDAC1 enzyme-inhibiting effective part of the willow flower is characterized in that it is selected from the n-butanol part of the willow flower, or a mixture of the n-butanol part and the water phase extraction part of the willow flower in any mass ratio.

The preparation method of the active part of the willow orchid inhibiting HDAC1 enzyme is characterized in that it comprises the following steps: crush the whole willow orchid, add an aqueous ethanol solution with a volume percentage concentration of 65% to 95% for reflux extraction, concentrate and dry the obtained extract, and obtain Extract; add distilled water to disperse the extract to obtain the extract dispersion, then use petroleum ether, ethyl acetate and n-butanol as solvents to extract the extract dispersion, and evaporate the solvent respectively to obtain the ethyl acetate part of willow orchid, willow orchid Part of n-butanol: Concentrate and dry the extracted extract dispersion to obtain the aqueous phase extraction part of Willowberry.

The reflux extraction is ultrasonic-assisted reflux extraction; the specific operation of ultrasonic-assisted reflux extraction is as follows: add the crushed willow orchid whole herb to ultrasonic extraction with a volume percentage concentration of 65% to 95% ethanol aqueous solution for 0.5 to 3 hours, filter, and add the volume percentage to the filter residue Concentration of 65% ~ 95% ethanol aqueous solution reflux extraction 1 ~ 3 times, combined extracts, that is.

The application of the active part of the willow orchid for inhibiting HDAC1 enzyme is characterized in that it is used in the preparation of drugs for inhibiting HDAC1 enzyme.

The preparation of the HDAC1 enzyme-inhibiting drug is prepared by mixing one or more of the mixtures of the ethyl acetate part of the willow flower, the n-butanol part of the willow flower and the aqueous phase extraction part of the willow flower with the pharmaceutical excipients. prepared oral or injectable formulations.

The injection preparation is liposome injection, nanoparticle injection or microsphere injection.

The oral preparation is powder, tablet, granule, capsule or solution.

The description for the present invention is as follows:

In the prior art, the routine use of Willow Orchid is to treat cold, fever and sores, and there is no related report that it has anti-tumor effect. The applicant found through a large number of studies that some extracts from Willow Orchid also have antibacterial and antitumor effects, but their efficacy is not obvious, and their specific effective parts cannot be determined. Therefore, what kind of solvent and what kind of preparation method can be used to obtain the effective part with the best effect of inhibiting HDAC1 enzyme activity is a problem that must be solved. Through research, the applicant found that: using the preparation method of the present invention, the ethyl acetate fraction, the n-butanol fraction, and the aqueous phase extraction fraction of Salixia, all of which have a good effect on inhibiting HDAC1 enzyme. Among them, the n-butanol moiety of Willow Orchid has the best effect on inhibiting HDAC1 enzyme activity.

In the preparation method, only reflux extraction can be used. Ultrasonic assisted reflux extraction is preferred, which has higher yield and extraction efficiency. The specific operation of ultrasonic-assisted reflux extraction is as follows: after ultrasonic extraction, filter to obtain ultrasonic extract and filter residue, add 65%~95% ethanol aqueous solution to the filter residue for reflux extraction, and combine ultrasonic extract and reflux extract. Further explanation, the specific operation of reflux extraction is as follows: directly add crushed medicinal materials or filter residue after ultrasonic extraction into the reflux heating device, add ethanol aqueous solution, heat extraction, let cold filter to obtain an extract and filter residue; Add ethanol aqueous solution to the mixture, heat, carry out the second reflux extraction, let it cool and filter to obtain the second extract and filter residue (the above is reflux extraction twice); combine multiple extracts to obtain the reflux extract. Preferably, the reflux extraction is performed for 0.5-2 hours each time; the ethanol aqueous solution added during each reflux extraction does not cover the surface of the medicinal material by 1cm-2cm. The temperature of reflux extraction is the conventional temperature of ethanol reflux extraction, which needs to be higher than the boiling point of ethanol, preferably 80°C~100°C.

In the preparation method, the extract is a crude extract, and different solvents are used to extract the extract dispersion, which refers to sequentially adding petroleum ether to the extract dispersion to extract and separate the petroleum ether extract, adding ethyl acetate to the extract dispersion Extract and separate to obtain ethyl acetate extract, add n-butanol to the extract dispersion to extract and separate to obtain n-butanol extract; take ethyl acetate extract and n-butanol extract to volatilize and remove the solvent respectively, and the obtained The ethyl acetate part and the n-butanol part of Willowberry; the remaining extract dispersion after extraction (the solvent is water), volatilizes and removes the solvent to obtain the aqueous phase extraction part of Willowberry. The aqueous phase extraction part can also be called the aqueous phase extract, and the aqueous phase extraction part is easily soluble in water; in contrast, petroleum ether, ethyl acetate and n-butanol are organic phases, and the petroleum ether part, ethyl acetate part and n-butanol The corresponding parts are easily soluble in petroleum ether, ethyl acetate and n-butanol. Among them, the ethyl acetate part of Willowberry contains alkaloids and flavonoids; the n-butanol part of Willowberry contains saponins and phenolic compounds; the aqueous phase extraction part of Willowberry contains polysaccharides and organic acid compounds. To evaporate the solvent is to evaporate the solvent. The solvent can be volatilized by heating at normal pressure to volatilize the solvent (petroleum ether, ethyl acetate and n-butanol). Preferably, the solvent is evaporated and concentrated under reduced pressure. This method has high efficiency and can prevent heat-sensitive components from losing their medicinal properties due to high temperature.

Preferred dosage forms are oral formulations or injection formulations. Oral preparations and injection preparations are the best routes of administration. The oral preparations described here are gastrointestinal administration preparations. Preferably, the oral preparations are sustained-release and controlled-release oral preparations. Others for parenteral administration, such as preparations for respiratory tract administration (sprays, aerosols, powder sprays); preparations for skin administration (external solutions, lotions, liniments, ointments, plasters, pastes) preparations, patches); film administration preparations (eye drops, nasal drops, ophthalmic ointment, gargle, sublingual tablets); cavity administration preparations (suppositories). The injection preparations can be conventional injection preparations. Classified according to the drug delivery system, preferably, the injection preparation of the present invention is liposome injection, nanoparticle injection or microsphere injection; other, the injection of the present invention can also be microcapsule injection, polymer micelle injection, microsphere injection Milk or submicroemulsion injections, submicron injections or gel injections, injections of the above drug delivery systems can prolong the circulation time of drug carriers in the body, prolong the residence time of drug-loaded particles at the absorption site, and control the burst release of drugs in the early stage of release effect. Pharmaceutical excipients include pharmaceutical carriers, as well as solvents, solubilizers, co-solvents, emulsifiers, suspending agents, clarifying agents, deflocculating agents, flavoring agents, colorants, preservatives, chemical sterilants, adsorbents, filter aids Agents, antioxidants, pH regulators, isotonic regulators, diluents, binders, wetting agents, disintegrants, lubricants, glidants, anti-adhesives, sustained release agents, controlled release agents, packaging One or more of coating materials, film-forming materials, and capsule materials.

Compared with the prior art, the beneficial effects of the active part, preparation method and application of the willow orchid of the present invention for inhibiting HDAC1 enzyme are:

1. The HDAC1 enzyme-inhibiting effective part of the willow orchid can effectively inhibit the activity of HDAC1 enzyme, and is used for preparing a drug for inhibiting HDAC1 enzyme, and has a good anti-tumor effect. In this field, Willow Orchid has the functions of diuresis and dampness, regulating qi and reducing swelling, promoting blood circulation and regulating menstruation. During the research, the applicant found that there is a shortage of raw materials for anti-tumor drugs, and found that willow orchid also has anti-tumor effects through research. Through the design of preparation methods and drug efficacy screening, the types and types of enzymes that will be inhibited by willow orchid were determined through research. The best effective part, the applicant has put in a lot of creative labor in the process of discovering and solving the above problems. For the first time, the applicant conducted drug efficacy screening on the HDAC1 enzyme, and determined through a large number of researches the ethyl acetate part, the n-butanol part of the willow flower, and the aqueous phase extraction part of the willow flower, or a mixture of two or more in any mass ratio All have good effect of inhibiting HDAC1 enzyme, HDAC1 enzyme survival rate is 18.94%~36.02%, and can be used to develop new anti-tumor Tibetan medicine. Since the effective fractions obtained by different solvent extractions have different inhibitory abilities to HDAC1 enzyme activity, the applicant determined through further research that: the n-butanol fraction of Willowberry has the best inhibitory effect on HDAC1 enzyme, and its HDAC1 enzyme survival rate is 18.94%. To sum up, the applicant discovered the new efficacy of willow orchid for the first time through research, determined the type of enzyme it inhibited through research, designed the preparation method and finally obtained the effective part that has a good inhibitory effect on HDAC1 enzyme, the above process It took a lot of creative work.

2. The preparation method of the active part of the willow orchid for inhibiting HDAC1 enzyme is convenient for extraction and has high extraction efficiency. In the preparation method, the applicant designed to add an aqueous ethanol solution with a volume percentage concentration of 65%~95% for ultrasonic-assisted reflux extraction, which improved the extraction efficiency; during the extraction, petroleum ether, ethyl acetate and n-butanol were used as solvents to extract the extract dispersion , the ethyl acetate fraction, the n-butanol fraction and the aqueous phase extraction fraction of Willowberry obtained by the above solvent extraction sequence have good inhibitory effect on HDAC1 enzyme.

3. The present invention expands the raw material channel for inhibiting HDAC1 enzyme, expands the use of willow orchid, and makes willow orchid develop into a new raw material for inhibiting HDAC1 enzyme medicine, which can significantly increase the added value of willow orchid.

detailed description

Embodiments 1 to 3 are specific implementations of the effective part and preparation method of the active part of Willow Orchid for inhibiting HDAC1 enzyme of the present invention, wherein Embodiment 1 is the best embodiment.

Example 1

The preparation method comprises the following steps: pulverizing the whole willow orchid, ultrasonically extracting with an aqueous solution of ethanol with a volume percentage concentration of 75% to 85% for 1 hour, and then filtering to obtain an ultrasonic extraction liquid and a filter residue, and adding 75% to 85% of the volume percentage concentration to the filter residue. Reflux extraction with ethanol solution for 3 times, each time for 0.5-1 hour, filter to obtain reflux extract, combine ultrasonic extraction and reflux extract, concentrate and dry to obtain extract; add distilled water to disperse extract to obtain extract dispersion, and then Use petroleum ether, ethyl acetate and n-butanol as solvents to extract the extract dispersion liquid, and after evaporating the solvent, obtain the petroleum ether part, ethyl acetate part, and n-butanol part of Willowberry; the remaining extract after extraction The dispersion liquid is concentrated and dried to obtain the aqueous phase extraction part of willow orchid.

Example 2

The preparation method comprises the steps of: pulverizing the whole willow orchid herb, ultrasonically extracting 85%-95% ethanol aqueous solution by volume percentage for 3 hours, then filtering to obtain the ultrasonic extract and filter residue, and adding 85%-95% volume percentage concentration to the filter residue Reflux extraction with ethanol solution for 3 times, each time for 1-1.5 hours, filter to obtain reflux extract, combine ultrasonic extraction and reflux extract, concentrate and dry to obtain extract; add distilled water to disperse extract to obtain extract dispersion, and then sequentially Use petroleum ether, ethyl acetate and n-butanol as solvents to extract the extract dispersion liquid, and after evaporating the solvent, obtain the petroleum ether part, ethyl acetate part, and n-butanol part of Willowberry; the remaining extract after extraction The dispersion liquid is concentrated and dried to obtain the aqueous phase extraction part of willow orchid.

Example 3

The preparation method comprises the following steps: pulverizing the whole willow orchid herb, ultrasonically extracting it with an aqueous solution of ethanol with a concentration of 65% to 75% by volume for 0.5 hours, and then filtering to obtain an ultrasonic extract and filter residue, and adding ethanol with a concentration of 65% to 75% by volume to the filter residue The solution was refluxed twice, each time for 1.5-2 hours, filtered to obtain the reflux extract, combined with the ultrasonic extract and the reflux extract, concentrated and dried to obtain the extract; the extract was dispersed with distilled water to obtain the extract dispersion, and then sequentially used Petroleum ether, ethyl acetate and n-butanol are used as the solvent to extract the extract dispersion liquid, and after the solvent is evaporated, the petroleum ether part, the ethyl acetate part of the willow flower and the n-butanol part of the willow flower are obtained; the remaining extract after the extraction is dispersed The solution was concentrated and dried to obtain the aqueous phase extraction part of willow orchid.

Performance Testing

Take 0.4mg of the dried petroleum ether fraction, the ethyl acetate fraction, the n-butanol fraction and the aqueous phase extraction fraction of the willow flower, respectively, and dissolve them in 100 µL of dimethyl sulfoxide (DMSO) to form DMSO solution, sonicated for 5 minutes. Then take 1 µL of the DMSO solution of the dissolved sample and add 99 µL of the buffer solution to obtain the buffer solution of the part of the petroleum ether part of the willow flower, the buffer solution of the part of the ethyl acetate part of the willow flower, and the n-butyric acid of the willow flower that inhibit HDAC1 enzymes. The buffer solution of the alcohol part and the buffer solution of the aqueous phase extraction part of willowflower. The formula of the buffer solution is: 50 mM/L Tris-HCl, 0.137 mM/L NaCl, 2.7 mM/L KCl, 1 mM/L MgCl ₂ , 0.01% Tween 20, and the pH of the buffer is 8.0.

1. Anti-HDAC1 enzyme experiment.

The main experimental materials are:

HDAC1 enzyme, product of Cisbio Bioassays;

Tris-HCl, Shanghai Jiemei Gene Pharmaceutical Technology Co., Ltd.;

EDTA, Shanghai Curie Biotechnology Co., Ltd.;

DTT, Shanghai Sangon Bioengineering Co., Ltd.;

BSA, Shanghai Jianglai Biotechnology Co., Ltd.;

DMSO, Shanghai Eka Biotechnology Co., Ltd.;

NaCl (analytical pure), Tianjin Hongyan Chemical Reagent Factory;

NaOH (analytical grade) was purchased from Hongyan Reagent Factory, Hedong District, Tianjin.

The main experimental instruments are:

FLUOstar Omega automatic multi-functional microplate reader, Guangzhou Bio-Gene Biotechnology Co., Ltd., Guangdong Province;

MP511 pH meter electric meter, Shanghai Sanxin Co., Ltd.;

BRAND 8-channel pipette gun, Hangzhou Huier Instrument Co., Ltd., Zhejiang Province;

500µL pipette gun, 50µL pipette gun, Shanghai Rongtai Biochemical Instrument Co., Ltd.;

Electronic balance XPE105, Mettler-Toledo Instrument Co., Ltd.

The active part of the willow orchid obtained in Example 1 for inhibiting HDAC1 enzyme was used as the test drug. The representative experiments are the inhibition of HDAC1 enzyme activity by the petroleum ether fraction, the ethyl acetate fraction, the n-butanol fraction, and the aqueous phase extraction fraction of Salixia. The method is to take another 4 µL of the petroleum ether fraction (label D), the buffer solution of the ethyl acetate fraction (label E), the buffer solution of the n-butanol fraction of the willow flower (label F) and the water of the willow flower respectively. For the buffer solution (label G) of the phase extraction site, the following operations were performed: put 4 µL of the buffer solution (D, E, F, G) into a 96-well plate, then add 2 µL of HDAC1 enzyme, and react at room temperature After 5 minutes, add 4 µL of H3(1-21) K9 substrate, and incubate in an incubator at 37°C for 60 minutes. Add 5 µL SA-XL665 and 5 µL H3K9me0 antibody, measure the value at 665nm, and obtain the absorbance of the experimental group.

Put 4 µL of buffer solution in a 96-well plate, then add 2 µL of HDAC1 enzyme, react at room temperature for 5 minutes, add 4 µL of H3(1-21) K9 substrate, and incubate in an incubator at 37°C for 60 minutes. Add 5µL SA-XL665 and 5µL H3K9me0 antibody, measure at 665nm, and get the absorbance of the control group. Calculate the survival rate of HDAC1 enzyme: Survival rate (%) = absorbance of the experimental group/absorbance of the control group × 100%.

Table 1 The activity test results of the HDAC1 enzyme-inhibiting effective parts of Willow Orchid

.

In Table 1, * P<0.01, it can be seen from Table 1 that the ethyl acetate fraction, the n-butanol fraction and the aqueous phase extraction fraction of Willowberry all have good inhibitory effect on HDAC1 enzyme.

2. Anti-tumor activity test.

The cytotoxic activity of the effective part of the inhibitor HDAC1 enzyme of willow orchid on human tumor cells was determined by MTT method. Get the appropriate amount of each part of the obtained willow orchid in Example 1, as a test compound, add DMSO to dissolve, and use the MTT method to measure the effects on human colon cancer HCT-8, human liver cancer Bel-7402 cells, human gastric cancer BGC-803 cells, and human lung cancer respectively. Growth inhibition rate of adenocarcinoma A549 cells and human ovarian cancer A2780 cells.

Experimental method: digest the cells in the logarithmic growth phase with 0.25% trypsin-EDTA, prepare a single-cell suspension with a certain concentration, and inoculate 800-2000 cells/well in a 96-well plate according to the difference in cell growth speed , add 100 µL of cell suspension to each well. After 24 hours, add fresh medium containing different concentrations of test compounds and corresponding solvent controls, add 100 µL to each well (DMSO final concentration <0.1%), set 5-7 dose groups for each test compound, and set at least 3 in each group. After culturing at 37°C for 72 h, discard the supernatant, add 100 µL of freshly prepared serum-free medium containing 0.5 mg/mL MTT to each well, continue culturing for 4 h, discard the supernatant, and add 200 µL to each well DMSO dissolves the MTT nail hairpin precipitate, oscillate and mix with a micro-oscillator, measure the optical density (OD) with a microplate reader at a reference wavelength of 450nm, and a detection wavelength of 570nm. The tumor cells treated with solvent control are used as the control group, and the following formula is used Calculate the inhibition rate of the test compound on tumor cells, and calculate the IC ₅₀ according to the intermediate effect equation: inhibition rate = (average OD value of the control group - average OD value of the treatment group) ÷ average OD value of the control group × 100%. The extract in the preparation method, as well as the obtained fractions of petroleum ether, ethyl acetate, n-butanol and aqueous phase extraction of willowberry were screened for tumor cytotoxic activity in vitro, and the results are shown in Table 2.

Table 2 Screening results of in vitro tumor cytotoxic activity

.

Note: 1), HCT-8 is human colon cancer cells, Bel-7402 is human liver cancer cells, BGC-823 is human gastric cancer cells, A549 is human lung cancer cells, and A2780 is human ovarian cancer cells. 2), >50 indicates no anti-cytotoxic activity. It can be seen from Table 2 that the ethyl acetate fraction, the n-butanol fraction and the aqueous phase extraction fraction of Willowberry do have good anti-tumor effects.

3. In vitro anti-tumor experiment of S ₁₈₀ tumor-bearing mice.

1. The anti-tumor experiment method of _S180 tumor-bearing mice in each part of Example 1 is as follows:

a) Kunming mice aged 5-6 weeks were randomly selected and divided into groups, 10 mice in each group, half male and half male in each group (separated cages); the group names were: blank control group, cyclophosphamide group and treatment group; The drug groups include: low, medium and high doses of willowberry petroleum ether parts, low, medium and high doses of willowberry ethyl acetate parts, low, medium and high doses of willowberry n-butanol parts, willowberry aqueous phase extraction Parts in low, medium and high dose groups, the mass ratio of 1:1 mixture of the n-butanol part and the aqueous phase extraction part of Willowberry in the low, medium and high dose groups, the quality of the aqueous phase extraction part and ethyl acetate part of Willowberry Compared with the 1:1 mixture of low, medium and high dose groups, the mass ratio of the 1:1 mixture of the n-butanol part and the ethyl acetate part of willow flower group to the low, middle and high dose groups; carry out skin disinfection on each mouse;

b) Inoculate 0.2ml of S ₁₈₀ tumor fluid subcutaneously on the right forelimb (the number of S ₁₈₀ tumor cells is in the range of 2.0×10 ⁶ to 2.2×10 ⁶ );

c) Administration 24 hours after inoculation; the blank control group was intragastrically administered 10mL/kg of normal saline for injection; the cyclophosphamide group was intraperitoneally injected with cyclophosphamide 20mg/kg; the administration group was prepared by intragastric administration of Example 1 The petroleum ether part of Willowberry, the ethyl acetate part of Willowberry, the n-butanol part of Willowberry and the aqueous phase extraction part of Willowberry, the mixture of n-butanol part and aqueous phase extraction part of Willowberry in a mass ratio of 1:1, the willowberry The mass ratio of the aqueous phase extraction part and the ethyl acetate part of the willow flower is 1:1 mixture, the mass ratio of the n-butanol part of the willow flower and the ethyl acetate part of the willow flower is a mixture of 1:1; after the first administration, the first weight of the mouse is weighed every other day; Each group is administered once a day, and the dosage of each administration is detailed in the table below, and the administration is continuous for 14 days;

d) One hour after the last administration, the mice were killed, the last body weight of the mice was weighed, the tumor pieces were peeled off and weighed, and the tumor inhibition rate was calculated. Tumor inhibition rate = (average tumor weight of the blank control group - average tumor weight of the treatment group) ÷ average tumor weight of the blank control group × 100%, and the calculation results of the tumor inhibition rate were entered in Table 3.

Table 3 Effects of various parts of Example 1 on mouse S ₁₈₀ solid tumor

.

2. S ₁₈₀ tumor-bearing mice in each part of Example 2 The method is the same as in Example 1, and the data is entered in Table 4.

Table 4 Effects of various parts of Example 2 on mouse _S180 solid tumor

.

3. The method of the anti-tumor experiment on S ₁₈₀ tumor-bearing mice at each site in Example 3 is the same as in Example 1, and the data is entered in Table 5.

Table 5 The impact of each part of Example 3 on the mouse _S180 solid tumor

.

According to the applicant's statistics: compared with the blank control group, there was no significant difference in the weight change of the mice (P>0.05). In Tables 3 to 5: S ₁₈₀ refers to mouse ascites tumor cells. In Tables 3 to 5, the higher the tumor inhibition rate of S ₁₈₀ , the better the antitumor effect. It can be seen from Tables 3 to 5 that: First, Examples 1 to 3 all have good antitumor effects, and when the same dose is used at the same site in Examples 1 to 3, there is no significant difference in the antitumor effects among the groups. Secondly, compared with the blank control group, the middle and high dose groups of the ethyl acetate part of the willow flower, the high dose group of the n-butanol part of the willow flower, the high dose group of the n-butanol part of the willow flower, and the middle and high dose of the aqueous phase extraction part of the willow flower Group, medium and high doses of the mixture of n-butanol fraction and the aqueous phase extraction fraction of Willow Orchard with a mass ratio of 1:1. group, the mixture of n-butanol fraction and ethyl acetate fraction of Willowberry at a mass ratio of 1:1, the middle and high dose groups, and the cyclophosphamide chemotherapy group, all of which can significantly inhibit the growth of S ₁₈₀ tumors.

Example 4

The effective part of Willowberry is prepared as a tablet, and the following steps are adopted: mix 300 mg of ethyl acetate part of Willowberry and 100 mg of starch, add 40 mg of starch paste with a mass percentage concentration of 10% to make a soft material, sieve to obtain wet granules, and dry to obtain Dry granules, whole granules, add 4 mg of magnesium stearate, mix evenly, and compress into tablets.

Example 5

The preparation method of the granule: mix 5 parts of the water-phase extraction part of willow orchid, 1 part of sucrose, and 3 parts of dextrin according to the weight ratio, add an appropriate amount of 2-5 parts of ethanol aqueous solution with a mass percentage of 95%, and stir while adding, to prepare To obtain the soft material, dry the soft material, pass through a 16-mesh sieve, and pack it separately.

Example 6

The micropill capsule is composed of the following raw materials in parts by weight: 30 parts of the n-butanol part of willowberry, 5 parts of lecithin, 5 parts of sodium taurocholate, and 30 parts of microcrystalline cellulose;

The preparation method of micropill capsules: Mix the n-butanol part of willowberry, lecithin, sodium taurocholate and microcrystalline cellulose according to the proportion, pour into ethanol aqueous solution and stir to obtain soft material, and pour the soft material into the extruder Middle extrusion, spheronization to obtain granules, extrusion speed 250r/min, spheronization speed 800r/min, spheronization time 20min, drying, passing through a 24-30 mesh sieve to obtain pellets, and filling the pellets into the capsule shell to obtain .

Example 7

The preparation method of liposome injection:

1) Under the protection of nitrogen, dissolve 50g of cholesterol succinate, 250g of distearoylphosphatidylethanolamine, 40g of soybean lecithin, 50g of poloxamer-188, and 10g of willowberry n-butanol in 1500ml with a volume ratio of 1:1 in the organic solvent of ethanol and n-butanol, stir to make it dissolve to obtain a suspension; the suspension is concentrated under reduced pressure to evaporate the organic solvent to obtain a phospholipid film;

2) Under the protection of nitrogen, add 8000ml of phosphate buffer solution with a pH of 6.8 to the phospholipid membrane, stir to elute the phospholipid membrane and fully swell and hydrate it, and filter through a 0.22μm microporous membrane to obtain the n-butanol fraction of Willowberry of liposomes;

3) Under sterile conditions, add 100g of trehalose to the liposomes in the n-butanol part of Willowberry, stir evenly, ultrasonicate for 0.5-1 hour, add water for injection to volume, and filter through a 0.22μm microporous membrane , filling to obtain the liposome injection of the n-butanol part of the willow tree.

Example 8

The preparation method of nanoparticle injection: Take 25g of the ethyl acetate part of willow flower and 100g of poloxamer 407 and dissolve it in 3000ml of absolute ethanol, add 30ml of 1mol/L zinc chloride ethanol aqueous solution, stir and mix, and ultrasonically treat for 0.5~1 hour Make it dissolve to obtain a mixed solution; concentrate the mixed solution under reduced pressure to evaporate and remove the solvent, put it in a refrigerator at -20°C for 2 hours; take it out and add water for injection to constant volume, ultrasonic treatment for 0.5-1 hour, and filter through a 0.22 μm microporous membrane filter and filling to obtain the nanoparticle injection of the ethyl acetate part of willowflower.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention to other forms. Any skilled person who is familiar with this profession may use the technical content disclosed above to change or modify the equivalent of equivalent changes. Example. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims (8)

1. willow herb suppression HDAC1 enzyme effective site, it is characterised in that: selected from willow herb ethyl acetate extract, willow herb n-butanol portion, The mixture of one or more any mass ratio in willow herb aqueous phase extraction position.

2. suppress HDAC1 enzyme effective site according to the willow herb described in claim 1, it is characterised in that: selected from willow herb n-butyl alcohol portion Position, or the mixture of any mass ratio of willow herb n-butanol portion and willow herb aqueous phase extraction position.

3. the preparation method of the willow herb suppression HDAC1 enzyme effective site described in claim 1, it is characterised in that include following step Rapid: willow herb herb is pulverized, add concentration of volume percent 65% ~ 95% ethanol water reflux, extract, by dense for gained extracting solution Contract, be dried, obtain extractum；Extractum is added distilled water dispersion, obtain extract dispersion liquid, use the most successively petroleum ether, ethyl acetate and N-butyl alcohol, as solvent extraction extract dispersion liquid, obtains willow herb ethyl acetate extract, willow herb n-butanol portion after flinging to solvent respectively； Extract dispersion liquid after extraction is concentrated, is dried, obtains willow herb aqueous phase extraction position.

4. the preparation method of HDAC1 enzyme effective site is suppressed according to the willow herb described in claim 3, it is characterised in that: described Reflux, extract, is ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: the willow herb after pulverizing is complete Grass adds concentration of volume percent 65% ~ 95% ethanol water ultrasonic extraction 0.5 ~ 3 hour, filters, and filtering residue adds volume hundred Proportion by subtraction concentration 65% ~ 95% ethanol water reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.

5. the application of the willow herb suppression HDAC1 enzyme effective site described in claim 1, it is characterised in that: in preparation suppression Application in terms of HDAC1 enzyme medicine.

The application of willow herb the most according to claim 5 suppression HDAC1 enzyme effective site, it is characterised in that: described preparation presses down HDAC1 enzyme medicine processed be by willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extraction position in one or The mixture of two or more any mass ratioes, the oral formulations being mixed with pharmaceutic adjuvant or ejection preparation.

The application of willow herb the most according to claim 6 suppression HDAC1 enzyme effective site, it is characterised in that: described injection Preparation is lipidosome injection, nanoparticle injection or micro-balloon injection.

The application of willow herb the most according to claim 6 suppression HDAC1 enzyme effective site, it is characterised in that: described is oral Preparation is powder, tablet, granule, capsule or solution.