CN1582956A - Composition of mycobacterium polysaccharide and its preparation - Google Patents
Composition of mycobacterium polysaccharide and its preparation Download PDFInfo
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Abstract
A polyopse composition composed of arabinose, mannose, glucose and galactose for treating leukocytopenia of cancer patient, chronic hepatitis and multiple immunopathies is extracted from the nonpathogenic mycobacteria.
Description
Technical field
The present invention relates to a kind of to treating the effective immunization therapy medicine of multiple disease.Particularly, the present invention relates to a kind of holosaccharide complex and preparation method of from non-pathogenic mycobacteria whole cell, extracting.
Background technology
Present studies show that, the polysaccharide in some fungus, antibacterial and Chinese medicine source has the effect of tangible enhancing human body immunity function.Wherein studying more is immunization and the application of relevant mycobacteria.The research of relevant mycobacteria immunization can roughly be summarised as two aspects.Be as immunological adjuvant on the one hand, for example by the bacillus calmette-guerin vaccine (BCG) of bacillus tuberculosis bovis preparation and the muramyldipeptide (MDP) that extraction prepares from the mycobacteria cell wall.This class material uses for a long time, and they belong to nonspecific immunity strengthening agent, except that being used for experimentation, also can be used for improving tumor patient and chronic infectious disease patient's immunologic function.
Be as immunotherapeutic agent on the other hand, wherein studying more is the extract of using mycobacteria, the report that uses cell wall extracts has been arranged, as CN94191293.0 a kind of immunotherapeutic composition has been described, it is a kind of preparation of not butyraceous modification mycobacteria cell wall extracts, some are invalid even act on other opposite material to goal of the invention but wherein contain, and can certain side effect be arranged to human body after use, as anaphylaxis.
Another example is that (SpecialSubstance Maruyama), was called ball mountain vaccine to Japanese S.S.M in the past.It is the extract of Bacillus tuberculosis green hill strain, and its main component is an AM, is the tentative medication as oncotherapy in early days in Japan, has the late tumor patient of prolongation life, eliminates the pain caused effect of tumor.Discover that further its antineoplastic mechanism is the parcel that can form collagen around tumor tissues, stops the diffusion of tumor, and finds that it can promote the generation of Hemopoietic factor, the leukocyte increasing level.Japanese health ministry was ratified the leukopenic medicine (medicine name Ancer-20) of this medicine as treatment back tumor patient in 1992.
Yet S.S.M is the polysaccharide material that is extracted by the Bacillus tuberculosis, because of the Bacillus tuberculosis has serious infectiousness to the people, causes its production process to have certain danger; And, make its production cycle long because the cultivation cycle of this bacterium is long.In addition, it is reported has 4.7% patient infusion part anaphylaxis to occur after S.S.M uses, and illustrates that said preparation has certain toxic and side effects, makes its application be subjected to a certain degree restriction.
Therefore, be necessary to study can shorten the production cycle, price economy and can make the side effect of clinical use reduce to Mycobacterium extract minimum even that be free from side effects.
Summary of the invention
According to above-mentioned needs, the present patent application people provides a kind of mycobacteria polysaccharides compound, thereby has solved an above-mentioned difficult problem.
One aspect of the present invention relates to the polysaccharides compound of a kind of mycobacteria, and it mainly contains arabinose, mannose, glucose and galactose.
In a kind of preferred embodiment, the molecular weight of above-mentioned mycobacteria polysaccharides compound is 2,000～20, between 000.
Described mycobacteria is Mycobacterium phlei preferably.
The invention still further relates to a kind of preparation method for preparing the mycobacteria polysaccharides compound, may further comprise the steps:
(1) subculture that will contain mycobacteria continues to be inoculated in the logical culture medium of Soviet Union and carries out amplification cultivation to growing into the Yellow-to-orange Mycoderma;
(2) polysaccharide is slightly carried: through boiling extraction, centrifugal going precipitated with above-mentioned culture, and acid-alkali treatment precipitates polysaccharide at low temperatures with polar organic solvent, obtains rough polysaccharide;
(3) refinishing polyose: above-mentioned raw sugar must be made with extra care polysaccharide through ion exchange column and hyperfiltration treatment postlyophilization;
In the preferred embodiment of preparation method of the present invention, above-mentioned polar organic solvent is an ethanol.
In the present invention, described mycobacteria polysaccharides compound or the type of service that contains the pharmaceutical compositions of this complex can be injection, tablet, pill, capsule, solution, suspending agent, Emulsion.
In the present invention, described mycobacteria polysaccharides compound or the route of administration that contains the pharmaceutical compositions of this complex comprise oral, percutaneous, vein or intramuscular injection.
In the present invention, described mycobacteria polysaccharides compound or the pharmaceutical compositions that contains this complex can use separately, also can be used in combination with other medicines.The medicine that is used in combination comprises antineoplastic agent.Described antineoplastic agent comprises cyclophosphamide, 5-fluorouracil, fortulon, peace Qu Xi, mitomycin, amycin, epirubicin, rubidomycin, vincristine, camptothecine, cisplatin, Le Shading, Xin Leer or paclitaxel etc.
In the present invention, the dosage scope of application of described mycobacteria polysaccharides compound is (0.02-5 μ g)/kg body weight/day.
The invention further relates to the pharmaceutical compositions that contains mycobacteria polysaccharides compound of the present invention.It is mycobacteria polysaccharides compound and pharmaceutically acceptable excipient that this complex contains active ingredient, and carrier or diluent are made into medicine with conventional method.
Confirm after deliberation, mycobacteria polysaccharides compound of the present invention compared with prior art have produce safer, the production cycle is shorter, use is without any side effects and range of application advantage widely, and low finished product cost.
The specific embodiment
The present invention is the mycobacteria with non-pathogenic----Mycobacterium phlei is a raw material, extracts polysaccharide component with biochemical method.
The present invention prepares by following technical process:
(1) strain is preserved and is identified
Strain is preserved with cultivating on the Luo Shi egg slant medium.Before increasing bacterium inoculation, earlier through the logical culture medium of the reviving cultivation of going down to posterity.Go down to posterity after the cultivation, the bacterial growth situation is examined and determine, and carry out the morphology calibrating through acidproof dyeing and Gram's staining.
Can use multiple mycobacteria, preferably use Mycobacterium phlei.
(2) subculture of assay approval is continued to be inoculated in the logical culture medium of Soviet Union and carry out 2 weeks of amplification cultivation.Because this bacterium is superficial growth, lead to liquid-based superficial growth yellowly to orange Mycoderma in Soviet Union.
(3) polysaccharide is slightly carried
Through boiling extraction, centrifugal going precipitated with above-mentioned culture, and acid-alkali treatment precipitates polysaccharide at low temperatures with ethanol and obtains rough polysaccharide.
(4) refinishing polyose
Rough polysaccharide must be made with extra care polysaccharide through anion-exchange column and hyperfiltration treatment postlyophilization, and its molecular weight is 2,000～20,000.
The key component that shows mycobacteria polysaccharide of the present invention by analysis is monosaccharide such as arabinose, mannose, glucose, galactose.
In the present invention, described mycobacteria polysaccharides compound or the pharmaceutical compositions that contains this complex can use with the form of injection, tablet, pill, capsule, solution, suspending agent, Emulsion.
In the present invention, described mycobacteria polysaccharides compound or the route of administration that contains the pharmaceutical compositions of this complex comprise oral, percutaneous, vein or intramuscular injection.
In the present invention, described mycobacteria polysaccharides compound or the pharmaceutical compositions that contains this complex can use separately, also can be used in combination with other medicines, for example be used in combination described antineoplastic agent such as cyclophosphamide, 5-fluorouracil, fortulon, peace Qu Xi, mitomycin, amycin, epirubicin, rubidomycin, vincristine, camptothecine, cisplatin, Le Shading, Xin Leer or paclitaxel etc. with antineoplastic agent.
The preferred using method of mycobacteria polysaccharide of the present invention is the single dose subcutaneous injection.To obtain the mycobacteria polysaccharides compound with method of the present invention and be dissolved in the injection normal saline, make mycobacteria polyoses injection (MPS), use for subcutaneous injection.Each 15 μ g (containing 15 μ g in the 1ml solution), the next day, inject once.The application time then decides according to kinds of Diseases and the degree of being in a bad way, and be one month a course of treatment, generally uses 1-3 the course of treatment.Also can intravenous injection, topical or oral administration.
Below will further set forth the present invention in the mode of embodiment.
Embodiment
Embodiment 1
This Mycobacterium phlei adopts Luo Shi egg slant medium to cultivate maintenance.Adopt the logical culture medium of Soviet Union to carry out amplification cultivation, every bottle of 110-120ml, after 35 ℃-37 ℃ 2 weeks of hot-house culture, culture bottle shows and grows the Yellow-to-orange Mycoderma.Liquid in the culture bottle under the Mycoderma is answered clear, the celadon that do not have or the growth of grey black pollutant, and stink or abnormal flavour do not have yet.Culture needs to guarantee its uncontaminated, no abnormal extraction that can be used for polysaccharide through checking by bottle.
Above-mentioned culture is concentrated in the big glass beaker, put in the boiling water heated and boiled 2 hours, cool off back 4000 rev/mins centrifugal 20 minutes, make bacterial sediment quarrel precipitation fully, supernatant is as clear as crystal.Collect supernatant, add glacial acetic acid, the glacial acetic acid amount is about and accounts for 1% of total amount of liquid, and is centrifugal with quadrat method, collects supernatant.This supernatant adds the sodium hydroxide of 1% amount again, and heating is 2 hours in 100 ℃ of water-baths, and is centrifugal with quadrat method, collects supernatant, 3 times of ethanol precipitations, and precipitate is the polysaccharide crude extract with ethanol, washing with acetone, oven dry.
Above-mentioned polysaccharide crude extract water dissolution.Last ion exchange column, fraction collector and ultraviolet surveillance instrument are collected automatically.The sample addition is 20-30ml.The each several part polysaccharide component of collecting is detected with the phenolsulfuric acid method, discard sugar-free and contain the part of pigment.
With the polysaccharide part of collecting, membrane ultrafiltration, it is dry to put into frozen vacuum dryer, is sub-packed in the bottle sealing, puts dry and cold place and preserves.
Use injecting normal saline, use content to be mixed with 15 μ g/ml, 45 μ g/ml mycobacteria polysaccharide solutions by prescription, small jar is pressed in packing, handles through autoclaving.Its shelf-life of mycobacteria polyoses injection through this processing was at least 2 years.
Embodiment 2
For a large amount of experimentation of warp that detects through embodiment 1 preparation shows that the present invention has extensive human body immunity improving function, effects such as leukocyte increasing.Human body is used without any side effects.
Engulf effect in the killing activity for check complex of the present invention strengthening mouse macrophage, carried out complex of the present invention is engulfed the killing activity influence to mouse macrophage experiment.Use the mice of 20-25g to experimentize.Animal is divided into two groups every group 10 at random.In experimental mice intraperitoneal injection complex 0.5ml of the present invention (50 μ g/ml), matched group intraperitoneal injection 0.5ml normal saline, behind the 16h, with (pH7.4) 3ml flushing mouse peritoneal of RPMI1640 liquid (please indicate the source), peritoneal irrigation liquid is placed in the tissue culture flasks, 37 ℃ hatch 30 minutes after, discard a bottle interior liquid, the adherent cell that obtains with 0.5mlRPMI1640 liquid rinsing bottle wall is this experiment source of macrophage.Make the cell suspension of 5 * 105/ml behind the numeration cell.Repel test check cell survival rate＞95% through trypan blue.
The clinical separation staphylococcus aureus strains that uses this laboratory to preserve after broth bouillon is cultivated 18h, is prepared into 2 * 10 with microbionation
8/ ml suspension is standby.
Mice S180 ascites cells (institute of oncology, Beijing provides) is inoculated into the subcutaneous growth of mice back takes out tumor, shred and grind with the cell dismembyator, adding RPMI1640 liquid system cell suspension. after cell suspension washed 3 times, counting cells was also made: 2 * 107/ml cell suspension. repel test check cell survival rate＞95% through trypan blue.
Reference literature (RobisonP etc., Infect.Immun1984; 43:744-752) method, (5 * 105/ml) suspensions add in the luminescence assays pipe with the 0.2ml macrophage, after putting 37 ℃ of water-bath 20min, add 20 μ lluminol, behind 37 ℃ of water bath heat preservation 10min, (LumiphotometerTD4000 Labosciene) measures luminous background with luminous photometer.Add then the staphylococcus aureus suspension (2 * 108/mi) or mice S180 ascites cells suspension (2 * 107/mi) 0.1ml, METHOD FOR CONTINUOUS DETERMINATION 30min behind the mixing observe the integrated value of (reducing background automatically) in the 30min.Measure the integrated value of two groups every mice sample during mensuration respectively, get its mean value computation.
Preparation of the present invention is subjected to staphylococcus aureus to stimulate active influence the in back to see Table 1 to mouse macrophage.
Table 1 preparation of the present invention is subjected to staphylococcus aureus to stimulate active influence the in back to mouse macrophage
Number of mice integrated value (cpm) compares for two groups
Group
(only) X ± S (t check)
Experimental group 10 37.92 ± 32.11
P＜0.01
Matched group 10 5.34 ± 5.38
The result shows, through the inductive mouse macrophage of injection preparation of the present invention with the staphylococcus aureus mechanism in, its luminous intensity (integrated value) is than the obvious enhancing of matched group (P＜0.01).
The applied chemistry luminescent method is measured the effect of macrophage to mice S180 ascites cells, also observes the activity that preparation of the present invention has strengthened macrophage, and experimental result is as shown in table 2.
Table 2 preparation of the present invention is subjected to tumor cell to stimulate active influence the in back to mouse macrophage
Number of mice integrated value (cpm) compares for two groups
Group
(only) X ± S (t check)
Experimental group 10 29.07 ± 27.22
P＜0.01
Matched group 10 5.48 ± 5.29
For understand strengthened active macrophage through preparation of the present invention can killing tumor cell, use dyestuff again and repel experimental technique, observed tumor cell at warp with without the situation (every group of each 3 mice of experimental group and matched group) of the mouse macrophage killing tumor cell of preparation injection of the present invention.Result's demonstration (experimental group) after preparation effect of the present invention kills and wounds 25.5% oncocyte (matched group kills and wounds 13.3%), points out preparation of the present invention to have and strengthens the effect that mouse macrophage kills and wounds mice S180 tumor cell.
Embodiment 3
Murine interleukin due to the elimination immunosuppressant (cyclophosphamide) reduces and promotes its rise
Animal: the LACA mice, male, 18～22g is available from Beijing Medical University's animal center.
Medicine: MPS is the aqueous injection of ampoule packing, is divided into 60 μ g/ml, 80 μ g/ml, 100 μ g/ml and 120 μ g/ml4 kind specifications.Hui Er blood (GRAN) is the ampoule injection of 75 P8, Japanese Kirin Beer Kabushiki Kaisha product, lot number: 483A.(Cydophosphamide CP) is every bottle of 20mg powder to cyclophosphamide, Shanghai No.12 Pharmaceutical Factory's product, lot number: 920317.
Experiment grouping and administration: every batch of experiment divides 7 groups, every group of 10 mices.The normal control group is healthy LACA mice, replaces administration with injecting normal saline; The cyclophosphamide group gives CP by 120mg/kg, in the 6th, 7 day, every day 1 intraperitoneal injection; The positive drug matched group is pressed 50 μ g/m with Hui Er blood
2(body surface area) calculates administration, i.e. every 3.9 μ g is in the 1st, 4 and 7 day subcutaneous injection 1 time; And MPS1 group～MPS4 group, be followed successively by 0.6,0.8,1.0 and 1.2mg/ (kgd) respectively, subcutaneous injection 1 time, the 1st～7 day successive administration.Above-mentioned positive drug matched group and each MPS group, all at the 6th, 7 day intraperitoneal injection of cyclophosphamide 120mg/kg, every day 1 time.
The bone marrow hematogenesis CFU-GM is cultivated and quantitative assay
Adopt double-deck soft agar culture method.Be the aseptic mouse femur of getting, flushing bone marrow prepares cell suspension, crosses 4.5 syringe needles, and counting nucleated cell number is diluted to 5.7 * 10 with cell suspension
6Ml
-1Standby.MLCM (CM) is prepared as: be liquid culture 7d with the healthy mice lung in advance, get supernatant, and the filtering with microporous membrane degerming, packing is standby, does target cell with the healthy mice medullary cell and measures the CM activity.Cultivating system is bottom RPMI 1640 5.0ml, horse serum 2.5ml, 3% agarose 1.0ml; Upper strata RPMl 1640 3.0ml, horse serum 1.4ml, CM0.6ml, bone marrow cell suspension 0.2ml, 3% agarose 0.5ml.Do bottom earlier, after the bottom cultivating system fully mixes, inject 1.0ml to each diameter 30mm plate, after waiting to coagulate, do the upper strata cultivating system, i.e. filling 1.0ml upper strata on bottom, after waiting to coagulate, little plate is placed the big plate of 100～200mm diameter, in 37 ℃ of 5%CO2, cultivate 5d.Counting hematopoietic cell colony (each colony should contain 〉=50 cells) under 40 power microscopes.Calculate the meansigma methods and the standard deviation of each group at last, and do statistical analysis.
Show normal mouse bone marrow per 2 * 10 through repeating 3 batches of result of experiment
5Individual nucleated cell can form 100～120 GM-CFU.And the cyclophosphamide group makes GM-CFU be subjected to a certain degree restriction, its suppression ratio be 47.0%～55.0% (with normal group relatively, P＜0.01).Hui Er blood has rising effect (comparing P＜0.001 with the cyclophosphamide group) to GM-CFU in this case.MPS promotes the effect difference of GM-CFU under various dose, a little less than effect relatively under the 0.6mg/kg dosage; 0.8mg/kg improving the GM-CFU effect down, dosage is similar to Hui Er blood; And during 1.0mg/kg, MPS is the strongest to the stimulation of GM-CFU, almost is equivalent to the level of 2 times of 0.6mg/kg groups; 1.2mg/kg group no longer increases GM-CFU, but still far above cyclophosphamide-a control group (P＜0.001).
Effect through experiment showed, that repeatedly MPS has more significantly stimulates the bone marrow hematogenesis CFU-GM, and dose-effect relationship is preferably arranged, its effect (0.6-1.0mg/kg dosage) the roughly activity level with Hui Er blood (0.19mg/kg) is suitable.
Embodiment 4
Mycobacteria complex of the present invention is to the clinical observation of the therapeutical effect of chronic hepatitis B
Object: totally 40 examples are clinical or pathology and are diagnosed as chronic viral hepatitis B and the carrier that HBV infects, and HBsAg.HBeAg is anti--three equal positive persons of HBc, the course of disease is more than 6 months.
Method: divide three groups to observe
First group of MPS+ Hepatitis B virus vaccine therapeutic alliance group 20 example, MPS (A) 20 μ g/l prop up.MPS (B) 2 μ g/l prop up, the next day replace 1 of subcutaneous injection, every month intramuscular injection Hepatitis B virus vaccine 30 μ g/l prop up four months courses of treatment totally four times again.
Second and third group is two groups of double blinding matched groups (being simple MPS group and placebo group) totally 20 examples, and the unified numbering of medicine is responsible for granting by the special messenger, and treatment is revealed the inside story after finishing, and the injected dose method and the course of treatment are all with first group.
Observation item: 3 monthly detection liver functions and HBV/m before the treatment, after treatment back every month and the drug withdrawal, part patient examines HBV-DNA, DNA-P and OKT4/8 ratio.All do not use other immunomodulators and antiviral agents during each group treatment.
For avoiding the error of detection technique, the serum specimen that reaches before the treatment after treating keeps, and the inspection of uniting.This group is selected the unite inspection (RPHA method) and with the accurate test kit contrast of import Holland prunus mume (sieb.) sieb.et zucc. of hepatitis provide and box, the cloudy coincidence rate unanimities of changeing of both HBeAg for use.
Observed result shows that it is 45% that the MPS+ Hepatitis B virus vaccine is united group eAg negative conversion rate, and simple MPS group eAg negative conversion rate is 40%, and placebo group is 20%.Learn processing by statistics for three groups, compare there was no significant difference (P＞0.05) between each group.But aspect the cloudy commentaries on classics of HBV-DNA, associating Hepatitis B virus vaccine group is 66.6%, simple MPS group is 40%, and placebo group is 0%, aspect cellular immunization OKT4/8 ratio improves, and associating Hepatitis B virus vaccine treatment group and simple MPS group, treatment back ascensionist is 7/9, account for 77.8%, placebo group does not have rising, thereby prompting MPS has reliable curative effect to improving cellular immune function.
Embodiment 5
Mycobacteria complex of the present invention is to the therapeutical effect of rheumatoid arthritis
Case is selected and grouping
All patients all meet Americanism damp disease association (ARA) diagnostic criteria in 1987: 1. deadlock in morning 1h at least,＞6 weeks, 2. 3 or 3 above arthralgia, in＞6 weeks, 3. wrist, the palm refer to, proximal interphalangeal joint is swollen,＞6 weeks, 4. symmetry swollen joint,＞6 weeks, 5. subcutaneous nodule, 6. hands X line picture changes (have at least osteoporosis and crack, joint narrow), 7. the rheumatoid factor positive (positive rate in the method therefor normal population is no more than 5%).Possess 4 in above-mentioned 7.
The main selection singly treated the rheumatoid arthritis in active stage patient that 2 months state of an illness do not have improvement with a kind of NSAID, possess 3 in following 5: 1. morning stiff time＞30min, 2. the average grip of both hands, patient male＜20kPa (150mmHg), female patients＜17.3kPa (130mmHg), 3. an arthralgia or a joint, tenderness number＞6,4. a joint, swollen joint number＞3,5. erythrocyte sedimentation rate (ESR, Wei Shi method)＞25mm/h.The X line is I～III phase by stages.Experimenter's medicine-less allergy history.Anemia of pregnant woman and women breast-feeding their children do not include this test in.The experimenter did not use SAARD class medicine in test in preceding 3 months, comprised MTX, penicillamine, golden preparation, hormone and Radix Tripterygii Wilfordii.Treatment less than 1 month and therapy discontinued person does not participate in efficacy evaluation.
Administrated method
A group case is accepted 6 months by a definite date mycobacteria polysaccharide and NSAID (non-steroidal anti-inflammatory drug) treatment earlier, and the mycobacteria polysaccharide of stopping using then is continue use methotrexate and NSAID (non-steroidal anti-inflammatory drug) treatment 6 months after 1 month instead with original NSAID (non-steroidal anti-inflammatory drug).B organizes methotrexate and the NSAID (non-steroidal anti-inflammatory drug) treatment that case elder generation accepts 6 months by a definite date, the methotrexate of stopping using then, and continuation uses the mycobacteria polysaccharide after 1 month instead with original NSAID (non-steroidal anti-inflammatory drug) and NSAID (non-steroidal anti-inflammatory drug) was treated 6 months.The used NSAID (non-steroidal anti-inflammatory drug) of each patient immobilizes in whole 12 months treatment, also remains unchanged substantially on the dosage.Mycobacteria polysaccharide usage: intramuscular injection is 2 times weekly, each 1 (every 15 μ g).Methotrexate usage: oral weekly 1 time (10mg) or intramuscular injection 1 time (10mg) weekly.
Efficacy evaluation
Efficacy evaluation: 1. produce effects: morning deadlock, swollen joint, articular pain, grip, erythrocyte sedimentation rate, c reactive protein, rheumatoid factor, have 4 progress＞50% at least in 7.2. effective: that 3 progress＞50% are arranged in above-mentioned 7.3. invalid: 3 progress persons of less than or the state of an illness person of increasing the weight of in above-mentioned 7.
The result
Gained the results are shown in Table 3 and table 4.
Table 3 MPS and MTX treat 6 months Comparison of therapeutic respectively
| Group | Produce effects | Effectively | Invalid | Total effective rate/% |
| The A group | 1 | ?11 | ?13 | ?48 |
| The B group | 3 | ?12 | ?10 | ?60 |
2 groups of equal n=25, through rank test u=0.931 3, P＞0.05
Table 4 A group and the contrast of B group treatment total effects
| Group | Produce effects | Effectively | Invalid | Total effective rate/% |
| The A group | 6 | ?11 | ????8 | ?68 |
| The B group | 12 | ?9 | ????14 | ?44 |
2 groups of equal n=25, through rank test u=3.317 9, P＞0.05
Proof uses the treatment of mycobacteria polysaccharide after 6 months the t lymphocyte subset group to be had dual regulation simultaneously.And its toxic and side effects than methotrexate still less, sees Table 5.
| Medicine | Sample number | Feel sick | Erythra | WBC reduces | PIT reduces | ALT raises | Total side effect rate % |
| MPS | ?26 | ?0 | ?1 | ?0 | ?0 | ?0 | ?3.8 |
| MTX | ?28 | ?3 | ?0 | ?3 | ?1 | ?2 | ?32.0 |
Although for the clear purpose of understanding, quite detailed description has been carried out in aforementioned invention, it is evident that and to carry out some changes and modification within the scope of the appended claims by the mode of illustration and embodiment.These changes and modification are also among scope of the present invention.
Claims (8)
1. a mycobacteria polysaccharides compound is characterized in that this polysaccharides compound contains arabinose, mannose, glucose and galactose.The molecular weight ranges of described polysaccharides compound is 2,000～20,000.
2. according to the described mycobacteria polysaccharides compound of claim 1, it is characterized in that described mycobacteria is a Mycobacterium phlei.
3. according to the described mycobacteria polysaccharides compound of claim 1, it is characterized in that the type of service of described mycobacteria polysaccharides compound is selected from injection, tablet, pill, capsule, solution, suspending agent and Emulsion.
4. according to the described mycobacteria polysaccharides compound of claim 1, it is characterized in that its route of administration is selected from oral, percutaneous, vein and intramuscular injection.
5. preparation method for preparing the mycobacteria polysaccharides compound may further comprise the steps:
(1) subculture that will contain mycobacteria continues to be inoculated in the logical culture medium of Soviet Union and carries out amplification cultivation to growing into the Yellow-to-orange Mycoderma;
(2) polysaccharide is slightly carried: through boiling extraction, centrifugal going precipitated with above-mentioned culture, and acid-alkali treatment precipitates polysaccharide at low temperatures with polar organic solvent, obtains rough polysaccharide;
(3) refinishing polyose: above-mentioned raw sugar must be made with extra care polysaccharide after ion exchange column and hyperfiltration treatment.
6. according to the described method of claim 5, it is characterized in that described strain is a non-pathogenic bacteria.
7. according to the described method of claim 5, it is characterized in that described strain is a Mycobacterium phlei.
8. pharmaceutical compositions is characterized in that containing mycobacteria complex and pharmaceutically acceptable excipient, carrier or the diluent of the claim 1 of effective dose.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103059159A (en) * | 2013-02-01 | 2013-04-24 | 黄河三角洲京博化工研究院有限公司 | Process for extracting mannan from beer yeast powder |
| CN103157108A (en) * | 2011-12-13 | 2013-06-19 | 宁云山 | An immunomodulator composition, and a pharmaceutical composition and applications thereof |
| CN105213432A (en) * | 2014-05-28 | 2016-01-06 | 重庆莱美药业股份有限公司 | Mycobacterium phlei oral administration nanometer grain and preparation method thereof |
| CN113444671A (en) * | 2021-08-04 | 2021-09-28 | 成都可恩生物科技有限公司 | Subculturing method of bacillus calmette-guerin strain capable of replacing culture medium of sutong potato |
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2003
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103157108A (en) * | 2011-12-13 | 2013-06-19 | 宁云山 | An immunomodulator composition, and a pharmaceutical composition and applications thereof |
| CN103157108B (en) * | 2011-12-13 | 2016-01-20 | 宁云山 | Immune modulator composition and pharmaceutical composition thereof and application |
| CN103059159A (en) * | 2013-02-01 | 2013-04-24 | 黄河三角洲京博化工研究院有限公司 | Process for extracting mannan from beer yeast powder |
| CN105213432A (en) * | 2014-05-28 | 2016-01-06 | 重庆莱美药业股份有限公司 | Mycobacterium phlei oral administration nanometer grain and preparation method thereof |
| CN105213432B (en) * | 2014-05-28 | 2019-04-19 | 重庆莱美药业股份有限公司 | Mycobacterium graminis oral administration nanometer grain and preparation method thereof |
| CN113444671A (en) * | 2021-08-04 | 2021-09-28 | 成都可恩生物科技有限公司 | Subculturing method of bacillus calmette-guerin strain capable of replacing culture medium of sutong potato |
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