CN102652792B

CN102652792B - Anti-cancer application of composition containing garlic oil, garlic total polysaccharide and garlic total saponin

Info

Publication number: CN102652792B
Application number: CN201110053280.9A
Authority: CN
Inventors: 刘丽梅; 鞠大宏; 柏冬; 刘梅洁; 王瑞海; 李玉梅
Original assignee: INSTITUTE OF BASIC THEORY CACMS
Current assignee: INSTITUTE OF BASIC THEORY CACMS
Priority date: 2011-03-04
Filing date: 2011-03-04
Publication date: 2014-03-26
Anticipated expiration: 2031-03-04
Also published as: CN102652792A

Abstract

The invention provides a composition consists based on garlic oil, garlic total polysaccharide and garlic total saponin as raw materials. The composition is integrated with water-soluble and fat-soluble effective components of garlic, so that the anti-cancer activity of the garlic is sufficiently played. The composition has excellent anti-tumor activity and has the effects of strengthening body resistance to eliminate pathogenic factors and reducing toxicity, has wide application prospect, and can be prepared into a tumor prevention medicament or health-care product with remarkable curative effect and small toxic or side effect. The invention also provides a method for preparing the composition. The method comprises the following steps: preparing the garlic oil from the garlic through double extraction; preparing garlic total polysaccharide from decoction dreg through water extraction and alcohol precipitation; and carrying out alcohol extraction and macroporous resin purification on the decoction dreg to obtain the garlic total saponin. The preparation method is simple to operate, low in cost and controllable in quality, realizes sufficient utilization of each part of garlic raw materials, and can be industrialized.

Description

The anticancer purpose of the compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin

Technical field

The invention belongs to field of medicaments, relate to a kind of compositions that comprises Bulbus Allii liposoluble constituent and Bulbus Allii water soluble ingredient composition, specifically, relate to the application of compositions in preparing antitumor drug and health product that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin, and the preparation method of said composition.

Background technology

Tumor is one of major disease of society harm humans health, has become the second-biggest-in-the-world cause of the death disease that is only second to cardiovascular and cerebrovascular disease.Treating malignant tumor scheme is generally Combined with Radiotherapy chemotherapeutic treatment after operation, but chemotherapeutics has certain toxic and side effects, Traditional Chinese Medicine Anti tumor medical instrument has unique advantage, has the effect of eliminating evil, righting or potentiation, attenuation, so Traditional Chinese Medicine Anti tumour medicine is the focus of research always.

Bulbus Allii cancer-resisting with a long history, its main component has organic compounds containing sulfur, amino acids, enzyme, saccharide, lipid, saponins, vitamin and trace element etc.Modern pharmacological research shows, the multiple efficacies such as that Bulbus Allii has is antibacterial, antiinflammatory, blood fat reducing, blood pressure lowering, antithrombotic, antitumor, defying age, antioxidation and enhancing human body immunity power.

One, the anti-cancer and kill cancer action of Bulbus Allii main component summary

Bulbus Allii belongs to immune stimulating type Chinese herbal medicine, can inducement interferon, suppress the generation of tumor necrosis factor, and reduce carcinogen effect, improve immune function of human body, aspect the preventing and treating of malignant tumor, there is wide DEVELOPMENT PROSPECT.The effect of Oleum Bulbus Allii and main component garlicin (also referred to as garlicin) thereof and diallyl disulfide and garlic polysaccharide, Bulbus Allii saponin is carried out to labor below:

1, immunization

Bulbus Allii has good immunoregulation effect, can increase the spleen weight of laboratory animal, increases phagocyte and T lymphocyte quantity, strengthens cytophagous phagocytic activity, improves lymhocyte transformation rate.Its active component Bulbus Allii extract can promote the lymphocytic propagation of T, improves the activity of NK cell, promotes the release of the cytokines such as IL-2, TNF, IFN, thus the defence of booster immunization system and function for monitoring.Especially, the garlicin containing in Bulbus Allii can also improve the cellular immune function of tumor patient.

2, the effect of prevention and treatment tumor

The diallyl sulfide that the Oleum Bulbus Allii extracting from Bulbus Allii contains has the effect that suppresses the growth of mankind's kinds of tumors.Particularly, the garlicin in diallyl sulfide (DATS, composite is garlicin), one of main component in Oleum Bulbus Allii, in oil, content is the highest.Garlicin can be blocked carcinogenic synthetic, suppresses carcinogenic activation and mutation, anti-distortion; Also tumor cell is had to direct killing effect, can grow, regulate cell cycle, promote cancer cell-apoptosis by anticancer.Another main component in diallyl disulfide in diallyl sulfide (DADS) Oleum Bulbus Allii, in oil, content occupies the second, finds at present, and it can suppress various human growth of tumour cell.In addition, the spirostanol saponin eruboside-B in Bulbus Allii has the anti-tumor in vivo effect similar to enoxolone, and some cell line is had to cytotoxicity, for example BC1, Lu1, Col2, KB, KB-V.Garlic polysaccharide can also the toxicity of ameliorate tumor medicine amycin (adriamycin, ADR) to heart.

3, antioxidation

Therefore at present because many oxide is all the inducible factor of cancer, research is thought, polyphenoils has certain effect to anticancer, has proved that some components in Bulbus Allii have very strong antioxidation, thereby has contributed to its antitumaous effect.In addition, garlic polysaccharide has the ability of removing ultra-oxygen anion free radical and hydroxyl radical free radical, and is better than garlicin under higher concentration, and the reducing power of garlicin is better than garlic polysaccharide.

In sum, at present the Oleum Bulbus Allii in Bulbus Allii and main component prevention thereof and antineoplastic action are had to play-by-play, Attenuation to the monomer antitumor action of the antioxidation of garlicin, garlic polysaccharide, Bulbus Allii saponin and garlic polysaccharide also has report, but three is not combined to the collaborative antineoplastic research report that carries out.

Two, the preparation method of Bulbus Allii main component summary

Patent 200610135316.7 relates to extraction process of garlic total saponin (allin) and garlic biological activity component compound and uses thereof, the problem of the main existence of this technology for do not illustrate contained saponin, bioactive ingredients be how to differentiate and content assaying method.Separately there are many pieces of patents to relate to the extracting method of garlic polysaccharide (as patent 2007100171321, CN1239719 and 200310117625.8) and Bulbus Allii quintessence oil (patent 200310117624.3,200610033685).But prior art only relates to Oleum Bulbus Allii, Bulbus Allii saponin or the garlic polysaccharide in independent extraction and application Bulbus Allii mostly.In fact, in Bulbus Allii, contain various active composition, be mainly divided into liposoluble constituent and water soluble ingredient two classes.Liposoluble constituent is mainly Oleum Bulbus Allii, and content is 0.2% left and right, water soluble ingredient aminoacid, protein, polysaccharide, saponin, vitamin, trace element etc., and content is 25% left and right.Yet in current Bulbus Allii medicine and health product, also do not combine the precedent of preparation Bulbus Allii liposoluble constituent and water soluble ingredient, mentioned component is not combined yet, the product being worth to give full play to the medicinal of Bulbus Allii or health care.

Summary of the invention

The anticancer purpose that the object of this invention is to provide a kind of compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin, the composition and method of making the same that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin described in another object of the present invention is to provide.

The object of the invention is to be achieved through the following technical solutions:

On the one hand, the invention provides a kind of compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin in the application for the preparation of preventing and/or treating in medicine, health product and/or the food of cancer.

Further, described Bulbus Allii main body of oil comprises: garlicin, diallyl disulfide, diallyl one sulfur, methacrylic trithio and methacrylic two sulfur etc., described Bulbus Allii total polysaccharides is heteropolysaccharide, and the composition after its hydrolysis comprises: fructose, glucose, arabinose, galactose, xylose and mannose, the main component of described garlic total saponin is steroidal saponin, it comprises: furostanol, spirostanol saponin, wherein, described furostanol comprises: proto-eruboside-B, proto-iso-eruboside-B, sativoside-B1 and proto-desgalactotigonin, described spirostanol saponin comprises: eruboside-B, iso-eruboside-B, sativoside-C, sativoside-R1, sativoside-R2, sativoside-B2, sativoside-B3, sativoside-B4 and sativoside-B5 etc., in Bulbus Allii, contain altogether in a word 25 kinds of saponin monomers.

Further, described cancer is selected from: the cancers such as gastric cancer, straight/colon cancer, bladder cancer, carcinoma of prostate, pulmonary carcinoma, nasopharyngeal carcinoma, hepatocarcinoma, osteocarcinoma, esophageal carcinoma, breast carcinoma, cervical cancer, cancer of pancreas, cerebroma, skin carcinoma, leukemia, lymphatic cancer and melanoma.

Further, described medicine is selected from following dosage form: the dosage forms such as injection, tablet, granule, capsule, soft capsule, drop pill, oral liquid, membrane, powder, externally used paste, suppository and pill.

Further, in described compositions, the ratio of Oleum Bulbus Allii, Bulbus Allii total polysaccharides, garlic total saponin is 1: 2.5: 1.25～1: 200: 50 by weight.

On the other hand, the invention provides a kind of method of preparing the compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin, it comprises the following steps:

1) method providing according to prior art (patent ZL200510083951.0) carries out that Bulbus Allii is two to be carried, and obtains Oleum Bulbus Allii, medicinal liquid and medicinal residues, and medicinal liquid retains;

2) Oleum Bulbus Allii adopts anhydrous sodium sulfate dehydration, obtains pure Oleum Bulbus Allii, then through cyclodextrin inclusion compound, obtains the clathrate of Oleum Bulbus Allii;

3) medicinal residues water extraction, obtains water extraction liquid;

4) by step 1) gained medicinal liquid and step 3) merging of gained water extraction liquid, reclaim under reduced pressure water, concentrated, concentrated solution adds ethanol precipitate with ethanol, spends the night, centrifugal, must precipitate and supernatant (being medicinal liquid I);

5) by step 4) gained precipitation is dry, obtains light yellow Bulbus Allii total polysaccharides; Or dissolving step 4) gained precipitation, centrifugal, supernatant is crossed resin column, and effluent concentrating under reduced pressure is dry, obtains yellow-white Bulbus Allii total polysaccharides;

6) by step 3) medicinal residues after water extraction drench dryly, add alcohol reflux, obtain medicinal liquid II;

7) combining step 4) gained medicinal liquid I and step 6) gained medicinal liquid II, decompression recycling ethanol is to without alcohol taste, and thin up is centrifugal, obtains clear liquid medicine III;

8) step 7) gained medicinal liquid III crosses macroporous resin, and washing and/or ethanol are washed successively, collects eluent, and decompression recycling ethanol is to without alcohol taste, dry, obtains brown or buff garlic total saponin;

9) by step 1) gained Oleum Bulbus Allii or step 2) gained Oleum Bulbus Allii clathrate, step 5) gained Bulbus Allii total polysaccharides and step 8) combination of gained garlic total saponin obtains the compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin.In addition, according to different dosage forms, form different prescription (prescription comprises the proportion requirement of three effective sites and suitable adjuvant), the different preparations of the compositions that can obtain comprising Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin.

Further, step 1) in, described two carrying as Bulbus Allii is pulverized, adds water insulation enzymolysis, and vapor distillation extracts, and carries out 1-3 time, adds 3-9 times of water at every turn, each 0.5-2h.

Further, step 2) in, the concrete steps of described Oleum Bulbus Allii enclose are: 65%-95% for Oleum Bulbus Allii (volume ratio) ethanol is in 1: 5-1: the ratio of 15 (mL/mL) is dissolved, in 25-45 ℃ of ultrasonic pond, with cyclodextrin inclusion compound 0.5-1.5h; Preferably, described cyclodextrin comprises alpha-cyclodextrin, beta-schardinger dextrin-, hydroxyethyl-β-cyclodextrin, HP-β-CD, beta-schardinger dextrin-methylate derivant and branched cyclodextrin etc., the concentration of cyclodextrin is 10% (mass volume ratio), Oleum Bulbus Allii: cyclodextrin is 1 by weight: 6-1: 12.

Further, step 3) in, described water extraction is 1-2 time, adds 3-9 times of water at every turn, each 0.5-2h.

Further, step 4), in, described reclaim under reduced pressure is carried out at not higher than 80 ℃ in temperature; Described concentrated solution, take Bulbus Allii (g): medicinal liquid (mL) is unit, and concentration is 1: 0.8-1.2, making the relative density of medicinal liquid at 60 ℃ is 0.9-1.10; The content of described ethanol in concentrated solution is 75-85% (volume ratio).

Further, step 5) in, the solvent of described dissolution precipitation is hot deionized water; Described resin is D316, HPD300L, D900, D318 or D941 type resin, is preferably D941 or D900 type resin; Described concentrating under reduced pressure carries out at not higher than 80 ℃ in temperature; Described being dried as drying under reduced pressure, spraying are dried or lyophilization, preferably, described drying under reduced pressure temperature is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

Further, step 6) in, the concrete steps of described reflux, extract, are: add doubly (mass ratio) 95% (volume ratio) alcohol reflux of 3-5, extract 1-2 time, each 0.5-1.5 hour.

Further, described step 7) comprise, merge medicinal liquid I, II, reclaim under reduced pressure is extremely without alcohol taste, and while making its 25 ℃, relative density is 1.00-1.10, thin up, amount of water is 0.5-0.9 times of medical material weight, centrifugal, obtains clear liquid medicine.

Further, step 8) in, medicinal liquid III is crossed to macroporous resin, described resin is preferably DM-130, AB-8 or HPD100, according to resin and crude drug weight ratio meter, applied sample amount is 1: 0.8-1.1, wash successively 4-6BV (Bed Volume, bed volume) and/or 30% (volume ratio) ethanol wash 2-5BV, 70%-95% (volume ratio) ethanol is washed 4-6BV, collect the rear ethanol elution of washing, or the ethanol elution after 30% (volume ratio) ethanol elution, at 70 ℃ of temperature <, decompression recycling ethanol is to without alcohol taste, drying under reduced pressure, spraying is dried or lyophilization, described drying under reduced pressure temperature is not higher than 60 ℃, the dry hothouse temperature of spraying is no more than 120 ℃, lyophilization cold hydrazine temperature is lower than-50 ℃.

Again on the one hand, the invention provides the compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin prepared by a kind of method described in basis.

Further, described Bulbus Allii main body of oil comprises: garlicin, diallyl disulfide, diallyl one sulfur, methacrylic trithio and methacrylic two sulfur etc.; Described Bulbus Allii total polysaccharides is heteropolysaccharide, and the composition after its hydrolysis comprises: fructose, glucose, arabinose, galactose, xylose and mannose; The main component of described garlic total saponin is steroidal saponin, it comprises: furostanol, spirostanol saponin, wherein, described furostanol comprises: proto-eruboside-B, proto-iso-eruboside-B, sativoside-B1 and proto-desgalactotigonin, described spirostanol saponin comprises: eruboside-B, iso-eruboside-B, sativoside-C, sativoside-R1, sativoside-R2, sativoside-B2, sativoside-B3, sativoside-B4 and sativoside-B5 etc.

Further again, in described Oleum Bulbus Allii, the total content of diallyl disulfide and garlicin is not less than 50%, and in Oleum Bulbus Allii clathrate, the utilization rate of Oleum Bulbus Allii is not less than 93%; In described Bulbus Allii total polysaccharides, Bulbus Allii total polysaccharides content is demarcated and is not less than 45% with glucose, and after the hydrolysis of Bulbus Allii total polysaccharides, each contents of monosaccharides summation is not less than 50%, and in described garlic total saponin, total saponin content is with saponin monomer proto-iso-eruboside-B (C ₅₇h ₉₆o ₃₀) for reference substance, demarcate and be not less than 40%.

The beneficial effect of the composition and method of making the same that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin provided by the invention is as follows:

The Oleum Bulbus Allii that comprises provided by the invention, the compositions of Bulbus Allii total polysaccharides and garlic total saponin has been filled up the fat-soluble and blank of water-soluble active ingredient aspect antitumor prevention and control field use in conjunction of Bulbus Allii, make full use of Bulbus Allii and extract Oleum Bulbus Allii medicinal residues afterwards, resulting compositions makes Bulbus Allii water solublity, the activity of fat-soluble effective ingredient cancer-resisting is fully played, product has significant anti-tumor activity, can reach strengthening vital QI to eliminate pathogenic factors, the effect of efficacy enhancing and toxicity reducing, can make antitumor drug, health product and food, and safety and low toxicity, have a extensive future.

The method of the compositions that preparation provided by the invention comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides, garlic total saponin be take fresh garlic as raw material, when extracting Oleum Bulbus Allii, extraction is separated to Bulbus Allii total polysaccharides and garlic total saponin, after purified by they combination, have simple to operate, product quality is controlled, be suitable for the advantages such as industrialization production.

Accompanying drawing explanation

Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:

Fig. 1 be embodiment 2 diallyl disulfide canonical plotting (standard curve equation: y=477.1029x-2.2526, correlation coefficient: 0.9999, the range of linearity: 0.368～7.36 μ g);

Fig. 2 be embodiment 2 diallyl trisulfide canonical plotting (standard curve equation: y=796.9455x-4.3282, correlation coefficient: 0.9999, the range of linearity: 0.2288～4.576 μ g);

Fig. 3 is Bulbus Allii saponin reference substance and the rear full wavelength scanner figure of sample colour developing of embodiment 3;

Fig. 4 be embodiment 3 Bulbus Allii saponin canonical plotting (standard curve equation: y=0.012x+0.002, correlation coefficient: 0.9995, the range of linearity: 19.44～58.32 μ g);

Fig. 5 be embodiment 4 glucose canonical plotting (standard curve equation: y=12.97x-0.078, correlation coefficient: 0.9990, the range of linearity: 17.8～71.2 μ g);

Fig. 6 is the standard solution chromatography of ions spectrogram of embodiment 4;

Fig. 7 is the sample ions chromatogram spectrogram of embodiment 4.

The specific embodiment

Below in conjunction with specific embodiment, and comparable data describes in further detail the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.In following embodiment, various processes and the method do not described in detail are conventional methods as known in the art.The source of agents useful for same, trade name and be necessary to list its constituent person, all indicate when occurring first, identical reagent used is if no special instructions, all identical with the content of indicating first thereafter.

embodiment 1: the compositions that preparation comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin

Bulbus Allii 15kg belt leather is ground into the granule that diameter is 3～5mm left and right, puts in multi-function extractor, adds 7 times of water gagings and extracts 2h, obtains volatile oil, medicinal liquid and medicinal residues.Collect the volatile oil Na in volatile oil device ₂sO ₄dehydration, obtains Bulbus Allii volatile oil 30.9mL (oil yield 0.206%, in Oleum Bulbus Allii, the content summation of diallyl disulfide and garlicin is 52.60%).Medicinal liquid retains.

Bulbus Allii slag adds 5 times of water gagings, extract 1 hour, by the medicinal liquid obtaining with above-mentionedly carry the medicinal liquid that oil obtains and merge, reclaim under reduced pressure water (80 ℃ of temperature <), concentrated 1: 1 (70 ℃ of relative densities 1.05), concentrated solution adds ethanol precipitate with ethanol, make to reach 85% (volume ratio) containing alcohol amount, placement is spent the night, centrifugal, must precipitate and medicinal liquid I precipitation drying under reduced pressure (60 ℃ of temperature <), milky garlic polysaccharide 2.24kg (paste-forming rate 14.93%, it is 61% as reference substance calculates content that total polysaccharides be take Fructus Vitis viniferae).

Medicinal residues drench dry, add 3 times of 95% (volume ratio) alcohol reflux 3 times, each 1 hour, obtain medicinal liquid II, merge medicinal liquid I, II, decompression recycling ethanol (70 ℃ of temperature <) is extremely without (25 ℃ of alcohol tastes, relative density 1.05), be diluted with water to 0.8 times (12000mL) extracting medical material, centrifugal, supernatant is crossed HPD100 macroporous resin, applied sample amount 1: 1.1, 30% (volume ratio) ethanol is washed 4BV, 95% (volume ratio) ethanol is washed 4BV, collect eluent, decompression recycling ethanol is extremely without alcohol taste (70 ℃ of temperature <), drying under reduced pressure (60 ℃ of temperature <), obtaining brown garlic total saponin extract 1.56kg (calculates by raw product, garlic total saponin paste-forming rate is 10.38%, in extract, total saponin content is with saponin monomer proto-iso-eruboside-B (C ₅₇h ₉₆o ₃₀) be demarcated as 42% for reference substance).

Get above-mentioned Oleum Bulbus Allii clathrate, Bulbus Allii total polysaccharides, garlic total saponin and carry out prescription:

1, the preparation of composition grain preparation

Granule prescription forms: Oleum Bulbus Allii: 1mL cyclodextrin clathrate 9.95g, and Bulbus Allii total polysaccharides: 45g, garlic total saponin: 17.5g, dextrin is appropriate, and aspartame is appropriate;

Preparation: mix, the ethanol of 95% (volume ratio) is done wetting agent, granulate, drying under reduced pressure (50 ℃ of temperature <), granulation 1000g, obtains Oleum Bulbus Allii, Bulbus Allii total polysaccharides, garlic total saponin composition grain altogether, and every gram of granule is containing Oleum Bulbus Allii 1mg, Bulbus Allii total polysaccharides 45mg, garlic total saponin 17.5mg.

2, the preparation of compositions Emulsion

Emulsion prescription forms: Oleum Bulbus Allii: 1g, Bulbus Allii total polysaccharides: 35g, garlic total saponin: 17.5g, tween 80: 1g;

Preparation: grind well, adding distil water, to 1000mL, is made Oleum Bulbus Allii, Bulbus Allii total polysaccharides, garlic total saponin compositions Emulsion, every milliliter of Emulsion is containing Oleum Bulbus Allii 1mg, garlic polysaccharide 35mg, garlic total saponin 17.5mg.

embodiment 2: the quality control of Oleum Bulbus Allii (with reference to patent ZL200510083951.0)

The content of garlicin and diallyl disulfide in the present embodiment employing liquid chromatography for measuring Oleum Bulbus Allii, assay method is:

1, instrument and material

High performance liquid chromatograph: HP1100 chromatograph of liquid (comprising quaternary pump, diode array detector, automatic sampler, HP chem workstation).

Chemical reagent: methanol, formic acid are chromatographically pure, water is distilled water, all the other reagent are analytical pure.Reference substance diallyl disulfide, diallyl trisulfide self-control, purity is all more than 98%.

2, chromatographic condition

Chromatographic column is Zorbax extend C18 post (4.6 * 250mm, 5 μ m); Mobile phase is methanol-0.17% (volume ratio) formic acid solution (80: 20); Flow velocity: 1mLmin-1; Detect wavelength: 240nm; Column temperature: 35 ℃.

3, the preparation of reference substance

Precision takes diallyl disulfide 30.44mg, and diallyl trisulfide reference substance 32.12mg is placed in 50mL volumetric flask, with dissolve with methanol and be diluted to scale, shakes up; Precision is drawn 0.6088mgmL respectively ^-1diallyl disulfide solution and 0.6424mgmL ^-1each 1.0mL of diallyl trisulfide solution in 10mL volumetric flask, with methanol, be diluted to scale, shake up, obtain 0.06088mgmL ^-1diallyl disulfide and 0.06424mgmL ^-1diallyl trisulfide mixed solution.

4, the preparation of sample solution

Get the about 2mg of Oleum Bulbus Allii sample that embodiment 1 makes, accurately weighed, be placed in 5mL measuring bottle, add methanol to scale, shake up, obtain.

5, the drafting of standard curve

Reference substance solution is

sample introduction

2,4,8,12,16,20 and 40 μ L respectively, by above-mentioned chromatographic condition, measure peak area integrated value, take peak area integrated value as vertical coordinate, and reference substance sample size is abscissa, and drawing standard curve calculates regression equation as illustrated in fig. 1 and 2.

6, sample determination

Accurate reference substance solution and each 10 μ L of need testing solution of drawing, inject high performance liquid chromatograph respectively, measure, and obtain.In Oleum Bulbus Allii, the content of diallyl disulfide, garlicin is respectively 20.33% and 39.06%, and the two summation is 59.39%.

embodiment 3: the quality control of garlic total saponin

The present embodiment adopts spectrophotometry instrument to measure the content of garlic total saponin, because at present domestic and international Bulbus Allii saponins compound is sold without reference substance, the inventor is separated to a saponin monomer proto-iso-eruboside-B from garlic total saponin, as reference substance, adopt UV-VIS spectrophotometry, take sulphuric acid-methanol as developer, set up garlic total saponin content assaying method, make product quality controlled.Through methodology checking, accurately and reliably, stability (RSD%=1.1%), precision (RSD%=1.0%), repeatability (RSD%=1.52%) are all good for the method, and average recovery is 102.59% (RSD/=1.4%, n=6.Through the method, detect, in garlic total saponin of the present invention, saponin content is not less than 40% in proto-iso-eruboside-B, and concrete assay method is as follows:

1, instrument and reagent

8453 ultraviolet-visible spectrophotometers (U.S., Agilent); HWSY11-K digital display thermostat water bath (mayor of Beijing bearing instruments and meters company); CX-250 ultrasonic washing unit (Tian Haishuanlong armarium company limited); Balance (German Sai Duolisi, P225D); Concentrated sulphuric acid and 95% (volume ratio) ethanol (being analytical pure, Beijing Chemical Plant); Proto-iso-eruboside-B (be called for short PIEB) (for assay, laboratory self-control, through NMR (Nuclear Magnetic Resonance) spectrum hydrogen compose ( ¹h-NMR), carbon is composed (13C-NMR) and its structure of mass spectrum (MS) Test Identification, HPLC normalization method test purity > 98%).

2, the preparation of reference substance solution

Precision takes garlic total saponin sample, and (60 orders, 38# post sample lot number: 20101129) 10mg puts in 25mL measuring bottle add the ultrasonic 30min of methanol to dissolve completely to sample, and standardize solution, filters and get final product.

3, the preparation of sample solution

Precision takes in Bulbus Allii saponin sample 3.64mg to the 10mL volumetric flask that embodiment 1 makes, and adds methanol to sample to dissolve completely, standardize solution and get final product.

4, the selection of coloration method and mensuration wavelength

Accurate 0.5mL reference substance solution and the 0.5mL need testing solution drawn, be placed in 10mL tool plug test tube, after 80 ℃ of water-baths volatilize, add sulphuric acid-methanol (7: 3) 5mL, shake up, 90 ℃ of water-bath 60min, ice-water bath 10min after taking out, take coordinative solvent as blank, working sample absorbance, as shown in Figure 3, after reference substance and sample colour developing, full wavelength scanner figure all has absorption maximum at 326nm place.

5, the drafting of standard curve

The accurate reference substance (0.324mg/mL) of drawing, dilution 1/10, accurate 0.6mL, 0.8mL, 1.0mL, 1.2mL and the 1.4mL of drawing is to tool plug test tube, after 80 ℃ of water-baths volatilize, add 70% (volume ratio) sulphuric acid-methanol 5mL, be placed in 60 ℃ of heating in water bath 60min, ice-water bath 10min after taking out, room temperature is placed after 15min, take corresponding solution as blank, measures absorbance under 326nm.The content of reference substance of take is abscissa, and absorbance is vertical coordinate, carries out linear regression, and as shown in Figure 4, calculating standard curve is y=0.012x+0.002, and correlation coefficient is 0.9995, shows that reference substance is good linear relationship within the scope of 19.44～58.32 μ g.

6, sample determination

Precision measures reference substance solution and each 1.0mL of sample solution respectively, be placed in tool plug test tube, 80 ℃ of water-baths volatilize solvent, respectively add the close plug of 70% (volume ratio) sulphuric acid-methanol 5mL, shake up, under 60 ℃ of water-baths, react 60min, ice bath 10min after taking out, room temperature is placed after 15min, take corresponding solution as blank, measures its absorbance under 326nm wavelength.

7, measurement result

The content of garlic total saponin is 42.00%.

embodiment 4: the quality control of Bulbus Allii total polysaccharides

1, instrument and reagent

8453 ultraviolet-visible spectrophotometers (U.S., Agilent), HWSY11-K digital display thermostat water bath (mayor of Beijing bearing instruments and meters company), CX-250 ultrasonic washing unit (Tian Haishuanlong armarium company limited); Balance (German Sai Duolisi, P225D); Phenol (analytical pure, Tianjin good fortune chemical reagent factory in morning), concentrated sulphuric acid (analytical pure, Beijing Chemical Plant), 95% (volume ratio) ethanol (analytical pure, Beijing Chemical Plant), D-anhydrous glucose reference substance is (for assay, Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110833-200503).

2, sample determination

(1) reference substance solution preparation: precision takes the about 12mg of anhydrous glucose reference substance, is placed in 25mL measuring bottle, adding distil water dissolves and standardize solution, obtains.

(2) drafting of standard curve: precision measures reference substance solution 1.0,2.0,3.0,4.0,5.0mL is placed in respectively 25mL measuring bottle, distilled water standardize solution.Precision measures 5 parts of each 1mL of above-mentioned reference substance solution in 10mL tool plug test tube respectively, add respectively 5% (mass volume ratio) phenol solution 1.0mL, after shaking up, add concentrated sulphuric acid 5.0mL, shake up rear boiling water bath 10min, take out and be placed in the cooling room temperature that is placed to of ice-water bath.The reagent blank of take is measured absorbance as reference at 485nm wavelength place, take absorbance as vertical coordinate, and concentration (mg/mL) is abscissa, drawing standard curve, as shown in Figure 5.

(3) preparation of sample solution: precision takes the about 30mg of polysaccharide sample that embodiment 1 makes, be placed in 10mL centrifuge tube, add 80% (volume ratio) alcoholic solution 8mL, sonic oscillation 60min, the centrifugal 15min of 3000rpm, abandoning supernatant, 80% (volume ratio) washing with alcohol 2～3 times for residue, add 8mL boiling water in centrifuge tube, ultrasonic 60min solution example, is transferred in 10mL measuring bottle after cooling, a small amount of distilled water wash centrifuge tube, cleaning mixture is incorporated in measuring bottle, and distilled water is settled to 10mL.Shake up filtration, get subsequent filtrate 1mL, to 25mL measuring bottle, add water standardize solution, be test sample solution, by operating under standard curve preparation, by standard curve, calculate polyoses content in test sample.

3, measurement result

In sample, take glucose meter polyoses content as 48.45% (n=2).

embodiment 5: the rear monosaccharide of employing chromatography of ions (ICS 3000) mensuration Bulbus Allii total polysaccharides hydrolysis content

1, instrument and reagent

Chromatographic column: CarboPac PA20 analytical column, 150*3mm, S/N 002823; CarboPac PA20 guard column, 50*3mm, S/N 002652

Detector: pulsed amperometric detector, Au electrode, sugared four potential waveforms of S/N 0050150

Leacheate forms and flow velocity: EG produces 1mMKOH, 0.45mL/min auto injection, 10 μ L

2, experimental technique

Get the sample 10mg that embodiment 1 makes, adding 2mL concentration is 2moL/L trifluoroacetic acid solution, is hydrolyzed 4 hours at airtight latter 80 ℃.Put to room temperature, standing 1 hour, precision measured supernatant 1mL, was placed in 50mL eggplant-shape bottle, 50 ℃ of evaporated under reduced pressure.Residue adds 50mL deionized water, after ultrasonic 10min dissolves, crosses 0.22 μ m filter membrane and RP post, sample introduction analysis.Above-mentioned solution dilutes 10 times again, for measuring content.

3, measurement result

Experimental result is as shown in table 1.

Table 1 chromatography of ions (ICS 3000) is measured Bulbus Allii total polysaccharides hydrolysis monosaccharide result

* wherein glucose and fructose content are the result of 10 times of dilutions

embodiment 6: Bulbus Allii effective site detects the WST of rat spleen lymphopoiesis impact

1, material

(1) Bulbus Allii different parts extract 1# (Oleum Bulbus Allii), the 2# (Bulbus Allii total polysaccharides) that tested medicine: embodiment 1 makes, 5# (garlic total saponin), 2#+5# (garlic total saponin extracts separated rear by 1: 1 biased sample with Bulbus Allii total polysaccharides) be # (Bulbus Allii total polysaccharides and garlic total saponin are without sample separation) (2+5), prepared and provide by Chinese department of Chinese medicine institute Chinese medical theory research, above-mentioned sample dissolves with DMSO (dimethyl sulfoxide), preparation before experiment;

Positive drug: ConA (Concanavalin A, con A, Sigma company);

(2) animal: SD rat, male, body weight (180 ± 10) g, clean level animal, laboratory animal room provides;

(3) reagent: lymphocyte separation medium (Sigma company), RPMI1640 culture medium (GIBCO company), hyclone (GIBCO company), DMSO (Sigma) etc., purchased from Beijing Ao Xintuopu Science and Technology Ltd.; Quick cell proliferation detecting kit (Bio-Vision company);

(4) instrument: full-automatic microplate reader, Thermo MK-3; CO ₂incubator, Revco 300T; Inverted microscope, OLYMPUS-IMT-2; Clean bench, BCN-1360B type, Beijing Dong Lianhaer instrument manufacturing company limited; Micropipettor, German Eppendorf; Table model high speed centrifuge TDL-40B type, Anting Scientific Instrument Factory, Shanghai; Electronic analytical balance, CP64 Shanghai Ao Haosi company.

2, method

Anesthesia SD rat, takes out spleen, spleen is shredded into unicellular (getting supernatant) and slowly add in the centrifuge tube that has added in advance lymphocyte separation medium, and the ratio of spleen suspension and separating medium is 1: 1.At 4 ℃ with the centrifugal 20min of 2000r/min, liquid phase is divided into three layers, with microsyringe, extract middle level lymphocyte and put into aseptic centrifuge tube, with PBS (Phosphate Buffered Saline, phosphate buffer) washing is 2 times, all with the centrifugal 10min of 2000r/min, abandons supernatant at every turn, with a small amount of RPIM1640 culture fluid (containing 10% serum), break up, by blood cell counting plate statistics, regulate cell initial concentration to 1 * 10 ⁶/ mL, gets 100 μ L and adds 96 well culture plates, in the 5%CO of 37 ℃ ₂in incubator, cultivate 24h.

Each 100 μ L of medicine 1#, the 2# of the different quality concentration that embodiment 1 is made, 5#, 2#+5#, (2+5) # and positive drug add experimental port, make final concentration difference 1.25,0.625,0.3125,0.156,0.078,0.039,0.0195,0.0098 and 0.005mg/mL, and set up blank group and positive controls simultaneously, blank group adds 100 μ L physiological saline solution, the ConA (final concentration is 5 μ g/mL) that adds 100 μ L in positive drug matched group, each concentration is established 3 multiple holes, cultivation 48 or 72h.Every hole adds the WST solution of 5mg/mL (according to quick cell proliferation detecting kit description preparation, 5 milliliters of electronics coupled reagent are joined in WST-1 powder, dissolve completely) 20 μ L, continue to cultivate 4h, in microplate reader, in 450nm wavelength, read each hole absorbance (A), with A value reflection lymphocytic proliferation rate, the results are shown in Table 2.

3, experimental result

As seen from Table 2, with matched group comparison, the compositions of Bulbus Allii total polysaccharides, garlic total saponin (2+5) #, 2#+5# have obvious facilitation (p < 0.01), wherein (2+5) # proliferation more obvious (p < 0.001) to Peripheral Blood T Lymphocytes of Rats propagation.Result shows, (2+5) # and (2#+5#) sample Peripheral Blood T Lymphocytes of Rats propagation is had to obvious facilitation.

Table 2 Bulbus Allii composition detects the WST of rat spleen lymphopoiesis (A) impact

A value	0.625mg/mL	0.156mg/mL	0.039mg/mL	0.0098mg/mL
					1# (Oleum Bulbus Allii)	0.398±0.004	0.404±0.019	0.399±0.013	0.395±0.005
2# (Bulbus Allii total polysaccharides)	0.383±0.004	0.409±0.018	0.411±0.006	0.401±0.005
					5# (garlic total saponin)	0.576±0.029	0.451±0.017	0.425±0.005	0.414±0.005
(2+5)#	0.637±0.009**	0.660±0.021**	0.644±0.062**	0.466±0.025*
					2#+5#	0.591±0.015*	0.462±0.013*	0.465±0.003*	0.456±0.014*
Cont	0.427±0.008
					ConA(5μg/mL)	0.603±0.054

Note: with matched group than * *: p < 0.001, *: p < 0.01

embodiment 7: the compositions that comprises Oleum Bulbus Allii, garlic total saponin and Bulbus Allii total polysaccharides is thin to tumor the inhibiting research of intracellular growth

1, material

(1) tested medicine and positive drug

Bulbus Allii different parts extract 1# (Oleum Bulbus Allii), the 2# (Bulbus Allii total polysaccharides) that tested medicine: embodiment 1 makes, 5# (garlic total saponin) are prepared and provide by Chinese department of Chinese medicine institute Chinese medical theory research.2# dissolves with DMEM (Dulbecco ' s Modified Eagle Medium), and 1#, 5# first dissolve with DMSO, then after dissolving with DMEM, with the filter filtration of 0.22 μ m, subpackage-20 ℃ preservation.

Positive drug: cisplatin is produced (lot number 905011CF) by Qilu Pharmaceutical Co., Ltd..

(2) stomach cancer cell line: MKN45 cell is purchased from Beijing consonance cell resource center; AGS, HGC-27 cell are purchased from Shanghai Life Sciences Research Institute, Chinese Academy Of Sciences.

(3) reagent: RPMI1640 culture medium (lot8109038 of GIBCO company); DMEM, Ham ' s F12 culture medium (Thermo company); Hyclone (GIBCO16000 company); Trypsin Solarbio company); MTT (Sigma); DMSO (Sigma) etc., purchased from Beijing Ao Xintuopu Science and Technology Ltd..

(4) instrument: full-automatic microplate reader, Thermo MK-3; CO ₂incubator, Revco 300T; Inverted microscope, OLYMPUS-IMT-2; Clean bench BCN-1360B type, Beijing Dong Lianhaer instrument manufacturing company limited; Micropipettor, German Eppendorf; Table model high speed centrifuge TDL-40B type, Anting Scientific Instrument Factory, Shanghai; Electronic analytical balance, CP64 Shanghai Ao Haosi company.

2, experimental technique

(1) cell culture processes:

MKN45 cell uses 1640 complete mediums cultivations, Ham ' the s F12 cultivation containing 10% (volume ratio) new-born calf serum for AGS, the HGC-27 cell containing 10% (volume ratio) new-born calf serum to cultivate with the DMEM that contains 10% (volume ratio) new-born calf serum, (in culture fluid, contain NaHCO3.2g, 100U/mL penicillin, 100 μ g/mL streptomycins), at the 5%CO of 37 ℃ ₂in incubator, be cultured to cell coverage rate 80～90% and go down to posterity when above, choose cell that growth conditions is good for experimentation.

(2) mtt assay (Zhang Juntian chief editor, modern pharmacology experimental program, China Concord Medical Science University of Beijing Medical University combined publication, in October, 1998 Beijing first impression, P819) measure Bulbus Allii different parts and extract composition to the inhibition of proliferation of human gastric cancer cell and measure medicine half-inhibition concentration (IC50):

Collect exponential phase MKN45, AGS, HGC-27 cell, with 2 * 10 ⁴the density in/hole adds 96 orifice plates, every hole 100 μ L.Each 100 μ L of the medicine of different quality concentration are added to experimental port, the final concentration of 1# is respectively 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.0395, 0.0198, 0.0099, 0.0049, 0.00248, 0.00124, 0.00064, 0.00032, 0.00016mg/mL, 2#, the final concentration difference 10 of 5#, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.0395, 0.0198, 0.0099, 0.0049mg/mL, and set up blank simultaneously, negative control group and positive controls, negative control hole inoculation equivalent cell, and to add containing final concentration be the complete culture solution of the DMSO of 0.1% (volume ratio), blank well is inoculating cell not, only adds equivalent complete culture solution, cisplatin is as positive controls, and its dosage is pressed quantity and converted, and whole mass concentration is 25,12.5,6.25,3.125,1.56,0.78,0.04,0.02 μ g/mL.Each concentration is established 3 multiple holes, cultivates 48h.Every hole adds the MTT solution of 5mg/mL, and ((3-(4 for tetrazole, 5-dimethylthbm) 1-2-yl)-2,5-diphenyl-tetrazolium bromide, with PBS, be diluted to 5mg/mL) 10 μ L, continue to cultivate 4h, remove supernatant, every hole adds the DMSO of 200 μ L, 10min is dissolved in concussion, under room temperature, place 10min, in microplate reader 570nm place's photometry density (OD) value, calculate cell inhibitory rate, draw cell inhibitory effect curve and measure medicine half-inhibition concentration (IC50).Calculate the formula of cell inhibitory rate: cell inhibitory rate (%)=(negative control group OD value-Experimental agents group OD value)/(negative control group OD value-blank group OD value) * 100%; The drug level of take is drawn concentration-response curve as abscissa, survival rate as vertical coordinate, obtains regression equation, draws the concentration (IC50) that suppresses 50% Growth of Cells, and experimental result is in Table 3-7.

The half casualty-producing concentrations (IC50) of table 3 Bulbus Allii effective site to people's gastric cancer MKN45 killing functions of immunocytes

Medicine grouping	IC50(mg/mL)
		1# (GO/ Oleum Bulbus Allii)	0.026
2# (TPG/ Bulbus Allii total polysaccharides)	3.66
		5# (TSG/ garlic total saponin)	0.37

Result shows, 1#, 5# sample are stronger to the lethal effect of tumor cell.

Table 4 Bulbus Allii effective site is on the impact of people's gastric cancer MKN45 cell proliferation (suppression ratio %)

Suppression ratio (%)	10mg/mL	2.5mg/mL	0.625mg/mL
				1#(GO)	89.0	89.3	77.6
2#(TPG)	75.2	44.9	37.9
				5#(TSG)	73.8	100.0	94.9

Result shows, 5#, 1# sample are stronger to the inhibitory action of tumor.

The contrast of table 5 Bulbus Allii effective site to the half casualty-producing concentrations (IC50) of MKN45, AGS, HGC, HeLa, HepG2 killing functions of immunocytes

Result shows, 1#, 5# sample are to gastric cancer MKN45, AGS, HGC cell, and 5# sample has lethal effect in various degree to cervical cancer HeLa, hepatoma Hep G 2 cells.

The contrast to people's gastric cancer MKN45 killing functions of immunocytes (IC50) of table 6 Bulbus Allii effective site and compositions thereof

Result shows, it is the strongest that 1#, 2#, 5# sample combination are killed and wounded the effect of people's gastric cancer MKN45 tumor cell, is better than the lethal effect of single position to tumor.The impact on people's gastric cancer MKN45 cell proliferation of table 7 Bulbus Allii different parts extract and compositions thereof

(suppression ratio %)

Suppression ratio (%)	1.25mg/mL	0.313mg/mL	0.078mg/mL	0.0198mg/mL
					1#(GO)	77.4	70.2	43.2	25.4
2#(TPG)	33.2	19.3	-19.6	-1.7
					5#(TSG)	88.6	55.6	5.0	19.9
1#+2#(GO+TPG)	69.6	71.4	31.8	20.3
					1#+5#(GO+TSG)	82.9	68.1	54.8	38.3
2#+5#(TPG+TSG)	90.5	56.0	26.7	15.3
					1#+2#+5#(GO+TPG+TSG)	84.9	76.5	58.3	43.7

Result shows, 1#, 2#, 5# sample combination are the strongest to the inhibitory action of people's gastric cancer MKN45 tumor cell, are better than the inhibitory action of single position to tumor.

the external growth inhibited effect to gastric carcinoma cells of embodiment 8 Bulbus Allii different parts extract

1, material

(1) tested medicine and positive drug:

Tested medicine: Bulbus Allii different parts extract 1#, 2# and 5#, standby and provide by Chinese department of Chinese medicine institute Basic Theories of Chinese Medicine skill institute system.2# dissolves with DMEM, and 1#, 5# first dissolve with DMSO, then after dissolving with DMEM, with the filter filtration of 0.22 μ m, subpackage-20 ℃ preservation.

(2) stomach cancer cell line: MKN45 cell is bought from Beijing consonance cell resource center; AGS, HGC-27 cell are purchased from Shanghai Life Sciences Research Institute, Chinese Academy Of Sciences.

(3) reagent: RPMI1640 culture medium (GIBCO company, lot8109038); DMEM and Ham ' s F12 culture medium (Thermo company); Hyclone (GIBCO16000 company); Trypsin Amresco company); MTT (Sigma company); DMSO (Sigma company) etc. are purchased from Beijing Ao Xintuopu Science and Technology Ltd..

(4) instrument: full-automatic microplate reader (Thermo MK-3); CO ₂incubator (Revco 300T); Inverted microscope (OLYMPUS-IMT-2); Clean bench BCN-1360B type (Beijing Dong Lianhaer instrument manufacturing company limited); Micropipettor (German Eppendorf); Table model high speed centrifuge TDL-40B type (Anting Scientific Instrument Factory, Shanghai); Electronic analytical balance CP64 (Shanghai Ao Haosi company).

2, experimental technique

(1) cell culture processes: MKN45 cell is with cultivating containing 1640 of 10% (volume ratio) new-born calf serum, AGS cultivates with Ham ' the s F12 containing 10% (volume ratio) new-born calf serum, HGC-27 cell is cultivated (in culture fluid, containing NaHCO3.2g, 100U/mL penicillin, 100 μ g/mL streptomycins), 37 ℃, 5%CO with the DMEM containing 10% (volume ratio) new-born calf serum ₂in incubator, be cultured to cell coverage rate 80～90% and go down to posterity when above, the good cell of growth conditions is for experimentation.

(2) mtt assay is measured Bulbus Allii different parts extraction composition to the inhibition of proliferation of human gastric cancer cell and is measured medicine half-inhibition concentration (IC50):

Collect exponential phase MKN45, AGS, HGC-27 cell, with 2 * 10 ⁴the density in/hole adds 96 orifice plates, every hole 100 μ L.Each 100 μ L of the garlic samples of different quality concentration are added to experimental port, the final concentration of 1# is respectively 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.0395, 0.0198, 0.0099, 0.0049, 0.00248, 0.00124, 0.00064, 0.00032, 0.00016mg/mL, 2#, the final concentration difference 10 of 5#, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.0395, 0.0198, 0.0099, 0.0049mg/mL, and set up blank group simultaneously, negative control group and positive controls, negative control hole inoculation equivalent cell, and to add containing final concentration be the complete culture solution of the DMSO of 0.1% (volume ratio), blank well is inoculating cell not, only adds equivalent complete culture solution, cisplatin is as positive controls, and its dosage is pressed quantity and converted, and whole mass concentration is 25,12.5,6.25,3.125,1.56,0.78,0.04,0.02 μ g/mL.Each concentration is established 3 multiple holes, cultivates 48h.Every hole adds 10 μ L (to use 5mg/mLMTT solution, with PBS, be diluted to 5mg/mL), continue to cultivate 4h, remove supernatant, every hole adds 200 μ LDMSO, and 10min is dissolved in concussion, room temperature 10min, in microplate reader 570nm place's photometry density (OD) value, calculate cell inhibitory rate, draw cell inhibitory effect curve and measure medicine half-inhibition concentration (IC50).Calculate the formula of cell inhibitory rate: cell inhibitory rate (%)=(negative control group OD value-Experimental agents group OD value)/(negative control group OD value-blank group OD value) * 100%; The drug level of take is drawn concentration-response curve as abscissa, survival rate as vertical coordinate, obtains regression equation, draws the concentration (IC50) that suppresses 50% Growth of Cells.

(3) screening of the best proportioning of the 1# based on orthogonal test, 2# and 5# use in conjunction

Orthogonal test is carried out five factors to 1#, 2# and 5#, each factor is established four levels and is investigated, be respectively 0, basic, normal, high dosage group, using stomach cancer cell metabolism MTT vigor (tumour inhibiting rate) as investigating index, according to the comprehensive grading of orthogonal test to determine the best compatibility concentration of 1#, 2# and use in conjunction.By the result of the IC50 of all 1#, 2# and 5# is carried out to statistical analysis, first the IC50 that determines 1#, 2# and 5# is middle dosage, according to the average level of high suppression ratio, determine the high dose of each medicine, low dosage dilutes with the dilution ratio centering dosage of height/middle dosage again.Orthogonal design is selected L ₁₆(4 ⁵) orthogonal table.

3, experimental result

(1) contrast of Bulbus Allii different parts extract 1#, 2#, the effect of 5# killing tumor cells

Bulbus Allii different parts extract 1#, 2#, 5# are as shown in table 8 to the median lethal dose(LD 50) (IC50) of people's gastric cancer MKN45, AGS, HGC-27 killing functions of immunocytes, and in three kinds of cells, the IC50 of 1# is all minimum, illustrates that its killing tumor cells effect is the strongest.The ascending order of different parts extract median lethal dose(LD 50) (IC50) is: 1# < 5# < 2#.

Table 8 Bulbus Allii different parts extract 1#, 2#, 5# are to people's gastric cancer MKN45, AGS, HGC-27 killing functions of immunocytes (median lethal dose(LD 50) (IC50))

(2) Bulbus Allii different parts extract 1#, 2#, 5# and the contrast of combination to gastric carcinoma cells lethal effect thereof

Bulbus Allii different parts extract 1#, 2#, 5# and combination thereof are as shown in table 9 to the IC50 of gastric carcinoma cells lethal effect, generally, in three kinds of cells, the IC50 of 1#, 1#+2#, 1#+5#, 1#+2#+5# is roughly in same level, and different cells are also slightly different to the reaction of same medicine.In MKN45 cell, the IC50 of 1#+2#+5# is a little less than other group, and the ascending order of median lethal dose(LD 50) (IC50) is: 1#+2#+5# < 1# < 1#+5# < 1#+2# < 5# < 2#+5#.In ags cell, the IC50 of 1# is minimum, and the ascending order of IC50 is: 1# < 1#+2# < 1#+5# < 1#+2#+5# < 5# < 2#+5#.In HGC cell, the IC50 of 1#+2# is minimum, and the ascending order of IC50 is: 1#+2# < 1#+2#+5# < 1# < 1#+5# < 5# < 2#+5#.

Table 9 Bulbus Allii different parts extract 1#, 2#, 5# and combination thereof are to people's gastric cancer MKN45, AGS, HGC-27 killing functions of immunocytes (median lethal dose(LD 50) (IC50))

The oral LD of Oleum Bulbus Allii mice ₅₀(Lethal Dose)=582.10 ± 62.87mg/kg, the oral LD of garlic total saponin mice ₅₀=17.09g/kg, the oral maximum dosage animal of Bulbus Allii total polysaccharides mice does not have death; From three effective site acute toxicities, Oleum Bulbus Allii, Bulbus Allii total polysaccharides, three effective sites of garlic total saponin are combined (1#+2#+5#) and are compared with same dosage Oleum Bulbus Allii 1#, inhibitory action to tumor is close, but may less (because the LD of total polysaccharides and total saponins of toxicity ₅₀be less than Oleum Bulbus Allii) infer thus, three part combinations together maximum advantage be righting, eliminating evil in, reduce the toxicity of Oleum Bulbus Allii, reduce zest.

3, Bulbus Allii extract 1#, 2#, the best proportioning screening of 5# use in conjunction to gastric carcinoma cells lethal effect

(1) Bulbus Allii extract 1#, 2#, the Orthogonal experiment results of 5# use in conjunction to the best proportioning screening of people's gastric cancer MKN45 killing functions of immunocytes, as shown in table 10,11.

Table 10 factor level table

Table 11 analysis of variance table

Soruces of variation	Sum of deviation square	Degree of freedom	Variance	F ratio	Significance
						Calibration model	6250.575	9	694.508	3.040	0.095
Distracter	58951.840	1	58951.840	258.050	0.000
						A	1350.525	3	450.175	1.971	0.220
B	1673.915	3	557.972	2.442	0.162
						C	3226.135	3	1075.378	4.707	0.051
Error	1370.705	6	228.451
						Summation	66573.120	16
Proofread and correct summation	7621.280	15

A R ²=0.820 (regulates R ²=0.550)

Variance analysis shows, on suppression ratio A, the B of people's gastric cancer MKN45 cell, C tri-factors there are no significant impact, influence factor's primary and secondary is sequentially C (5#) > B (2#) > A (1#), each factor is got optimum level, determines to be A to MKN45 killing functions of immunocytes three's best proportioning ₄b ₄c ₃, i.e. 1#: 2#: 5#=1: 2.5: 1.25.

(2) Bulbus Allii extract 1#, 2#, the Orthogonal experiment results of 5# use in conjunction to the best proportioning screening of people's gastric cancer ags cell lethal effect, as shown in table 12.

Table 12 factor level table

Table 13 analysis of variance table

Soruces of variation	Sum of deviation square	Degree of freedom	Variance	F ratio	Significance
						Calibration model	1788.194	9	198.688	0.835	0.612
Distracter	28844.085	1	28844.085	121.253	0.000
						A	579.100	3	193.033	0.811	0.532
B	618.924	3	206.308	0.867	0.508
						C	590.170	3	196.723	0.827	0.525
Error	1427.302	6	237.884
						Summation	32059.581	16
Proofread and correct summation	3215.497	15

A R ²=0.556 (regulates R ²=-0.110)

Variance analysis shows, on there are no significant the impact of suppression ratio A, the B of people's gastric cancer ags cell, C tri-factors.Influence factor's primary and secondary is sequentially B (2#) > A (1#) > C (5#), and each factor is got optimum level, determines the best of ags cell lethal effect three than being A ₂b ₄c ₂, i.e. 1#: 2#: 5#=1: 13.3: 3.3.

(3) Bulbus Allii extract 1#, 2#, the Orthogonal experiment results of 5# use in conjunction to the best proportioning screening of people's gastric cancer HGC killing functions of immunocytes, as shown in table 14.

Table 14 factor level table

Table 15 analysis of variance table

Soruces of variation	Sum of deviation square	Degree of freedom	Variance	F ratio	Significance
						Calibration model	5397.129	9	599.681	7.060	0.014
Distracter	47613.208	1	47613.208	560.560	0.000
						A	299.100	3	99.700	1.174	0.395
B	161.911	3	53.970	0.635	0.619
						C	4936.117	3	1645.372	19.371	0.002
Error	509.632	6	84.939
						Summation	53519.969	16
Proofread and correct summation	5906.761	15

A R ²=0.914 (regulates R ²=0.784)

Variance analysis shows, suppression ratio C factor to people's gastric cancer HGC cell has a significant impact, other factors there are no significant impact, influence factor's primary and secondary is sequentially C (5#) > A (1#) > B (2#).Each factor is got optimum level, determines the best of HGC killing functions of immunocytes than being A ₃b ₄c ₂, i.e. 1#: 2#: 5#=1: 200: 50.

the inhibitory action of embodiment 9 garlic samples to mice S180 transplanted tumor

The present embodiment has been studied garlic samples to the tumor-inhibiting action of mice-transplanted tumor model (selecting murine sarcoma S180 model), to determine the anti-tumor in vivo effect of garlic samples and to strengthen immune effect, main testing index is the weight of animals, tumour inhibiting rate, half hemolytic dose, lymphocyte transformation, macrophage phagocytic chicken red blood cell rate.

1, materials and methods

(1) tested material: 2# (Bulbus Allii total polysaccharides) and 5# (garlic total saponin) drug powder are provided by Chinese department of Chinese medicine institute's Chinese medical theory research institute's quality analysis of Chinese medicine chamber.

(2) laboratory animal: ICR mice, 18-20g, male, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., animal quality certification SCXK (capital) 2007-0001, gives conventional normal feedstuff before experiment starts and adapts to 5 days～7 days.

(3) feeding environment: raise in barrier environment, temperature is 21～25 ℃, relative humidity 40～70%.

(4) tumor inoculation, grouping and administration: every the right axil subcutaneous vaccination of animal S180 sarcoma cell 2 * 10 ⁶, after 24h, random packet, 10 every group, be respectively blank group (to commensurability normal saline), model group (to the solvent control with commensurability), the high, medium and low dosage group of Bulbus Allii oral administration, continuous 10 days, observe tumour inhibiting rate and immune indexes.

(5) reagent and instrument:

Reagent: RPMI1640 culture medium dry powder is purchased from Gibco company; Penicillin (Penicillin G SodiunSalt), streptomycin (Streptomycin Sulfate), Hepes, L-glutaminate, pancreatin, Sodium Pyruvate (Pyruvic Acid) etc. are purchased from Amresco company; MTT is purchased from Promega company; Bacteria lipopolysaccharide (LPS) is Sigma company product; Hyclone (FBS) is Hangzhou Ilex purpurea Hassk.[I.chinensis Sims company product; Sheep red blood cell (SRBC) (SRBC) is purchased from Department Of Medicine, Peking University's animal center; Chicken red blood cell (CRBC) is from animal medicine institute of China Agricultural University; Complement, Dou Shi reagent, Giemsa dyestuff are China Medical Sciences Academy Medical Plants Institute's preparation.

Instrument: CO ₂incubator (NAPCO); Inverted fluorescence microscope (Olympus, model C K40); Bio-Tek MQX200 type microplate reader; Centrifuge (German HERAEUS Labofuge 400R).

(6) Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell test:

Conventional separating mouse abdominal cavity cell liquid, Cell sap and 1% (volume ratio) chicken red blood cell equal-volume is mixed, and adding hyclone to make its final concentration is 10% (volume ratio).Cell mixing drop is added in the slide sealing ring by 3% (mass ratio) agar closed edge, cultivates 20min for 37 ℃, finish rear rapidly with normal saline not attached cell wash out, fixing 1min in methanol solution, Giemsa liquid dyeing 15min, distilled water flushing is clean, dries.Microscopic counting phagocytic rate (engulfing the shared percentage ratio of macrophage of chicken red blood cell in every 100 macrophages).

(7) mice spleen lymphocytes proliferation reaction test:

1h after last administration, after the de-neck of mice is put to death, sterile working takes out respectively and respectively organizes the spleen of mice and make splenocyte suspension, and is made into 5 * 10 ⁶/ mL, add 96 porocyte culture plates, every hole 100 μ L, add complete RPMI 1640+LPS solution (20 μ g/mL) again or do not add stimulant 1640 culture medium solution 100 μ L, put 37 ℃ of cultivation 72h in incubator, every hole adds MTT (5mg/mL) 20 μ L, cultivate again 4h, first sucking-off supernatant gently, every hole adds 100 μ L acid isopropyl alcohol cell pyrolysis liquids again, and incubated overnight is fully to dissolve MTT.Next day, by microplate reader, at 570nm place, detect OD value.

(8) mice serum hemolysin test:

Mice serum, complement, sheep red blood cell (SRBC) mix with certain proportion, hatch 30 minutes for 37 ℃, add Dou Shi reagent, detect OD value by microplate reader at 570nm place.

2, result

(1) tumor-inhibiting action of garlic samples 5# to murine sarcoma S180 model

The tumor-inhibiting action of table 16 garlic samples 5# to murine sarcoma S180 model

Group	Dosage (g/kg)	N (only)	Tumor heavy (g)	Tumour inhibiting rate (%)
					Model group		10	1.09±0.47
5#	0.875	10	1.01±0.35	6.62％
						1.75	10	0.85±0.38	21.23％
	3.5	10	0.62±0.41	42.85％

Table 16 explanation garlic total saponin has certain tumor-inhibiting action, has dose-effect relationship; Heavy dose of group has good inhibitory action to S180 solid tumor.

(2) impact of Bulbus Allii 2# sample on the tumor-inhibiting action of murine sarcoma S180 and immunologic function

The tumor-inhibiting action of table 17 garlic samples 2# to murine sarcoma S180 model

Group	Dosage (g/kg)	Tumor heavy (g)	Tumour inhibiting rate (%)
				Model		0.60±0.35
2#	0.75	0.58±0.28	3.81％
					1.5	0.45±0.25	25.17％
	3.0	0.32±0.47	46.94％

Table 17 explanation Bulbus Allii total polysaccharides has certain tumor-inhibiting action, has dose-effect relationship; Heavy dose of group has good inhibitory action to S180 solid tumor.

The immunologic function test of table 18 Bulbus Allii 2# sample to mice S180

Group	Dosage	Half hemolytic dose	Lymphopoiesis	Phagocytic rate
					Model		0.092±0.042	0.879±0.062	15.0±4.6
2#	1.5	0.072±0.015	0.854±0.056	15.0±5.0
						3.0	0.111±0.094	0.844±0.064	9.0±6.7
	6.0	0.185±0.132 ^a	0.783±0.066	33.0±14.9 ^b

A: compare P < 0.05, b with model: compare P < 0.01. with model

Table 18 explanation Bulbus Allii total polysaccharides high dose has obvious facilitation to engulfing of the generation of serum antibody and peritoneal macrophage.

The tumor-inhibiting action of table 19 Bulbus Allii effective site 1# to murine sarcoma S180 model

In table 19 explanation Oleum Bulbus Allii, dosage group has good inhibitory action to S180 solid tumor, and toxicity appears in high dose group.

The tumor-inhibiting action of table 20 Bulbus Allii effective site combination to murine sarcoma S180 model

Table 20 explanation Oleum Bulbus Allii, Bulbus Allii total polysaccharides, garlic total saponin (1#+2#+5#) compositions low dose group have good inhibitory action to S180 solid tumor, and toxicity appears in middle and high dosage group.

Claims

1. the compositions being comprised of Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin, is characterized in that, described compositions is prepared by following steps:

1) Bulbus Allii is two carries, and obtains Oleum Bulbus Allii, medicinal liquid and medicinal residues, and medicinal liquid retains;

3) medicinal residues water extraction, obtains water extraction liquid;

4) step 1) gained medicinal liquid and step 3) gained water extraction liquid are merged, reclaim under reduced pressure water, concentrated, concentrated solution adds ethanol precipitate with ethanol, spends the night, centrifugal, must precipitate and supernatant, i.e. medicinal liquid I;

5) step 4) gained precipitation is dry, obtain light yellow Bulbus Allii total polysaccharides; Or dissolving step 4) gained precipitation, centrifugal, supernatant is crossed resin column, and effluent concentrating under reduced pressure is dry, obtains yellow-white Bulbus Allii total polysaccharides;

6) medicinal residues after step 3) water extraction are drenched and done, add alcohol reflux, obtain medicinal liquid II;

9) by step 1) gained Oleum Bulbus Allii or step 2) gained Oleum Bulbus Allii clathrate, step 5) gained Bulbus Allii total polysaccharides and the combination of step 8) gained garlic total saponin, obtain the compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin,

Wherein, the main composition of described Oleum Bulbus Allii comprises: garlicin, diallyl disulfide, diallyl one sulfur, methacrylic trithio and methacrylic two sulfur; Described Bulbus Allii total polysaccharides is heteropolysaccharide, and the composition after its hydrolysis comprises: fructose, glucose, arabinose, galactose, xylose and mannose; The main component of described garlic total saponin is steroidal saponin, it comprises: furostanol, spirostanol saponin, wherein, described furostanol comprises: proto-eruboside-B, proto-iso-eruboside-B, sativoside-B1 and proto-desgalactotigonin, described spirostanol saponin comprises: eruboside-B, iso-eruboside-B, sativoside-C, sativoside-R1, sativoside-R2, sativoside-B2, sativoside-B3, sativoside-B4 and sativoside-B5.

2. compositions according to claim 1, is characterized in that, in step 1), described two carrying as Bulbus Allii is pulverized, adds water insulation enzymolysis, and vapor distillation extracts, and carries out 1-3 time, adds 3-9 times of water at every turn, each 0.5-2h;

Step 2) in, the concrete steps of described Oleum Bulbus Allii enclose are: Oleum Bulbus Allii 65%-95%(volume ratio) ethanol is in 1:5-1:15(mL/mL) ratio dissolve, in 25-45 ℃ of ultrasonic pond, with cyclodextrin inclusion compound 0.5-1.5h;

In step 3), described water extraction is 1-2 time, adds 3-9 times of water at every turn, each 0.5-2h;

In step 4), described reclaim under reduced pressure is carried out at not higher than 80 ℃ in temperature; Described concentrated solution, take Bulbus Allii (g): medicinal liquid (mL) is unit, and concentration is 1:0.8-1.2, and making the relative density of medicinal liquid at 60 ℃ is 0.9-1.10; The content of described ethanol in concentrated solution is 75-85%(volume ratio);

In step 5), the solvent of described dissolution precipitation is hot deionized water; Described resin is D316, HPD300L, D900, D318 or D941 type resin; Described concentrating under reduced pressure carries out at not higher than 80 ℃ in temperature; Described being dried as drying under reduced pressure, spray dry or lyophilization;

In step 6), the concrete steps of described reflux, extract, are: the 95%(volume ratio that adds 3-5 times of mass ratio) alcohol reflux, extracts each 0.5-1.5 hour 1-2 time;

Step 7) comprises, merges medicinal liquid I, II, and reclaim under reduced pressure is extremely without alcohol taste, and while making its 25 ℃, relative density is 1.00-1.10, thin up, and amount of water is 0.5-0.9 times of medical material weight, centrifugal, obtains clear liquid medicine; And

In step 8), medicinal liquid III is crossed to styrene type macroporous resin, according to resin and crude drug weight ratio meter, applied sample amount is 1:0.8-1.1, wash successively 4-6BV and/or 30%(volume ratio) ethanol washes 2-5BV, 70%-95%(volume ratio) ethanol is washed 4-6BV, collect the rear ethanol elution of washing, or 30%(volume ratio) ethanol elution after ethanol elution, at temperature <70 ℃, decompression recycling ethanol is extremely without alcohol taste, drying under reduced pressure, spraying is dried or lyophilization, described drying under reduced pressure temperature is not higher than 60 ℃, the dry hothouse temperature of spraying is no more than 120 ℃, lyophilization cold hydrazine temperature is lower than-50 ℃.

3. compositions according to claim 2, it is characterized in that, described cyclodextrin comprises alpha-cyclodextrin, beta-schardinger dextrin-, hydroxyethyl-β-cyclodextrin, HP-β-CD, beta-schardinger dextrin-methylate derivant and branched cyclodextrin, the concentration of cyclodextrin is 10% mass volume ratio, Oleum Bulbus Allii: cyclodextrin is 1:6-1:12 by weight.

4. compositions according to claim 2, is characterized in that, resin described in step 5) is D941 or D900 type resin.

5. compositions according to claim 3, is characterized in that, resin described in step 5) is D941 or D900 type resin.

6. compositions according to claim 2, is characterized in that, the temperature of drying under reduced pressure described in step 5) is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

7. compositions according to claim 3, is characterized in that, the temperature of drying under reduced pressure described in step 5) is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

8. compositions according to claim 4, is characterized in that, the temperature of drying under reduced pressure described in step 5) is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

9. compositions according to claim 5, is characterized in that, the temperature of drying under reduced pressure described in step 5) is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

10. compositions according to claim 2, is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.

11. compositionss according to claim 3, is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.

12. compositionss according to claim 4, is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.

13. compositionss according to claim 5, is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.

14. compositionss according to claim 6, is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.

15. compositionss according to claim 7, is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.

16. compositionss according to claim 8, is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.

17. compositionss according to claim 9, is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.

18. according to the compositions described in any one in claim 1-17, it is characterized in that, in described compositions, the ratio of Oleum Bulbus Allii, Bulbus Allii total polysaccharides, garlic total saponin is 1:2.5:1.25～1:200:50 by weight.

19. compositionss according to claim 1, is characterized in that, in described Oleum Bulbus Allii, the total content of diallyl disulfide and garlicin is not less than 50%, and in Oleum Bulbus Allii clathrate, the utilization rate of Oleum Bulbus Allii is not less than 93%; In described Bulbus Allii total polysaccharides, Bulbus Allii total polysaccharides content is demarcated and is not less than 45% with glucose, and after the hydrolysis of Bulbus Allii total polysaccharides, the summation of each contents of monosaccharides is not less than 50%; In described garlic total saponin, total saponin content be take saponin monomer proto-iso-eruboside-B and is demarcated and to be not less than 40% as reference substance.

20. according to the compositions described in any one in claim 1-19 in the application for the preparation of preventing and/or treating in medicine, health product and/or the food of cancer.

21. application according to claim 20, is characterized in that, in described compositions, the ratio of Oleum Bulbus Allii, Bulbus Allii total polysaccharides, garlic total saponin is 1:2.5:1.25～1:200:50 by weight.

22. according to the application described in claim 20 or 21, it is characterized in that:

Described Bulbus Allii main body of oil comprises: garlicin, diallyl disulfide, diallyl one sulfur, methacrylic trithio and methacrylic two sulfur;

Described Bulbus Allii total polysaccharides is heteropolysaccharide, and the composition after its hydrolysis comprises: fructose, glucose, arabinose, galactose, xylose and mannose;

The main component of described garlic total saponin is steroidal saponin, it comprises: furostanol, spirostanol saponin, wherein, described furostanol comprises: proto-eruboside-B, proto-iso-eruboside-B, sativoside-B1 and proto-desgalactotigonin, described spirostanol saponin comprises: eruboside-B, iso-eruboside-B, sativoside-C, sativoside-R1, sativoside-R2, sativoside-B2, sativoside-B3, sativoside-B4 and sativoside-B5.

23. application according to claim 20, it is characterized in that, described cancer is selected from: gastric cancer, straight/colon cancer, bladder cancer, carcinoma of prostate, pulmonary carcinoma, nasopharyngeal carcinoma, hepatocarcinoma, osteocarcinoma, esophageal carcinoma, breast carcinoma, cervical cancer, cancer of pancreas, cerebroma, skin carcinoma, leukemia, lymphatic cancer and melanoma.

24. application according to claim 20, is characterized in that, described medicine is selected from following dosage form: injection, tablet, granule, capsule, soft capsule, drop pill, oral liquid, membrane, powder, externally used paste, suppository and pill.

25. 1 kinds of methods of preparing the compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin, is characterized in that, said method comprising the steps of:

3) medicinal residues water extraction, obtains water extraction liquid;

9) by step 1) gained Oleum Bulbus Allii or step 2) gained Oleum Bulbus Allii clathrate, step 5) gained Bulbus Allii total polysaccharides and the combination of step 8) gained garlic total saponin, obtain the compositions that comprises Oleum Bulbus Allii, Bulbus Allii total polysaccharides and garlic total saponin.

26. methods according to claim 25, is characterized in that:

In step 1), described two carrying as Bulbus Allii is pulverized, adds water insulation enzymolysis, and vapor distillation extracts, and carries out 1-3 time, adds 3-9 times of water at every turn, each 0.5-2h;

Step 2), in, the concrete steps of described Oleum Bulbus Allii enclose are: Oleum Bulbus Allii with 65%-95% volume ratio ethanol in 1:5-1:15(mL/mL) ratio dissolving, in 25-45 ℃ of ultrasonic pond, with cyclodextrin inclusion compound 0.5-1.5h;

In step 4), described reclaim under reduced pressure is carried out at not higher than 80 ℃ in temperature; Described concentrated solution, take Bulbus Allii (g): medicinal liquid (mL) is unit, and concentration is 1:0.8-1.2, and making the relative density of medicinal liquid at 60 ℃ is 0.9-1.10; The content of described ethanol in concentrated solution is 75-85% volume ratio;

In step 6), the concrete steps of described reflux, extract, are: add 3-5 times of mass ratio 95% volume ratio alcohol reflux, extract 1-2 time, each 0.5-1.5 hour;

In step 8), medicinal liquid III is crossed to styrene type macroporous resin, according to resin and crude drug weight ratio meter, applied sample amount is 1:0.8-1.1, wash successively 4-6BV and/or 30% volume ratio ethanol is washed 2-5BV, 70%-95% volume ratio ethanol is washed 4-6BV, collect the rear ethanol elution of washing, or 30% ethanol elution after volume ratio ethanol elution, at temperature <70 ℃, decompression recycling ethanol is extremely without alcohol taste, drying under reduced pressure, spraying is dried or lyophilization, described drying under reduced pressure temperature is not higher than 60 ℃, the dry hothouse temperature of spraying is no more than 120 ℃, lyophilization cold hydrazine temperature is lower than-50 ℃.

27. methods according to claim 26, it is characterized in that, described cyclodextrin comprises alpha-cyclodextrin, beta-schardinger dextrin-, hydroxyethyl-β-cyclodextrin, HP-β-CD, beta-schardinger dextrin-methylate derivant and branched cyclodextrin, the concentration of cyclodextrin is 10% mass volume ratio, Oleum Bulbus Allii: cyclodextrin is 1:6-1:12 by weight.

28. methods according to claim 26, is characterized in that, resin described in step 5) is D941 or D900 type resin.

29. methods according to claim 27, is characterized in that, resin described in step 5) is D941 or D900 type resin.

30. methods according to claim 26, is characterized in that, the temperature of drying under reduced pressure described in step 5) is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

31. methods according to claim 27, is characterized in that, the temperature of drying under reduced pressure described in step 5) is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

32. methods according to claim 28, is characterized in that, the temperature of drying under reduced pressure described in step 5) is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

33. methods according to claim 29, is characterized in that, the temperature of drying under reduced pressure described in step 5) is not higher than 60 ℃, described spray-dired preheater temperature is 280 ℃, its hothouse temperature is 120 ℃, and described cryodesiccated cold hydrazine temperature is-50 ℃, and its baking temperature is 25 ℃.

34. according to the method described in any one in claim 26-33, it is characterized in that, styrene type macroporous resin described in step 8) is DM-130, AB-8 or HPD100.