CN101214285B - Use of giant knotweed rhizome extract in preparing product for resisting AIDS virus and hepatitis B - Google Patents
Use of giant knotweed rhizome extract in preparing product for resisting AIDS virus and hepatitis B Download PDFInfo
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- CN101214285B CN101214285B CN2008100323233A CN200810032323A CN101214285B CN 101214285 B CN101214285 B CN 101214285B CN 2008100323233 A CN2008100323233 A CN 2008100323233A CN 200810032323 A CN200810032323 A CN 200810032323A CN 101214285 B CN101214285 B CN 101214285B
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Abstract
The present invention relates to purposes of giant knotweed extract. The in vitro anti-HIV experiment shows that resveratrol 3-O-Beta-D- glucoside 2''-sulfate sodium or potash salt and resveratrol 3-O-Beta-D- glucoside 4''-sulfate sodium or potash salt show significant activity of resisting hepatitis B virus; resveratrol 3-O-Beta-D- glucoside 2''-sulfate sodium or potash salt also has significant HIV inhibited effect. The virus infectious diseases such as AIDS and hepatitis B poses a serious threat to human's health and life and hepatitis B is one of the major diseases affecting human's health. The present invention provides a lead compound for doing R&D of novel anti-HIV and hepatitis B virus medicines and also a novel medicine source for clinic application, thus having a significant value to develop and utilize the medical plant resources of our country.
Description
Technical field
The present invention relates to medicine and food technology field; Specifically relate to the purposes of Chinese medicine extract; More particularly relate to the purposes that the Chinese medicine extract Rhizoma Polygoni Cuspidati extract is used to prepare anti AIDS virus and anti-hepatitis B virus product, specifically relate to Chinese medicine extract Rhizoma Polygoni Cuspidati extract resveratrol 3-O-β-D-glucoside 2 again " sulfate group sodium or potassium salt (be called for short: chemical compound 1) with resveratrol 3-O-β-D-glucoside 4 '-sulfate group sodium or potassium salt (abbreviation: chemical compound 2) be used to prepare the purposes of anti AIDS virus and anti-hepatitis B virus product.
Background technology
(1) research overview of Rhizoma Polygoni Cuspidati
Rhizoma Polygoni Cuspidati is Polygonaceae (Polygonaceae) arsesmart Polygonum cuspidatum Sieb.et Zucc. rhizome.Be born in mountain valley, small stream side or bank, for perennial shrub shape draft, up to more than 1 meter more.
Rhizoma Polygoni Cuspidati is a Chinese medicine very famous in the Polygonaceae.The traditional Chinese medical science thinks that the Rhizoma Polygoni Cuspidati nature and flavor are bitter, flat, have and dispel the wind, dampness removing, removing blood stasis with potent drugs, the effect of stimulating the menstrual flow; Can dispelling the wind and dampness pathogens, bones and muscles pain, jaundice due to damp-heat, the stranguria with turbid discharge leukorrhagia, women's amenorrhea, lochiostasis, the skin ulcer blood that leaks down, injury from falling down is scalded diseases such as malignant boil tinea disease.Rhizoma Polygoni Cuspidati is distributed in middle part and southern areas such as Jiangsu, Zhejiang, Jiangxi, Fujian, Shandong, Henan, Shaanxi, Hubei, Yunnan, Sichuan, Guizhou, and all can excavate spring and autumn.
Owing to it is found that Rhizoma Polygoni Cuspidati has good clinical practice in recent years, become new research focus gradually.Mainly contain chemical constituents such as stilbene, anthraquinone, flavonoid in the Rhizoma Polygoni Cuspidati, and have physiologically active widely.The inventor had once carried out systematic study for the water soluble part of Rhizoma Polygoni Cuspidati, separated to have obtained a series of new chemical constituents.Noval chemical compound comprises sodium or potassium salt (Xiao K., Xuan L., the Xu Y. of the diphenylethylene compounds of being with sulfate group; Bai D.Stilbene glycosidesulfates from Polygonum cuspidatum.J.Nat.Prod.2000,63 (10): 1373-1376), stilbene glycoside dimer (Xiao K.; Xuan L., Xu Y., Bai D.; Zhong D., Wu H., Wang Z.; Zhang N.Dimeric stilbeneglycosides from Polygonum cuspidatum.Europ.J.Org.Chem.2002:564-568); The plain constituents of phenylpropyl alcohol (Xiao K., Xuan L., Xu Y., Bai D., Zhong D.Constituents from Polygonum cuspidatum.Chem.&Pharm.Bull.2002,50 (5): 605-608).Also comprise in addition some compound known etc. (Xiao Kai, Xuan Lijiang, Xu Yaming etc. the chemical constitution study of Rhizoma Polygoni Cuspidati. Chinese Pharmaceutical Journal .2003,38 (1): 12-14; Xiao Kai, Xuan Lijiang, Xu Yaming etc. the water soluble ingredient research of Rhizoma Polygoni Cuspidati, Chinese herbal medicine .2003,34 (6): 496-498).(" sulfate) (sodium orpotassium trans-resveratrol-3-O-β-D-glucopyranoside-4 '-sulfate) is for separating two kinds of diphenylethylene compounds that obtain from Rhizoma Polygoni Cuspidati; be two kinds of native compounds, its molecular formula is all C with chemical compound 2 for sodium orpotassium trans-resveratrol-3-O-β-D-glucopyranoside-2 for chemical compound 1 wherein
20H
21O
11SNa or C
20H
21O
11SK, its chemical structural formula is following:
Chemical compound 1 chemical compound 2
M＝Na?or?K
Modern age, pharmacological experiment showed that Rhizoma Polygoni Cuspidati had antiviral activity preferably, external various bacteria and virus was had inhibition and deactivation.But for example its to herpes simplex virus, the influenza Asia first type capital 68-1 of section virus and dust II type etc. all have inhibitory action (cloudy strong, the Guo Li bow. Chinese medicine modern study and clinical (1) [M]. Beijing: Xueyuan Press .1993,434-439; Namba T, Kurokawa M, Kadota S; Shiraki K.Development of antiviral therapeutic agents from traditionalmedicines.Yakugaku Zasshi, 1998,118 (9): 383-400); Rhizoma Polygoni Cuspidati and Radix Astragali coupling to herpes simplex virus have good coordinate repression (Wang Zhijie, Cheng Zhongmin, Fang Xueyun. Rhizoma Polygoni Cuspidati Radix Astragali coupling anti-I type herpesvirus pharmacodynamic analysis. CHINA JOURNAL OF CHINESE MATERIA MEDICA; 1999,24 (3): 176-180.).External activity (the Chang JS that has shown stronger anti-HBV of Rhizoma Polygoni Cuspidati ethanol extract and water extract; Liu HW; Wang KC, Chen MC, Chiang LC et al.Ethanol extract of Polygonumcuspidatum inhibits hepatitis B virus in a stable HBV-producing cell line.Antiviral Research; 2005,66:29-34.).Rhizoma Polygoni Cuspidati water extract can suppress the gp120 on HIV-1 surface and the CD of cell surface external
4In conjunction with; Suppress HIV-1 and infect the MT-4 cell, and the reverse transcriptase of HIV-1 is had significant deactivation (Jiang Y, SanoK; Molimato S; Et al.Anti-HIV-1 activity of Polygonum cuspidatum extract in vitro.JapaneseArchives of Sexually Transmitted diseases, 1994,5 (1): 138-146.); Rhizoma Polygoni Cuspidati water extract can suppress (the Jiang Yan such as splenomegaly, immunosuppressant and viremia of the model of AIDS Mus that LP-BM5 virus causes in vivo; Wang Hongxia; Bao Zuoyi; Deng. estimate the antivirus action of Rhizoma Polygoni Cuspidati water extract with the Mus model of AIDS. Chinese virusology, 1998,13 (4): 306-311.).
(2) extraction separation method of Chinese herbal medicine effective ingredients commonly used
1, solvent extraction method
(1) principle: solvent extraction method is according to the dissolution properties of various compositions in solvent in the Chinese herbal medicine, select for use the active component dissolubility big, to not needing the little solvent of stripping composition dissolubility, and the method that effective ingredient is dissolved out in the medical material tissue.When solvent was added in the herbal raw material (needing suitably to pulverize), solvent had dissolved solable matter owing to diffusion, osmosis penetrate in the cell through cell wall gradually; And cause the concentration difference inside and outside the cell; So intracellular concentrated solution is constantly to external diffusion, solvent constantly gets into again in the medical material histiocyte, so repeatedly comes and goes; When solution concentration reaches dynamic equilibrium inside and outside cell; This saturated solution is leached, continue repeatedly to add novel solvent, just can be bordering on complete stripping or the stripping of big portion to desirable ingredients.The dissolubility of medicinal herb components in solvent is directly relevant with solvent property.Solvent can be divided into hydrophilic organic solvent and lipotropy organic solvent, and dissolved material also has hydrophilic and lipophilic difference.Hydrophilic radical is many in the organic compound molecule structure, and its polarity is negligent of oil greatly; The hydrophilic radical that has is few, and its polarity is little and be negligent of water.The character of each kind solvent, equally also relevant with its molecular structure.Like this, the inventor just can remove to estimate their this type of character and the solvent of selecting for use through to the medicinal herb components structural analysis.Generally speaking,, bigger dissolubility will be arranged therein, i.e. the rule of so-called " similar mixing " as long as this character of the hydrophilic of medicinal herb components and lipotropy and solvent is suitable.This is to select appropriate solvent in Chinese herbal medicine, to extract one of foundation of required composition.
(2) choice of Solvent: the key of utilization solvent extraction method is to select appropriate solvent.Solvent is selected suitably, just can be more successfully the composition of needs be extracted.Selective solvent will be noted following 3 points: 1. solvent is big to the effective ingredient dissolubility, and is little to the impurity dissolubility; 2. solvent can not play chemical change with the composition of Chinese medicine; 3. solvent want economical, be easy to get, safe in utilization etc.Common extraction solvent can be divided into following three types:
1. water: water is a kind of strong polar solvent.Hydrophilic composition in the Chinese herbal medicine can both be gone out by water-soluble like the not too big polysaccharide of inorganic salt, saccharide, molecule, tannin, aminoacid, protein, acylate, alkaloid salt and glycoside etc.In order to increase the dissolubility of some composition, also often adopt sour water and aqueous alkali as extracting solvent.
2. hydrophilic organic solvent: just general said and the miscible organic solvent of water, ethanol), methanol (but also claims: another name for), acetone etc., the most frequently used with ethanol (claim not only: like ethanol.Alcoholic acid solubility property is relatively good, and is stronger to the penetration capacity of Chinese herbal medicine cell.Outside hydrophilic composition isolating protein, phlegmatic temperament, pectin, starch and the part polysaccharide etc., big multipotency dissolves in ethanol.Be insoluble in the low-polarity component of water, the dissolubility in ethanol is also bigger.Can also adopt Different concentrations of alcohol to extract according to the character that is extracted material.More less than water consumption with ethanol extraction, extraction time is short, and it is also few to dissolve the water-solubility impurity that.Ethanol is organic solvent, though inflammable, toxicity is little, low price, and convenient sources has a locking equipment can reclaim repeatedly and use, and alcoholic acid extracting solution is difficult for moldy metamorphism.Owing to these reasons, be one of always the most frequently used method with the method for ethanol extraction.The character of methanol is similar with ethanol, boiling point lower (64 ℃), but toxic, should note during use.
3. lipophilic organic solvent: the organic solvent that just general said and water can not be miscible, like petroleum ether, benzene, chloroform, ether, ethyl acetate, dichloroethanes etc.These choice of Solvent performances are strong, can not or be not easy to propose hydrophilic impurities.But this kind solvent volatility is big, how inflammable (except the chloroform), generally poisonous, price is more expensive, equipment requirements is higher, and they penetrate plant tissue ability a little less than, often need to extract repeatedly for a long time could extract complete.If contain more water in the medical material, just be difficult to leach its effective ingredient with this kind solvent, therefore, when extracting herbal raw material in a large number, directly using this kind solvent has certain limitation.
(3) method for distilling: use the solvent extraction medicinal herb components, infusion process commonly used, percolation, decocting method, reflux extraction and continuous backflow extraction method etc.Simultaneously, factors such as the degree of grinding of raw material, extraction time, extraction temperature, appointed condition also can both influence extraction efficiency, must take in.
1. (be called for short: infusion process): the dipping genealogy of law is packed herbal powder or fragment in the proper container, adds The suitable solvent (like ethanol, rare alcohol or water), and the dipping medical material is with the stripping method of composition wherein to flood extraction method.This law is relatively simple, but leaching rate is relatively poor, and is solvent like water, and its extracting solution is prone to moldy metamorphism, must note adding suitable preservatives.
2. (be called for short: percolation): percolation is that herbal powder is contained in the percolator to the percolation extraction method, constantly adds novel solvent, makes it penetrate medical material, flows out a kind of leaching method of leachate from top to bottom from the percolator bottom.When moving down when solvent infilters medicated powder, stripping composition proportion strengthens, its position is just replaced in the solution on upper strata or rare immersion, causes good concentration difference, and diffusion energy is carried out preferably, so leaching effect is superior to infusion process.But should control flow velocity, in oozing transient, on powder, replenish novel solvent at any time, make till effective ingredient fully leaches in the medical material.Maybe extremely shallow or when oozing the volume that gushes liquid and being equivalent to heavy 10 times of crude drug when oozing the dropping liquid color, just can think and extract basically fully.The rare leachate that in mass production, often will collect is as the usefulness of the solvent of another batch new raw material.
3. (be called for short: decocting method): decocting method is traditional leaching method that China uses the earliest to decoct extraction method.Used container is generally pottery, sand jar or copper, enamel ware, should not use iron pan, in order to avoid the medicinal liquid variable color.Preferably stir often during straight fire heating, in order to avoid that local medical material is heated is too high, burnt easily the paste.Big reaction pot, big copper pot, barrel are adopted in the pharmaceutical factory that steam-heating apparatus is arranged more, or feed Steam Heating in the pond of cement block.It is interconnection through pipeline also can several to be decocted device, fries in shallow oil continuously and soaks.
4. heating and refluxing extraction method: use the organic solvent heating extraction, need to adopt the reflux device, in order to avoid the solvent evaporates loss.When operating in a small amount, can on round-bottomed flask, connect reflux condenser.The powder charge material is about 20%～60% of capacity in the bottle, and solvent soaked the about 1～2cm in medical material surface.Reflux in water-bath, the general maintenance, seethed with excitement 3～6 hours, puts cold filtration, and solubilizer in medicinal residues is made second and third time reflux and is made an appointment with half an hour respectively again, or to carrying till the most effective ingredient basically.This method extraction efficiency is high than cold-maceration, the continuous extractions that adopt in the mass production more.
5. continuous backflow extraction method: use volatile organic solvent and extract Chinese herbal medicine effective ingredients, no matter small test or large-scale production, all with continuous extraction for well, and need lessly with quantity of solvent, the extraction composition is also more complete.Laboratory fat-extraction device commonly used or title apparatus,Soxhlet's.Continuous extraction generally needs several hours ability to extract fully.It is longer to extract the composition heated time, meets the labile composition of thermally labile and should not adopt this method.
2, separation and purification process:
Resulting extracts of Chinese herbal medicine of said extracted method or extract remain mixture, need further remove impurity, separate and make with extra care.
(1) solvent segregation: generally be with above-mentioned total extract, select three for use, the solvent of four kind of opposed polarity, by low polarity to high polarity proceed step by step extraction separation.Aqueous extract or ethanol extract are jelly often, are difficult to be dispersed in the low polar solvent, so can not extract fully; Can admix an amount of inert filler, like kieselguhr or fiber powder etc., low temperature or natural drying then; After the pulverizing; To select for use solvent to extract successively, make each constituent in the total extract again, obtain according to the difference of its dissolubility in the opposed polarity solvent separating.Utilize the Chinese herbal medicine chemical constituent, the dissolubility in the opposed polarity solvent carries out separation and purification, is the most frequently used method.
(2) solvent extraction:
1. extraction: the solvent extraction extraction is called for short extraction again, is to utilize the difference of each composition partition coefficient in two kinds of immiscible solvents in the mixture and reach isolating method.If each composition partition coefficient in solvent differs big more during extraction, then separation efficiency is high more; If the effective ingredient in aqueous extract is lipophilic material; The general lipotropy organic solvent of using more; Extract like benzene, chloroform or ether, if effective ingredient is to be partial to hydrophilic material, indissoluble is separated in lipophilic solvent; Lipophilic solvent, for example ethyl acetate, butanols etc. a little less than just need using instead.Can also in chloroform, ether, add an amount of ethanol or methanol to increase its hydrophilic.When extracting flavones ingredient, how with ethyl acetate and water extraction.Extract the strong saponin of hydrophilic then multiselect extract with n-butyl alcohol, isoamyl alcohol and water.But, the common organic solvents hydrophilic is big more, and the effect of doing extraction with water is just bad more, and is because more hydrophilic impurities is followed, very big to the further refining influence of effective ingredient.
2. counter current continuous extraction method: be a kind of successive solvent extraction.Its device can have one, several or more extracting tube.Fill the contact surface during with increase solvent extraction in the pipe with little porcelain circle or little rustless steel wire ring.If a kind of infusion of Chinese herbal medicine need extract with the benzene lighter than water, ethyl acetate etc., then need water extracting liquid is contained in the extracting tube, and benzene, ethyl acetate are stored in the high-level container.Extract whether complete, but sample thief is analysed with thin layer chromatography, ply of paper and chromogenic reaction or precipitation are checked.
3. counter-current distribution method: counter-current distribution method is claimed CCD method, counter-current distribution or countercurrent distribution again.Counter-current distribution method is consistent with solvent counter-current extraction principle, but the application of sample amount is certain, and continuous in the solvent of a constant volume, reaches the separation of mixture through repeatedly being shifted the extraction distribution.
4. drop counter-current distribution method: the drop counter-current distribution method is claimed the droplet countercurrent chromatography method again.Be improved solvent extraction on the counter-current distribution method basis in recent years.To the same basically counter-current distribution method of the selection of solvent system, but requirement can separate at short notice, and can generate effective drop.Because mobile phase forms drop, in thin distribution extracting tube, contacting effectively with immobile phase, rubbing constantly forms new surface, promotes the distribution of solute in solvent, so its separating effect is often good than counter-current distribution method.
(3) macroporous adsorbent resin method: macroporous adsorbent resin is the one type of organic polymer adsorbent that grows up the sixties in 20th century; Have the good adsorption performance, be applied to the development of the extraction separation and the new Chinese medicine of Chinese herbal medicine chemical constituent surplus in the of nearly ten over year gradually.
Macroporous adsorbent resin is for absorption and screen the parting material that principle combines.Its adsorptivity is because the result of Van der Waals force or generation hydrogen bond.The screening principle is because itself cellular structure determines.Because absorption and screening principle, organic compound separates through certain solvent elution on macroporous adsorbent resin according to the difference of absorption affinity and the size of molecular weight.This make organic compound especially the purification of water soluble compound be able to simplify greatly.The skeleton of macroporous adsorbent resin is generated by styrene and divinylbenzene polycondensation, because the adding of modifier, the polarity of macroporous adsorbent resin changes, and according to the surface nature of resin, that adsorbent resin generally is divided into is nonpolar, three types of Semi-polarity and polarity.
Nonpolar adsorption resin be by the very little monomer-polymer of dipole moment make not with the adsorbent resin of any functional group.Typical example is the adsorbent resin of styrene-divinylbenzene system, like D101, XAD-1, DiaionHP-10 macroporous adsorbent resin.
The Semi-polarity adsorbent resin refers to contain the adsorbent resin of ester group, like an acrylic ester or a crosslinked analog copolymer such as methacrylate and double methyl methacrylate.It is on the basis of nonpolar macroporous adsorption resin, adds acrylic acid methyl ester. or acrylonitrile polycondensation and forms, like the AB-8 macroporous adsorbent resin of the domestic frequent use of China.
Polar Adsorbent Resin is meant that amide-containing, itrile group, phenolic hydroxyl group etc. are nitrogenous, the adsorbent resin of oxygen, sulfur polar functionalities base.In addition, be called strong Polar Adsorbent Resin to the ion exchange resin of ligand groups such as nitrogenous, oxygen, sulfur sometimes, the boundary distinguish of strong Polar Adsorbent Resin and ion exchange resin.Polar macroporous adsorption resin can be formed by methyl methacrylate, acrylamide or the polycondensation of sulfoxide class, like the Diaion HP 2MG of Mitsubishi chemical industry, the XAD-10 of U.S. Rohm-hass company, XAD-9 macroporous adsorbent resin.
Compare with other adsorbent with active carbon, macroporous adsorbent resin has a lot of advantages, and is higher like the adsorptive selectivity to certain material; Physical and chemical stability and mechanical strength are better; Description is more, can change resin physics or chemical constitution as required; Adsorbent resin is generally spherical particle, and fluid resistance is less or the like.Thereby be widely used in chemical industry, medicine and other fields, more and more about macroporous adsorbent resin in recent years at natural product extraction applications in separation research report.Macroporous adsorbent resin centering herbal chemistry composition such as alkaloid, flavone, saponin, coumarin and some other glycoside compositions all have certain adsorption.Absorbability to sugar is very poor, and is stronger to the absorbability of pigment.
(4) sedimentation method: be in extracts of Chinese herbal medicine, to add some reagent to make the generation deposition, with the method that obtains effective ingredient or remove impurity.Like lead salt precipitation: lead salt precipitation is one of classical way of separating some medicinal herb components.Because lead acetate and Lead monosubacetate in water and alcoholic solution, can generate the lead salt or the complex salt deposition of indissoluble with multiple medicinal herb components, so this character capable of using makes effective ingredient separate with impurity.Then lead salt deposition is suspended in the novel solvent, passes to hydrogen sulfide gas, make and decompose and transfer insoluble vulcanized lead to and precipitate.
(5) salting out method: salting out method is in the water extract of Chinese herbal medicine, adds inorganic salt to finite concentration, or the state that reaches capacity, and can make the dissolubility of some composition in water reduce deposition and separate out, and separate with the big impurity of water solublity.The inorganic salt that work commonly used is saltoutd has sodium chloride, sodium sulfate, magnesium sulfate, ammonium sulfate etc.
(6) dialysis: dialysis is to utilize small-molecule substance in solution, can pass through semipermeable membrane, and macromolecular substances can not reach isolating method through the character of semipermeable membrane.Otherwise also can macromolecular impurity be stayed in the semipermeable membrane, and micromolecular material is got in the outer solution of film through semipermeable membrane, and separation and purification in addition.
(7) crystallization, recrystallization and Steppecd crystallization: identify the Chinese herbal medicine chemical constituent, study its chemical constitution, must at first medicinal herb components be prepared into the pure article of monomer.At normal temperatures, the character of material own is the chemical compound of liquid, can carry out separation and purification with fractionating process or chromatography respectively.Generally speaking, the Chinese herbal medicine chemical constituent is solid material at normal temperatures mostly, all has the general character of crystalline solid, can reach the purpose of separation and purification according to the difference of dissolubility with crystallization process.
3, conventional drying method
(1) vacuum drying: be based on such ultimate principle: water saturation vapour pressure and temperature are closely related; Under vacuum state; The boiling point lowering of water; I.e. operation operation at low temperatures just under vacuum can be avoided the destruction of nutritional labeling such as vitamin etc. at high temperature, has improved rate of drying simultaneously.Vacuum drying is widely used in industries such as food, pharmacy, chemical industry, and China also develops and introduced various vacuum dryers, and its version is varied.Form commonly used mainly contains box type vacuum exsiccator, bipyramid formula vacuum desiccator, belt vacuum desiccator etc.These traditional Minton dryers mainly adopt heating such as hot blast, steam or electricity, utilize conduction of heat, convection current or radiation theory that heat is passed to material inside from the outside.It is low that vacuum drying has a baking temperature, and anoxia relatively in the hothouse can be avoided fat oxidation, and series of advantages such as pigment brown stain are suitable for the drying of heat sensitivity food material, and equipment cost, dry expense are also relatively low in addition.
(2) spray drying: be that fluidization technique is used for the exsiccant a kind of method of liquid material.Because of being wink-dry, be specially adapted to heat sensitive material, so the products obtained therefrom quality is good, keep original color, smell and taste, and be prone to dissolving.The research that utilizes spray drying to prepare microcapsule is carried out; It is that heart material is suspended in the solution of dress material; Through centrifugal atomizer it is sprayed in the thermal current; The product of gained is the microcapsule that dress material bag heart material forms, and this microcapsule powder can be used in direct compression, also can prepare capsule, syrup or suspensoid.
(3) lyophilization: be that the dry liquid material is frozen into solid, under the low-temperature reduced-pressure condition, utilize the distillation performance of icing, make the low-temperature material dehydration and reach exsiccant a kind of method.Because material is dry under high vacuum and cryogenic conditions, so the drying of some extremely thermo-labile article is well suited for.Wang Dalin has reported a kind of spraying ventilation lyophilizing new technique; Be to utilize cold air or nitrogen as medium; The scars of flowing through rapidly make water sublimate, the product microgranule that makes of spraying lyophilizing is little, fast drying, time are short, evenly, good fluidity, and the good instant capacity of tool.In recent years, plaster material and the exsiccant research of sticky material have been obtained bigger progress, fluidization technology, spraying technique, inert carrier technology then grow up on this research basis.Rotatingandflashstreamingdrier, thermojet pneumatic drier, inert carrier drying machine all are fit to the drying of heat sensitive material and plaster material.These new achievements in research are used for Chinese medicine preparation production, with the technical merit of improving Chinese medicine processing greatly, enhance productivity.
(4) far infrared heating drying method: be a new dry technology; Its drying principles is to change electric energy into far infrared radiation, thereby by the molecule absorption of medical material, produces resonance; Cause the vibration and the rotation of molecule and atom; Cause the object heating,, finally reach exsiccant purpose through thermal diffusion, evaporation and chemical change.Far-infrared ray drying can be saved electric energy 20%～50%, and effect is better.
(5) micro-wave drying method: be the new technique that develops rapidly a sixties in 20th century; Microwave drying is actually through eddy-current heating and medium heating; Make moisture and fat in the thing that is dried absorb microwave energy to some extent, thereby and change it into heat and reach exsiccant purpose.But microwave drying killing microorganisms and mycete, and has disinfective action.The microwave heating installation of China's production at present has 915mhz and two frequencies of 2450mhz.
(3) research overview of AIDS and hepatitis B
1, general introduction
Disease of viral infection is AIDS and hepatitis B serious threat human health and life particularly.Present AIDS is popular being becoming increasingly rampant in the whole world; AIDS associating Planning Department of the United Nations announces; By the end of the end of the year 2005 whole world HIV (humanimmunodeficiency virus, be called for short: HIV) the infected reach more than 6,500 ten thousand (1. WHO:http: //www.unaids.org/en/KnowledgeCentre/HIVData/GlobalReport/D efault.asp; 2. Liu Jia, peak, Lu Fengmin, the popular present situation in the Zhuan Hui .HIV/AIDS whole world. infectious disease information .2006,19 (5): 228-232.).AIDS also just spreads with surprising speed in the Asia, and the China's AIDS popularity is also very serious, and the infected about 1,000,000 is arranged at present; And annual estimate 2010 and will reach 1,000 ten thousand with 30% speed increment, be equivalent to whole Belgian population (1. Fu Ji China. the popular present situation of AIDS and problem that faces and challenge. the .2007 of preventive medicine forum; 13 (3) I0001-I0002,2. Portsmouth S, Stebbing J; Keyi X, Jianping Z, Guohua P.HIV and AIDS in the People ' s Republic of China:a collaborative review.Int J STD AIDS; 2003,14 (11): 757-61.).
Hepatitis B is one of principal disease that influences human health.The number that the whole world infected HBV approximately surpasses 2,000,000,000, have at present 3.5 hundred million chronic viral hepatitis B patients (referring to 1. Fung, S.K.; Luk, A.S., Viral hepatitis.Curr.Opin.Gastroenterol.2003; 20:241-247; 2. McMahon BJ.Epidemiology and natural history ofhepatitis B.Semin Liver Dis, 2005,25 (Suppl 1): 3-8).Chinese then be the higher country of viral hepatitis sickness rate; The people infected hepatitis B virus more than 700,000,000, and total infection rate reaches 57%, had that the people carries hepatitis B virus more than 100,000,000; More than existing chronic viral hepatitis B patient 30,000,000 people, and annual acute hepatitis b new cases reach 3,000,000 examples.60%～70% liver biopsy pathology is reported as chronic persistent hepatitis or chronic active hepatitis in so-called " asymptomatic carrier "; Secular hepatitis B virus infection will cause liver cirrhosis, part to develop into hepatocarcinoma; Be one of underlying cause of death (Bi Shengli. to the viral hepatitis of problem further investigation China. China's experiment and clinical virology magazine; 2002,16 (1): 5-6.).
2, antiviral drugs
Since AIDS was popular, the research and development of antiviral drugs had got into fast-developing period, and new medicine constantly appears on the market.Mainly contain following several kinds: nucleoside medicine, like acyclovir (acyclovir), Cymevan (ganciclovir, ganciclovir), lamivudine (lamivudine) etc.; The non-nucleoside medicine, ribavirin (vibarvirin) and amantadine (amantadine); Other types medicine such as alpha-interferon etc. (α-interferon).Above-mentioned several types of medicines can the part relief of symptoms, prolongs life cycle, but significant disadvantages is also arranged.Be that they mostly have serious toxicity, and because virus is easy to variation, so drug resistance can occur very soon; Because serious toxicity, the patient treatment compliance is poor, can not adhere to, further promotes chemical sproof generation; Drug price is expensive; They mostly are to active virus of duplicating effectively, and can not remove latency or non-replicative phase virus, and are prone to recurrence after the drug withdrawal.This make their application receive very big restriction (precious referring to 1. make pottery wearing. AIDS virus resisting drug research progress. Chinese Journal of New Drugs, 2002,11 (11): 842-846; 2. Gulick RM.Newantiretroviral drugs.Clin Microbiol Infect, 2003,9 (3): 186-193; 3. Laurence J.HIV therapeuticsin children and adults and their side effects.AIDS Read, 2004,14 (1): 6-7; 4. Bourinbaiar AS; Jirathitikal V.Low-cost anti-HIV compounds:potential application for AIDS therapy indeveloping countries.Curr Pharm Des, 2003,9 (18): 1419-1431; 5. Thomas C; Nelson M, Stebbing J.HIV and Hepatitis B:A Review.AIDS Patient Care and STDs.2003,17 (12): 623-633.).
3, external anti AIDS virus model
Adopt chemical compound to induce host cell to form plasmodial inhibition experiment to HIV.
HIV all can infect human T lymphocyte, mononuclear phagocyte and neurocyte in vivo and in vitro.In general, the method for cellular level can be used for screening and studying the chemical compound that suppresses virus replication arbitrary target spot in the cycle.It is exactly a cell model that is commonly used to screen HIV-resistant activity that chemical compound induces host cell to form plasmodial inhibition experiment to HIV.It is C8166 that the human T lymphocyte is adopted in our external anti AIDS virus experiment.HIV infects the C8166 cell and produces big syncytial cell and expansionization effect, causes cell death in 4～6d.
Utilize the mtt assay testing drug for to HIV-1
IIIBThe inhibition of cytopathogenic effect (CPE) is active, confirms its influence to C8166 cell survival and propagation.Under inverted microscope, observe with counting and contain the chemical compound of different diluted concentrations and do not contain the syncytial cell number in the testing compound culture hole; Calculate suppression ratio and EC50 (Zheng Yongtang. the in-vitro screening of inverase and research method. Chinese Journal of New Drugs; 2002,11 (8): 596-600.).
4, anti AIDS virus model in the body
Comprise primates and non-human primate animal model.Primates comprises that human immunodeficiency virus I type (chimpanzee), II type (baboon), simian immunodeficiency virus (are called for short: model such as SIV).This type of non-human primate animal animal model belongs to due to the retrovirus slow virus infection.Wherein to study maximum be to infect and make the morbific virus of ungulate: sheep lung adenoma-demyelinating leukoencephalitis virus (is called for short: MVV), goat joint-encephalitis (is called for short: CAEV), equine infectious anemia virus (is called for short: EIAV) (be called for short: BIV) (referring to Yang Chuanbo with newfound bovine immunodeficiency virus; Xu Xingran; Cai Jiali. people's AIDS-like disease Research of Animal Model for Study progress. animal medicine progress .2005,26 (3): 46-50.).
5, anti-hepatitis B virus cell model
Select the model of HBV transgenosis cell strain HepG2-2.2.15 cell for use for experiment.Owing to lack better cell and animal model, can property tumor cell line HepG2-2.2.15 be the main cell model that is used for the anti-HBV medicine of in-vitro screening at present in the liver source of external long-term cultivation.The HepG2-2.2.15 cell is with the transfer vector plasmid transfection HepG-2 cell that contains HBV genome (2 HBV heads are connected to the tail direction with tail to the tail dimer); Screen through G418; The clone's ability stably excreting HBsAg, HBeAg, HBcAg and the Dane granule that obtain; And can detect various intermediate duplication bodies such as DNA and RNA in the cell, s1 and preceding S2 before can also secreting.This cell strain can be stablized and duplicates the HBV virion, is a very sophisticated external model, extensively is used to the in-vitro screening and the evaluation of anti-HBV medicine.(referring to 1. Sell MA, Chen ML, Acs G.Production of hepatitis B virus particles in HepG2 cells.Proc.Natl.Acad.Sci.U.S.A.1987,84,1005-1009; 2. Sells MA; Zelent AZ; Shvartsman M, Acs G.Replicative intermediates ofhepatitis B virus in HepG2 cells that produce infectious virions.J.Virol.1988,62:2836-2844; 3. Lampertico P; Maher JS; Gerber MA.Development and application of an in vitro model forscreening anti-hepatitis B virus therapeutics.Hepatology, 1991,13:422-426.).
6, anti-hepatitis B virus body inner model
Use NOD.CB17-Prkdc
Scid/ J HBV transgenic mouse model.NOD.CB17-Prkdc
Scid/ J HBV transgenic mouse model is application hepatitis B virus (HBV) animal model (Larkin J comparatively widely; Clayton M; Sun B, Perchonock CE, et al.Hepatitis B virus transgenic mouse model of chronic liver disease.Nat.Med.; 1999,5:907-912; Morrissey DV; Lockridge JA, Shaw L, Blanchard K; Et al.Potent andpersistent in vivo anti-HBV activity of chemically modified siRNAs.Nat.Biotechnol., 23 (8): 1002-1007.).This model is that HBV carrier tail vein high-pressure injection is gone into NOD.CB17-Prkdc
Scid/ J Mus, this Mus is an immunodeficient mouse, lacks sophisticated T cell and B cell, and the also most of disappearance of the activity of natural killer cell.The pWTD of dimer end to end of total length HBV gene is used as the carrier tail vein high-pressure injection of HBV and goes into.This HBV transgenic mouse is invaded the hepatitis that the hepatitis of profit can simulating human with mononuclear cell, can be used to study the immunizing composition that causes the hepatitis B morbidity and the antigen target of virus.
Therefore the traditional Chinese herbal medicine that combines China is therefrom excavated effectively, low toxicity, inexpensive antiviral drugs particularly anti AIDS virus and hepatitis B virus medicaments, has very important significance.
Through the document retrieval etc., up to the present, find to have these two kinds of chemical compounds as yet at the report that is used to prevent, diagnose, detect, protect, treat and study aspect HIV and hepatitis B virus and the directly related disease thereof.
Summary of the invention
The technical problem that will solve required for the present invention is the effect that discloses a kind of Chinese medicine Rhizoma Polygoni Cuspidati extract anti AIDS virus and anti-hepatitis B virus; Promptly this extract is used to prepare anti AIDS virus and anti-hepatitis B virus product activity component cpd 1 and chemical compound 2, to overcome the above-mentioned defective that prior art exists.
That is to say that the present invention is intended to the activity of clear and definite a kind of Rhizoma Polygoni Cuspidati extract in anti AIDS virus and hepatitis B virus application facet through researchs such as cell and zooperies, promptly the present invention relates to a kind of new purposes of Rhizoma Polygoni Cuspidati extract.
Described Rhizoma Polygoni Cuspidati extract is meant resveratrol 3-O-β-D-glucoside 2 " sulfate group sodium or potassium salt (be called for short: chemical compound 1) with resveratrol 3-O-β-D-glucoside 4 '-sulfate group sodium or potassium salt (abbreviation: chemical compound 2).
Chemical compound 1 in the Rhizoma Polygoni Cuspidati extract all is active component of anti AIDS virus and anti-hepatitis B virus with chemical compound 2; Its occupation mode be comprise respectively separately use, both merge and use or unite a kind of in the use etc. with other chemical substances; Preferred use separately respectively all can be used in preparation anti AIDS virus and anti-hepatitis B virus product.
Described anti AIDS virus and anti-hepatitis B virus product are meant one or more of the product that is used for preventing, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof; Described AIDS is to comprise a kind of in HIV-1 type AIDS and the HIV-2 type AIDS, preferred HIV-1 type AIDS.
Described anti AIDS virus and anti-hepatitis B virus product all are to comprise a kind of in medicine and the field products such as food, are to comprise in medicine, reagent or the food etc. one or more, preferred agents.
(1) technical conceive
The independent development original new drug is a present urgent task of China; China's Chinese medicine and pharmacy has a long history; Also accumulated rich experience with Chinese herbal medicine prevention and treatment disease aspect; Therefore from Chinese medicine, seeking effective active component or find that its new purposes all is effectively quick approach, also is the place of the advantage of Chinese original new drug development.
The inventor carries out the chemical constitution study of system through the extract to single Rhizoma Polygoni Cuspidati medical material, screens and prove the activity and the purposes of this extract.Thereby the inventor infers preventing, diagnose, protecting and the active clinical drug effects in aspect such as treatment AIDS and hepatitis B of Rhizoma Polygoni Cuspidati; Also should mainly be through the active site Rhizoma Polygoni Cuspidati extract particularly the drug effect of chemical compound 1 and chemical compound 2 bring into play, result of study also prove and confirmed Rhizoma Polygoni Cuspidati extract particularly chemical compound 1 have significant pharmacologically active with chemical compound 2.
AIDS and hepatitis B have a strong impact on the health of Chinese population; Development treatment AIDS and hepatitis B medicament, especially prevent, diagnose, protect and treat aspect products such as AIDS and hepatitis B particularly medicine have remarkable social benefit, economic benefit.
(2) pharmacologically active of Rhizoma Polygoni Cuspidati extract
The present invention has carried out many-sided test to Rhizoma Polygoni Cuspidati extract in the activity of prevention, diagnosis, protection, treatment and aspects such as research AIDS and hepatitis B.
Supplying test agent all is to take conventional method for preparing to obtain; Chemical compound 1 content in the resulting Rhizoma Polygoni Cuspidati crude extract is general＜and 50%, chemical compound 2 content are general＜60%; But through the Rhizoma Polygoni Cuspidati crude extract is carried out purification; Can access the Rhizoma Polygoni Cuspidati extract behind the purification, the chemical compound 1 wherein and the purity of chemical compound 2 all can be more than 95%, i.e. the defined Rhizoma Polygoni Cuspidati extract of the present invention.
That is to say; Adopting the main component chemical compound 1 of Rhizoma Polygoni Cuspidati extract is raw material with chemical compound 2; Or the Rhizoma Polygoni Cuspidati medical material that direct employing contains chemical compound 1 and chemical compound 2 is a raw material; Perhaps directly adopting the Rhizoma Polygoni Cuspidati crude extract that contains chemical compound 1 and chemical compound 2 is raw material, can both directly or indirectly be used to prepare antidepressant product.Rhizoma Polygoni Cuspidati extract preferably uses with pure basically form, like the purity of chemical compound 1 and chemical compound 2 all >=95%.
The in vitro tests of Rhizoma Polygoni Cuspidati extract AIDS and hepatitis B prevention, diagnosis, protection and therapeutic activity
1, the HIV-resistant activity of chemical compound 1 and chemical compound 2
It is C8166 that the human T lymphocyte is adopted in external anti AIDS virus experiment.Utilize the mtt assay testing drug for to HIV-1
IIIB(be called for short: inhibition CPE) is active, and the result shows that 1 couple of HIV of chemical compound induces the C8166 cell to form syncytium has obvious inhibiting activity, HIV is had significant inhibitory effect, its CC for cytopathogenic effect
50＞2000 μ g/mL, EC
50Be 153.42 μ g/mL, therapeutic index is＞13.04 (seeing table 1 and table 2).And this compound water soluble is fine, the very low nontoxic in other words lead compound that can be used as treatment AIDS of cytotoxicity.
The toxic action of table 1, chemical compound 1 and 2 pairs of C8166 cells of chemical compound
| Chemical compound | Concentration | Cell survival rate | ±SD | CC 50(μg/mL) |
| Chemical compound 1 | 2000 | 76.81 | 5.26 | ＞2000 |
| 400 | 116.71 | 6.24 | ||
| 80 | 111.04 | 4.27 | ||
| 16 | 108.42 | 13.37 | ||
| 3.2 | 111.57 | 6.37 | ||
| 0.64 | 106.10 | 0.09 | ||
| Chemical compound 2 | 2000 | 68.20 | 7.96 | ＞2000 |
| 400 | 114.06 | 4.79 | ||
| 80 | 110.03 | 10.07 | ||
| 16 | 88.89 | 13.46 | ||
| 3.2 | 90.30 | 11.08 | ||
| 0.64 | 68.91 | 4.86 | ||
| AZT | 2000 | 35.69 | 1.78 | 1288.24 |
| 400 | 88.80 | 1.74 | ||
| 80 | 99.17 | 4.16 | ||
| 16 | 100.38 | 3.44 | ||
| 3.2 | 102.10 | 5.98 | ||
| 0.64 | 106.68 | 5.73 |
Table 2, chemical compound 1 and 2 couples of HIV-1 of chemical compound
IIIBInduce the cytopathic inhibitory action of C8166
| Chemical compound | Concentration (μ g/ml) | Suppression ratio (%) | ±SD | EC 50(μg/mL) | Therapeutic index (TI) |
| Chemical compound 1 | 200 | 58.72 | 4.86 | 153.42 | ＞13.04 |
| 40 | 5.81 | 6.45 | |||
| 8 | -0.92 | 12.00 | |||
| Chemical compound 2 | 200 | 17.13 | 6.11 | ＞200 | |
| 40 | 7.034 | 5.53 | |||
| 8 | 0.00 | 4.00 | |||
| AZT | 1 | 100 | 0 | 0.0049 | 262906.12 |
| 0.2 | 100 | 0 | |||
| 0.04 | 91.30 | 6.15 | |||
| 0.008 | 66.18 | 4.66 | |||
| 0.0016 | 13.04 | 5.80 |
2, the external anti-HBV of chemical compound 1 and chemical compound 2 is active
External anti-hepatitis B virus experiment selected HBV transgenosis cell strain HepG2-2.2.15 cell is the model of experiment.This cell strain can be stablized and duplicates the HBV virion, extensively is used to the in-vitro screening of anti-HBV medicine.Experimental result shows (seeing table 3, table 4): chemical compound 1 all has remarkable inhibiting activity to duplicating of HBV DNA with chemical compound 2, and chemical compound 1 and chemical compound 2 can also obviously suppress the secretion of HBV surface antigen HBsAg.Further research shows that this effect has certain agent effect relationship.And find this chemical compound under experimental concentration, the growth of pair cell does not have tangible toxicity.
Table 3, chemical compound variable concentrations are to HBV DNA copy number (log in the HepG2-2.2.15 cell
10Copies/mL) influence
| Chemical compound | Concentration (μ M) | ||
| 0.2uM | 1uM | 5uM | |
| Chemical compound 1 | 3.58±0.37 * | 3.14±0.34 ** | 2.51±0.31 *** |
| Chemical compound 2 | 3.20±0.26 * | 2.92±0.23 ** | 2.38±0.36 *** |
| H 2O | 4.14±0.22 | ||
| DMSO | 4.17±0.24 | ||
*Compare with the DMSO group P＜0.05;
*Compare with the DMSO group P＜0.01;
* *Compare with the DMSO group P＜0.001.
Table 4, chemical compound variable concentrations are to the excretory influence of HBV HbsAg (ng/mL) in the HepG2-2.2.15 cell
| Chemical compound | Concentration (μ M) | ||
| 0.2uM | 1uM | 5uM | |
| Chemical compound 1 | 147±26 * | 121±30 ** | 110±23 *** |
| Chemical compound 2 | 141±22 * | 132±25 ** | 124±29 *** |
| H 2O | 185±35 | ||
| DMSO | 206±40 | ||
*Compare with the DMSO group P＜0.05;
*Compare with the DMSO group P＜0.01;
* *Compare with the DMSO group P＜0.001.
3, anti-HBV is active in the body of chemical compound 1 and chemical compound 2
Anti-hepatitis B virus in the body (is called for short: HBV) the active NOD.CB17-Prkdc of application
Scid/ J HBV transgenic mouse model.Discover that chemical compound 1 and chemical compound 2 can significantly reduce the copy number (seeing table 5) of HBV DNA in the model mouse body, reduce the level (seeing table 6) of HbsAg in the serum.Research also finds, chemical compound 1 and chemical compound 2 present certain agent effect relationship (seeing table 7 and table 8) in the intravital anti-HBV activity of transgenic mice.
Table 5, chemical compound are to HBV DNA copy number (log in the transgenic mouse
10Copies/mL) influence
| Chemical compound | 3?day?after?last?dose | 6?day?after?last?dose | ||
| 1mg/kg | 10mg/kg | 1mg/kg | 10mg/kg | |
| Chemical compound 1 | 6.83±0.40 * | 6.12±0.36 ** | 6.84±0.37 * | 6.17±0.43 ** |
| Chemical compound 2 | 6.50±0.34 * | 6.14±0.35 ** | 6.70±0.28 * | 6.16±0.29 ** |
| PBS | 7.41±0.45 | 7.22±0.31 | ||
*In the identical period, compare with the PBS group P＜0.05;
*Compared with the PBS group in the identical period P＜0.01.
Table 6, chemical compound are to the excretory influence of HBV HbsAg (ng/mL) in the transgenic mouse
| Chemical compound | 3?day?after?ast?dose | 6?day?after?last?dose | ||
| 1mg/kg | 10mg/kg | 1mg/kg | 10mg/kg | |
| Chemical compound 1 | 609±30 * | 522±36 ** | 1016±44 * | 911±40 ** |
| Chemical compound 2 | 614±34 * | 510±25 ** | 1030±0.48 * | 923±59 ** |
| PBS | 624±37 | 1126±61 | ||
*In the identical period, compare with the PBS group P＜0.05;
*In the identical period, compare with the PBS group P＜0.01.
Table 7, chemical compound various dose are to HBV DNA copy number (log in the transgenic mouse
10Copies/mL) influence
| Chemical compound | Dosage (mg/kg) | ||
| 10 | 20 | 40 | |
| Chemical compound 1 | 6.20±0.30 * | 5.87±0.38 ** | 5.16±0.43 *** |
| Chemical compound 2 | 6.36±0.35 * | 5.72±0.31 ** | 5.11±0.40 *** |
| PBS | 7.51±0.45 | ||
*Compare with the PBS group P＜0.05;
*Compare with the PBS group P＜0.01;
* *Compare with the PBS group P＜0.001.
Table 8, chemical compound various dose are to the excretory influence of HBV HbsAg (ng/mL) in the transgenic mouse
| Chemical compound | Dosage (mg/kg) | ||
| 10 | 20 | 40 | |
| Chemical compound 1 | 533±40 * | 480±46 ** | 395±39 *** |
| Chemical compound 2 | 550±45 * | 501±31 ** | 430±40 *** |
| PBS | 618±32 | ||
*Compare with the PBS group P＜0.05;
*Compare with the PBS group P＜0.01;
* *Compare with the PBS group P＜0.001.
(3) method for distilling of Rhizoma Polygoni Cuspidati extract
The Rhizoma Polygoni Cuspidati that the present invention is produced with Chinese Jiangsu is a raw material, adopts solvent extraction and multiple chromatography method, separates obtaining this two kinds of native compounds.Adopt that ultraviolet light is general, methods such as infrared spectrum, mass spectrum, proton nmr spectra and carbon spectrum, the structure of having confirmed these two kinds of chemical compounds is resveratrol 3-O-β-D-glucoside 2 " sulfate group sodium or potassium salt and resveratrol 3-O-β-D-glucoside 4 '-sulfate group sodium or potassium salt.
Rhizoma Polygoni Cuspidati extract of the present invention particularly chemical compound 1 can prepare through following method with chemical compound 2:
The method for preparing of the said Rhizoma Polygoni Cuspidati extract of the present invention comprises the steps:
(1) extract: it is some to get the Rhizoma Polygoni Cuspidati raw material, extracts, and gets extracting solution, i.e. Rhizoma Polygoni Cuspidati crude extract;
(2) separation and purification:, promptly get Rhizoma Polygoni Cuspidati extract with this crude extract separation and purification.
Described method for distilling comprises all operable methods well known in the art such as solvent extraction method.
The method for distilling of solvent extraction method mentioned above is the conventional method for distilling of this area; That is to say and comprise ultrasonic extraction commonly used, dipping extraction method, percolation extraction method, decoct in extraction method, heating and refluxing extraction method or the continuous backflow extraction method etc. one or more, preferred heating and refluxing extraction method or percolation extraction method; Extraction time can be once or repeatedly.Simultaneously, factors such as the degree of grinding of raw material, extraction time, extraction temperature, appointed condition also can both influence extraction efficiency, must take in.Various method for distilling comprise all technical data of ins and outs, all can be referring to relevant teaching material and relevant technical literature etc.
The routine that the employed extraction solvent of solvent extraction method mentioned above is this area is extracted solvent, that is to say to comprise that three types of common water, hydrophilic organic solvent or lipophilic organic solvents etc. extract one or more of solvent;
Described water is to comprise a kind of in water, sour water or the aqueous alkali etc.;
Described hydrophilic organic solvent is general said and the miscible organic solvent of water; Comprise in ethanol, ethanol water, methanol or the acetone etc. one or more; In preferred alcohol or the Different concentrations of alcohol aqueous solution etc. one or more, further preferred Different concentrations of alcohol aqueous solution;
Described lipophilic organic solvent is the organic solvent that generally said and water can not be miscible; Comprise in petroleum ether, benzene, chloroform, ether, ethyl acetate, dichloromethane or the dichloroethanes etc. one or more; In preferred petroleum ether, chloroform, ether, ethyl acetate, dichloromethane or the dichloroethanes etc. one or more, one or more in further preferred chloroform, ether, ethyl acetate or the dichloromethane etc.
The resulting Rhizoma Polygoni Cuspidati crude extract of said extracted method needs further separation and purification.
Described isolation and purification method is all operable methods well known in the art; Comprise solvent segregation; Solvent extraction (comprise in extraction, counter current continuous extraction method, counter-current distribution method or the drop counter-current distribution method etc. one or more), macroporous adsorbent resin method, the sedimentation method; Salting out method, one or more in column chromatography or crystallization and recrystallization and the Steppecd crystallization etc.; These methods all are known technologies of this area, at needs further in the conclusive evidence, are easy to find all technical data that comprise ins and outs from relevant teaching material and relevant technical literature etc.
In order to make product property stable, easy to use and preserve, also can increase the step of an optimization, i.e. step 3:
(3) drying.
Described drying means is an operable method well known in the art, comprises that atmosphere pressure desiccation is like in oven for drying, hypobaric drying method, boulton process, spray drying method, freeze-drying, far infrared heating drying method or micro-wave drying method etc. one or more.
In the specific implementation, need adopt wherein suitable method, and choose measures necessary such as appropriate condition to reach the set goal according to existing fund, technology and relevant requirement.And these methods also all are known technologies of this area, at needs further in the conclusive evidence, all are to be easy to find all technical data that comprise ins and outs from relevant teaching material and relevant technical literature etc.
(4) purposes of Rhizoma Polygoni Cuspidati extract
1, general introduction
The purpose of this invention is to provide a kind of product that is used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof, comprise in medicine, reagent, the food etc. one or more, preferred agents.
Through pharmacologically active screening proof, Rhizoma Polygoni Cuspidati extract is the active site of its prevention, diagnosis, detection, protection, treatment and research AIDS and hepatitis B and directly related disease thereof.
Show that through experimentation Rhizoma Polygoni Cuspidati extract is external can to suppress HIV and hepatitis B virus, can significantly suppress the intravital hepatitis B virus of mice.Completed acute toxicity testing proves; The mouse stomach administration surpasses 1.0g/kg to the maximum tolerated dose of this active site; Be equivalent to more than 200 times of clinical recommended drug dosage; Show that this effective site is safe and reliable, solved complicated component in the Chinese medicine compound, active constituent content is low and has contained the problem of toxic component.
In sum; The inventor has carried out theory study to Rhizoma Polygoni Cuspidati extract; Through a large amount of particularly secular pharmacology tests of experimentation, find that the Rhizoma Polygoni Cuspidati extract of being addressed has the activity of significant prevention, diagnosis, detection, protection, treatment and research AIDS and hepatitis B and directly related disease thereof.Therefore, Rhizoma Polygoni Cuspidati extract and compositions thereof can be used for preparing anti AIDS virus and anti-hepatitis B virus product, are the medicine that feedstock production forms with Rhizoma Polygoni Cuspidati extract of the present invention preferably.
2, the method for using of Rhizoma Polygoni Cuspidati extract and compositions thereof and requirement
Rhizoma Polygoni Cuspidati extract of the present invention can be united use separately or with other active component; Comprise and be used to prepare the product that is used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof; Comprise medicine, reagent or food etc., especially medicine.
Aspect concrete use, Rhizoma Polygoni Cuspidati extract of the present invention can use separately, can also use with other many chemical substances.These chemical substances biologically active or have the function of treatment disease whether no matter; Comprise miscellaneous function like collaborative amplification, antagonism or alleviate the side effect etc. of Rhizoma Polygoni Cuspidati extract, these chemical substances are to comprise in pharmaceutically acceptable carrier, food, natural product, chemical synthetic drug or the human medication etc. one or more; Preferably include in pharmaceutically acceptable carrier or the food etc. one or more; Further preferred pharmaceutically acceptable carrier.
" the pharmaceutically acceptable carrier " that this paper uses comprises one or more in any He all physiology suitable solvent, disperse medium, afterbirth, antibacterial and antifungal, isotonic agent or the absorption delay agent etc.The example of pharmaceutically acceptable carrier comprises one or more water, saline, PBS, glucose, glycerol or ethanol etc. and in the compositions one or more thereof.In many cases, in said composition, preferably include isotonic agent, for example, sugar, such as in the polyhydric alcohol of mannitol, sorbitol, sorbitol or the sodium chloride etc. one or more.Pharmaceutically acceptable carrier can also comprise a spot of auxiliary substance, one or more in wetting agent or emulsifying agent, antiseptic or the buffer etc. for example, and they have strengthened the effect duration or the effectiveness of this Rhizoma Polygoni Cuspidati extract.
See that from concrete classification said pharmaceutically acceptable carrier is meant the pharmaceutical carrier that the medicine and pharmacology field is conventional, comprises excipient, like in starch or the water etc. one or more; Lubricant is like in glycerol or the magnesium stearate etc. one or more; Disintegrating agent is like microcrystalline Cellulose etc.; Filler is like in starch or the lactose etc. one or more; Bonding agent is like in pregelatinized Starch, dextrin, cellulose derivative, alginate, gelatin or the polyvinylpyrrolidone etc. one or more; Osmotic pressure regulator is like in glucose, sucrose, sorbitol or the mannitol etc. one or more; Diluent is like water etc.; Disintegrating agent is like in agar, calcium carbonate or the sodium bicarbonate etc. one or more; Absorption enhancer is like quaternary ammonium compound etc.; Surfactant is like hexadecanol etc.; Absorption carrier is like in Kaolin or the soap clay etc. one or more; Lubricant is like in Pulvis Talci, calcium stearate, magnesium stearate or the Polyethylene Glycol etc. one or more; In addition, can also in compositions, add other adjuvant, like in flavouring agent or the sweeting agent etc. one or more.
For example; With the dissolving of active component Rhizoma Polygoni Cuspidati extract, suspendible or (for example be emulsifiable in the suitable aqueous solvent; In distilled water, normal saline or the Green's solution etc. one or more) or in the oil-based solvent (for example; Vegetable oil is one or more in olive oil, Oleum sesami, Oleum Gossypii semen, Semen Maydis oil or the propylene glycol etc. for example) in; Can make ejection preparation; Wherein can contain dispersant (for example, one or more in polyoxyethylene sorbitan monoleate, polyoxyethylene hardened castor oil 60, Polyethylene Glycol, benzyl alcohol, chlorobutanol or the phenol etc.), osmotic pressure regulator (for example, one or more in sodium chloride, glycerol, D9-mannose, D-sorbitol or the glucose etc.) in the solvent.In this case; If necessary; Can add additive, for example solubilizing agent (for example, polyoxyethylene sorbitan monoleate, polyoxyethylene castor oil, polyvidone, Polyethylene Glycol 40 Oleum Ricini or general youth Buddhist nun restrain one or more among F-68 etc.), stabilizing agent are (for example; Human serum albumin etc.), analgesic (for example, one or more in procaine hydrochloride or the lignocaine etc.) etc.
According to the invention and Rhizoma Polygoni Cuspidati extract can also unite use with the form of compositions, particularly with other chemical substance such as medicine animal especially mammal is comprised that people or other animals treat compositions for use or similar compositions.Said mammal; Comprise in people, mice, rat, sheep, monkey, cattle, pig, horse, rabbit, dog, chimpanzee, baboon, Adeps seu carnis Rhiopithecus roxellanae, macaque or the Rhesus Macacus etc. one or more; In preferred people, mice, rat, monkey, pig, rabbit or the dog etc. one or more, one or more in further preferred people, rat or the monkey etc.For example, can with Rhizoma Polygoni Cuspidati extract of the present invention add be suitable for to curee's Pharmaceutical composition in.Usually, this Pharmaceutical composition comprises Rhizoma Polygoni Cuspidati extract of the present invention and pharmaceutically acceptable carrier.
The compositions of Rhizoma Polygoni Cuspidati extract particularly pharmaceutical composition can have various forms, comprises in the dosage forms such as liquid, semisolid and solid for example one or more; Wherein said pharmaceutical composition comprises that the Rhizoma Polygoni Cuspidati extract of treating effective dose is an active component, and one or more pharmaceutically acceptable carriers.
The pharmaceutical composition of Rhizoma Polygoni Cuspidati extract can adopt conventional production method well known in the art to process various dosage forms, and active component is mixed with one or more carriers, is made into required dosage form then.Described dosage form comprises one or more in tablet, capsule, granule, suspensoid, Emulsion, solution, syrup or the injection etc., takes one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more), the mucosa dialysis etc. to carry out prevention, diagnosis, detection, protection, treatment or the scientific research of AIDS and hepatitis B and directly related disease thereof.
It is 0.5%～99% active component Rhizoma Polygoni Cuspidati extract that pharmaceutical composition preferably contains weight ratio, further preferably contains weight ratio and be 1%～95% active component Rhizoma Polygoni Cuspidati extract, most preferably contains weight ratio and be 5%～90% active component Rhizoma Polygoni Cuspidati extract.
The pharmaceutical composition of Rhizoma Polygoni Cuspidati extract generally must be aseptic and stable under the production condition of storage.Can said composition be mixed with solution, microemulsion, dispersion liquid, liposome or other is suitable for the ordered structure of high drug level.Through with a kind of of this Rhizoma Polygoni Cuspidati extract of aequum and required mentioned component or combine to add in the appropriate solvent and then carry out aseptic filtration and prepare aseptic parenteral solution.Generally speaking, prepare dispersion liquid through this Rhizoma Polygoni Cuspidati extract being added in the aseptic solvent that contains basic disperse medium and required above-mentioned other composition.Under the situation of the sterile powder that is used to prepare aseptic parenteral solution, the method for preparing of recommendation is vacuum drying and lyophilized preparation.For example, through passing through to keep required granular size such as the coating of lecithin, under the situation of dispersion liquid and, can keeping the adequate liquidity of solution through using surfactant.Through comprising that in said composition the medicament (for example Monostearate or gelatin) that postpones to absorb can reach the prolongation absorption of injectable composition.
When being used for the patient, Rhizoma Polygoni Cuspidati extract dosage of the present invention is 5～20mg/kgd, and this dosage or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.
Rhizoma Polygoni Cuspidati extract of the present invention and Pharmaceutical composition thereof can comprise the Rhizoma Polygoni Cuspidati extract of the present invention of " treatment effective dose " or " prevention effective dose "." treatment effective dose " is meant at the dosage of necessity and effectively reaches the amount of required therapeutic effect under the time.The treatment effective dose of Rhizoma Polygoni Cuspidati extract can cause that at this individuality the factors such as ability of required reaction change according to the patient's condition, age, sex and body weight and this Rhizoma Polygoni Cuspidati extract such as individuality.The treatment effective dose also refers to that the useful therapeutic effect of this Rhizoma Polygoni Cuspidati extract surpasses the amount of its any toxicity or harmful effect." prevention effective dose " is meant the amount that under necessary dosage and time, effectively reaches required preventive effect.Because preventive dose is used for the ill preceding or early stage curee of disease, the prevention effective dose is usually less than the treatment effective dose.The typical non-limiting scope of the treatment of Rhizoma Polygoni Cuspidati extract of the present invention or prevention effective dose is 5～20mg/kg, more preferably 5～10mg/kg.Should note; Dose value will change according to disease type of desiring to alleviate and seriousness; That is to say when being used for the patient that Rhizoma Polygoni Cuspidati extract dosage of the present invention or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.In addition; Should understand; For any specific curee; Should along with the time according to individual need and give with or supervision give the professional judgement adjustment given dose system with the people of said compositions, and the dosage range that this paper sets is merely illustrative, the scope or the practice of compositions that can't the requirement for restriction protection.
That is to say, need be according to object, route of administration, institute's disease of treat and the situation etc. of treatment, variation Rhizoma Polygoni Cuspidati extract of the present invention at every turn and/or dosage or the consumption of every day.For example, give mammal through vein, adult (like body weight 60kg) especially, the single dose of said Rhizoma Polygoni Cuspidati extract is about 50～400mg, preferably about 100mg, preferred administration every day 1～3 time.Can adjust dosage unit, to propose the best required reaction of arch (for example, treatment or prevention are replied).For example, can single heavy dose of administration can give several divided doses or reduce or increase dosage in proportion according to the urgency of treatment situation in a period of time.The non-intestinal compositions that preparation is easy to the unified dosage unit form of administration and dosage is especially favourable.The dosage unit form that this paper uses refers to be suitable for the physical separation unit of dosage unit of the mammalian subject of desire treatment; The calculating that each unit contains scheduled volume is used for together producing with required pharmaceutical carrier the active matter Rhizoma Polygoni Cuspidati extract of required therapeutic effect.The specification of dosage unit form of the present invention; Mixing the interior of this technology that is used for treating the individual sensitivity Rhizoma Polygoni Cuspidati extract by the following specific characteristic of confirming and directly depending on following (a) this Rhizoma Polygoni Cuspidati extract and the particular treatment of desiring to reach or preventive effect with (b) in restriction.
3, the pharmaceutical dosage form of Rhizoma Polygoni Cuspidati extract and compositions thereof and route of administration
The product that is used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof of Rhizoma Polygoni Cuspidati extract of the present invention and preparation of compositions thereof, wherein the product according to the requirement of beverage, food technology field preparation can be used in prevention, protection and treats AIDS and hepatitis B and directly related disease thereof; Can be used in patient's treatment or health care according to the product of the requirement of medical technical field preparation; Can either directly be used to prepare the medicine of treatment or health care separately; Also can mix with many chemical substances or make up, directly or indirectly be used to prepare the medicine of treatment or health care.Chemical substance described here identical with described in this joint preceding text.
In the present invention, required material comprises raw material of the present invention, above-mentioned matching used chemical substance etc., all should adopt the material of food stage or pharmaceutical grade according to practical situation and needs.
Rhizoma Polygoni Cuspidati extract of the present invention and compositions thereof can be used the whole bag of tricks administration known in the art, although route of administration/administering mode of in many therapeutic use, recommending is spray or oral administration.But the technical staff will appreciate that route of administration/administering mode changes with required result.In some practical implementation, this reactive compound can together prepare for example empty release formulation with the carrier of protecting this chemical compound to avoid rapid release, comprises that graft transmission system, transdermal paste one or more in transmission system or the microcapsule transmission system etc.In addition, can also use biodegradable, biocompatible polymer, for example one or more in ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or the polylactic acid etc.Prepare the equal patent applied for of many methods of this preparation or be generally those skilled in the art and know (Sustained andControlled Release Drug Delivery Systems for example; J.R.Robinson edits, Marcel Dekker, Inc.; New York, 1978).
Rhizoma Polygoni Cuspidati extract of the present invention and compositions thereof, one or more modes in administered through oral, rectum or the parenteral etc. are applied to the patient who needs this treatment usually.
Be used for when oral, can be made into conventional solid preparation such as in tablet, powder, granule or the capsule etc. one or more.When implementing, Rhizoma Polygoni Cuspidati extract of the present invention can be together oral with for example inert diluent or assimilable edible carrier.This Rhizoma Polygoni Cuspidati extract (with its composition altogether, if desired) can also be wrapped in hard or soft shell gelatin capsules, is pressed into tablet or directly adds in curee's the meals.About oral therapeutic administration, can said Rhizoma Polygoni Cuspidati extract be added with excipient and uses with one or more forms in edible tablet, buccal tablet agent, lozenge, capsule, suspension, syrup or wafer or the like.
For to give Rhizoma Polygoni Cuspidati extract of the present invention outside the parenterai administration, possibly together give to this Rhizoma Polygoni Cuspidati extract coating or with this Rhizoma Polygoni Cuspidati extract with the material that prevents its inactivation.Can also the reactive compound that replenish be added in the said composition.Other medicine that in the specific implementation, Rhizoma Polygoni Cuspidati extract of the present invention and one or more can be used to treat disease is prepared altogether and/or is given altogether.Thisly unite use, can utilize this medicine that gives primely, therefore avoid possible toxicity or the complication relevant with various monotherapies than low dosage.
Process in liquid preparation such as water preparation, oil-suspending agent or other liquid preparation one or more, like in syrup, tincture or the elixir etc. one or more; When being used for parenteral, can be made in solution, water preparation or the oiliness suspending agent etc. of injection one or more.
Above medicine or pharmaceutical composition can use various approach; In described type of service; Preferred form is that oral formulations (like in tablet, coated tablet, capsule, solution or the suspension etc. one or more), non-intestinal give one or more in the dosage form (like in injection, ointment or the patch etc. one or more) etc.; Further one or more in preferred tablet, capsule or the injection etc., a kind of in special preferred tablet or the injection.
In addition; The employed medicinal raw material of Rhizoma Polygoni Cuspidati extract comprises that Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. also can directly be used to prepare the product that is used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof in some cases separately; Also can mix with many chemical substances or make up, directly or indirectly be used to prepare the product that is used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof with the form of compositions.Chemical substance described here identical with described in this joint preceding text.
For example; The employed medicinal raw material of Rhizoma Polygoni Cuspidati extract comprises that the powder of Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. is used to prepare and is used for the particularly various dosage forms of medicine of the prevention of AIDS and hepatitis B, diagnosis, protection and treatment product; Or the employed medicinal raw material of the Rhizoma Polygoni Cuspidati extract powder that comprises Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. and relevant adjuvant are used to prepare the product various dosage forms of medicine especially that are used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof; Or the employed medicinal raw material of the Rhizoma Polygoni Cuspidati extract powder that comprises Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. and the relevant preparation product such as the medicine that are used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof is used to prepare the various dosage forms of the product such as the medicine that are used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof together; Or the employed medicinal raw material of the Rhizoma Polygoni Cuspidati extract powder that comprises Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. and relevant ancillary drug are used to prepare the various dosage forms of the product such as the medicine that are used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof together; Like in tablet, capsule or the suspensoid etc. one or more, preferred capsule.
One of described method is that the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract is comprised that the powder fill of Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. is a capsule; Two of method product such as medicine that to be powder that the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract is comprised Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. be used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof with relevant preparation fill together are capsule, and three of method is powder that the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract is comprised Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. with relevant ancillary drug fill together is capsule; Four of method is that the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract is comprised that it is tablet that the powder of Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. is directly pressed by conventional method with relevant adjuvant together; Five of method is the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract to be comprised that product such as medicine that the powder of Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc., relevant preparation be used to prevent, diagnose, detect, protect, treat and study AIDS and hepatitis B and directly related disease thereof are directly pressed by conventional method with relevant adjuvant together be that tablet, six of method are that the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract is comprised that the powder of Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc., relevant ancillary drug directly press to tablet by conventional method etc. with relevant adjuvant together.
Except that six kinds of above-mentioned basic skills; Can also select the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract to comprise other forms of Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc. or the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract is carried out after method well known in the art handles, prepare the product that contains the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract such as the medicine of various dosage forms.But; It should be noted that; When the employed medicinal raw material of above-mentioned direct use Rhizoma Polygoni Cuspidati extract comprises Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc.; Should be earlier according to the dosage requirement of employed Rhizoma Polygoni Cuspidati extract, the employed medicinal raw material of Rhizoma Polygoni Cuspidati extract that obtains required use of converting comprises the consumption of Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract etc.
In sum, Rhizoma Polygoni Cuspidati extract of the present invention and compositions thereof can be used for preventing, diagnose, detect, protect, treating and study the product of AIDS and hepatitis B and directly related disease thereof, preferred agents and food, further preferred agents.
(4) technological speciality
The present invention is to being that the Rhizoma Polygoni Cuspidati extract of main component has been expanded new medical usage with the stilbene glucoside, also for prevention, diagnosis, detect, protection, treatment and research AIDS and hepatitis B and directly related disease thereof provide a kind of new medicament sources.Rhizoma Polygoni Cuspidati extract safety and low toxicity of the present invention; Pharmacological action is stronger, and its raw material sources are abundant, inexpensive, and preparation technology is simple; Yield is high, can be used for preparing the product of prevention, diagnosis, detection, protection, treatment and research AIDS and hepatitis B and directly related disease thereof.
The present invention studies Rhizoma Polygoni Cuspidati extract targetedly, and the Rhizoma Polygoni Cuspidati extract pharmacological action is stronger, and it is safe in utilization, and one-object-many-purposes has been brought into play effect to greatest extent; The Rhizoma Polygoni Cuspidati extract stable in properties uses the quality of the pharmaceutical preparations of preparation stable, is more suitable for the suitability for industrialized production of anti-AIDS and hepatitis B product; The effect of prevention, diagnosis, detection, protection, treatment and research AIDS and hepatitis B and directly related disease thereof is obvious, and the scope of application is wide especially, therefore applies easily, can have a tremendous social and economic benefits in the short period of time.
In a word; Active adaption of the present invention modern medical service and the job demand of scientific research field and the needs of human nature service; The present invention provides new source for new anti-HIV and the anti-HBV product of research and development; Resources of medicinal plant to developing China has important value, is the safe raw material that is used to prevent, diagnose, detect, protect, treat and study AIDS and aspects such as hepatitis B and directly related disease thereof.
The specific embodiment
The present invention has studied the new pharmacological action of existing Rhizoma Polygoni Cuspidati extract and new purposes; A kind of raw material that can be used in preparation AIDS and products such as hepatitis B prevention, diagnosis, protection and treatment is provided, has been convenient to the safe handling in medical industry and fields such as relevant industries such as food, beverage.
(1) method for preparing of Rhizoma Polygoni Cuspidati extract
For further setting forth the particularly method for distilling of chemical compound 1 and chemical compound 2 of above-mentioned Rhizoma Polygoni Cuspidati extract, explain by way of example below.
For example, the method for preparing of the said Rhizoma Polygoni Cuspidati extract of the present invention can for:
(1) extracts: by routine the Rhizoma Polygoni Cuspidati pulverizing medicinal materials is become coarse powder,, collect 5～30 times of medical material volume percolates, be extracting solution with 10～95% ethanol percolate extraction.
Or: by routine the Rhizoma Polygoni Cuspidati medical material of pulverizing is carried with 20～95% ethanol heat, the solvent volumetric usage is about 4～8 times of crude drug first; Maybe with the Rhizoma Polygoni Cuspidati medical material of pulverizing with 20～95% ethanol percolate extraction, the solvent volumetric usage is about 4～12 times of crude drug; Get extracting solution; With extracting liquid filtering, concentrated, the centrifugal or filtration of concentrated solution;
(2) separation and purification: it is 0.5～10: 1 (medical material weight: liquor capacity), be sample liquid that extracting solution is concentrated to concentration.(nonpolar or low pole macroporous resin is for being the polystyrene type porous adsorbent resin of bridging materials with styrene through nonpolar or low pole macroporous resin adsorption with sample liquid; Like D101, D201, ZTC-1, AB-8,1300-1 type macroporous resin etc.) or MCI gel CHP20P column chromatography or Sephadex LH-20 column chromatography, remove impurity with water wash; The moisture lower alcohol eluting of reuse, lower alcohol is C such as methanol, ethanol or propanol
1～C
5Alcohols, its concentration are 10～70%, and volumetric usage is 5～24 times of resin bed volume, collect the lower alcohol eluent, are concentrated into suitable volumes; Further use MCI gel CHP20P column chromatography, remove impurity with water wash; The moisture lower alcohol eluting of reuse, lower alcohol is C such as methanol, ethanol or propanol
1～C
5Alcohols, its concentration are 10～70%, and volumetric usage is 5～24 times of resin bed volume, collect 10%～50% eluting position, are concentrated into suitable volumes; Further use Toyopearl HW-40F column chromatography again, remove impurity with water wash; The moisture lower alcohol eluting of reuse, lower alcohol is C such as methanol, ethanol or propanol
1～C
5Alcohols, its concentration are 10～60%, and volumetric usage is 5～24 times of resin bed volume, collect 10%～30% eluting position, are concentrated into suitable volumes, are the Rhizoma Polygoni Cuspidati extract that mainly contains chemical compound 1 and chemical compound 2; Further again column chromatography can obtain isolated pure compound 1 and pure compound 2 respectively.
(2) authentication method of Rhizoma Polygoni Cuspidati extract
Integrated application mass spectrum of the present invention (be called for short: MS), proton nmr spectra (be called for short:
1H-NMR), carbon-13 nmr spectra (be called for short:
13C-NMR) and the nuclear magnetic resonance, NMR two-dimensional spectrum (be called for short: 2D-NMR) wait analysiss technology that chemical compound in the Rhizoma Polygoni Cuspidati extract in the Rhizoma Polygoni Cuspidati extract 1 and chemical compound 2 are carried out the structure evaluation.
Chemical compound 1 has following physicochemical property:
Molecular formula C
20H
21O
11SNa or C
20H
21O
11SK, MW:492 or 508; The white amorphous powder, soluble in water and aqueous methanol; [α]
24D-60 ° (c 0.19 MeOH); Ultraviolet spectra UV (MeOH) λ max:215,233 (sh), 306,319nm; Infrared spectrum IR (KBr) vmax:3406,1609,1539,1514,1447,1258,1173,1072,997cm
-1Proton nmr spectra (400 MHz, CD
3OD+D
2O) δ: 7.03 (1H, t, 2.0, H-2), 6.71 (1H, t, 2.0, H-4), 6.85 (1H, t, 2.0, H-6); 7.57 (2H, d, 8.7, H-2 ', 6 '), 7.00 (2H, d, 8.7, H-3 ', 5 '), 7.04 (1H, d; 16.3, H-α), 7.23 (1H, d, 16.3, H-β), 5.25 (1H, d, 7.7, H-1 "), 4.50 (1H, dd; 7.7,9.0, H-2 "), 3.97 (1H, dd, 9.0,9.4, H-3 "), 3.74 (1H, dd, 9.4,9.6; H-4 "), 3.74 (1H, m, H-5 "), 4.14 (" α), 3.94 (1H, overlapped, H-6 are " β) for 1H, dd, 11.2,1.8, H-6.Carbon-13 nmr spectra (100MHz, CD
3OD+D
2O) δ: 141.6 (C-1), 107.9 (C-2), 160.4 (C-3), 104.7 (C-4), 159.3 (C-5), 108.9 (C-6); (130.7 C-1 '), 129.2 (C-2 ', 6 '), 116.8 (C-3 ', 5 '); (158.2 C-4 '), 129.2 (C-6 '), 126.8 (C-α), 130.3 (C-β), 100.8 (C-1 "); 81.5 (C-2 "), 77.2 (C-3 "), 71.3 (C-4 "), 78.0 (C-5 "), 62.0 (C-6 ").Mass spectrum FABMS m/z 547 [M+K]
+, 531 [M+Na]
+, 508 [M]
+(referring to Xiao K., Xuan L., Xu Y., Bai D.Stilbene glycoside sulfates from Polygonum cuspidatum.J.Nat.Prod.2000,63 (10): 1373-1376).
Chemical compound 2 has following physicochemical property:
Molecular formula C
20H
21O
11SNa or C
20H
21O
11SK, MW:492 or 508; The white amorphous powder, soluble in water and aqueous methanol; [α]
24D-70.6 ° (c 0.08 MeOH); Ultraviolet spectra UV (MeOH) λ max:211,229 (sh), 301,312nm; Infrared spectrum IR (KBr) vmax:3406,1597,1504,1444,1263,1165,1074,1047,871,843cm
-1Proton nmr spectra (400MHz, D
2O) δ: 6.78 (1H, t, 2.1, H-2), 6.48 (1H, t, 2.1, H-4), 6.68 (1H, t, 2.1, H-6); 7.48 (2H, d, 8.7, H-2 ', 6 '), 6.29 (2H, d, 8.7, H-3 ', 5 '), 6.89 (1H, d; 16.4, H-α), 7.00 (1H, d, 16.4, H-β), 4.98 (1H, d, 7.6, H-1 "), 3.56 (overlapped); 3.63 (1H, dd, 9.2,8.7, H-3 "), 3.51 (1H, dd, 8.7,9.4, H-4 "), 3.59 (1H; m, H-5 "), 3.95 (1H, dd, 12.5,2.0, H-6 " α), 3.76 (1H, 12.5,5.7, H-6 is " β).Carbon-13 nmr spectra (100MHz, D
2O) δ: 142.5 (C-1), 109.2 (C-2), 160.7 (C-3), 106.1 (C-4), 159.5 (C-5), 110.9 (C-6); (137.4 C-1 '), 130.5 (C-2 ', 6 '), 124.4 (C-3 ', 5 '); (153.3 C-4 '), 130.5 (C-6 '), 130.5 (C-α), 131.1 (C-β), 103.0 (C-1 "); 75.6 (C-2 "), 78.3 (C-3 "), 72.2 (C-4 "), 78.8 (C-5 "), 63.3 (C-6 ").Mass spectrum FABMS m/z 547 [M+K]
+, 531 [M+Na]
+, 508 [M]
+(referring to Xiao K., Xuan L., XuY., Bai D.Stilbene glycoside sulfates from Polygonum cuspidatum.J.Nat.Prod.2000,63 (10): 1373-1376).
(3) the practical dosage form of using always
The present invention prepares injectable powder and generally adopts conventional freeze-drying, as solvent, the steps include: to get Rhizoma Polygoni Cuspidati extract with water, adds excipient, is dissolved in water, and adds active carbon, filtration sterilization, and plug is partly rolled in fill, and lyophilization, tamponade are rolled lid and are got final product.Used excipient is selected from one or more in mannitol, gelatin hydrolysate, glucose, lactose, the dextran etc.Every bottle contains Rhizoma Polygoni Cuspidati extract 10～100mg.
The present invention prepares injectable powder also can adopt spray drying method, as solvent, the steps include: to get Rhizoma Polygoni Cuspidati extract with water, adds or do not add excipient (excipient is the same), is dissolved in water, and adds active carbon, filtration sterilization, and spray drying, aseptic subpackaged, tamponade is rolled lid and is got final product.Every bottle contains Rhizoma Polygoni Cuspidati extract 10～100mg.
When the present invention prepared small-volume injection, preparation got final product as solvent with water for injection, also can add appropriate amount of auxiliary materials, and adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzyl benzoate, the dimethyl acetylamide.Every contains Rhizoma Polygoni Cuspidati extract 10～100mg.
The present invention prepares glucose infusion liquid or sodium chloride transfusion; With water for injection as solvent; Add the preparation of an amount of glucose or sodium chloride and get final product, also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzyl benzoate, the dimethyl acetylamide.Every bottle contains Rhizoma Polygoni Cuspidati extract 10～100mg.
The present invention prepares oral formulations such as tablet, capsule, granule, oral liquid, and adjuvant can be lactose, starch, dextrin, stearate etc., presses the routine techniques preparation.
In the present invention, the instance of the above-described specific embodiment and the following stated all is in order to set forth the present invention better, is not to be used for limiting scope of invention.
Through embodiment the present invention is described in detail below.
Embodiment 1, anti AIDS virus test
The active material of the C8166 cell model test external anti-HIV-1 of medicine, method and step
(1) reagent
HEPES, MTT, DMF, penicillin (Penicillin), streptomycin sulfate (Streptomycin sulfate), glutamine (Glutamine) are all available from Sigma company; (2-ME 2-Mercaptoethanol) is the Bio-Rad Company products to 2 mercapto ethanol.RPMI-1640 and newborn calf serum are the Gibco Company products.
(2) culture medium
The RPMI-1640 complete medium contains 10% newborn calf serum, 2mM L-glutaminate, 10mM HEPES, 50 μ M 2 mercapto ethanols, 100,000 IU penicillins, 100 μ g/ml streptomycins.
(3) cell and virus
The human T lymphocyte is C8166 and HIV-1 experiment strain HIV-1
IIIBBy Britain Medical Research Council, AIDS Reagent Project is so kind as to give.All cells is all cultivated with the RPMI-1640 complete medium that contains 15% calf serum with virus.Prepare HIV-1 by conventional method
IIIB, titration also calculates viral TCID
50After the packing of virus stock solution, put-70 ℃ of preservations.Cell and virus frozen and recovery by conventional method.
(4) HIV-1 infectious titration
HIV-1
IIIBCarry out titration by the said method improvement of Johnson&Byington (1990), be summarized as follows: the HIV-1 stock solution is done 4 times of dilutions on 96 orifice plates, 10 gradients, 6 repeating holes of every gradient are provided with control wells 6 holes simultaneously.Every hole adds C8166 cell 50 μ l, and every hole final volume is 200 μ l.37.5 ℃ CO
2Cultivate.Added fresh RPMI-1640 complete medium 100 μ l on the 3rd day, under inverted microscope, observed the inductive cytopathic effect of HIV-1 (CPE) in every hole, and whether had the formation of syncytium (Syncytium) to confirm in the 7th day with every hole; Press the TCID that the Reed&Muench method is calculated virus
50(50%Tissue culture infection dose).
(5) to the toxicity test of C8166 cell
4 * 10
5/ ml C8166 cell suspension 100ul mixes with different drug solutions to be measured, establishes three repeating holes.The not control wells of drug is set simultaneously, 37.5 ℃ of CO
2Cultivated 3 days, and adopted the MTT colorimetry to detect cytotoxicity.The ELx800 ELIASA is measured the OD value, and the mensuration wavelength is 595nm, and reference wavelength is 630nm.Calculate CC
50Value (50% Cytotoxicconcentration), the drug level during promptly to 50% normal T lymphocyte series C8166 toxigenicity.
(6) inhibition of HIV-1IIIB cytopathogenic effect (CPE) is tested
With 8 * 10
5/ ml C8166 cell 50ul/ hole is inoculated on the 96 porocyte culture plates that contain 100 μ l/ hole gradient doubling dilution medicines, adds the HIV-1 of 50 μ l then
IIIBThe dilution supernatant, 1300 TCID50/ holes.If 3 repeating holes.The not normal cell control wells of drug is set simultaneously.The positive medicine contrast of AZT.37.5 ℃ CO
2Cultivated 3 days, plasmodial formation is counted in (100 *) under the inverted microscope.EC
50(50%Effective concentration) forms 50% o'clock drug level for suppressing syncytium.
(7) computing formula
Draw dose-effect curve according to experimental result, calculate the 50% valid density (EC that chemical compound suppresses virus by the Reed&Muench method
50), 50% cell growth inhibiting concentration (CC
50) and the active therapeutic index TI of anti-HIV-1 value (Therapeutic index) be: TI=CC
50/ EC
50
1. cell growth survival rate (%)=experimental port OD value/control wells OD value * 100.
2. the cytopathogenic suppression ratio of HIV-1 (%)=(1-experimental port syncytium number/control wells syncytium number) * 100
Experimental result and conclusion: 1 couple of HIV of chemical compound induces the C8166 cell to form syncytium has obvious inhibiting activity, HIV is had significant inhibitory effect, its CC
50＞2000 μ g/mL, EC
50Be 153.42 μ g/mL, therapeutic index is＞13.04 (seeing table 1 and table 2).And this compound water soluble is fine, the very low nontoxic in other words reactive compound that can be used as treatment AIDS of cytotoxicity.
Embodiment 2, the test of external anti-hepatitis B virus
(1) material and reagent
The HepG2-2.2.15 of cell: HBV-DNA clone transfection human liver cancer cell is made up in 1987 by U.S. The Mount Sinai MedicalCenter.Dimethyl sulfoxine (is called for short: DMSO), kanamycin, MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt) be all available from U.S. Sigma company; RPMI 1640 culture medium and hyclone, U.S. Gibco Company products; ELISA HBsAg detection kit: Bio/Rad Company products; The viral DNA level detects with ABI Prism 7000 Sequence Detection appearance (Applied Biosystems).
(2) test method
This experiment is at the HepG2-2.2.15 of In vitro culture (2*10
6/ add the candidate drug of variable concentrations in ml); Drug effect is after one week; Detect the copy number of cell HBV DNA and the concentration of HBV surface antigen HBsAg through quantitative PCR and Elisa method respectively; Come Preliminary screening that HBV is had inhibiting medicine. the growth curve of HepG2-2.2.15 under the candidate drug effect has also been observed in this research, finds candidate drug under experimental concentration, and the growth of pair cell does not have tangible toxicity.
Hepatitis B virus DNA is analyzed:
Viral DNA extracts from 50 μ L culture medium according to description with QIAmp 96 DNA Blood kit (Qiagen).The viral DNA level detects with ABI Prism 7000 Sequence Detection appearance (Applied Biosystems).Quantitatively PCR in real time uses following primer and probe sequence: forward primer 5 '-CCTGTATTCCCATCCCATCGT-3 ' (HBV nucleotide 2006-2026), reverse primer 5 '-TGAGCCAAGAGAAACGGACTG-3 ' (HBV nucleotide 2063-2083) and probe FAM 5 '-TTCGCA AAATACCTATGGGAGTGGGCC-3 ' (HBV nucleotide 2035-2062).Contain the genomic psHBV-1 carrier of total length HBV and be used as the standard curve how many copy numbers are the every mL culture medium of calculating contain.
Hbs antigen (be called for short: HBsAg) enzyme-linked immunosorbent assay (be called for short: ELISA):
The level of HbsAg detects with Genetic Systems/Bio-Rad HBsAg ELISA detection kit, and the cell of untransfected HBV carrier is used as the background of analysis.
Experimental result and conclusion: chemical compound 1 has remarkable inhibiting activity with duplicating of 2 couples of HBV DNA of chemical compound, and chemical compound 1 and chemical compound 2 can also obviously suppress the secretion of HBV surface antigen HBsAg.Further research shows that this effect has certain agent effect relationship (seeing table 3, table 4).And find this chemical compound under experimental concentration, the growth of pair cell does not have tangible toxicity.
Anti-hepatitis B virus test in embodiment 3, the body
(1) animal and reagent
Mice is adopted the 5-6 NOD.CB17-Prkdc in week
ScidThe female Mus of/J (Jackson Laboratory) is about every 20g.
(2) test method
0.3 μ g pWTD HBV vector plasmid is gone into NOD.CB17-Prkdc through tail vein high-pressure injection
Scid/ J Mus (JacsonLaboratory).Chemical compound 1 and chemical compound 2 behind tail vein high-pressure injection HBV plasmid the 7th day and the 10th day are distinguished the dosage of lumbar injection 1mg/kg or 10mg/kg.HBV DNA copy number is measured with quantitative PCR in real time, uses log
10Copies/mL (± s.e.m.) expression.The serum HBsAg level is analyzed with the ELISA method, with ng/ml (± s.e.m.) expression (n=5).2 doses of effect relationship researchs of chemical compound 1 and chemical compound.
The HBV DNA analysis:
Viral DNA extracts from 50 μ L Mus serum according to description with QIAmp 96 DNA Blood kit (Qiagen), and the HBV dna level is measured with ABI Prism 7000 sequenators (Applied Biosystems).Quantitatively PCR in real time detects with following primer and probe: forward primer 5 '-CCTGTATTCCCATCCCATCGT-3 ' (HBV nucleotide 2006-2026), reverse primer 5 '-TGAGCCAAGAGAAACGGACTG-3 ' (HBV nucleotide 2063-2083) and probe FAM 5 '-TTCGCA AAATACCTATGGGAGTGGGCC-3 ' (HBV nucleotide 2035-2062).Contain the genomic psHBV-1 carrier of total length HBV and be used as the standard curve how many copy numbers the every mL serum of calculating contains.
The hbs antigen enzyme-linked immunosorbent assay:
The level of HBsAg detects according to description with Genetic Systems/Bio-Rad HBsAg ELISA detection kit, and the cell of untransfected HBV carrier is used as the background of analysis.
Experimental result and conclusion: chemical compound 1 and chemical compound 2 can significantly reduce the copy number of HBV DNA in the model mouse body, reduce the level (seeing table 5, table 6) of HbsAg in the serum.Research also finds, chemical compound 1 and chemical compound 2 present certain agent effect relationship (seeing table 7, table 8) in the intravital anti-HBV activity of transgenic mice.
The preparation of embodiment 4, Rhizoma Polygoni Cuspidati extract
Available from the Chinese medicine Rhizoma Polygoni Cuspidati 10Kg of Shanghai City medical material company, the acetone percolation with 60% extracts three times, concentrates and boils off acetone, and deposition is filtered, and removes most of liposoluble substance.Remainder is after being concentrated into proper volume; Aqueous precipitation part liposoluble substance again; After being concentrated into small size, MCI gel CHP20P column chromatography in the gradation is earlier with the sugar of water elution with the removal considerable part; The 10-80% methanol-eluted fractions is used in the back, and reuse 80%-100% methanol-eluted fractions is to remove a large amount of emodins-8-O-β-D-glucoside; The 50-80% methanol-eluted fractions partly goes up the LH-20 column chromatography; Use earlier water elution; Use the methanol gradient elution of 10-80% then, 30% methanol-eluted fractions component separates obtaining the required chemical compound of 50mg 1,70mg chemical compound 2 through various chromatography method MCI gel CHP20P, LH-20, ODS C-18 integrated application; Through analysis of physical and chemical property, wave spectrum analysis (mainly be that ultraviolet light is general, infrared spectrum, mass spectrum, proton nmr spectra and carbon spectrum) is confirmed structure.
The preparation of embodiment 5, Rhizoma Polygoni Cuspidati extract injectable powder
Get Rhizoma Polygoni Cuspidati extract 30g, add dextran 30g, add 500ml water for injection, stir and make its dissolving; Add the injection water to 2000ml, add the 3.0g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Be filled in the aseptic cillin bottle, every bottle of 2ml partly rolls plug; Lyophilization, tamponade is rolled lid and is got final product again.
The preparation of embodiment 6, Rhizoma Polygoni Cuspidati extract injectable powder
Get Rhizoma Polygoni Cuspidati extract 60g, add 500ml water for injection, stir and make its dissolving; Add the injection water to 1000ml, add the 1g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Lyophilization gets sterilized powder, is distributed into 1000 bottles.
The preparation of embodiment 7, Rhizoma Polygoni Cuspidati extract injectable powder
Get Rhizoma Polygoni Cuspidati extract 40g, add lactose 50g, add 100ml water for injection, stir and make its dissolving; Add the injection water to 1000ml, add the 1.5g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Spray drying gets sterilized powder, is distributed into 1000 bottles.
The preparation of embodiment 8, Rhizoma Polygoni Cuspidati extract injectable powder
(1) prescription
Injection Rhizoma Polygoni Cuspidati extract semi-finished product 150g
Mannitol 400g
Water for injection adds to 10000ml
Process 10000 bottles altogether
(2) preparation technology
Take by weighing the recipe quantity Rhizoma Polygoni Cuspidati extract by above prescription, join in an amount of water for injection, stir and make its dissolving; Add recipe quantity mannitol, stir and make dissolving fully, add to the full amount of water for injection; Add 0.1% needle-use activated carbon of amount of liquid, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Plug is partly rolled in fill; Lyophilization, lid is rolled in tamponade.Make 9735 bottles altogether, yield rate is 97.35%.
The preparation of embodiment 9, Rhizoma Polygoni Cuspidati extract small-volume injection
Get Rhizoma Polygoni Cuspidati extract 5g, add 100ml water for injection, stir and make its dissolving; Add the injection water to 1000ml, with 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 10ml, sterilization gets final product.
The preparation of embodiment 10, Rhizoma Polygoni Cuspidati extract small-volume injection
Get Rhizoma Polygoni Cuspidati extract 10g, add propylene glycol 30g, add 200ml water for injection, stir and make its dissolving; Add the injection water to 1000ml, add the 1.5g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 5ml, sterilization gets final product.
The preparation of embodiment 11, Rhizoma Polygoni Cuspidati extract glucose infusion liquid
Get Rhizoma Polygoni Cuspidati extract 2g, add Polyethylene Glycol 10g, add glucose 500g, add 2000ml water for injection, stir and make its dissolving; Add the injection water to 5000ml; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 100ml, sterilization gets final product.
The preparation of embodiment 12, Rhizoma Polygoni Cuspidati extract glucose infusion liquid
Get Rhizoma Polygoni Cuspidati extract 2g, add glucose 250g, add 1000ml water for injection, stir and make its dissolving; Add the injection water to 5000ml; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 250ml, sterilization gets final product.
The preparation of embodiment 13, the transfusion of Rhizoma Polygoni Cuspidati extract sodium chloride
Get Rhizoma Polygoni Cuspidati extract 1g, add sodium chloride 90g, add 1000ml water for injection, stir and make its dissolving; Add the injection water to 10000ml; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 250ml, sterilization gets final product.
The preparation of embodiment 14, Rhizoma Polygoni Cuspidati extract tablet
(1) prescription
Rhizoma Polygoni Cuspidati extract 1000.0g
Microcrystalline Cellulose 1170.0g
Pregelatinized Starch 690.0g
Lactose 125.0g
The 5%PVP dehydrated alcohol is an amount of
Magnesium stearate 15.0g
Process 10000 altogether
(2) preparation technology takes by weighing the principal agent and the adjuvant of recipe quantity respectively by above prescription; Progressively increase behind the method mix homogeneously according to making soft material, system granule, drying, processes such as granulate under the formulation and technology item by equivalent; Calculated the heavy back of sheet with single punch tablet machine and 10.5mm shallow concave punch tabletting; Control nude film hardness 5～7kg makes 9698 in tablet altogether, and yield rate is 96.98%.Adopt lift-over nebulization coating, art for coating is following:
The preparation of coating solution: gastric solubleness thin film dress material: 85G61235 is provided by Shanghai Colorcon Coating Technology Co., Ltd
Art for coating: will treat that (hardness 5～7kg) is put into coating pan to the coating nude film; Start agitating device and air blast heater, when treating that the nude film temperature rises to 40 ℃, last 1/3 place that begins to open spray gun alignment tab bed sprays into the coating solution coating; 38～42 ℃ of control strip bed tempertaures; Gas pound pressure 6kg, the coating solution flow velocity is 50mL/min, coating membrane heavily account for coated tablet heavy 3%.
The preparation of embodiment 15, Rhizoma Polygoni Cuspidati extract tablet
Get Rhizoma Polygoni Cuspidati extract 100g, microcrystalline Cellulose 80g, lactose 15g, pregelatinized Starch 60g sieve, and mix homogeneously with an amount of 10%PVP alcoholic solution system soft material, is granulated, and drying adds magnesium stearate 3g, granulate, and tabletting is processed 1000.
The preparation of embodiment 16, Rhizoma Polygoni Cuspidati extract capsule
(1) prescription
Rhizoma Polygoni Cuspidati extract 1000.0g
Microcrystalline Cellulose 1000g
Carboxymethyl starch sodium 140g
Dehydrated alcohol is an amount of
Pulvis Talci 80g
Process 10000 capsules altogether
(2) preparation technology gets in crude drug Rhizoma Polygoni Cuspidati extract and the prescription other adjuvant respectively by above prescription and crosses 100 mesh sieves respectively; Put 60 ℃ of oven dry, take by weighing recipe quantity Rhizoma Polygoni Cuspidati extract and microcrystalline Cellulose, the carboxymethyl starch sodium equivalent method mix homogeneously that progressively increases, with an amount of dehydrated alcohol system soft material; 30 mesh sieves are granulated; 50～60 ℃ of dryings 2 hours with 30 mesh sieve granulate, add the Pulvis Talci and the carboxymethyl starch sodium mix homogeneously of recipe quantity.
Claims (12)
1. the application of Rhizoma Polygoni Cuspidati extract in preparation anti AIDS virus and anti-hepatitis B virus product; And Rhizoma Polygoni Cuspidati extract is the unique active component in this anti AIDS virus and the anti-hepatitis B virus product, and this Rhizoma Polygoni Cuspidati extract is meant resveratrol 3-O-β-D-glucoside 2 " sulfate group sodium or potassium salt and resveratrol 3-O-β-D-glucoside 4 '-sulfate group sodium or potassium salt.
2. the application of the compositions of Rhizoma Polygoni Cuspidati extract in preparation anti AIDS virus and anti-hepatitis B virus product; And Rhizoma Polygoni Cuspidati extract is the unique active component in this anti AIDS virus and the anti-hepatitis B virus product, and this Rhizoma Polygoni Cuspidati extract is meant resveratrol 3-O-β-D-glucoside 2 " sulfate group sodium or potassium salt and resveratrol 3-O-β-D-glucoside 4 '-sulfate group sodium or potassium salt.
3. the application of the medicinal raw material of Rhizoma Polygoni Cuspidati extract in preparation anti AIDS virus and anti-hepatitis B virus product; And Rhizoma Polygoni Cuspidati extract is the unique active component in this anti AIDS virus and the anti-hepatitis B virus product, and this Rhizoma Polygoni Cuspidati extract is meant resveratrol 3-O-β-D-glucoside 2 " sulfate group sodium or potassium salt and resveratrol 3-O-β-D-glucoside 4 '-sulfate group sodium or potassium salt.
4. the application of the medicinal raw material of Rhizoma Polygoni Cuspidati extract according to claim 3 in preparation anti AIDS virus and anti-hepatitis B virus product is characterized in that the medicinal raw material of described Rhizoma Polygoni Cuspidati extract is Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract.
5. the application of the compositions of the medicinal raw material of Rhizoma Polygoni Cuspidati extract in preparation anti AIDS virus and anti-hepatitis B virus product; And Rhizoma Polygoni Cuspidati extract is the unique active component in this anti AIDS virus and the anti-hepatitis B virus product, and this Rhizoma Polygoni Cuspidati extract is meant resveratrol 3-O-β-D-glucoside 2 " sulfate group sodium or potassium salt and resveratrol 3-O-β-D-glucoside 4 '-sulfate group sodium or potassium salt.
6. the application of the compositions of the medicinal raw material of Rhizoma Polygoni Cuspidati extract according to claim 5 in preparation anti AIDS virus and anti-hepatitis B virus product is characterized in that the medicinal raw material of described Rhizoma Polygoni Cuspidati extract is Rhizoma Polygoni Cuspidati medical material or Rhizoma Polygoni Cuspidati crude extract.
7. according to each described application of claim 1～6, it is characterized in that described anti AIDS virus and anti-hepatitis B virus product are meant the product that is used to prevent, treat AIDS and hepatitis B.
8. the application of Rhizoma Polygoni Cuspidati extract according to claim 7 is characterized in that, described AIDS is HIV-1 type AIDS or HIV-2 type AIDS.
9. the application of Rhizoma Polygoni Cuspidati extract according to claim 8 is characterized in that, described AIDS is HIV-1 type AIDS.
10. the application of Rhizoma Polygoni Cuspidati extract according to claim 7 is characterized in that, described anti AIDS virus and anti-hepatitis B virus product are medicine or food.
11. the application of Rhizoma Polygoni Cuspidati extract according to claim 10 is characterized in that, described anti AIDS virus and anti-hepatitis B virus product are medicines.
12. application according to each described Rhizoma Polygoni Cuspidati extract of claim 1～6; It is characterized in that the resveratrol 3-O-β-D-glucoside 2 in described this Rhizoma Polygoni Cuspidati extract " sulfate group sodium or potassium salt and resveratrol 3-O-β-D-glucoside 4 '-purity of sulfate group sodium or potassium salt is all >=95%.
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| CN102273494B (en) * | 2011-08-15 | 2013-07-10 | 高海利 | Giant knotweed multifunctional plant growth regulator and application thereof |
| CN102552437B (en) * | 2012-01-16 | 2013-12-11 | 李泽琳 | Chinese medicinal composition for treating acquired immune deficiency syndrome and preparation method thereof |
| CN102718812A (en) * | 2012-05-22 | 2012-10-10 | 中国人民解放军第四军医大学 | Method for purifying stilbene glucoside in Lysimachia clethroide duby |
| CN102718811A (en) * | 2012-05-22 | 2012-10-10 | 中国人民解放军第四军医大学 | Preparation method of stilbene glycoside in polygonum cuspidatum |
| TWI542304B (en) * | 2014-02-19 | 2016-07-21 | Microwave heating insulation pad and the heating pad with the insulation package | |
| CN107501431A (en) * | 2017-09-19 | 2017-12-22 | 李仲昆 | A kind of water-soluble giant knotweed polyoses extract and preparation method thereof and purposes |
| CN109481694A (en) * | 2018-12-20 | 2019-03-19 | 药大制药有限公司 | A kind of antiviral conjugate of Ribavirin-resveratrol, preparation method and application |
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| CN1176113A (en) * | 1996-09-09 | 1998-03-18 | 徐向田 | Compound bufonin of bufo gargarizans |
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