CN1305468C - Bolengsu compound and its prepn, medicine composition and use - Google Patents

Bolengsu compound and its prepn, medicine composition and use Download PDF

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CN1305468C
CN1305468C CNB03123965XA CN03123965A CN1305468C CN 1305468 C CN1305468 C CN 1305468C CN B03123965X A CNB03123965X A CN B03123965XA CN 03123965 A CN03123965 A CN 03123965A CN 1305468 C CN1305468 C CN 1305468C
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sex
baill
characterized
extract
described
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CNB03123965XA
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CN1486691A (en
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袁海龙
李仙义
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中国人民解放军第三○二医院
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Abstract

The present invention discloses an extraction of herpetospermumpeduncul-osum (Sex.) baill. And herpetin of cucurbitaceous plant, and also relates to a preparation method for the extraction of herpetospermumpeduncul-osum (Sex.) baill. And the herpetin, a medicine composition of the extraction of herpetospermumpeduncul-osum (Sex.) baill., the herpetin and a derivative of the herpetin, and a purpose of the herpetin, the derivative of the herpetin and the medicine composition as medicine, particularly a purpose for preparing antivirus medicine, hepatic injury resistant medicine and antineoplastic medicine. The present invention is verified by a cell experiment and an animal experiment, and herpetospermumpeduncul-osum (Sex.) baill. Has the advantages of high reactivity and little toxicity.

Description

Bolengsu chemical compound and method for making thereof and its pharmaceutical composition and purposes

Technical field

The present invention relates to cucurbitaceous plant ripple rib Herpetospermumpedunculosum (Sex.) Baill. extract and Bolengsu, the preparation method of ripple rib extract and Bolengsu, the pharmaceutical composition of ripple rib extract and Bolengsu and derivant thereof, ripple rib extract and Bolengsu and derivant thereof and pharmaceutical composition thereof are as the purposes of medicine, especially prepare antiviral, anti-liver injury, the purposes in the anti-tumor drug.Confirm that through cell experiment and zoopery Bolengsu has the advantage that activity is high, toxicity is little.

Background technology

Chronic viral hepatitis is the common transmittable disease of serious harm people ' s health.Only hepatitis B patient just accounts for 5% of world population according to statistics.Sickness rate height in China's viral hepatitis occupies first place in the world, and promptly about 80% hepatitis concentrates on China.The existing Patients with Viral Hepatitis of China reaches more than 3,000 ten thousand people at present, has every year nearly 500,000 people to die from and the hepatitis B diseases associated.Therefore, chronic viral hepatitis particularly the treatment of hepatitis B be the difficult problem of world the world of medicine always.

Because huge hepatitis B crowd demands urgently obtaining medical treatment, therefore, the medicine of chronic hepatitis is various in style on the China market at present, according to incompletely statistics, and kind surplus Chinese and western drugs has reached 700, but the medicine that curative effect is sure, side effect is little is few in number.Although the popularization of hepatitis vaccine prevents and treats hepatitis and plays huge impetus, hepatitis still increases with the speed in every year nearly 1,000,000.Therefore, the development novel structure, it is still very urgent to act on sure novel antihepatitis drug.Lamivudine is the medicine of the hepatitis virus resisting of generally acknowledging in the existing medicine, but lamivudine costs an arm and a leg, and requires the patient to take medicine for a long time, and be indefinite the course of treatment, and knock-on is strong and bigger Liver and kidney toxicity arranged after patient's drug withdrawal.Also do not have to kill hepatitis virus now and can protect hepatocellular medicine again.

Cucurbitaceous plant Herpetospermum pedunculosum (Sex.) Baill. ripple rib is a kind of plant that is grown in Tibet, does not see the medicinal report to it.

Summary of the invention

In order to overcome the weak point of prior art, the object of the present invention is to provide the antiviral of cucurbitaceous plant Herpetospermum pedunculosum (Sex.) Baill. ripple rib, anti-chemical damage, anti-tumor application.

Another object of the present invention is to provide the extract of cucurbitaceous plant Herpetospermumpedunculosum (Sex.) Baill. ripple rib.

Another purpose of the present invention is to provide cucurbitaceous plant Herpetospermumpedunculosum (Sex.) Baill. ripple rib preparation method of extract.

Another order of the present invention is to provide the pharmaceutical composition that contains ripple rib extract, and it comprises as carrier commonly used in the extract of cucurbitaceous plant Herpetospermum pedunculosum (Sex.) the Baill. ripple rib of active ingredient and the pharmaceutical field.

The antiviral that another purpose of the present invention is cucurbitaceous plant Herpetospemumpedunculosum (Sex.) Baill. ripple rib extract is provided and contains its pharmaceutical composition, anti-chemical damage, anti-tumor application.

Another purpose of the present invention is to provide a kind of new Bolengsu chemical compound.

Another purpose of the present invention is to provide a kind of preparation method of new Bolengsu.

Another purpose of the present invention is to provide the Bolengsu pharmaceutical composition, and it comprises as carrier commonly used in the Bolengsu chemical compound of active ingredient and isomer and the pharmaceutical field.

Another purpose of the present invention is to provide Bolengsu derivant and compositions thereof as antiviral, anti-chemical damage, the application of antitumor drug.

One aspect of the present invention relates to the Bolengsu shown in general formula (I), and relevant enantiomer, diastereomer, polymorph, ester, salt and solvation form:

The present invention also comprises the derivant of general formula compound I, their stereoisomer, their polymorph, their pharmaceutically acceptable salt, their pharmaceutically acceptable solvate.

The present invention relates to shown in general formula I acceptable derivates or prodrug on the chemical compound drug effect, the meaning is any acceptable salt of chemical compound shown in general formula I, the salt of ester and ester; Perhaps other derivant of The compounds of this invention, its when giving the receptor, can be direct or indirect chemical compound of the present invention is provided.Especially preferred derivant and prodrug are, when this chemical compound gives mammal, can increase The compounds of this invention stability, and/or can increase the The compounds of this invention bioavailability (for example, make the chemical compound of oral administration be easier to be absorbed into blood), and/or can increase this chemical compound in the distribution that is relevant to the biological compartment of these species (for example, liver, tumor).Prodrug is passing through linking to each other of covalent bond of any and carrier, and when this prodrug gave mammalian subject, it discharged in vivo as active this chemical compound of general formula I.Preferred prodrug comprises, but is not limited to, and increases water solublity or the group by biomembranous active permeability and connects the derivant that the structure as general formula I forms.The preparation of the prodrug of compound of Formula I is the substituent group that exists in the chemical compound by being modified at, and with such method, this modification is can be isolating for this chemical compound, no matter is with in the conventional method or body.Prodrug comprises that hydroxyl connects any substituent group in the chemical compound of general formula I, when giving mammalian subject, forms hydroxyl freely after the separation.The embodiment of prodrug comprises, but is not limited to, the acetylation of hydroxyl in the compound of Formula I, and formylated, etc.

The various polymorphs of the chemical compound shown in general formula I constitute part of the present invention and can prepare by the compound crystal under different conditions.For example, use different normally used solvents or their mixture to come crystallization; Crystallization under different temperature; Various pattern is cooled off; In crystallization process, from very near very slow scope.Polymorph can be followed progressive or cooling fast obtains by heating or melting compound.The existence of polymorph can NMR, IR, and the scanning of heat difference, X-light powder diffraction or other technology are proved conclusively.

Chemical compound of the present invention can be modified by connecting similar functional group increases the biological property of selection.This being modified at is known in the art, including but not limited to, those increases penetrate into specific biological compartment (for example liver), increase oral or intravenous bioavailability, increase dissolubility and allow to change metabolism by drug administration by injection, change excretory speed, or the like.

In order to finish the object of the invention, the present invention takes following technical scheme:

The extract of cucurbitaceous plant ripple rib Herpetospermum pedunculosum (Sex.) Baill. and the preparation of Bolengsu

Root, stem, leaf, the seed of crude drug cucurbitaceous plant ripple rib Herpetospermumpedunculosum (Sex.) Baill., its seed Herpetospermum caudigerum Wall. preferably, drying and suitable pulverizing in order to the contact area that increases with solvent, are raised the efficiency.

The extraction solvent of crude drug can make the mixture of water, alcohols or water and alcohols.Preferred alcohols, especially low-molecular-weight alcohol comprises methanol, ethanol, isopropyl alcohol, butanols etc., more preferably ethanol.During extraction quantity of solvent be former medicine weight 2-20 doubly.Can soak before extracting.Extraction can be static or down dynamic, preferably under dynamic condition.In order to improve the efficient of extraction, can use ultrasound wave etc.The temperature of extracting be from room temperature (for example 20 ℃) to the scope of solvent refluxing temperature in, preferably under the temperature of backflow.Extraction can be carried out continuously or intermittently, can repeat 1-4 time during intermittent extraction.

Behind the last EOS, merging filtrate, elimination medicinal residues.Filtrate is concentrated into the extractum that does not have solvent at normal pressure or decompression heating under dynamical state.

After the removing oil of extractum petroleum ether, ethyl acetate extraction.Reclaim ethyl acetate, get dry powder.

Dry powder carries out purification, carries out column chromatography, can pass through macroporous resin, ion exchange resin, adsorpting column chromatography, preferred adsorpting column chromatography, and more preferably silica gel column chromatography is the effective site that chromatography obtains.Elution system can be sieved with thin layer chromatography, selects to make the solvent system of the Rf value of separated component at 0.2-0.3.Preferably use dichloromethane: 50: 1,30: 1,10: 1,5: 1,1: 1 gradient elution of acetone, collect eluate in 10: 1,5: 1,1: 1, get the Bolengsu crude product.

The Bolengsu crude product is again through HPLC preparation, chemical compound that can be shown in general formula I, and purity is more than 98%.

The present invention also provides the pharmaceutical composition of extract of the present invention, chemical compound, and it comprises medicine effective dose, as extraction of active ingredients thing, chemical compound and pharmaceutically acceptable carrier.

Usually pharmaceutical composition of the present invention contains extract of the present invention, the chemical compound of 0.1-95% weight.

The pharmaceutical composition of extract of the present invention, chemical compound can be according to method preparation well known in the art.When being used for this purpose, if desired, extract of the present invention, chemical compound and one or more solids or liquid medicine excipient and/or adjuvant can be combined, make and can be used as suitable administration form or the dosage form that people's medicine or veterinary drug use.

Extract of the present invention, chemical compound or contain its pharmaceutical composition can the unit dosage form administration, route of administration can be intestinal or non-intestinal, as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum etc., preferred oral administration.

Extract of the present invention, chemical compound or the route of administration that contains its pharmaceutical composition can be drug administration by injection.Injection comprises intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection etc.

Form of administration can be liquid dosage form, solid dosage forms.As liquid dosage form can be true solution class, colloidal type, particulate formulations, emulsion dosage form, mixed suspension form.Other dosage forms are tablet, capsule, drop pill, aerosol, pill, powder, solution, suspensoid, Emulsion, granule, suppository, lyophilized injectable powder etc. for example.

Extract of the present invention, chemical compound can be made ordinary preparation, also can be slow releasing preparation, controlled release preparation, targeting preparation and various particulate delivery system.

For the unit form of administration is made tablet, can be extensive use of various carrier well known in the art.Example about carrier is, for example diluent and absorbent are as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, carbamide, calcium carbonate, kaolin, microcrystalline Cellulose, aluminium silicate etc.; Wetting agent and binding agent are as water, glycerol, Polyethylene Glycol, ethanol, propanol, starch slurry, dextrin, syrup, Mel, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethyl cellulose, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.; Disintegrating agent, for example dry starch, alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.; Disintegrate inhibitor, for example sucrose, glyceryl tristearate, cocoa butter, hydrogenation wet goods; Absorption enhancer, for example quaternary ammonium salt, sodium lauryl sulphate etc.; Lubricant, for example Pulvis Talci, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, Polyethylene Glycol etc.Tablet further can also be made coated tablet, for example sugar coated tablet, thin membrane coated tablet, ECT, or double-layer tablet and multilayer tablet.

For example, can be extensive use of various carrier well known in the art for pill is made in the administration unit.Example about carrier is, for example diluent and absorbent are as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, Kaolin, Pulvis Talci etc.; Binding agent is as arabic gum, Tragacanth, gelatin, ethanol, Mel, liquid sugar, rice paste or batter etc.; Disintegrating agent is as agar powder, dry starch, alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.

For example for capsule is made in the administration unit, effective ingredient extract of the present invention, chemical compound are mixed with above-mentioned various carriers, and the mixture that will obtain thus places hard gelatine capsule or soft capsule.Also effective ingredient extract of the present invention, chemical compound can be made microcapsule, be suspended in and form suspensoid in the aqueous medium, in the hard capsule of also can packing into or make injection and use.

For example, extract of the present invention, chemical compound are made injection preparation, as solution, suspensoid solution, Emulsion, lyophilized injectable powder, this preparation can be moisture or non-water, can contain acceptable carrier, diluent, binding agent, lubricant, antiseptic, surfactant or dispersant on a kind of and/or multiple pharmacodynamics.Can be selected from water, ethanol, Polyethylene Glycol, 1 as diluent, the isooctadecanol of ammediol, ethoxylation, the isooctadecanol of polyoxyization, Polyoxyethylene Sorbitol Fatty Acid Esters etc.In addition, ooze injection, can in injection preparation, add proper amount of sodium chloride, glucose or glycerol, in addition, can also add conventional cosolvent, buffer agent, pH regulator agent etc. in order to prepare etc.These adjuvants are that this area is commonly used

In addition, as needs, also can in pharmaceutical preparation, add coloring agent, antiseptic, spice, correctives, sweeting agent or other material.

For reaching the medication purpose, strengthen therapeutic effect, medicine of the present invention or pharmaceutical composition can be with any known medication administrations.

The dosage of extract of the present invention, compound medicine compositions depends on many factors, for example to prevent or treat the character and the order of severity of disease, the sex of patient or animal, age, body weight, personality and individual reaction, route of administration, administration number of times, therapeutic purposes, therefore therapeutic dose of the present invention can have large-scale variation.In general, the using dosage of Chinese materia medica composition of the present invention is well known to a person skilled in the art.The actual drug quantity that can be according to the present invention be contained in the last preparation in extract, the compound composition, in addition suitable adjustment to reach the requirement of its treatment effective dose, is finished prevention of the present invention or therapeutic purposes.The consumption of the suitable dose scope extract of the present invention of the every day of extract of the present invention, chemical compound is the 0.001-100mg/Kg body weight, is preferably the 0.01-60mg/Kg body weight, and more preferably the 0.05-10mg/Kg body weight most preferably is the 0.1-1mg/Kg body weight.Above-mentioned dosage can the single dose form or be divided into several, for example two, three or four dosage form administrations this be subject to administration doctor's clinical experience and comprise the dosage regimen of using other treatment means.Each treats that required accumulated dose can be divided into repeatedly or by the dose administration.Extract of the present invention or compositions can be taken separately, or merge use and adjust dosage with other treatment medicine or symptomatic drugs.

Bolengsu of the present invention has good antivirus action, especially hepatitis virus resisting effect.Give Bolengsu after experiment shows duckling infected duck hepatitis B virus (DHBV), one day 2 times 10 days inhibition effects to the hepatitis b virus infected clear middle DHBVDNA level of Sanguis Anas domestica of infected duck of Bolengsu 0.08g/kg have significance effect, non-toxic reaction; 0.16g/kg the group inhibitory action is also stronger.Compare with the positive drug lamivudine, 0.08g/kg group inhibitory action in the time of 10 days is stronger, and knock-on is slow.Above experimental result shows: Bolengsu has stronger inhibitory action to hepatitis B virus.

Pharmacological evaluation shows: Bolengsu adds in the 2.2.15 cell culture fluid can obviously suppress duplicating and expressing of HBsAg and HBeAg, illustrates that chemical compound of the present invention has inhibitory action to hepatitis B virus.

Bolengsu of the present invention has stronger function for protecting liver and reducing enzyme activity.To disposable ip carbon tetrachloride (CCl 4) in the induced mice acute liver damage experiment, each administration group rat blood serum GPT, GOT value all are lower than model control group, Bolengsu height, middle dosage group and model group relatively have significant difference, illustrate that Bolengsu is to CCl 4The induced mice acute liver damage has therapeutical effect preferably.To disposable ip carbon tetrachloride (CCl 4) rising of induced mice serum glutamic pyruvic transminase (GPT), glutamic oxaloacetic transaminase, GOT (GOT) also has stronger inhibitory action.Illustrate that Bolengsu of the present invention has stronger function for protecting liver and reducing enzyme activity, especially to chemical substance, CCl particularly 4The hepatic injury that causes, Bolengsu have good protective effect.

Bolengsu of the present invention has stronger antitumor action.Respectively with human hepatoma cell strain BEL-7402, BEL-7404, and human colon cancer cell strain HCT be object of study, with culture medium and the cell co-cultivation that contains different diluted concentration Bolengsus, the effect of employing mtt assay detection of drugs is to the inhibitory action of cancerous cell.The result shows: Bolengsu is respectively 1.44,1.64 to 50% suppression ratio of BEL-7402, BEL-7404, is 2.31 to 50% suppression ratio of HCT.Illustrate that The compounds of this invention has the obvious suppression effect to the different carcinoma cell in-vitro growth.

Description of drawings

The preparation technology figure of cucurbitaceous plant Herpetospermum pedunculosum (Sex.) Baill. ripple rib extract (is crude drug with the Herpetospermum caudigerum Wall.) and Bolengsu.

The specific embodiment

The experiment of the following examples and pharmaceutically active is used for further specifying the present invention, but this and do not mean that any limitation of the invention.

The preparation of embodiment 1 cucurbitaceous plant Herpetospermum pedunculosum (Sex.) Baill. ripple rib extract and Bolengsu

The seed Herpetospermum caudigerum Wall. 5kg of material ripple rib gets it filled, pulverize, soak with 95% ethanol (5 times of amounts), alcohol reflux 3 times, each 1h, merge extractive liquid,, be concentrated into and do not have the alcohol flavor, leave standstill and remove oil reservoir, after petroleum ether (one times of amount) is sloughed residual oil, ethyl acetate (one times of amount, one times of amount, one times of amount) extraction three times.Reclaim ethyl acetate, get dry powder.This part is used dichloromethane successively through silica gel column chromatography repeatedly: 50: 1,30: 1,10: 1,5: 1,1: 1 gradient elution of acetone, collect eluate in 10: 1,5: 1,1: 1, the Bolengsu crude product.

It is more than 98% that the Bolengsu crude product is prepared into Bolengsu (HPLC condition) purity through HPLC.Idiographic flow is seen accompanying drawing.

C 13-NMR、C 13-H 1cosy: C number Chemical shift (δ ppm) The H number ????1 ????146.79 ????C ????2 ????113.39 ????CH ????3 ????134.51 ????C ????4 ????117.30 ????CH ????5 ????146.06 ????C ????6 ????55.79 ????CH 3 ????7 ????129.16 ????C ????8 ????54.36 ????CH ????9 ????63.90 ????CH 2 ????10 ????88.05 ????CH ????11 ????133.69 ????C ????12 ????118.83 ????CH ????13 ????115.14 ????CH ????14 ????146.53 ????C ????15 ????148.02 ????C ????16 ????53.38 ????CH 3 ????17 ????109.69 ????CH ????18 ????32.97 ????CH 2 ????19 ????42.96 ????CH ????20 ????72.53 ????CH 2 ????21 ????83.07 ????CH ????22 ????53.07 ????CH ????23 ????59.48 ????CH 2 ????24 ????134.74 ????C ????25 ????118.83 ????CH ????26 ????115.01 ????CH ????27 ????148.09 ????C ????28 ????55.41 ????CH 3 ????29 ????144.37 ????C ????30 ????109.59 ????CH

Pharmacological evaluation

Hepatitis virus resisting effect in the body of experimental example 1 Bolengsu

1.1 the reagent lamivudine, Britain Glaxo Wellcome company limited B022306.A- 32P-dCTP (the auspicious biotechnology of Beijing good fortune engineering company, lot number: 981206); Nick translation medicine box (Promega company, lot number: 990426); DHB is DHB DNA (DHBV-DNA) strong positive Shanghai sheldrake serum (medical biotechnology institute of Chinese Academy of Medical Sciences Viral Laboratory provides ,-70 ℃ of preservations)

1.2 animal 1 age in days Beijing dimension duck is available from Nangyuan District, Beijing duckery

1.3 method 1 age in days Beijing duck, clear through the positive Sanguis Anas domestica of lower limb shin intravenous injection Shanghai sheldrake DHBV-DNA, every 0.3ml infects and got blood in back 7 days, and separation of serum detects DHBVDNA content in the serum.Duckling serum is divided into 5 groups at random with duck after DHBV is positive after testing, three dosage groups of virus control group, lamivudine group (50mg/kg), Bolengsu (160,80,40mg/kg), 6 every group.Gastric infusion, 1.5ml/, every day 2 times.Matched group gives with the volume normal saline, successive administration 10 days.Respectively at 5 days (T after the Drug therapy 5), 10 days (T 10) and drug withdrawal after 3 days (P 3) get blood from duck shin vein, separation of serum is pressed nick translation test kit description method, uses 32P labelling DHBVDNA probe, and make the clear dot blot hybridization of Sanguis Anas domestica, autoradiography diaphragm speckle, microplate reader is measured OD value (optical filter is 490nm), calculating serum DHBV-DNA optical density, with hybridization spot OD value as specimen DHBV-DNA level value.

Shine the horizontal rejection ratio of the group clear DHBV-DNA of Sanguis Anas domestica when table 1 Bolengsu treatment group and viral infection Medicine Dosage g/kg Number of animals (only) Suppression ratio (%) ????T 5 ????T 10 ????P 3 Virus control group Bolengsu ? ? ? 0.04 0.08 ? ????6 ? ????6 ????6 ? ????-0.81 ? ????18.24* ????24.99* ? ????15.65 ? ????34.88* ????62.48** ? ????-8.56 ? ????23.06 ????45.85**

Lamivudine ????0.16 ????0.05 ????6 ????6 ????21.66* ????45.27** ????58.45** ????56.92** ????37.68** ????27.35*

Statistical disposition: drug treatment group HBV-DNA suppression ratio and virus control group compare (t check in groups) with the DHBV-DNA suppression ratio of time. *P<0.05, **P<0.01, ***P<0.001。

Experimental result shows that one day 2 times 10 days inhibition effects to the hepatitis b virus infected clear middle DHBVDNA level of Sanguis Anas domestica of infected duck of Bolengsu 0.08g/kg have significance effect, non-toxic reaction; 0.16g/kg the group inhibitory action is also stronger, with 0.08g/kg group there was no significant difference.Compare with the oral inhibitory action to DHBVDNA of positive drug lamivudine, 0.08g/kg group inhibitory action in the time of 10 days is stronger, and knock-on is slow, but does not have significant difference on the statistics.Above experimental result shows: Bolengsu has good therapeutical effect to the duck hepatitis B virus infection duck, and is better than the positive drug lamivudine.

The hepatitis virus resisting effect of experimental example 2 Bolengsus

2.1 reagent

DMEM (U.S. Gibco company product); Hyclone (U.S. Gibco company product); G-418 (U.S. Sigma company product); HBeAg, HBsAg immobile phase radioimmunoassay box (Beifang Inst. of Immune Reagents, Chinese Isotopes Co.'s product);

2.2.2.2.15 cell the has been transfection full genome of HBV (Aayw subunit), can secrete HBsAg, HBeAg, the female oncocyte of the particulate people liver of HBV-DNA system, U.S. Mount sinai medical center makes up, the cultivation of going down to posterity of medical biotechnology institute of Chinese Academy of Medical Sciences Viral Laboratory)

2.3 method is with 100,000 every milliliter 2.2.15 cell inoculation 24 well culture plates, every hole 1ml, 37 ℃ of 5%CO 2Cultivated 24 hours, and added the following 2 times of dilution test medicinal liquids of non-toxic concn, 5 dilution factors are respectively 4,2,1,0.5,0.25mg/ml, every concentration 3 holes, 37 ℃ of 5%CO 2Cultivate, collect the 8th day pastille culture fluid ,-20 ℃ of stored frozen.Measure with reference to solid phase radioimmunoassay box description, measure every hole cpm value with the γ calculating instrument.HBsAg, the HBeAg positive and negative control are established in experiment.

Table 2 Bolengsu in the 2.2.15 cell to the influence of HBsAg Drug level mg/ml ????cpm(x±s) Suppression ratio % 0.006 0.003 0.0015 cell contrast ????2158±176 ????1774±63 ????3206±216 ????7069±245 ? ????63.02 **????69.60 **????44.15 *? ?

Compare * * p<0.01 * p<0.05 with cell

Table 3 Bolengsu in the 2.2.15 cell to the influence of HBeAg Drug level mg/ml ????cpm(x±s) Suppression ratio % 0.006 0.003 0.0015 cell contrast ????1073±213 ????1224±120 ????1507±185 ????2753±123 ? ????61.04 ????55.53 ????45.26 ? ?

Compare * * p<0.01 * p<0.05 with cell

Experiment shows: Bolengsu added the 2.2.15 cell culture 8 days, obviously suppressed the secretion of HBsAg and HBeAg, can suppress duplicating and expressing of 2.2.15 cell HBsAg, HBeAg.The compounds of this invention illustrates that in the 2.2.15 cell culture hepatitis B virus being had inhibitory action it has stronger inhibitory action to hepatitis B virus.

The function for protecting liver and reducing enzyme activity of experimental example 3 Bolengsus

3.1 positive control drug: 99J15043L01 bifendate, Beijing XieHe medicine Factory, lot number: 010309;

3.2 reagent: glutamate pyruvate transaminase (GPT), glutamic oxaloacetic transaminase, GOT (GOT) are measured test kit, be the safe clinical reagent company limited of Beijing northization product E agles MEM dry powder, G-418 (Geneticin), yeast t-RNA, E.C. 3.4.21.64, U.S. GIBCO company product;

3.3 animal: Kunming mouse, body weight 18-22g, all male and female half and half are provided by Military Medical Science Institute's animal center, and the animal quality certification number is respectively the moving word BDW98012 of medical officer and the moving word of medical officer BDW95001 number.The zoopery condition: two grade standards, the quality certification number: medical officer moves word B98006.

3.4 instrument BS-224 type biochemical analysis analyzer, Beijing biochemical instrument factory.

3.5 getting 60 of healthy mices, method is divided into six groups at random, i.e. normal control group, CCl 4(0.02ml/kg) three dosage groups of model group, bifendate group 0.029g/kg, Bolengsu (0.0166,0.0083,0.0042g/kg), 10 every group.Every day, the ig administration was 2 times, and matched group and model group are given the distilled water with same volume, behind 7 medicines of successive administration, and except that the normal control group, ip in mice 0.1%CCl 4(olive oil is a solvent) 0.02ml/g, after the modeling 40 hours, mice socket of the eye venous blood collection was surveyed serum GPT, GOT value, sees Table 4.

Table 4. Bolengsu causes the influence of chmice acute hepatic injury to CCl4 Group Dosage (g/kg) Number of animals (only) ??GPT ??(U·L -1) ??GOT ??(U·L -1) Matched group model group bifendate Bolengsu ? ? ????0.029 ????0.0166 ????0.0083 ????0.0042 ????10 ????10 ????10 ????10 ????10 ????10 ??10.90±0.85 ??152.31±20.34 ##??30.71±2.28 **??34.20±10.12 **??36.22±10.03 **??62.75±8.21 * ??49.59±5.731 ??162.92±9.08 ##??70.14±8.06 *??72.43±9.11 *??69.12±8.97 *??86.28±10.23 *

Compare with the normal control group, * p<0.05 * * p<0.01 is compared with model group in #p<0.05 ##p<0.01

The result shows that each administration group rat blood serum GPT, GOT value all are lower than model control group, and Bolengsu height, middle dosage group and model group relatively have significant difference, illustrate that Bolengsu is to CCl 4The induced mice acute liver damage has therapeutical effect preferably.

Experimental example 4 antitumor actions

4.1 method respectively with human hepatoma cell strain BEL-7402, BEL-7404, and human colon cancer cell strain HCT be object of study, with culture medium and the cell co-cultivation that contains different diluted concentration Bolengsus, adopt the inhibitory action of mtt assay detection of drugs effect to cancerous cell.Revise [1] slightly according to literature method, target cell is adjusted to 1 * 10 with complete culture solution 5/ mL is inoculated in respectively in 96 well culture plates, and every hole 100 μ L add different pastille serum of 100 μ L and blank serum then respectively, and cumulative volume 200 μ L establish 7 parallel holes for every group, at 37 ℃, 5%CO 2, cultivate 48h under the saturated humidity condition after, every hole adds 5mL MTT 10 μ L, carries out chromogenic reaction after continuing to cultivate 4h, add 20% dimethyl sulfoxide, 100 μ L cessation reactions, concussion 10min, at DG3022-A type enzyme-linked immunosorbent assay instrument, wavelength 570nm surveys absorbance (A) value.The cytotoxic activity computing formula is:

Result: see Table 5.

Table 5. Bolengsu is to the suppression ratio (%) of growth of tumour cell Cell strain Concentration (μ g/ml) ??1.25 ??2.50 ????5.00 ????10.00 ??20.00 ??IC50 ?BEL-7402 ?BEL-7404 ?HCT ??34.90 ??12.30 ??32.00 ??41.90 ??60.00 ??18.00 ????100.00 ????100.00 ????72.00 ????98.80 ????98.50 ????100.00 ??100.00 ??100.00 ??100.00 ??1.44 ??1.64 ??2.31

The result shows: Bolengsu is respectively 1.44,1.64 to 50% suppression ratio of BEL-7402, BEL-7404, is 2.31 to 50% suppression ratio of HCT.Illustrate that chemical compound of the present invention has the obvious suppression effect to the different carcinoma cell in-vitro growth.

Claims (12)

1, the application of cucurbitaceous plant ripple rib Herpetospermum pedunculosum (Sex.) Baill. in the anti-hepatic lesions medicine of preparation, wherein said hepatic lesions is meant hepatovirus, hepatic injury and liver tumor.
According to the application of claim 1, it is characterized in that 2, described virus is hepatitis virus.
According to the application of claim 2, it is characterized in that 3, described hepatitis virus is a hepatitis B virus.
According to the application of claim 1, it is characterized in that 4, described hepatic injury is chemical hepatic injury.
5, the extract of cucurbitaceous plant Herpetospermum pedunculosum (Sex.) Baill. ripple rib, it is characterized in that, described extract prepares by following method: with the solvent extraction of cucurbitaceous plant Herpetospermumpedunculosum (Sex.) Baill. ripple rib, merge extractive liquid,, concentrate, the petroleum ether removing oil, ethyl acetate extraction, concentrate dry powder, dry powder is through silica gel column chromatography repeatedly, use dichloromethane successively: acetone 50: 1,30: 1,10: 1,5: 1,1: 1 gradient elution was collected 10: 1,5: 1,1: 1 eluate gained.
6, cucurbitaceous plant Herpetospermum pedunculosum (Sex.) Baill. ripple rib preparation method of extract, it is characterized in that, the solvent extraction of cucurbitaceous plant Herpetospermum pedunculosum (Sex.) Baill. ripple rib, merge extractive liquid,, concentrate, the petroleum ether removing oil, ethyl acetate extraction, concentrate dry powder, dry powder is through silica gel column chromatography repeatedly, use dichloromethane successively: 50: 1,30: 1,10: 1,5: 1,1: 1 gradient elution of acetone, collect eluate in 10: 1,5: 1,1: 1.
According to the preparation method of claim 6, it is characterized in that 7, described solvent is an ethanol.
8, the pharmaceutical composition of a kind of cucurbitaceous plant Herpetospermum pedunculosum (Sex.) Baill. ripple rib extract, it is characterized in that, comprise as carrier commonly used in the extract of the described cucurbitaceous plant Herpetospermumpedunculosum of the claim 5 of active ingredient (Sex.) Baill. ripple rib and the pharmaceutical field.
9, cucurbitaceous plant Herpetospermumpedunculosum as claimed in claim 5 (Sex.) Baill. ripple rib extract is in the anti-hepatovirus of preparation, anti-chemical damage, the application in the anti-liver tumor medicine.
According to the application of claim 1, it is characterized in that 10, described virus is hepatitis virus.
According to the application of claim 2, it is characterized in that 11, described hepatitis virus is a hepatitis B virus.
According to the application of claim 1, it is characterized in that 12, described hepatic injury is chemical hepatic injury.
CNB03123965XA 2003-05-29 2003-05-29 Bolengsu compound and its prepn, medicine composition and use CN1305468C (en)

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CN100333736C (en) * 2004-07-26 2007-08-29 杨东平 Medication for treating ascites of liver, cirrhosis of liver and liver cancer in early stage
CN100389785C (en) * 2006-04-04 2008-05-28 钱毓洲 Pedunculate herpetospermum seed extract, its dripping pill and their preparing method and application
CN101311173B (en) * 2007-05-25 2010-07-28 成都中医药大学 Herpe-lactone, preparation method and use thereof
CN102000136B (en) * 2010-11-25 2012-04-18 西南大学 Method for extracting components having antibacterial activity from fruit of pedunculate herpetospermum
CN102140101B (en) * 2011-03-24 2013-04-10 中国人民解放军第三〇二医院 Method for preparing Herperione, application thereof, capsule thereof, preparation method and application of capsule
CN105311637B (en) * 2014-08-04 2019-07-09 西南民族大学 A kind of pharmaceutical composition containing Bolengsu
WO2019213807A1 (en) * 2018-05-07 2019-11-14 袁海龙 Lignan active ingredient composition of herpetospermum caudigerum wall and preparation method therefor and application and dosage form thereof

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