CN1101833A - Medicine of homologous fructosan mixture, producing process and use thereof - Google Patents

Abstract

The white or slightly yellow homologous fructosan mixture containing glucose and fructose with molecular weight of 666-1962 is prepared by extracting active components from achyranthes root, and serves for enhancing immunity, inhibiting tumor growing and transfer, increasing number of leucocytes, and protecting and restoring liver functions, so it is used as a medicine for tumor, hepatitis and AIDS.

Description

Medicine of homologous fructosan mixture, producing process and use thereof

The present invention is a kind of energy enhancing human body immunity function, treats hepatitis and improve leukocytic medicine, and this medicine is good especially as the auxiliary treatment of tumour patient, and this medicine can separate from the Chinese medicine Radix Achyranthis Bidentatae, extract.

The understanding that people act in immunology polysaccharide arises from 1917 years, Dochez and Avery find that streptococcus pneumoniae secretes a kind of special solable matter in its growth course, and this material can be induced an immunoreation originally and produce special antibody (serum globulin) as immunity.Afterwards, Heidelberger, Avery and Goebel had proved that this was a polysaccharide (pneumococcal polysaccharide) material, and this also is the antigenicity substance of first found non-protide.

Later studies have shown that having only relative very little decisive position in the polysaccharide chain is that antagonist is narrow spectrum, if having identical decisive position in the different polysaccharide chains, just can produce identical immunoreation.And since early fifties in this century, people have found that pleasantly surprisedly a large amount of sugar chain component bands main bio information and protein, fat, phospholipid and nucleic acid combine and are participating in immunologic process and other life process, more make people that the important function of sugar chain in immunoreation had more and more deep impression.

In recent years to polysaccharide as antigen and immunogenic research, in identification and classification to antibacterial, to immunoreactive darker understanding, the active site in determining antibody-antigen-reactive, and detect and human body diseases that the opposing microorganism causes in brought into play enormous function.

Nature, people have more keen interest to human immunity.Recent two decades comes, generally acknowledged with immunotherapy treatment cancer by countries in the world, immunotherapy stimulates human body nonspecific defense function exactly, particularly stimulates patient's immune system to reach the purpose of treatment disease under the situation of body's immunological function damaged.The surgical operation that its superiority compares, X-ray therapy and chemotherapy are self-evident.The immunologic function that improves antibody is equally very necessary to healthy person, can help to resist disease, slow down aging.AIDS (AIDS) harm makes people to the serious consequence of immunodeficiency more deep understanding arranged, and therefore, seeking the medicine with good immunoregulation effect is pressing for of contemporary medical science.Scientists is at first sought immunopotentiating agent from the metabolite of microorganism and antibacterial, found muramyldipeptide (Muramyl dipeptide) in succession, corynebacterium parvum vaccine (Corybacterium parvum), and Picibanil active drugs such as (OK-432), but because antibacterial and metabolite thereof have shortcomings such as the strong and side effect of antigenicity is big, scientists turns to again and excavates immunotherapy medicaments in the plant, West Germany particularly, Japan, state such as Australia and India scholar makes great efforts that primary study has antibiotic from the traditional medicine of this country, detoxifcation plant amedica such as sets upright to Immune Effects.And the most valued immunopotentiating agent is the polysaccharide compound that extracts from Chinese herbal medicine.

Radix Achyranthis Bidentatae (Achyranthes bidentata BI) is a kind of Chinese medicine of China, has the effect of " invigorating the liver and kidney, bone and muscle strengthening; it is tingle to end the waist knee joint, dispelling malignant blood, broken crux; control the carbuncle malignant boil " according to ancient medical book record, and these effects and modern age, raise immunity was consistent clinically.People had once isolated compositions such as saponin, alkaloid and sterone from Radix Achyranthis Bidentatae, and had carried out preliminary pharmacological research.Separation and Extraction from the crude drug Radix Achyranthis Bidentatae such as Tian Gengyuan (CN 88105524.7) has obtained a kind of Inokopolyose; the pharmacological research that the sugared component of 100% purity is carried out shows that it has the enhancing humoral immunization; the effect of cellular immunization; and can protect mouse liver cell to avoid the effect of carbon tetrachloride murder by poisoning and anti-mice S-180 meat cancer; this Inokopolyose number-average molecular weight is the polysaccharide that 1.0:0.66 formed less than 10000 by glucose and mannose mole ratio.Because it is actually rare to have the small-molecular weight polysaccharide of remarkable increase immunologic function, be still that people pay close attention to so from Radix Achyranthis Bidentatae, further extract active ingredient, the especially medicine of small-molecular weight polysaccharide.

The invention provides a kind of levan class that belongs to has energy enhancing human body immunity function and improves leukocytic medicine, this medicine is not only effectively sick to treatment hepatitis, and be particularly suited for ancillary drug as tumour patient, can improve patient's immunologic function and leukocyte.Also be suitable for preventing the generation and the development of senile disease, thereby with slow down aging.Also can be used for simultaneously the treatment of AIDS.Medicine of the present invention is a kind of chemical compound with following structure:

Medicine of the present invention also can be the chemical compound of following structure:

The mixture of the homologue of the levan that medicine of the present invention is made up of glucose and these two kinds of monosaccharide of fructose, we abbreviate Abps or AbE as.

Drug molecule weight range of the present invention is 666-2124, and mean molecule quantity is 1300-1500.The recommendation molecular weight ranges is 666-1962, and mean molecule quantity is 1300-1400.Outward appearance is the loose shape solid of white or little yellow, easily moisture absorption.At ultra-violet (UV) band 202nm one absworption peak is arranged.

Measuring its sugared content with anthrone method is 100%, and results of elemental analyses is nonnitrogenous, phosphorus, and sulfur shows no aminoacid, peptide, phospholipid and sulfuric acid ester.Show with Conductometric Titration Method and not contain carboxyl.Thereby be a neutral sugar.

The purified postlyophilization of this medicine is TLC and is analyzed.Thin plate is: aluminium sheets silica gel 60F254 (MERCK), 2*10cm2; Developing solvent: n-butyl alcohol: pyridine: water (1: 1: 1).Thin plate launches through developing solvent earlier, is placed in the air to dry up, and point sample again behind the triplicate with the colour developing of sulphuric acid methanol solution, is more well Rf=0.50.

Medicine of the present invention is analyzed with C-18 reversed-phase column (μ BONDAPAKTM, water), and mobile phase is 15% MecN-H ₂, flow velocity 1ml/min, 20 ℃ of column temperatures carry out efficient liquid phase chromatographic analysis with the differential detector and are sharp-pointed unimodal (Fig. 1).When increasing the fine ratio of second in the eluant, when accounting for 15% volume, still be one unimodal, and when increasing the fine ratio of second again, retention time is elongated as second is fine, the peak type is widened.We can also see that the Abps through Sephadex G-50 purification also contains the non-sugared component of minute quantity from figure, so we come purification Abps again with the anti-phase preparative column of C-18.The Abps of 50mg is used Kaseisorb LC NH ₂Suger, Waters (4.6mm I.D.250mm) post, with acetonitrile: water (65: 35) is eluting, flow velocity 0.7me/min, wet 20 ℃ of post, the differential detector carries out efficient liquid phase chromatographic analysis, and Apbs is a very wide bag.Be divided into ten pipes through sephahex G-50 by elution order, can see that Abps is the mixture (Fig. 2) of the different oligosaccharide of long section.Use Spherisorb NH ₂Post (ID4.6mm, 30cm.5 the μ Dalian Chemistry and Physics Institute), eluent is that second is fine: water (50: 50), flow velocity 1ml/min, the differential detector, Abps is a bag peak (Fig. 3), when add 2 gram Polyethylene Glycol (W.M.3,000-4 in above-mentioned eluant 500ml, 500) time, had and differentiated (Fig. 4) preferably, Abps is a mixture really as can be seen, is divided into nine compositions in the drawings.

Medicine of the present invention is analyzed through fast atom bombardment MALDI-MS (FAB-MS), selects glycerol and thioglycerol mixed-matrix for use, and the molecular weight side is decided scope and reached 4,000, occurs the accurate quasi-molecular ions 2095M[12 of maximum molecule sugar+133(Cs on the collection of illustrative plates ⁺)], and a series of mass numbers differ hexose of 162() fragment ion peak 1933,1771,1609,1448(M+1), 1285,1124(M+1), 961 etc. (Fig. 5).

At 100C, 1N sulphuric acid tube sealing two hours is with brium carbonate neutralization, the centrifugal solid of removing, concentrating under reduced pressure with Abps.It is complete to detect hydrolysis with TLC, condition: thin plate: silica gel 60F254, MERCK; Developing solvent: n-butyl alcohol: pyridine: water (6: 4: 3).Making HPLC analyzes, use Carbohydrate Analysis post (3.9mm * 30cm), eluant: second is fine: water (65: 35), flow velocity 0.5ml/min, the time high-efficient liquid phase chromatogram (Fig. 6) of complete hydrolysis product of Abps and the standard sample of glucose, mannose and fructose compare (Fig. 7), find out that Abps is made up of fructose and glucosyl group.

We are the same with the HPLC(condition) ratio of these two kinds of glycosyls is carried out quantitatively.Since fructose under acid-hydrolyzed condition more than the glucose instability, the relatively mild condition of having selected to compare, the sulphuric acid of 0.2N, 50 ℃ of hydrolysis 0.5 hour.It is complete through the TLC(condition as above) to detect hydrolysis.The glucose and the fructose water solution of preparation series of standards concentration are made standard curve with peak area to injection volume (μ g), record the glycosyl ratio of sucrose: glucose: fructose (1.0: 1.0), and with the correctness of check the method.To Abps and through the tee pipe Abps that Sephadex G-50 is divided into, be quantitative glycosyl analysis and FAB-MS respectively and analyze, the result is listed as into following table (table 1):

Table 1.Abps and Abps through Sephadex G-50 divide the glycosyl mol ratio of three components and the maximum m/z value of FAB-MS

Analyze through Sephadex G-50 HPLC quantitative analysis FAB-MS

Isolating component Glc:Fru M+133 (Cs+)

Part1 1:8.7 2095 (12 sugar)

Part2 1:6.0 1609 (9 sugar)

Part3 1:4.6 1125 (6 sugar)

Abps 1:7.0 2095 (12 sugar)

Abps is the different levan of a degree of polymerization as can be seen from the above table

According to top analysis, we have known that Abps is the different fructan mixt of length of a chain.And the unusual feature of the 13C nuclear magnetic resonance, NMR of levan.Be apparent that most the formant of the β-fructofuranose base C-2 that concentrates on 103.80-104.80ppm and the formant of the six carbon atom that is positioned at terminal α-glucopyranosyl sugar ring.The inulin type that fructosyl connects in the mode of β-(2-1) and fructosyl are compared with the levan type that the mode of β-(6-1) is connected, and the chemical shift of corresponding each carbon all has little difference on the glycosyl.

Inulin is with the increase of the degree of polymerization, and the resonance signal intensity of carbon atom reduces on the terminal glucose base, and up to disappearance, and chemical shift does not almost change, and just C-1+ slightly moves to low; And fructosyl is along with away from the sucrose base, and it is also little that the chemical shift of each carbon accordingly changes, and just compares with the fructosyl that connects glucosyl group, and the chemical shift of corresponding each carbon has small variation, C-3, and C-5, the variation of C-6 is little; C-2, C-1 slightly shift to High-Field, and C-4 slightly shifts to low.

The 13C nmr spectrum of Abps has tangible levan feature (Fig. 8), can belong to the chemical shift (table 2) of its each type carbon atom according to document.

Levan with long chain type is compared, and the signal of the glucosyl group sugar ring carbon atom of Abps is very strong, shows that Abps is the short chain type.Levan with straight chain type is compared, and the resonance signal more complicated of Abps fructosyl carbon atom, existing 2, the 1 inulin types that connect have the fructosyl sugar of the levan type of (6-1) connection to encircle the resonance signal of carbon again, show that Abps is a branching type.We also find 13C nuclear magnetic resoance spectrum and the levan component among the Bromegrass (Acta chem.Scand, 44,158,1990) of Abps, the spectrogram of (with or without-1,6-)-2,6 short-movie section fructan mixts that connect of Fru-(2-side chain)

The chemical shift (ppm) of the 13C nuclear magnetic resonance, NMR of table 2. Abps

The carbon atom chemical shift

C-1 60.99 61.92 62.06

C-2 103.82 103.96 104.12 104.31

104.42 104.61 104.81 105.16

C-3 77.02 77.12 77.28 77.62

C-4 74.88 74.99 75.23 75.54

75.81 76.00

C-5 80.91 81.77

C-6 62.89 63.11 63.83 63.97

C-1* 92.75 93.13

C-2* 71.75

C-3* 73.29

C-4* 69.85

C-5* 73.24

C-6* 60.63

* number the carbon atom of expression glucopyranosyl sugar ring

Quite similar.Just from the C-5 of fructosyl, the signal of C-3 etc. can find out significantly that the intensity contrast of two groups of signals (1 substituted type and 6 substituted types of fructofuranose base) separately is just in time opposite, and the main chain that therefore can infer Abps is 1, the 2 levan type that connects.

Classical methylation analysis is a most effectual way of analyzing polysaccharide glycosidic bond link position up to now.The exhaustive methylation of polysaccharide has several methods (Carbohydr Res., 131,209.1984）。The Hakomori method is the most frequently used method, adds a certain amount of sodium hydride in exsiccant dimethyl sulfoxine, and 50 ℃ keep several hours up to there not being hydrogen to emit, obtain the anionic dimethyl sulfoxide solution of two methylsulfinyl.Adding is through the anhydrous dimethyl sulfoxide solution of fine exsiccant Abps, and nitrogen protection was bathed the formula supersound process one hour, made that the hydroxyl on the sugar all becomes sodium alkoxide, added iodomethane again and obtained methylated polysaccharide.Use the infrared detection hydroxyl, if also there is not exhaustive methylation, behind the sample drying, repeat above-mentioned steps, do not had hydroxyl up to infrared detection, key is an anhydrous condition, and permethylated Abps uses earlier the formic acid partial hydrolysis, then with the thorough hydrolysis of sulphuric acid, because permethylated polysaccharide often is insoluble to mineral acid and is dissolved in formic acid.Then with sodium borohydride reduction, reuse acetic anhydride-pyridine acetylation obtains the derivant of the sugar alcohol of part methyl partial acetylation at last.Use gas chromatography-mass spectrometry analysis.When analyzing with the OV-18 packed column, A appears, B, C, four peaks of D; Each peak with the contrast of standard spectrogram, obtains following result (table 3) through EI mass spectral analysis (Fig. 9-12).

Table 3.Abps methylation analysis result

GC peak O-methyl sugar of bonding

A 1,3,4,6 fructose, furan type-(2-

B 2,3,4,6-glucose, pyranoid form-(1-

C 3,4,6--1) fructose, furan type-(2-

D 3,4--1,6) fructose, furan type-(2-

A on the gas chromatogram, B, C, the area ratio at four peaks of D is 3.7: 2.6: 4.5: 1.Because if the levan of straight chain type, promptly the area at D peak is zero, the area of A equals the area of B so; If with the levan of a side chain, promptly the area at D peak is 1, needs the A peak of 2 areas and the B peak of 1 area so.So we can infer that the levan of existing straight chain type contains the levan of branched chain type again among the Abps, and the content of straight chain type levan is about: 1.6/2.6=61%, then branched chain type is about 39%.

Infrared spectrum also is used for the structural analysis of polysaccharide, and some the specific function groups in the carbohydrate structure all present some special INFRARED ABSORPTION, also is used to study the configuration and structure and the conformation of some polysaccharide with the conjugate of sugar of glycosidic bond.From the infrared spectrum (Figure 13) of Abps, we only can learn the group that does not have special replacement among the Abps.

Levan solution in the plant all is levorotation, and optical rotation is between-19 to-55.The optical rotation that we record Abps is-18 °--between 35 °, (C 1.00, H as-31.2 ° ₂O) ,-18.3 ° of (C1.5, H ₂O) be in above-mentioned scope.

The object of the invention also provides a kind of method for preparing said medicine.

Method of the present invention is that the crude drug Radix Achyranthis Bidentatae is soaked in water, and soak is dialysed through water, and the dialysis solution centrifugalize is concentrated and dry; Or the dialysis solution organic solvent secondary fractional precipitation through dissolving each other in water again, drying; Also soak can be used the organic solvent secondary fractional precipitation that dissolves each other with water, precipitate with water-soluble Jie after, the separation of dialysing.The wherein said organic solvent that dissolves each other with water can be the alcohol or the ketone of low carbon chain, as methanol, ethanol, propanol, acetone.The organic solvent that described and water dissolves each other and the volume ratio first order of Radix Achyranthis Bidentatae soak or dialysis solution are 0.3-3: 1, and the second level is 0.5-5: 1.

Described in the present invention concentrate and dry, preferably adopt cryoconcentration or drying, as vacuum drying, vacuum concentration, lyophilization, low temperature spray drying etc. are to keep the activity of medicine.

Adopt above-mentioned three kinds of methods of the present invention, the yield of medicine is 4-10 part of crude drug weight.

In order to obtain pure medicine, can also be purified with the column chromatography method of routine.As adopt dextran gel Sephadex G-50 column chromatography, and be that eluant separates with water, collect single sugared peak, get final product through concentrating again.

Medicine of the present invention is the mixture of a homologous fructosan thing, mean molecule quantity is 1300-1500, the maximum molecular weight that FAB-MS shows is a 1962(12 sugar), classical methylation analysis reaches the analysis to the first glycosides of glycosyl after the thorough hydrolysis of exhaustive methylation, prove that it has the structure of top general formula, also can confirm from 13C nuclear magnetic resonance, NMR and polarimetric analysis data.

Medicine of the present invention has the leukocytic function of raising to the person, because the treatment tumor is often used heavy dose of chemotherapeutics, thereby hemopoietic function of bone marrow usually occurs and be suppressed, especially leukocytic minimizing, the carrying out of influence treatment, by the clinical proof of AbE, no matter to malignant tumor patient, leukemia, or viral hepatitis, acute hepatitis and chronic persistent hepatitis all have obvious leukogenic effect, and clinical symptoms improvement, spirit, appetite, weak and sleep all are clearly better.Do not need to improve hemogram by blood transfusion or leucocyte transfusion.Use medicine of the present invention, chronic hepatitis patient HBsAg and HBeAg can turning out cloudy property, and toxic hepatitis patient GPT can recover.And have the antibody cell number increase in the spleen that impels of enhancing human body immunity function, improve serum immune globulin, activate the phagocytic function of reticuloendothelial system, increase SC C _aContent, promote the generation of interleukin-I and interleukin II, promote the effect of T cell and B cell, can protect and recover liver function, can be used as a kind of immunotherapy medicaments and be used for diseases such as tumor, hepatitis and AIDS.

Medicine of the present invention not only has immunocompetence, and it is little to have a molecular weight, good water solubility, and human body easily absorbs, and not only can be used for orally, also can be used for injection.Method for producing medicines of the present invention is easy, and raw material is easy to get, and is convenient to suitability for industrialized production.

To help further to understand method of the present invention by following embodiment.

Instrument and analytical method used in this description are summarized as follows.

1.13C-nuclear magnetic resonance, NMR: use the Brucker-AMX-600 instrument.Sample is solvent with the heavy water, and 20 ℃, with 1, the 4-dioxazole is made external standard (δ 67.64ppm).

2.FAB-mass spectrum: use VG QUATTRO MS/MS instrument.Sample be dissolved in glycerol and thioglycerol mixed-matrix, bombard with cesium ion.

3. optically-active: use Perkin-Elmer 241MC instrument.

4. infrared spectrum: use Shimadzu IR-440 instrument.

5. ultra-violet absorption spectrum (resorcinol method): use Perkin Elmer Lambda 5 instruments.

6.GC/MS(methylation analysis): use VG QUATTRO MS/MS mass spectrograph, HP5890II GC system, capillary column: SE-54(0.25mm I.D.*15m), column temperature: 160-190 ℃/6min.

7. high pressure liquid chromatography:

Use high-pressure pump: Model 6000(Waters Assoc.), detector: I.R.(R401 Waters), monitor: Waters 730 Data Module., mobile phase: twice distillation water, second fine (HPLC level), before the use all through ultrasonic degas and ultrafiltration.

8. number-average molecular weight:

According to Raoult's law, Abps has better water solubility and suitable molecular weight, can measure its mean molecule quantity with the vpo method.The aqueous solution of preparation Abps (C=12.5245g/kg reads 1/3,1/2,2/3, the △ T during 1C, and the mapping extrapolation promptly gets 0 → 0 o'clock △ T, calculates the number-average molecular weight of Abs.

Embodiment 1

To clean 10 kilograms of Radix Achyranthis Bidentatae sections, and add the 30-50 kg of water and soaked 30-40 hour, the elimination residue must soak the 22-30 kilogram.

Get 10000 milliliters of soaks, in the time of 0-15 ℃, add acetone 0.3-3 doubly, a large amount of precipitations produce, and disgorging gets supernatant, adds 5000-50000 milliliter acetone in supernatant again, produce a large amount of white precipitates, take out precipitation, with the suitable quantity of water dissolving, the tap water dialysis is after 40-50 hour, remove insoluble matter, lyophilization gets loose shape solid 120 gram, the i.e. Abps of little yellow.Mean molecule quantity 1300-1400.

Embodiment 2

With the soak 1000ml of embodiment 1, water dialysis 30-50 hour, dialysis solution concentrates in the time of＜80 ℃, and drying obtains the thick Abps of 150 grams.Mean molecule quantity 1300-1500.

Embodiment 3

With the dialysis solution of embodiment 2, the acetone that adds 1500ml produces precipitation, gets supernatant and adds 1500ml acetone, produces white precipitate, takes out heavy liquid, and lyophilization obtains the loose shape solid Abps of 10 grams.Mean molecule quantity 1300-1400.

Embodiment 4

Abps with embodiment 3 uses dissolved in distilled water, and last spehadexG-50 chromatographic column is made eluent with water, detects sugared peak with sulfuric acid-phynol method, collects the collection liquid of single peak position, and the cryoconcentration drying obtains 8 of refining Abps and restrains.Mass spectrometry results such as Fig. 5.

Embodiment 5

110 case ductus arteriosus interventional therapy matter leukocyte are in the middle and advanced stage tumor of 1.9～3.9K/ml, hepatocarcinoma 50 examples wherein, gastric cancer 16 examples, colon cancer 20 examples, pulmonary carcinoma 12 examples, ovarian cancer 12 examples, year makes 29-81 year, gives AbE gram every day 1, divide the secondary clothes, two weeks had a blood test and resemble after the medication, were a course of treatment all around.

Clinically show that medication two all leukocyte rising 1K/ml persons are 67 examples, reach the above person of 4K/ml (X=5.57K/ml) after 4 weeks, account for 107 examples, account for 97.2%.The patient once more after the chemotherapy leukocyte still descend, but can go up again with chemotherapy medication simultaneously, this medicine all can use in radiation treatment and chemotherapy.

Embodiment 6

4 routine leukemia, year makes 8-10 year, two week of medication the back leukocyte promptly go up, 1 routine monocytic leukemia, 2K/ml is reduced in radiotherapy for 51 times, overcomes every day 0.5 three times, rises to 8K/ml after all around, guarantees hocketing of chemotherapy.Abnormal liver function after another routine acute myeloblastic leukemia chemotherapy, leukopenia, the back liver function is normal around taking medicine, leukocyte 3-5K/ml, clinical symptoms is improved, spirit, appetite, weak and sleep all is clearly better.

Embodiment 7

Acute hepatitis 22 examples, chronic persistent hepatitis 14 examples, after week, leukocyte all obviously raises with AbE medicine 3-4, rises to 6.84K/ml and 5.45K/ml by average 4.77K/ml and 4.4K/ml before the treatment.GPT70% descends in the 10 routine chronic hepatitis treatments, and 4-8 week liver function recovery is normal, and gamma Globulin descends 2%, and showing increases protein content in the liver, stablizes the cured effect of hepatocyte.To the 2 routine chronic hepatitis long time token thing positives, take medicine 12 week back HBsAg and the cloudy commentaries on classics of HBeAg, recurrence so far.

Claims (10)

1, a kind of medicine that contains the homology mixture of levan is characterized in that having following structure:

Wherein: R=OH; A, b, c, d, o, p, q=0 → 9;

a+b+c+d+o+p+q=1→9。

2, medicine as claimed in claim 1 is characterized in that having following structure,

3, medicine as claimed in claim 1 or 2 is characterized in that molecular weight ranges is 666-2124, is recommended as 666-1962, and mean molecule quantity is 1300-1500, is recommended as 1300-1400.

4, medicine production method as claimed in claim 1 or 2 is characterized in that the crude drug Radix Achyranthis Bidentatae is soaked in water, the dialysis of soak water, and the dialysis solution centrifugalize, concentrated and dry; The perhaps organic solvent secondary fractional precipitation that above-mentioned dialysis solution is dissolved each other through water again, drying; Also above-mentioned soak can be used organic solvent two utmost point fractional precipitations that dissolve each other with water, precipitate with water dissolution after, water dialysis, the dialysis solution centrifugalize concentrates and dry.

5, medicine production method as claimed in claim 4 is characterized in that the organic solvent that described and water dissolve each other and the volume ratio of Radix Achyranthis Bidentatae soak or dialysis solution, and the first order is 0.3-3:1, and the second level is 0.5-5:1.

6, medicine production method as claimed in claim 4 is characterized in that organic solvent that described and water dissolve each other is the alcohol of low carbon chain or ketone, dimethyl sulfoxide.

7, medicine production method as claimed in claim 4 is characterized in that described concentrate or drying is vacuum concentration, vacuum drying or lyophilization.

8, as claim 4,5 or 6 described medicine production methods, it is characterized in that purifying with column chromatography, concentrate.

9, the purposes of medicine as claimed in claim 1 or 2 is characterized in that the immunotherapy medicaments that can be used as tumor, hepatitis, rises the Pseudobulbus Bletillae (Rhizoma Bletillae) AIDS.

10, the purposes of medicine as claimed in claim 1 or 2 is characterized in that the medicine of raise immunity.

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