CN1712517A - Asexual reproducing bacterial fermentative filtrate of cordyceps, fermenting substance and use thereof - Google Patents
Asexual reproducing bacterial fermentative filtrate of cordyceps, fermenting substance and use thereof Download PDFInfo
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- CN1712517A CN1712517A CN 200410025517 CN200410025517A CN1712517A CN 1712517 A CN1712517 A CN 1712517A CN 200410025517 CN200410025517 CN 200410025517 CN 200410025517 A CN200410025517 A CN 200410025517A CN 1712517 A CN1712517 A CN 1712517A
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Abstract
Asexual reproductive bacterial fermented filtrate of Chinese caterpillar fungus, fermenting substance and its use are disclosed. The process is carried out by solid-liquid separating the fermented product consisting of mycelium, fermented filtrate and other metabolic products, membrane separating, solid-liquid mixing, refining, spraying, drying and obtaining fermenting substance, or dissolvent separating, chromatograph column separating, purifying and drying to composite product or compound. It can be used for immune anomalous diseases and aftercare, to prevent effort syndrome, tonify lung and nourish kidney.
Description
Technical field
The present invention relates to a kind of Chinese Cordyceps sinensis asexual generation strain fermentation product, particularly a kind of Chinese Cordyceps sinensis asexual generation strain fermentation thing and wherein ferment filtrate, with and application in preparation protective foods and medicine.
Background technology
Chinese medicine---Chinese Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.), have another name called Cordyceps, among the peoplely be called for short Chinese caterpillar fungus usually again, being Chinese Cordyceps sinensis asexual generation bacterial classification---cordyceps sinensis (Hirsutella hepiali Chen et Shen sp.Nov) colonizes on the bat moth larvae body and Mycophyta Eumycota, Ascomycotina Ascomycotina, gang pyrenomycetes Pyrenomycetes, ergot Zoopagales Clavicipitales, kind of Clavicipitaceae Clavicinitaccae Cordyceps Cordyceps of forming.Cordyceps sinensis is that China is exclusive, only is distributed in high mountain steppe and alpine scrub band between China's Qinghai-Tibet Platean height above sea level 3000-5000 rice.Because of the singularity of its ecological habit makes it rare precious.
Cordyceps sinensis is a kind of Cordyceps.Cordyceps sinensis fungus has been reported more than 300 kinds in the whole world, and China reports nearly 90 kinds.The fungi of Cordyceps can colonize on larva, pupa, adult even the cocoon of various insects, and the Chinese caterpillar fungus form presents in different poses and with different expressions.Therefore " Chinese caterpillar fungus " is Cordyceps generalized general designation, and except that Cordyceps sinensis, other all Chinese caterpillar funguses all can not be referred to as Cordyceps sinensis.
As protective foods and the history in existing more than 1,000 year of medicine, Cordyceps sinensis and genseng, pilose antler are called the top grade of " reason weakness " with being described as Chinese medicine three big " treasured " by ancient Chinese medicine doctor Cordyceps sinensis in the Chinese nation.Nearly millennium comes, the effect that the Chinese materia medica document of China has all been put down in writing Cordyceps sinensis return through.The Pharmacopoeia of the People's Republic of China put down in writing its effect for " invigorate the lung and the kidney, hemostasis and phlegm.Be used for that chronic cough void is breathed heavily, labor is coughed hemoptysis, impotence and seminal emission, soreness of waist and knee joint." along with going deep into that modern medicine is studied, the effect of Cordyceps sinensis and pharmacological action mechanism are opened.Especially by dysimmunity (allergic immune reaction) state is regulated immunostimulant that embodies or immunosuppressive action down under different states.
Therefore, utilize and (promptly on the artificial synthetic medium, can grow the stroma the same with natural cordyceps by biological assay, thecasporous evaluation) Chinese Cordyceps sinensis asexual generation bacterial classification, as Chinese Cordyceps sinensis asexual generation bacterial classification cordyceps sinensis (Hirsutella hepiali Chen et Shensp.Nov.), cultivate the tunning that is produced through microbial fermentation, provide a kind of similar to natural cordyceps even more pretend the product of usefulness, be used for substituting natural cordyceps in health care, prevention and treatment are gone up and are used, and will have crucial meaning (referring to Chinese patent 97110448.4).
The 1980s, one of inventor has finished the microbial liquid fermentation culture of this bacterial classification, and utilized mycelium, drying is made medicine, can partly substitute Cordyceps sinensis, its medicine---hundred make capsule (authentication code: the accurate word Z10910036 of traditional Chinese medicines), be extensive use of clinically.But that kind since the restriction of condition ferment filtrate and effective constituent are not wherein further studied, ferment filtrate discarded need not, wasted a large amount of resources, the while has been polluted environment again.Therefore, Chinese Cordyceps sinensis asexual generation strain fermentation filtrate is developed and utilized, very positive meaning will be arranged the protection of resources effective utilization and environment by the present invention.
Summary of the invention
The object of the present invention is to provide a kind of new Chinese Cordyceps sinensis asexual generation strain fermentation thing and ferment filtrate wherein, with and clinical application.
The objective of the invention is to realize by following technical proposal:
A kind of ferment filtrate of Chinese Cordyceps sinensis asexual generation bacterial classification, it is made by following method, the method comprising the steps of 1): Chinese Cordyceps sinensis asexual generation bacterial classification cordyceps sinensis is in the substratum top fermentation, obtains mycelium and contains fermented supernatant fluid and the ferment filtrate of wherein each kind of meta-bolites; It is characterized in that this method also comprises the following steps 2): the tunning in the step 1) after going out mycelium, solid-liquid separation and/or membrane sepn is obtained this ferment filtrate.
After membrane sepn, also can further carry out the solvent separation and separate the effective constituent that obtains ferment filtrate of the present invention with the chromatograph post.
Ferment filtrate and isolated mycelium mixing homogenate that above-mentioned various method steps is made, and spraying drying and make fermented product of the present invention.
Fermented product of the present invention, ferment filtrate can be applied to regulate immunology.Particularly, can prepare and be used for immune allergic disorder (IV, III, II, the I type) as graft-rejection, systemic lupus erythematous, the idiopathic thrombocytopenia purpura, diseases such as allergic immune reaction such as allergic rhinitis and asthma, and respiratory system, kidney, liver, dysimmunity disease and operative results such as the rejection after the organ transplantation, the prevention of fatigue syndrome, treatment and healthcare product, also can be used for preparation invigorates the lung and the kidney, hemostasis and phlegm, chronic cough void is breathed heavily, the labor spitting of blood of coughing, impotence and seminal emission, soreness of waist and knee joint, the prevention of weakness, treatment and healthcare product.
For the ease of using, can above-mentioned fermented product, ferment filtrate is purified, be dried to composite product or single product, adopt known method, make various formulations, as tablet, capsule, pill, powder, granule, lozenge, paste, patch, syrup, mixture or aerosol.
Description of drawings
Fig. 1 shows given the test agent and the dexamethasone restraining effect to Sephadex (plug send out treat gram think) G 200 inducing mouse abdominal cavity acidophilias and neutrophil accumulation reaction.
Fig. 2 shows given the test agent and the dexamethasone restraining effect to the mouse peritoneal total white blood cells.
Embodiment
The preparation of embodiment 1 ferment filtrate of the present invention
China's Cordyceps sinensis asexual generation bacterial classification cordyceps sinensis liquid medium within top fermentation obtains fermented supernatant fluid, mycelium and various meta-bolitess wherein; Fermentation process is an aerated culture in slant strains cultivation, shake-flask seed cultivation, one-level, the cultivation of secondary seed jar, the liquid fermentation tank fermentation, and 10-18 ℃, pH6.0-7.5 cultivated respectively 30-60,10,6,6,6-12 days.Separation method is a tunning through (or without) solid-liquid separation, and application method includes but not limited to means such as sheet frame whizzer, press filtration, also can use membrane sepn to substitute the former or carry out method such as membrane sepn again after it to make ferment filtrate of the present invention.The ferment filtrate that obtains is a brown yellow transparent liquid, pH value 5.0-7.0, and wherein all amount of dry matters are 0.1-2.0%, comprise materials such as polysaccharide and organic compound.
The fermenting process of above-mentioned Chinese Cordyceps sinensis asexual generation bacterial classification cordyceps sinensis sees also Chinese patent 97110448.4.
The preparation of embodiment 2 ferment filtrates of the present invention
The conventional solvent of utilization separates and waits the chromatograph post to separate (containing membrane sepn) to obtain the ferment filtrate that effective constituent is rich in the present invention on embodiment 1 basis.
The preparation of embodiment 3 fermented products of the present invention
The ferment filtrate of the present invention of the foregoing description 1 and 2 is concentrated, and concentrated solution mixes with mycelium, homogenate, be spray dried to Chinese Cordyceps sinensis asexual generation strain fermentation thing.
With the product that obtains in the foregoing description 1,2,3, purified, be dried to composite product or single product, adopt known method, add suitable ancillary component, make various formulations, as tablet, capsule, pill, powder, granule, lozenge, paste, patch, syrup, mixture, aerosol or injection.
Experimental example 1
Get 50 of ICR mouse, male and female half and half, body weight 20 ± 2 gram is divided into 5 groups, is respectively blank group, model group, 1,2 groups in sample and positive control drug Dexamethasone group (DXM).
Specimen preparation:
Given the test agent 1,2 is respectively fermentation mycelium powder and the fermented liquid of Chinese Cordyceps sinensis asexual generation bacterial classification cordyceps sinensis (Hirsutella hepiali Chen et Shen sp.Nov), sample 1 oral dosage is 3g/kg/d, consistent with the subliminal dose 3g/d of Chinese medicine Cordyceps sinensis record, experimental result shows: during the fermentation mycelium dry powder 3g/d of oral Chinese Cordyceps sinensis asexual generation bacterial classification, have anti-inflammatory, anti-allergic effects; The ferment filtrate 22.2ml/kg/d of sample 2 oral these bacterial classifications, when being the zymophyte powder of this bacterial classification of this batch production 3g, the concentrated solution of fermented liquid that produces this bacterial classification of 22.2ml simultaneously (is calculated as follows: this wholesale yeast-like fungi powder yield 0.54%, promptly by obtaining in 555 milliliters the Cordyceps fermented liquid, button removes 20% mycelium volume, fermentating liquid volume is 444 milliliters, be concentrated into 1/20 then, be 22.2ml) so, per kilogram is oral to Cordyceps fungus powder 3g and the comparison that is meant two portion of product in same fermentating liquid volume to 22.2ml, and its biological activity has comparability.
Dosage and approach:
Sample 1 (3g/kg/d); Sample 2 (22.2ml/kg/d).Cause inflammation preceding every day of gastric infusion 1 time, for three days on end.Cause scorching back gastric infusion every day 1 time, continuous 2 days.The volume of gastric infusion is 0.5ml/10g.Blank group does not deal with; Model group physiological saline 0.5ml/10g irritates stomach, every day 1 time, continuous 5 days; Positive control drug dexamethasone (DXM, 1mg/kg/d) abdominal injection, every day 1 time, continuous 5 days.
Animal causes inflammation:
Cause inflammation before the 4th administration.Blank group does not deal with.Model group, administration group and positive control drug group are all used Sephadex G 200 (0.1mg/ml) abdominal injection, every mouse 2mg/kg.
Peritoneal lavage
Adopt the cervical vertebra dislocation method to put to death mouse after causing scorching 48h in the abdominal cavity.Every mouse peritoneal injection irrigating solution 5ml gently rubs moments later sucking-off peritoneal lavage fluid.
White blood cell count(WBC)
Peritoneal lavage fluid is shaken up the back take out 40 μ l, with 8 times of white corpuscle diluted, take a morsel and drip on cell counting count board, at microscopically counting total white blood cells.
Arneth's count
Behind the centrifugal 10min of peritoneal lavage fluid low temperature 2000rpm, abandoning supernatant is played even back smear with pipettor.After treating that smear is at room temperature done, the Wright 5～6min that dyes.Carefully dyestuff is rinsed well with tap water, under 40 times of mirrors of opticmicroscope, carried out Arneth's count.
Fig. 1 shows given the test agent and the dexamethasone restraining effect to Sephadex G 200 inducing mouse abdominal cavity acidophilias and neutrophil accumulation reaction.Statistical procedures: x ± SD; Compare with the blank group
###Compare with model group P＜0.001
*P＜0.05,
*P＜0.01.
Fig. 2 shows given the test agent and the dexamethasone restraining effect to the mouse peritoneal total white blood cells.Statistical procedures: x ± SD; Compare with the blank group
##Compare with model group P＜0.01
*P＜0.05,
*P＜0.01.
Result: Sephadex G 200 (2mg/kg) abdominal injection causes the gathering of mouse peritoneal inflammatory cell, raises 2.5 times approximately with blank group comparison total white blood cells, and wherein acidophilia and neutrophil leucocyte ratio significantly rise, more about 30 times with the blank group.These results show that Sephadex G 200 mainly induces acidophilia and neutrophil accumulation.This aggreation all can obviously be suppressed by given the test agent.The inhibition acidophilia of given the test agent and neutrophil accumulation action intensity are sample 2＞1, with model group significant difference (P＜0.05～0.01) are arranged more all, see Fig. 1.The action intensity that given the test agent suppresses total white blood cells is a sample 2＞1, and wherein sample 2 more all has significant difference (P＜0.01) with model group, sees Fig. 2.Positive control drug dexamethasone abdominal injection all has remarkable restraining effect to acidophilia and neutrophil accumulation and total white blood cells.
Conclusion
1, the inhibition acidophilia of given the test agent and neutrophil accumulation action intensity and model group more all have significant difference, sample 2 (P＜0.01)＞1 (P＜0.05).
2, the action intensity and the model group of given the test agent 2 inhibition total white blood cellses relatively are significant difference, and action intensity and model group that sample 1 suppresses total white blood cells compare there was no significant difference, action intensity, sample 2 (P＜0.05)＞sample 1 (P＞0.05).
Experimental example 2
Rat allogeneic skin graft experiment also proves the activity of anti-repulsion of ferment filtrate of Chinese Cordyceps sinensis asexual generation bacterial classification cordyceps sinensis greater than fermentation mycelium, and hardening, incrustation, blue or green black phenomenon do not appear in cutify.
Experimental example 3 tunning acute toxicity test in mice:
10 of Kunming mouses, Chinese Cordyceps sinensis asexual generation strain fermentation thing and ferment filtrate be with the dosage of 20g/kg, one day three times gastric infusions, fasting is 12 hours before the administration, the animal dead rate is 0 as a result, does not see other toxic reaction, and the maximum tolerated dose of mouse (MTD) is 20g/kg.
By above-mentioned disclosed content as seen, Chinese Cordyceps sinensis asexual generation strain fermentation product of the present invention is regulated and therapeutic action the dysimmunity disease is significant, and have no side effect, the preparation method is simple, be easy to large-scale industrial production, be expected to solve this medicine resource of Chinese Cordyceps sinensis obtains suffered restriction from nature problem.
Claims (7)
1, a kind of ferment filtrate of Chinese Cordyceps sinensis asexual generation bacterial classification, it is made by following method, the method comprising the steps of 1): Chinese Cordyceps sinensis asexual generation bacterial classification cordyceps sinensis obtains mycelium and contains fermented supernatant fluid and the ferment filtrate of wherein each kind of meta-bolites in the substratum top fermentation; It is characterized in that this method also comprises the following steps 2): the tunning in the step 1) after going out mycelium, solid-liquid separation and/or membrane sepn is obtained this ferment filtrate.
2, ferment filtrate according to claim 1 is characterized in that also carrying out the solvent separation and separates with the chromatograph post after membrane sepn.
3, a kind of fermented product of Chinese Cordyceps sinensis asexual generation bacterial classification, it is by ferment filtrate and isolated mycelium mixing homogenate according to claim 1 or 2, and spraying drying and making.
4, the application of the ferment filtrate of Chinese Cordyceps sinensis asexual generation bacterial classification according to claim 1 and 2 in preparation immunoregulation druge or healthcare products.
5, application according to claim 4 is characterized in that this medicine or healthcare products are tablet, capsule, injection, pill, powder, granule, lozenge, paste, patch, syrup, mixture or aerosol-type.
6, the application of the fermented product of Chinese Cordyceps sinensis asexual generation bacterial classification according to claim 3 in preparation immunoregulation druge or healthcare products.
7, application according to claim 6 is characterized in that this medicine or healthcare products are tablet, capsule, injection, pill, powder, granule, lozenge, paste, patch, syrup, mixture or aerosol-type.
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| CN 200410025517 CN1712517A (en) | 2004-06-25 | 2004-06-25 | Asexual reproducing bacterial fermentative filtrate of cordyceps, fermenting substance and use thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102835652A (en) * | 2012-09-17 | 2012-12-26 | 江苏神华药业有限公司 | Method for extracting cordyceps hirsutella sinensis mycelia on basis of membrane technology |
| CN102835651A (en) * | 2012-09-17 | 2012-12-26 | 江苏神华药业有限公司 | Method for extracting cordyceps cephalosporin mycelia on basis of membrane technology |
-
2004
- 2004-06-25 CN CN 200410025517 patent/CN1712517A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102835652A (en) * | 2012-09-17 | 2012-12-26 | 江苏神华药业有限公司 | Method for extracting cordyceps hirsutella sinensis mycelia on basis of membrane technology |
| CN102835651A (en) * | 2012-09-17 | 2012-12-26 | 江苏神华药业有限公司 | Method for extracting cordyceps cephalosporin mycelia on basis of membrane technology |
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