CN101269110B - Radix astragali fermentation liquor for inhibiting cancer cell growth and fermentation method - Google Patents
Radix astragali fermentation liquor for inhibiting cancer cell growth and fermentation method Download PDFInfo
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Abstract
The invention relates to a method of fermenting astragalus using Cordyceps militaris and consists of the following steps: firstly, Cordyceps militaris is activated; secondly, Cordyceps militaris is added into a culture medium containing astragalus for a fermentation culture to obtain a fermented product; thirdly, the fermented product is conducted a centrifugal treatment and the obtained supernatant fluid is a fermented fluid.
Description
Technical field
The present invention relates to the fermentation liquid and the fermentation process thereof of the growth of a kind of anticancer, especially a kind of with Cordyceps militaris (L.) Link. fungus fermentation liquid and the fermentation process thereof that Radix Astragali obtains that ferment.
Background technology
In medical progressive epoch now, cancer still is the disease that causes death, and it is characterized by the undesired agglomerate of the malignant tissue that over-drastic cell division causes.Cancer cell does not have the suffered restriction of normal cell growth, can invade and capture the proper space of leaving other cell for.
The treatment for cancer method comprises chemotherapy, operative treatment, radiation cure, and the combination of these Therapeutic Method.Wherein, but the chemical compound of one or more anticancer growth is used in chemotherapy usually.Though researched and developed many cancer chemotherapy medicaments at present, but still needed more effective chemotherapy.
At present, Chinese herbal medicine continues, extensively carries out for many years for the research that suppresses cancer.Radix Astragali is the root of perennial leguminous herbaceous plant Radix astragali, and is of a great variety, mainly originates in ground such as Gansu, Shaanxi, the Inner Mongol, Hebei, Shanxi.Wherein, with film pod Radix astragali Astragalus membranaceus (Fisch) Bunge and Inner Mongol Radix astragali Astragalus mongholicus Bunge better quality.Now, known Radix Astragali has the various clinical function, except raise immunity, also have promote hemopoietic, promote the growth of all kinds of blood cell cell, antiplatelet aggregation, resist myocardial ischemia, bring high blood pressure down, multiple functions such as diuresis, defying age and hepatoprotective, anti-inflammatory, antibiotic, antiviral.
Radix Astragali comprises at the document aspect the treatment cancer: scholars such as Rittenhouse discover with the tumor cell that the meeting secretory substance suppresses macrophage, the Radix Astragali water extract has immunosuppressive effect to renal cancer cell Renca, transitional cell bladder carcinoma cell line MBT-2, and can make the macrophage immunity functional rehabilitation of common cultivation.Scholars such as Satonori so that cancer drug BBN are carcinogenic with mice, make its cytohormone secretion, immunity degradation, in feedstuff, add the Radix Astragali extract then, found that: when concentration reaches 10mg/kg B.W., the incidence rate of tumor is reduced to 50%, and the increased activity of T cell (T cell), NK cell (Nature killer cell), the growing amount of IL-2, IFN-γ obviously increases.Thereby show that Radix Astragali can reach the effect that suppresses cancer by the generation of activation poisoning cell and cytohormone.
In addition, present known Cordyceps contains chemical constituents such as ucleosides, steroid, aminoacid, peptide class, mannitol, saccharide, organic acid, inorganic elements, vitamins, protein and polyamines class.Cunningham people such as (1951) finds that from the fermenting process of preparing thing of Cordyceps militaris (L.) Link. a kind of composition is the nucleoside antibiotic (cordycepin) of 3 '-deoxyadenosine, and it is called cordycepin.Most scholars think that the effect of the anti-curing cancers of Cordyceps is in close relations with the relative equilibrium of its raise immunity, adjusting body immune system, also there is the scholar to think that it is the mechanism of Worm grass anticancer that cordycepin suppresses nucleic acid synthetic, because Cordyceps militaris (L.) Link. originally has certain effect in direct anticancer aspect.
Summary of the invention
The technical problem to be solved in the present invention is: by in conjunction with Cordyceps militaris (L.) Link. and Radix Astragali, provide a kind of product with anticancer growth function, and a kind of method of obtaining aforementioned product.
In order to solve the problems of the technologies described above, the invention provides a kind of method of the Radix Astragali that ferments, it comprises: activation Cordyceps militaris (L.) Link. fungus (Cordyceps militaris); Cordyceps militaris (L.) Link. fungus is added one contain in the culture medium of Radix Astragali and carry out fermentation culture, to obtain tunning; And with the tunning centrifugal treating, obtaining supernatant is fermentation liquid.
The present invention also provides a kind of fermentation liquid that is made by said method, and it has the function of anticancer growth.
The present invention also further provides a kind of compositions that prevents and/or treats cancer, and it comprises fermentation liquid and a kind of pharmaceutically acceptable carrier (carrier) or excipient that effective dose makes as stated above.
Description of drawings
For goal of the invention of the present invention, feature and advantage are become apparent more, below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, wherein:
Figure 1 shows that the IC of the fermentation liquid of Cordyceps militaris (L.) Link. fungus standing for fermentation Radix Astragali to cancerous cell
50Test result.
Fig. 2 a is depicted as the influence of unleavened Radix Astragali to the cancerous cell survival rate.
Fig. 2 b is depicted as by fermentation Radix Astragali to the influence of cancerous cell survival rate.
Fig. 3 a-c is depicted as the fermentation liquid that obtains under the different fermentations temperature to cancerous cell IC
50Test result.
Figure 4 shows that the test result of Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid for CT26 cancerous cell survival rate.
Figure 5 shows that of the influence of Cordyceps militaris (L.) Link. Radix Astragali fermentation liquid for the BALB/c mouse body weight of inoculation CT26 cancerous cell.
Figure 6 shows that Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for leukocytic influence in the BALB/c mouse blood of inoculation CT26 cancerous cell.
Fig. 7 a-b is depicted as the influence of Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid for the BALB/c mouse spleen of inoculation CT26 cancerous cell.
Be respectively Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid shown in Fig. 8 a-b for the BALB/c mouse gross tumor volume of inoculation CT26 cancerous cell and the influence of weight.
The specific embodiment
The invention provides a kind ofly with the ferment method of Radix Astragali of Cordyceps militaris (L.) Link. fungus, it comprises: activation Cordyceps militaris (L.) Link. fungus (Cordyceps militaris); Cordyceps militaris (L.) Link. fungus is added one contain in the culture medium of Radix Astragali and carry out fermentation culture, to obtain tunning; And with the tunning centrifugal treating, obtaining supernatant is fermentation liquid.
Wherein, activation step is inoculated in a culture medium for the Cordyceps militaris (L.) Link. fungus that will be stored in the inclined-plane preservation pipe, under 15-30 ℃, is preferably in 25 ℃ and cultivates down, to produce new mycelia.In a better embodiment of the present invention, Cordyceps militaris (L.) Link. fungus is Cordyceps militaris BCRC32219.Culture medium is the culture medium of any suitable conk, for example, glucose digesting protein agar culture medium GPA (glucose peptone agar, GPA), potato agar culture medium (potato dextrose agar, PDA) or Martin's agar culture medium (Martin agar), preferable with the PDA solid medium.Subsequently, new mycelia is implanted activation in a large number in the culture fluid, cultivate down, be preferably in 25 ℃ and cultivate down at 15-30 ℃; Concussion was cultivated 3-6 days, was preferably 5 days.Culture fluid is the culture medium of any suitable conk, for example, yeast Fructus Hordei Germinatus extract fluid medium (yeast malt extract broth, YM), potato glucose fluid medium (dextrose broth, PDB), or glucose digesting protein fluid medium (glucose peptone broth GPB), preferably can be the YM fluid medium.
Then, will contain in the culture medium of Radix Astragali with the 1-15% access through activatory Cordyceps militaris (L.) Link. fungus.Wherein, Radix Astragali is not limit kind, and is preferable with Inner Mongol Radix Astragali (Astragalus mongholicus Bunge) or film pod Radix Astragali (Astragalusmembranaceus (Fisch) Bunge).In a better embodiment of the present invention, the composition proportion of culture medium is: the water of the Radix Astragali of about 5-15% (w/w) and about 85-95% (w/w), the fermentation culture temperature is preferable with 15-30 ℃.In another better embodiment of the present invention, the fermentation culture mode is for leaving standstill cultivation, and fermented incubation time is preferable with 1-6 week.
Fermentation obtains tunning after finishing, and with this tunning centrifugal treating, gets supernatant, obtains fermentation liquid.Subsequently, can also be with filtering fermentation liquor and lyophilization.
The fermentation liquid that is made by said method has the ability of anticancer growth, and its suppressible cancerous cell comprises: colorectal cancer cells, hepatoma carcinoma cell, stomach cancer cell, breast cancer cell, blood cell, ovarian cancer cell, cervical cancer cell, skin cancer cell, testicular cancer cell, prostate gland cancer cell, pancreatic cancer cell, renal cancer cell, brain cancer cell, esophageal cancer cell, transitional cell bladder carcinoma cell line, lung carcinoma cell and nasopharyngeal carcinoma cell.Wherein, better to the inhibition effect of colorectal cancer cells, hepatoma carcinoma cell and breast cancer cell.
In addition, experimentize with the mice of this fermentation liquid to the inoculation tumor cell, the result shows: this fermentation liquid not only can not impact the body weight of mice, and can increase the leukocyte number of mice, promotes its immunity.In addition, this fermentation liquid is for the various organs of mice, and for example heart, liver, lungs, kidney there is no ill effect.With the mice of the fermentation liquid dosage feeding inoculated tumour cell of 100mg/kg/ days and 200mg/kg/ days, find that it has the effect of significant inhibition tumor.
The present invention prevents and/or treats the compositions of cancer, comprises fermentation liquid and a kind of pharmaceutically acceptable carrier or excipient that effective dose makes as stated above.Wherein, the cancer that can prevent and/or treat comprises colorectal cancer, hepatocarcinoma, gastric cancer, breast carcinoma, leukemia, ovarian cancer, cervical cancer, skin carcinoma, carcinoma of testis, carcinoma of prostate, pancreatic cancer, renal cancer, the brain cancer, esophageal carcinoma, bladder cancer, pulmonary carcinoma and nasopharyngeal carcinoma.In an embodiment of the invention, the cancer that can prevent and/or treat is preferably colorectal cancer, hepatocarcinoma or breast carcinoma.
The administering mode of above-mentioned composition can be oral, non-oral, via sucking spraying (inhalation spray) or by implanting reservoir modes such as (implanted reservoir).The form of compositions can be solid-state, semisolid and vacuum lyophilization (lyophilized) powder, or liquid dosage form, comprise tablet, suppository, pill, capsule, medicated powder, suspension medicinal liquid, Emulsion, medicine oil, liquid medicine, spray, ointment and injection etc.Preferably can the applying unit dosage form, and can accurately determine dosage by simple approach.
Embodiment
With the ferment preparation of fermentation liquid of Radix Astragali of Cordyceps militaris (L.) Link. fungus
Cordyceps militaris link bacterial strain Cordyceps militaris BCRC32219 is seeded to the YM liquid culture based on 25 ℃ of incubators with the activation of PDA solid medium, and the 100rpm concussion was cultivated 5 days; With 10% kind of former culture medium (20g Radix Astragali+200mL water) that contains 10% Inner Mongol Radix Astragali (Astragalus mongholicus Bunge) that is seeded to, under 25 ℃, leave standstill and cultivate 1-5 week again; Fermentation liquid is after sterilization, and with 10000rpm centrifugal treating 20 minutes, the supernatant that obtains was a fermentation liquid; Subsequently, with the filtration of bleeding of Whatman No.1 filter paper, gained filtrate is standby with the freezer dryer lyophilizing with fermentation liquid.
The fermentation liquid of Cordyceps militaris (L.) Link. fungus standing for fermentation Radix Astragali is to the IC of cancerous cell
50Test
The fermentation liquid of different fermentations time of embodiment 1 gained is cooked 503nhibiting concentration (IC to hepatoma carcinoma cell Hep G2
50) (hereinafter to be referred as IC
50) test, as shown in Figure 1: the fermentation liquid in 1 week of fermenting is to the IC of hepatoma carcinoma cell Hep G2
50Be about 950ppm; Along with the increase of fermentation time, fermentation liquid is to the IC of hepatoma carcinoma cell Hep G2
50Lower thereupon; Arrived fermentation during 4 weeks, fermentation liquid is to the IC of hepatoma carcinoma cell Hep G2
50Reduce to about 44ppm.
Unleavened Radix Astragali and through the influence of the Radix Astragali of Cordyceps militaris (L.) Link. fungus fermentation to the cancerous cell survival rate
With the fermentation liquid in 14 weeks of fermentation of undressed Radix Astragali powder, embodiment after filtration the powder of lyophilization gained be deployed into variable concentrations respectively, test its influence respectively to hepatoma carcinoma cell Hep G2 survival rate, the result is shown in Fig. 2 a and 2b.Can find out that by Fig. 2 a undressed Radix Astragali there is no inhibitory action for hepatoma carcinoma cell Hep G2 and produces under variable concentrations.Can find out that by Fig. 2 b through the ferment Radix Astragali in 4 weeks of Cordyceps militaris (L.) Link. fungus, then suppressing is all appearred in hepatoma carcinoma cell Hep G2 the effect of its growth under variable concentrations, and concentration is high more, it is obvious more to suppress effect.
The fermentation liquid of different fermentations temperature is to cancerous cell IC
50Test
The method of the method for making of fermentation liquid and embodiment 1 is similar, but fermentation temperature is changed to 15 ℃, 20 ℃ and 25 ℃ of three kinds of temperature respectively, and it was 4 weeks that fermentation time is then all ordered.(Fig. 3 a), breast cancer cell MCF-7 (Fig. 3 b) and hepatoma carcinoma cell Hep G2 (Fig. 3 c) be IC to stomach cancer cell AGS respectively with the fermentation liquid of above-mentioned different fermentations temperature
50Test.By Fig. 3 a-c as can be seen, fermentation temperature is that 25 ℃ the ability of fermentation liquid anticancer is the strongest, to the IC of three kinds of cancerous cell
50All the fermentation liquid than 15 ℃ and 20 ℃ is low.In addition, hepatoma carcinoma cell HepG2 is the most responsive to fermentation liquid, and the fermentation liquid of three different fermentations temperature is to its IC
50All be lower than 35ppm.
Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is the active anticancer test in mouse model
The ferment fermentation liquid in 4 weeks of embodiment 1 gained is carried out following each experiment:
(1) Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for the BALB/c mouse preliminary experiment of inoculation CT26 cancerous cell, and Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for CT26 cancerous cell IC
50Test
Before carrying out zoopery, must understand the inhibition effect of ferment filtrate earlier for the growth of CT26 mice colorectal cancer cell, experimentize with the above-mentioned ferment filtrate freeze-dried powder of variable concentrations.Can be known by inference by Fig. 4, Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for the IC of CT26 cell
50For about 20ppm.Therefore, on the feeding concentration of follow-up BALB/c mouse experiment, be designed to three concentration of 20ppm, 100ppm and 200ppm, and with phosphate buffered solution (Phosphate buffered saline, PBS) (hereinafter to be referred as PBS) is the control group, and cancer chemotherapy medication 5-FU (15ppm) is the positive control group.
(2) Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for the influence of the BALB/c mouse body weight of inoculation CT26 cancerous cell
As shown in Figure 5, mice is not too significantly change of body weight in 21 days process of raising, and the body weight of experimental group, control group and normal group slowly increases to about 27 grams by first day 25 grams; Aspect experimental group, raise in 21 days and there is no any experiment mice death, as can be known Cordyceps militaris (L.) Link. Radix Astragali ferment filtrate under this concentration for mice and avirulence; And positive control group part, the mice body weight obviously descends, because 5-FU itself is a kind of cancer chemotherapy medication, mechanism of action is synthetic for suppressing DNA, also produce inhibitory action for normal soma in the anticancer growth, institute is so that the mice weight loss.
(3) Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for leukocytic influence in the BALB/c mouse blood of inoculation CT26 cancerous cell
Begin to gather the eye socket blood of mice in the previous day of mouse inoculation CT26 cancerous cell, counting leukocyte number, observing the influence that immunity is subjected in the mouse experiment process, the result as shown in Figure 6: except that positive control group 5-FU, all the other each leukocyte numbers of organizing all have lifting; And 5-FU group white blood cell count to fall now be synthetic because himself can suppress DNA, more obvious for the fast immunocyte performance of growth cycle.
(4) Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for the influence of the BALB/c mouse spleen of inoculation CT26 cancerous cell
Spleen is the peripheral lymphoid organ of human body maximum, is positioned on the sanguimotor path, filtering blood is arranged and the antigen of invading in the blood is played functions such as immunoreation.Get its spleen weighing after mice is butchered, and calculate cell number.The mice of inoculated tumour except that the positive control group, compared to its spleen weight of normal group (Fig. 7 a) and cell number (Fig. 7 b) all have remarkable increase, supposition to be because inoculated tumour is given birth to the inner immunostimulation that produces of body for mice, cause the hypertrophy of spleen; On the other hand, positive control group 5-FU causes spleen weight to reduce because it suppresses the side effect of synthetic DNA, and cell number does not then have significant difference with normal group.
(5) Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for the influence of the BALB/c mouse internal organs of inoculation CT26 cancerous cell
Except spleen, take out other internal organs of mice in the lump and observed, its result is as shown in table 1.
Can find that by table 1 aspect the weight of heart, liver, lungs and kidney, normal group and experimental group do not have significant difference, have only each organ of 5-FU group all to have significantly and reduce.
Table 1
| Cardiac weight (g) | Liver weight (g) | Lungs weight (g) | Kidney weight (g) | |
| Normal group | 0.158±0.006 | 1.846±0.146 | 0.185±0.022 | 0.479±0.010 |
| PBS | 0.145±0.011 | 1.993±0.224 | 0.209±0.014 | 0.488±0.034 |
| 20mg/kg | 0.148±0.015 | 1.998±0.155 | 0.218±0.023 | 0.461±0.043 |
| 100mg/kg | 0.148±0.007 | 1.936±0.174 | 0.219±0.035 | 0.469±0.036 |
| 200mg/kg | 0.154±0.023 | 1.884±0.232 | 0.189±0.022 | 0.465±0.065 |
| 5-FU | 0.104±0.022 | 0.981±0.340 | 0.151±0.028 | 0.279±0.055 |
(6) Cordyceps militaris (L.) Link. fungus Radix Astragali fermentation liquid is for the BALB/c mouse gross tumor volume of inoculation CT26 cancerous cell and the influence of weight
Began to measure the gross tumor volume size of mice behind the inoculation cancerous cell on the tenth day, gross tumor volume calculates by following formula: [length (mm) * width (mm)
2] ÷ 2, the result is shown in Fig. 8 a.
By Fig. 8 a as can be seen: at 5-FU prescription face, the volume of mouse tumor is very little to have significant difference with experimental group, has best tumor suppression effect; Feeding 20mg/kg/ days group and PBS group do not have notable difference, and the expression feeding did not suppress the effect of tumor growth in 20mg/kg/ days.Improve feeding concentration to 100mg/kg/ days and 200mg/kg/ days, there were significant differences compared to the PBS group can to find inhibition effect for tumor, and suppression ratio is respectively 48.89% and 43.81% in the time of the 21st day; 100mg/kg/ days and 200mg/kg/ days do not have significant difference for two groups, and Radix Astragali Cordyceps militaris (L.) Link. fermentation liquid can effectively suppress tumor growth under this concentration as can be known.
Tumor weight part, mice were butchered behind inoculated tumour on the 22nd day, weighing in addition after tumor is taken out, and the result is shown in Fig. 8 b: at 5-FU prescription face, there were significant differences for the weight of mouse tumor and experimental group, tumor weight meansigma methods minimum; Feeding 20mg/kg/ days group and PBS group does not have significant difference, and when concentration was increased to 100mg/kg/ days and 200mg/kg/ days, there were significant differences compared to the PBS group to find the weight of tumor, and suppression ratio is respectively 39.48% and 31.21%.
Though the present invention discloses as above by the specific embodiment; but it is not in order to qualification the present invention, any those skilled in the art, without departing from the spirit and scope of the present invention; all can do to change and conversion, so protection scope of the present invention should be as the criterion with defining of claim.
Claims (10)
1. the method for the Radix Astragali that ferments, it comprises:
Activation Cordyceps militaris (L.) Link. fungus (Cordyceps militaris);
Cordyceps militaris (L.) Link. fungus is added one contain in the culture medium of Radix Astragali and carry out fermentation culture, to obtain tunning; With
With the tunning centrifugal treating, obtaining supernatant is fermentation liquid.
2. the method for fermentation Radix Astragali according to claim 1 is characterized in that: it further comprises filtering fermentation liquor and lyophilization.
3. the method for fermentation Radix Astragali according to claim 1, it is characterized in that: described Cordyceps militaris (L.) Link. fungus is Cordyceps militaris BCRC32219, and described Radix Astragali is Inner Mongol Radix Astragali (Astragalusmembranaceus var.mongholicus Astragalus mongholicus Bunge) or film pod Radix Astragali (Astragalus membranaceus (Fisch) Bunge).
4. the method for fermentation Radix Astragali according to claim 1 is characterized in that: the composition of described culture medium comprises the Radix Astragali of about 5-15% (w/w) and the water of about 85-95% (w/w).
5. the method for fermentation Radix Astragali according to claim 1 is characterized in that: described fermentation culture is to leave standstill to cultivate about 1-6 week under about 15-30 ℃.
6. a fermentation liquid is obtained by the described method of claim 1, and it has the ability of anticancer growth.
7. fermentation liquid according to claim 6 is characterized in that: described cancerous cell is colorectal cancer cells, hepatoma carcinoma cell, stomach cancer cell or breast cancer cell.
8. compositions that prevents and/or treats cancer, it comprises and contains fermentation liquid and a kind of pharmaceutically acceptable carrier (carrier) or the excipient that effective dose is made by the described method of claim 1.
9. the compositions that prevents and/or treats cancer according to claim 8 is characterized in that: described cancer is colorectal cancer, hepatocarcinoma, gastric cancer or breast carcinoma.
10. the compositions that prevents and/or treats cancer according to claim 8 is characterized in that: its administering mode be oral, non-oral, via sucking spraying (inhalation spray) or by implanting reservoir (implanted reservoir).
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