Mushroom ferment pure protein with antineoplastic activity, extracting method and formulation

A technology of anti-tumor activity and shiitake mushrooms, which is applied in the field of medical technology products, can solve the problems that the components of drug source substances cannot play a better role, and the components of crude protein are complicated, and achieve the effect of directly killing tumor cells without toxic side effects

Inactive Publication Date: 2009-01-14
DALIAN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the composition of crude protein is complex, so that the composition of drug source substances cannot play a better role, so it is particularly important to find specific pharmacologically active protein monomers from it

Method used

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  • Mushroom ferment pure protein with antineoplastic activity, extracting method and formulation
  • Mushroom ferment pure protein with antineoplastic activity, extracting method and formulation
  • Mushroom ferment pure protein with antineoplastic activity, extracting method and formulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the preparation of mushroom fermented liquid

[0040] (1) Preparation of Lentinus edodes Fermentation Medium

[0041] glucose

1g

fresh brewer's yeast

8g

sucrose

6g

whole milk powder

1g

Peptone

1g

Potassium dihydrogen phosphate

0.3g

Vitamin B1

0.01g

Vitamin B2

0.01g

[0042] Add the above items into 1000ml of distilled water, mix them and put them in a water bath at 50°C to dissolve them completely, adjust the pH to 7.5, divide them into 300ml Erlenmeyer flasks, each bottle is 250ml, and cover with 4 layers of gauze and a layer of kraft paper to seal. , sterilized for 15 minutes, cooled for later use.

[0043] (2) Take the mycelium of shiitake mushroom C91-3 that grew well on the 7th day, and inoculate it into the above-mentioned medium, and inoculate 1 g of mycelium in each bottle.

[0044] (3) Culture on a shaker at room temperature, at 75 rpm for the f...

Embodiment 2

[0046] Embodiment 2: Preparation of fermented crude protein from mushrooms

[0047] (1) Slowly add ammonium sulfate powder to the fermentation broth at 4°C while stirring with a magnetic stirrer until the ammonium sulfate is supersaturated and stop adding. After standing still for 20 minutes, centrifuge in a low temperature centrifuge (5,000g / min, 4°C) for 30 minutes. Discard the supernatant and collect the pellet.

[0048] (2) Put the precipitate obtained above into a dialysis bag, use deionized water as a dialysis buffer, and dialyze at 4° C. to remove residual ammonium sulfate. Nessler's reagent is used to test whether there is NH4+ residue. After adding Nessler's reagent, the solution turns yellow and there is NH4+ residue, otherwise it is colorless. The buffer was changed every 30 min until all NH4+ was removed.

[0049] (3) Preserve the desalted protein (i.e. crude protein) solution in a -20°C refrigerator; or concentrate and freeze-dry the desalted crude protein solu...

Embodiment 3

[0050] Embodiment 3: Preparation of mushroom fermented pure protein A and freeze-dried powder

[0051] (1) Take 400mg of lyophilized crude protein fermented with mushrooms, dialyze in 0.01M Tris-HCl buffer solution with pH 8.0 at 4°C until the ion balance between the inside and outside of the dialysis membrane is reached, centrifuge the dialysate at 3,000g / min for 10min at 4°C, and filter Impurities, Coomassie brilliant blue to determine the protein concentration of the dialysate.

[0052] (2) The sample was transferred to a DEAE-Cellulose column (5×20 cm), and the loading amount was 100 mg. First, use 3 column volumes of 0.01MTris-HCl buffer to elute the unadsorbed protein until the eluate that flows out is detected by Coomassie Brilliant Blue to contain no protein, adjust the flow rate of the eluate to 1ml / min, and then use 0- 0.5M NaCl (2×100ml) was used to gradiently elute the bound protein, and the eluate was collected by an automatic fraction collector. After the chrom...

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Abstract

Disclosed is lentinus edodes fermented pure protein with anti-tumor activity as well as an extracting method and a preparation thereof. The protein is obtained through fermentation, salting-out, dialysis, drying and column chromatography of mycelia of lentinus edodes C91-3; the protein is light brown or earthy yellow powder which can be dissolved in water and has specific fragrance, the pH value of aqueous solution is 5.0 to 8.0, the molecular weight is 1 KD to 90 KD, and the protein contains 18 types of amino acids and various proteins. The experiment proves that in the anti-tumor experiment in vitro, the tumor restraining rate of the lentinus edodes fermented pure protein LFP91-3-A to H22 and S180 in 72 hours can achieve 59.93 percent and 80.13 percent; lentinus edodes fermented pure protein LFP91-3-A has an inducing effect on H22 cell telomerase of which the activity is obviously restrained and apoptotic. The tumor restraining rate of lentinus edodes fermented pure protein LFP91-3-B to S180 can achieve 76.7 percent; the experiment proves that the strain fermented pure protein has relatively strong effect of directly killing tumor cells. The lentinus edodes fermented pure protein has no toxic side effect through animal acute toxicity experiments and long-term toxicity experiments.

Description

Technical field: [0001] The invention relates to a product in the technical field of medicine, in particular to pure protein fermented from shiitake mushrooms with antitumor activity, an extraction method and a preparation thereof. Background technique: [0002] Using active protein as a drug has the characteristics of high activity, strong specificity, clear biological function and favorable clinical application. Since the 1970s, the continuous update of protein microanalysis technology and the establishment of various biological activity determination methods have greatly promoted the research of active proteins. In recent years, the research on peptide proteins has progressed rapidly. With the deepening of the work, more and more people pay attention to some natural proteins with physiological and pharmaceutical activities. The research on protein drugs has always been an active field in drug research. How to discover active polypeptide protein substances from natural an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C07K1/14C07K1/16C07K14/37A61K38/16A61K9/48A61K9/14A61K9/20A61K9/08A61P35/00C12R1/645
Inventor 黄敏李星云钟民涛宁安红王晓丽
Owner DALIAN MEDICAL UNIVERSITY
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