CN107441126B - Macro-fungus composition capable of improving NK cell tumor killing activity and preparation method thereof - Google Patents
Macro-fungus composition capable of improving NK cell tumor killing activity and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/076—Poria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
- A61K36/066—Clavicipitaceae
- A61K36/068—Cordyceps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/074—Ganoderma
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
Abstract
The invention provides a macro fungus composition capable of improving NK cell tumor killing activity and a preparation method thereof, belonging to the fields of health food and traditional Chinese medicine. The large-scale fungus composition is prepared from ganoderma lucidum, mushroom, poria cocos, needle mushroom, hericium erinaceus and cordyceps sinensis fermentation fungus powder according to a certain proportion. The product prepared by the invention can effectively improve the killing activity of NK cell tumor and has obvious effect on improving the immunity of the organism.
Description
Technical Field
The invention belongs to the field of health food and traditional Chinese medicine, and particularly relates to a large fungus composition capable of improving NK cell tumor killing activity and a preparation method thereof.
Background
Natural killer cells (NK cells for short) are one of the important components of the body's innate immune system, and can kill target cells without specific antigen stimulation, thus having the functions of immune clearance and immune monitoring. Only activated NK cells can diffuse and infiltrate into the infection source or tumor tissues to play an anti-tumor role. NK cells can also secrete various cytokines such as TNF-alpha, TNF-beta, IFN-gamma and the like in coordination with the anti-tumor effect. The activation of NK cells is regulated by surface receptors, the receptors transmit inhibitory or activating signals according to different structural functions to regulate the activity of the NK cells, so as to regulate the anti-tumor function of a body, and the activity of the NK cells in the body is proved to be related to various tumors, for example, the immune clearance and immune monitoring functions of the NK cells of a lung cancer patient are inhibited to a certain extent. Lung cancer has been found to be associated with decreased expression of granzyme B, perforin, IFN- γ, and these three substances are partially synthesized and secreted by NK cells (G Hodeg, J Barnawi, C Jurisevic, et al long cancer associated with a degraded expression of protein, granzyme B and Interferon (IFN) - γ by profiling lung tissue T cells, Natural Killer (NK) T-like and NK cells [ J ] cin Exp Immunol 2014, 178(1): 79-85.); the absolute number of NK cells in breast cancer patients did not change, but the proportion of poorly differentiated and non-cytotoxic NK cell subsets increased (Mamessier E, Bourgin C, Olive D. Whenbreaker cancer cells started to fed the ecological process of NK cells off [ J ]. Oncoinmmoniology, 2013, 2(12): E26688.); the activity of NK cells of colorectal cancer patients is obviously reduced; the activity of NK cells gradually diminishes as the tumor grows; the activity of NK cells of patients with metastasis after 2 weeks of chemotherapy is also obviously reduced (17), and the like, the NK cells play an important role in the formation of large tumors, meanwhile, the direct action of NK cells in the growth and metastasis of tumors is also proved, and the activated NK cells can inhibit the formation and metastasis of tumors (Ito M, Hiramatsu H, Kobayashi K, et al.NOD/SCID/gamma (c) (null) mouse: an excellent recipient mouse model for expression of human cells [ J ] Blood,2002,100(9): 3175-.
Disclosure of Invention
The invention aims to provide a large fungus composition with an activity of improving NK cell tumor killing and a preparation method thereof.
The invention comprises the following steps:
the invention relates to a macro fungus composition capable of improving NK cell tumor killing activity, which is characterized by comprising the following raw materials in parts by mass: 30-90 parts of lucid ganoderma, 30-90 parts of mushroom, 30-90 parts of poria cocos, 90-150 parts of needle mushroom, 90-150 parts of hericium erinaceus and 10-20 parts of cordyceps sinensis fermentation fungus powder.
Preferably, the feed comprises the following raw materials in parts by mass: 40-80 parts of lucid ganoderma, 40-80 parts of mushroom, 40-80 parts of poria cocos, 100-130 parts of needle mushroom, 100-140 parts of hericium erinaceus and 12-18 parts of cordyceps sinensis zymocyte powder
Preferably, the feed comprises the following raw materials in parts by mass: 50 parts of lucid ganoderma, 50 parts of mushroom, 50 parts of poria cocos, 110 parts of needle mushroom, 110 parts of hericium erinaceus and 15 parts of cordyceps sinensis fermentation fungus powder.
Preferably, the feed comprises the following raw materials in parts by mass: 30g of lucid ganoderma, 30g of mushroom, 30g of tuckahoe, 90g of needle mushroom, 90g of hericium erinaceus and 10 parts of cordyceps sinensis zymocyte powder.
The preparation method of the large-scale fungus composition with the NK cell tumor killing activity improving function is characterized by mixing the ganoderma lucidum, the mushroom, the poria cocos, the needle mushroom and the hericium erinaceus which are combined and matched with each other with water in an amount which is 8-30 times of the weight of the ganoderma lucidum, the mushroom, the poria cocos, the needle mushroom and the hericium erinaceus, decocting and extracting for 1-5 hours, extracting for 1-5 times, filtering, combining extracting solutions, drying the extracting solutions to form dry paste, crushing, combining with cordyceps sinensis fermentation fungus powder, and uniformly mixing to obtain the large-scale fungus composition.
Preferably, the temperature for drying the extract is preferably 35-50 ℃.
Preferably, the ganoderma lucidum, the shiitake mushroom, the poria cocos, the needle mushroom and the hericium erinaceus are crushed before being mixed with water; the particle size of the crushed raw material is 1-10 mm.
Preferably, the obtained macrofungi composition is mixed with auxiliary materials and prepared into mixture, tablets, capsules, granules, pills, syrup and soft extract oral preparations. .
The invention has the following innovations:
1. the macro fungus composition with the function of improving NK cell tumor killing activity is obtained through a large number of experiments, and is a multi-family macro fungus combination with definite specific curative effect, the formula comprises macro fungi of multiple families of Polyporaceae, Pleurotaceae, Tricholomataceae, Hericium Erinaceus and Hepialidae, specific polysaccharides and other functional components contained in fungi of various families, and the macro fungus composition is optimally matched and is novel in matching. The large fungi selected by the invention, namely ganoderma lucidum, mushroom, tuckahoe, flammulina velutipes, hericium erinaceus and cordyceps sinensis, contain polysaccharide components, but the main configuration and the composition of the polysaccharide in different fungi are different, for example, the main chain of the lentinan is a 1-3 beta-D-glucose sugar chain, the side chain is composed of a beta-D- (1-6) and a D-glucose polymer connected with the beta-D- (1-3) chain, the pachyman has fewer side chains, the side chains are connected with each other by the beta-D- (1-6), and different monosaccharide compositions and spatial configurations determine that the immune active sites of the functional polysaccharide are different in selective combination. The ganoderma lucidum triterpene, the lentinacin, the cordyceps adenosine, the needle mushroom functional protein, the poria triterpene, the hericium erinaceus diterpene and the like are respectively characteristic anti-tumor active ingredients and play roles in different action mechanisms, and different types of large-scale fungi are matched and combined in an optimal scheme to realize function optimization.
2. Experiments prove that the compatibility proportion of each component is the optimal proportion considering both the efficacy and the process.
3. The preparation process of the composition is reasonable and simple, can be used for industrial transformation, and ensures the practicability of the composition.
Detailed Description
The invention is further illustrated with reference to the following examples:
example 1
The invention is prepared by weighing the following medicinal components in parts by weight: 30g of lucid ganoderma, 30g of mushroom, 30g of poria cocos, 90g of needle mushroom, 90g of hericium erinaceus and 10g of cordyceps sinensis zymocyte powder.
The preparation process comprises the following steps: decocting Ganoderma, Lentinus Edodes, Poria, needle mushroom, and Hericium Erinaceus with 10 times of water for 3 times, each time for 1 hr, filtering, mixing extractive solutions, drying the extractive solution to obtain dry extract, pulverizing, mixing with Cordyceps fermented powder, mixing, and making into capsule.
Example 2
The invention is prepared by weighing the following medicinal components in parts by weight: 500g of lucid ganoderma, 500g of mushroom, 500g of poria cocos, 1100g of needle mushroom, 1100g of hericium erinaceus and 150g of cordyceps sinensis zymocyte powder.
The preparation process comprises the following steps: adding 10 times of water into Ganoderma, Lentinus Edodes, Poria, needle mushroom, and Hericium Erinaceus, decocting for 2 times (each time for 2 hr), filtering, mixing extractive solutions, drying the extractive solution to obtain dry extract, pulverizing, mixing with Cordyceps fermented powder at a certain ratio, mixing, and tabletting.
Example 3
The invention is prepared by weighing the following medicinal components in parts by weight: 1000g of lucid ganoderma, 1000g of mushroom, 1000g of tuckahoe, 2200g of needle mushroom, 2200g of hericium erinaceus and 300g of cordyceps sinensis zymocyte powder.
The preparation process comprises the following steps: adding 15 times of water into Ganoderma, Lentinus Edodes, Poria, needle mushroom, and Hericium Erinaceus, decocting for 3 times, each time for 1 hr, filtering, mixing extractive solutions, drying the extractive solution to obtain dry extract, pulverizing, mixing with Cordyceps fermented powder at a certain ratio, mixing, and granulating.
The pharmacological experiment result of the invention is as follows:
1. material
Animal(s) production
Male SD (S, prague-Dawley) rats, 6-8 weeks old, and 180-220 g in weight, were purchased from the university of Guilin laboratory animal center and housed in the Specific Pathogen Free (SPF) animal laboratory center.
Cells
The mouse lymphoma cell strain Yac-1 is purchased from Shanghai cell bank of Chinese academy of sciences.
Experimental sample
Composition group sample preparation: 30g of lucid ganoderma, 30g of shiitake mushroom, 30g of poria cocos, 90g of needle mushroom and 90g of hericium erinaceus are mixed with 10 times of water, decocted for 3 times, 1 hour each time, filtered, combined with extracting solutions, dried into dry paste, crushed, combined with 10g of cordyceps sinensis fermentation fungus powder, mixed uniformly, one tenth of mixed paste powder in weight is taken, 90ml of water is added, ultrasonic extraction is carried out, and the volume of the extracting solution is fixed to 100ml, so that the Chinese medicine preparation is obtained.
Preparing a ganoderma lucidum aqueous humor sample: adding 10 times of water into 30g of lucid ganoderma, decocting for 3 times, 1 hour each time, filtering, combining extracting solutions, and fixing the volume to 1000ml to obtain the traditional Chinese medicine.
Sample preparation for each negative group: removing corresponding negative medicinal materials, and preparing samples with the same composition by the other preparation methods.
Primary reagent
RPMI-1640 medium, penicillin-streptomycin (P-S) double antibody purchased from Gibco; fetal Calf Serum (FCS) was purchased from hangzhou sijiqing corporation; rat mononuclear cell isolate (density 1080g/L) was purchased from Tianjin, N.P.company; IV collagenase DNase I, trypan blue (trypanblue), RNase and PI dyes are purchased from Sigma; dynabeads FlowComp Flexi (110.60D); phycoerythrin (PE) labeled mouse anti-rat NKR-P1 monoclonal antibody (MR 6804, IgG 1), Fluorescein Isothiocyanate (FITC) labeled mouse anti-rat CD 3 monoclonal antibody (MR 5301, IgM) purchased from Caltag; cytotox96 Non-Radioactive cytoxicity Assay kit, purchased from Promega.
Major instrumentation superclean bench, suzhou clarification plant company; CO 22Thermostated cell culture chambers, Heraeus products; beckmancoulter desk refrigerated centrifuge and FACSCalibur flow cytometer, products of becton dickinson; wellscank MK3 microplate reader, product Labsystems Dragon.
2 method
2.1 isolation and identification of rat spleen NK cells
Rats were anesthetized by conventional intraperitoneal injection of 1% sodium pentobarbital. Laparotomy, and remove spleen. The spleen was ground and passed through a 100, 300 mesh stainless steel screen to remove debris and connective tissue. The obtained filtrate was gently added to the rat mononuclear lymphocyte isolate in equal proportions and centrifuged at 500 Xg for 25min at 37 ℃. Collecting the intermediate whiteMembranous layer, washed 2 times with complete RPMI-1640 medium and B cells removed by nylon trichome, resuspended to obtain rat spleen mononuclear cells (MNC). The obtained rat spleen MNC were purified using the Dynabeads FlowComp Flexi system. Based on specific markers (CD) on the surface of rat NK cells3-And NKR-P1+) Negative sorting was performed by DSB-X-conjugatedantai-CD 3, and positive sorting was performed by DSB-X-conjugatedantai-NKR-P1. Purified cells (rat NK cells defined as CD 3-NKR-P1) were analyzed by flow cytometry+Cell), purity of cell>90 percent. Cell viability and cell exclusion rate were determined by trypan blue staining>95％。
2.2 cell culture
The mouse lymphoma Yac-1 cells are cultured in complete RPMI-1640 medium at 37 ℃ with 5% CO2And (4) carrying out suspension culture in a constant-temperature cell culture box, carrying out passage for 1 time every 2-3 d, and using the culture medium when the cell growth activity is good. The purified rat spleen NK cells were suspension cultured in complete RPMI-1640 medium of pH 7.2, treated with 100. mu.L of each of the same doses of different drug solutions, and then incubated at 37 ℃ with 5% CO2And (5) performing suspension culture in a constant-temperature cell incubator, and collecting cells and supernatant for later use in 4h, 8 h, 12h and 16h respectively.
2.3 flow cytometry for detecting proliferation rate of NK cells in spleen of rat
Collected rat spleen NK cells were resuspended to 1X 10 in complete RPMI-1640 medium9cells/L, 0.5mL and 2mL of 75% frozen ethanol were added, overnight at 4 ℃. Centrifuging at 1200r/min for 5min at 2d, and discarding the supernatant. After 2 times of PBS washing, 50. mu. LRNase (5g/L) and 450. mu. LPI dye (50mg/L) were added, and the mixture was placed at 4 ℃ in the dark for 30min and analyzed on a computer. The experiment was repeated 3 times. Cell Proliferation Index (PI) ═ S + G2/M)/(G0/G1+ S + G2/M) × 100%.
2.4 detection of killing Activity of rat spleen NK cells by Lactate Dehydrogenase (LDH) method
NK cell sensitive logarithmic proliferation phase mouse lymphoma Yac-1 cells with complete RPMI-1640 medium heavy suspension to 1 x 108cells/L as target cells, rat spleen NK cells in complete RPMI-1640 mediumResuspending to 1X 109cells/L as effector cells. In a 96-well cell culture plate, 50 mul of effective target cell suspension is added into each well of an experimental group (the effective target ratio is 20: 1), a target cell natural release control group, a maximum release control group, an effector cell natural release control group and a blank control group are arranged at the same time, and each sample is provided with 3 multiple wells. Centrifuging at 250 Xg for 4min to make effective target cells contact sufficiently, standing at 37 deg.C and 5% CO2Incubate in the constant temperature cell incubator for 4 h. The control group with maximum release of target cells was added with 10 μ LLysisSolution per well 45min in advance. Centrifuge at 250 Xg for 4 min. 50 μ L of supernatant was pipetted per well and transferred to another 96-well cell culture plate. 50 μ L of Lreconstitustubstratemix was added to each well and incubated for 30min at room temperature in the dark. 50 μ L of stop solution was added to each well and the A value was read at 490nm in the microplate reader for 1 h. The experiment was repeated 3 times. NK killing activity (%) ═ (experimental group a value-effector cell spontaneous release group a value-target cell spontaneous release group a value)/(target cell maximum release group a value-target cell spontaneous release group a value) × 100%.
Results
1. Effect on proliferation of NK cells in rats
The detection results of the flow cytometry are shown in table 1, and it can be seen from table 1 that the composition has significant influence on rat NK cell proliferation, the proliferation rate is significantly improved compared with the blank group, the proliferation rate of the ganoderma lucidum water extract group and each deficient negative group is also significantly improved compared with the blank group, but the proliferation rate of the composition under the same culture time is similar to that of the needle mushroom negative group and the mushroom negative group, but is significantly improved compared with other groups.
TABLE 1 flow cytometer test results
| Group of | Proliferation rate of NK cells of each group after 12h of culture |
| Blank group | 10.08±0.91 |
| Composition set | 23.78±1.31 |
| Ganoderma lucidum water extract group | 18.33±1.69 |
| Ganoderma lucidum negative group | 19.48±1.19 |
| Negative group of shiitake mushrooms | 22.13±1.91 |
| Poria cocos negative group | 19.56±1.01 |
| Needle mushroom negative group | 23.01±1.81 |
| Hericium erinaceus negative group | 20.92±1.01 |
| Cordyceps sinensis negative group | 18.55±1.76 |
2. Effect on the rate of killing of Yac-1 cells by rat NK cells
The killing activity of rat spleen NK cells on Yac-1 cells is detected by applying a Lactate Dehydrogenase (LDH) method, and the results show that the composition has a significant influence on the killing activity of rat NK cells in a culture environment with the pH of 7.2, and the killing activity in the same culture time is significantly enhanced compared with that in other groups.
TABLE 2 killing Activity of rat spleen NK cells against Yac-1 cells
Comprehensive analysis of the above experimental results shows that the formula of the large fungus composition with the function of improving NK cell tumor killing activity is the optimal formula.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A macro fungus composition capable of improving NK cell tumor killing activity is characterized by comprising the following raw materials in parts by mass: 30g of lucid ganoderma, 30g of mushroom, 30g of poria cocos, 90g of needle mushroom, 90g of hericium erinaceus and 10g of cordyceps sinensis fermentation powder, wherein the preparation method of the large fungus composition capable of improving NK cell tumor killing activity comprises the following steps: 30g of lucid ganoderma, 30g of shiitake mushroom, 30g of poria cocos, 90g of needle mushroom and 90g of hericium erinaceus are mixed with 10 times of water, decocted for 3 times, 1 hour each time, filtered, combined with extracting solutions, dried into dry paste, crushed, combined with 10g of cordyceps sinensis fermentation fungus powder, mixed uniformly, one tenth of mixed paste powder in weight is taken, 90ml of water is added, ultrasonic extraction is carried out, and the volume of the extracting solution is fixed to 100ml, so that the Chinese medicine preparation is obtained.
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