A Grifola frondosa polysaccharide-protein complex with anti-tumor activity and its preparation

Abstract

The present invention relates to the field of biopharmaceutical technology, and specifically discloses a Grifola frondosa polysaccharide-protein complex with anti-tumor activity. The present invention also discloses a method for preparing the Grifola frondosa polysaccharide-protein complex with anti-tumor activity, including: extracting Grifola frondosa fruiting bodies by wet grinding, combining extracts, ultraviolet treatment for 1-2 minutes under stirring, the ultraviolet power is 13-17w, concentrating, and vacuum drying to obtain a crude product; purifying the crude product to obtain a purified material; adding dopamine hydrochloride and 2,2,6,6-tetramethylpiperidinoxide to an ethanol aqueous solution and stirring, adding 1,3,5-trimethylbenzene and continuing to stir, centrifuging, drying, washing, drying, and then mixing with the purified material and polyethylene glycol 400 and adding to chloroform, ultrasonication at room temperature, adding hydrogenated soybean lecithin, lecithin, and cholesterol to mix, rotary evaporation, adding phosphate buffer to hydrate, ultrasonication, and whole grains.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a Grifola frondosa polysaccharide-protein complex with anti-tumor activity and a preparation method and application thereof. Background Art

[0002] Malignant tumors remain a major disease that seriously threatens human life.

[0003] Chemotherapy is currently one of the most effective means of treating cancer. Surgery and radiotherapy are local treatments, which are only effective for tumors at the treatment site. They are difficult to effectively treat potential metastatic lesions and cancers that have already undergone clinical metastasis. Chemotherapy is a systemic treatment method, and chemotherapy drugs will be distributed throughout the body's organs and tissues through blood circulation.

[0004] At present, chemotherapy is widely used in the clinical treatment of cancer patients, but it will inevitably damage normal human cells while killing cancer cells, resulting in a variety of adverse reactions, affecting the survival time and quality of life of cancer patients. At present, enhancing the efficacy of chemotherapy drugs, improving the quality of life of patients, and prolonging the survival time of cancer patients has always been a hot topic in the field of anti-tumor drug research.

[0005] Plant polysaccharides are a kind of ingredients extracted and purified from natural plants. They have become a research hotspot in the anti-tumor field due to their wide biological activity, good compatibility with the human body, and few toxic and side effects.

[0006] Grifola frondosa is a large fungus that can be used as both food and medicine. Grifola frondosa polysaccharide is a substance with a high content in Grifola frondosa and is also one of its main functional ingredients. Grifola frondosa polysaccharide can currently be extracted from Grifola frondosa fruiting bodies, mycelium and fermentation broth. Domestic and foreign studies have confirmed that Grifola frondosa polysaccharide has anti-cancer effects in vitro, but there has been no report showing that Grifola frondosa polysaccharide has the effect of enhancing the efficacy of chemotherapy. Summary of the invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a Grifola frondosa polysaccharide-protein complex with anti-tumor activity and a preparation method and application thereof.

[0008] A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity comprises the following steps:

[0009] S1. Add deionized water to the fruiting bodies of Grifola frondosa, grind for 10-20 min, add to deionized water, stir for 10-30 min, cool to -20--40°C, keep warm for 1-2 h, raise to 30-40°C, ultrasonically treat for 1-2 h, let stand for 10-30 min, cool to -50--60°C, keep warm for 1-2 h, return to room temperature, and filter to obtain extract a;

[0010] S2, add water to the filter residue obtained in S1, ultrasonically treat for 1-2h, and filter to obtain extract b; combine extract a and extract b, treat with ultraviolet light for 1-2min under stirring, the ultraviolet power is 13-17w, concentrate, add ethanol and stir for 10-30min, let stand for 5-10h, filter, collect the precipitate, and vacuum dry to obtain a crude product;

[0011] S3, add ethanol to the crude product and stir for 10-20 min, let stand for 3-5 h, collect the precipitate, vacuum dry, add ethanol and stir for 10-20 min, let stand for 5-10 h, collect the precipitate, vacuum dry, dialyze with a 3000Da dialysis bag for 70-80 h, and vacuum dry to obtain a purified material;

[0012] S4. Add dopamine hydrochloride and 2,2,6,6-tetramethylpiperidinoxide to an ethanol aqueous solution and stir for 1-2 hours. Add 1,3,5-trimethylbenzene and continue stirring for 5-15 minutes. Adjust the pH value of the system to 10-12 and continue stirring for 1-2 hours. Centrifuge, remove the supernatant, freeze-dry, wash 1-3 times, freeze-dry, mix with the purified material and polyethylene glycol 400, add to chloroform, ultrasonicate at room temperature for 10-30 minutes, add hydrogenated soybean lecithin, lecithin and cholesterol, mix, rotary evaporate, add phosphate buffer and ultrasonicate, and use a microporous filter membrane to granulate.

[0013] Preferably, in S1, the grinding pressure is 0.05-0.15 MPa, and the grinding speed is 1000-5000 r / min.

[0014] Preferably, in S1, the ultrasonic frequency is 5-12 kHz.

[0015] Preferably, in S1, the Grifola frondosa fruiting bodies and deionized water are ground in a mass ratio of 10-20:5-10.

[0016] Preferably, in S2, the ultrasonic frequency is 8-15 kHz.

[0017] Preferably, in S2, the treatment distance of the UV lamp in the UV box is 20±0.5 cm.

[0018] Preferably, in S2, the volume is concentrated to 30-50% of the original volume.

[0019] Preferably, in S4, the mass ratio of dopamine hydrochloride, 2,2,6,6-tetramethylpiperidinoxide, 1,3,5-trimethylbenzene, purified material, polyethylene glycol 400, hydrogenated soybean lecithin, lecithin, and cholesterol is 10-15: 5-10: 1-10: 10-20: 30-50: 1-2: 5-10: 5-10.

[0020] Preferably, in S4, the mass fraction of the ethanol aqueous solution is 20-40%, and the pH value of the phosphate buffer is 7.1-7.5.

[0021] Preferably, in S4, phosphate buffer is added and ultrasonic treatment is performed for 4-6 min, and the ultrasonic power is 260-320w.

[0022] Preferably, in S4, the pore size of the microporous filter membrane is 0.45 μm and 0.22 μm.

[0023] A Grifola frondosa polysaccharide-protein complex with anti-tumor activity is prepared by adopting the method for preparing the Grifola frondosa polysaccharide-protein complex with anti-tumor activity.

[0024] The application of the above-mentioned Grifola frondosa polysaccharide-protein complex with anti-tumor activity in the preparation of drugs for treating gastric cancer and / or laryngeal cancer.

[0025] Beneficial effects:

[0026] The present invention can not only effectively inhibit tumor growth and enhance the body's immune response, but also significantly enhance the chemotherapy effect.

[0027] The invention uses the fruiting bodies of Grifola frondosa as raw materials, and sequentially grinds and low-temperature freezes the products. The polysaccharides and glycoproteins in the fruiting bodies of Grifola frondosa have good dissolution effects, and the chemotherapeutic activity thereof is extremely high after ultraviolet treatment. The product not only enhances the immune response of the tumor body, but also effectively increases the number of total T lymphocytes and CD8+T cells in peripheral blood; meanwhile, the product can effectively inhibit the proliferation of gastric cancer cells and laryngeal cancer cells, and has good anti-tumor activity on both gastric cancer cells and laryngeal cancer cells, and is particularly effective in inhibiting the activity on gastric cancer cells, with less toxic and side effects.

[0028] The obtained Grifola frondosa polysaccharide-protein complex has good anti-tumor activity, can effectively improve drug efficacy, enhance its tumor inhibition effect, and reduce toxic side effects through targeted regulation of the distribution of active ingredients, and shows good stability when stored at 4°C for 12 months, has no adverse reactions when used for a long time, can enhance the efficacy of chemotherapy drugs, has clinical application prospects, and has the prospect of being developed into an anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the changes in particle size, leakage rate, oxidation index and stability coefficient of the Grifola frondosa polysaccharide-protein complex obtained in Example 5 within 12 months of storage.

[0030] Figure 2 This is a comparison chart of the inhibition of human gastric cancer cell line SGC-7901 and human laryngeal cancer cell line Hep-2 in vitro by the Grifola frondosa polysaccharide-protein complex obtained in Example 5 and Comparative Examples 1-2.

[0031] Figure 3 This is a comparison chart of the tumor inhibition rates of the Grifola frondosa polysaccharide-protein complex obtained in Example 5 and Comparative Examples 1-2 against human gastric cancer cell line SGC-7901 and human laryngeal cancer cell line Hep-2 in vivo.

[0032] Figure 4 This is a comparison chart of CD3 / CD45 and CD8 / CD4 in mice implanted with the human gastric cancer cell line SGC-7901 by intragastric administration of the Grifola frondosa polysaccharide-protein complex obtained in Example 5 and Comparative Examples 1-2.

[0033] Figure 5 It is a comparison chart of the tumor inhibition rates of the etoposide group, Example 5 group, Comparative Example 1 group, and Comparative Example 2 group on human gastric cancer cell line SGC-7901 and human laryngeal cancer cell line HEp-2 in vivo. DETAILED DESCRIPTION

[0034] The present invention will be further explained below in conjunction with specific embodiments.

[0035] Example 1

[0036] A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity comprises the following steps:

[0037] S1. Add 5 kg of deionized water to 10 kg of Grifola frondosa fruiting bodies and grind for 10 min at a grinding speed of 1000 r / min and a grinding pressure of 0.05 MPa. Add to 30 kg of deionized water and stir at a speed of 1000 r / min for 10 min. Reduce to -20 ° C., keep warm for 1 h, increase to 30 ° C., perform ultrasonic treatment for 1 h at an ultrasonic frequency of 5 kHz, let stand for 10 min, reduce to -50 ° C., keep warm for 1 h, return to room temperature, and filter to obtain an extract. a ;

[0038] S2. Add 50 kg of water to the residue obtained in S1 and perform ultrasonic treatment for 1 hour at an ultrasonic frequency of 8 kHz. Filter and obtain an extract. b ; Combined extracts a With extract b , sent into a UV box for treatment for 1 min under stirring, wherein the UV lamp power in the UV box is 13w, the distance is 20±0.5cm, concentrated to 30% of the original volume, added 100kg of ethanol and stirred for 10min, allowed to stand for 5h, filtered, the precipitate was collected, and vacuum dried to obtain a crude product;

[0039] S3, add 100 kg of ethanol to the crude product and stir for 10 min, let stand for 3 h, collect the precipitate, vacuum dry, add 100 kg of ethanol and stir for 10 min, let stand for 5 h, collect the precipitate, vacuum dry, send it into a 3000 Da dialysis bag and dialyze it with running water for 70 h, and vacuum dry it to obtain a purified material;

[0040] S4, add 10kg dopamine hydrochloride and 5kg 2,2,6,6-tetramethylpiperidinoxide to 50kg 20% ​​ethanol aqueous solution, stir at 1000r / min for 1h, add 1kg 1,3,5-trimethylbenzene, continue stirring for 5 minutes, adjust the pH value of the system to 10-12, continue stirring for 1 hour, centrifuge, remove the supernatant, freeze-dry, wash once with deionized water and anhydrous ethanol alternately, freeze-dry, then mix with 10kg purified material and 30kg polyethylene glycol 400 and dissolve in an appropriate amount of chloroform, ultrasonicate at room temperature for 10 minutes, the ultrasonic frequency is 5kHz, add 1kg hydrogenated soybean lecithin, 5kg lecithin, 5kg cholesterol and mix, rotary evaporate, add pH=7.1-7.5 phosphate buffer solution for hydration for 30 minutes, ultrasonicate for 4 minutes, the ultrasonic power is 260w; pass through 0.45μm and 0.22μm microporous filter membranes in turn for whole particles, place in a cillin bottle, fill with nitrogen and seal for storage.

[0041] Example 2

[0042] A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity comprises the following steps:

[0043] S1. Add 10 kg of deionized water to 20 kg of Grifola frondosa fruiting bodies and grind for 20 min at a grinding speed of 5000 r / min and a grinding pressure of 0.15 MPa. Add to 50 kg of deionized water and stir at a speed of 2000 r / min for 30 min. Reduce to -40 ° C., keep warm for 2 h, increase to 40 ° C., ultrasonically treat for 2 h at an ultrasonic frequency of 12 kHz, stand for 30 min, reduce to -60 ° C., keep warm for 2 h, return to room temperature, and filter to obtain an extract. a ;

[0044] S2. Add 100 kg of water to the residue obtained in S1 and perform ultrasonic treatment for 2 h at an ultrasonic frequency of 15 kHz. Filter and obtain an extract. b ; Combined extracts a With extract b , sent into a UV box for treatment for 2 minutes under stirring, wherein the UV lamp power in the UV box is 17w, the distance is 20±0.5cm, concentrated to 50% of the original volume, added 300kg of ethanol and stirred for 30min, allowed to stand for 10h, filtered, the precipitate was collected, and vacuum dried to obtain a crude product;

[0045] S3, add 200 kg of ethanol to the crude product and stir for 20 min, let stand for 5 h, collect the precipitate, vacuum dry, add 200 kg of ethanol and stir for 20 min, let stand for 10 h, collect the precipitate, vacuum dry, send it into a 3000 Da dialysis bag and dialyze it with running water for 80 h, and vacuum dry it to obtain a purified material;

[0046] S4, add 15kg dopamine hydrochloride and 10kg 2,2,6,6-tetramethylpiperidinoxide to 150kg 40% ethanol aqueous solution, stir at 2000r / min for 2h, add 10kg 1,3,5-trimethylbenzene, continue stirring for 15 minutes, adjust the pH value of the system to 10-12, continue stirring for 2 hours, centrifuge, remove the supernatant, freeze-dry, wash with deionized water and anhydrous ethanol alternately for 3 times, freeze-dry, then mix with 20kg purified material and 50kg polyethylene glycol 400 and dissolve in an appropriate amount of chloroform, ultrasonicate at room temperature for 30 minutes, the ultrasonic frequency is 12kHz, add 2kg hydrogenated soybean lecithin, 10kg lecithin, 10kg cholesterol and mix, rotary evaporate, add pH=7.1-7.5 phosphate buffer solution for hydration for 30 minutes, ultrasonicate for 6 minutes, the ultrasonic power is 320w; pass through 0.45μm and 0.22μm microporous filter membranes in turn for whole particles, place in a cillin bottle, fill with nitrogen and seal for storage.

[0047] Example 3

[0048] A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity comprises the following steps:

[0049] S1. Add 9 kg of deionized water to 12 kg of Grifola frondosa fruiting bodies and grind for 12 min at a grinding speed of 4000 r / min and a grinding pressure of 0.08 MPa. Add to 45 kg of deionized water and stir at a speed of 1300 r / min for 25 min. Reduce to -25 ° C., keep warm for 100 min, raise to 33 ° C., ultrasonically treat for 100 min at an ultrasonic frequency of 7 kHz, stand for 25 min, reduce to -53 ° C., keep warm for 100 min, return to room temperature, and filter to obtain an extract. a ;

[0050] S2. Add 70 kg of water to the residue obtained in S1 and perform ultrasonic treatment for 100 min at an ultrasonic frequency of 9 kHz. Filter and obtain an extract. b ; Combined extracts a With extract b , sent into a UV box for treatment for 1.5 min under stirring, wherein the UV lamp power in the UV box is 16w, the distance is 20±0.5cm, concentrated to 35% of the original volume, added 240kg of ethanol and stirred for 15min, allowed to stand for 9h, filtered, the precipitate was collected, and vacuum dried to obtain a crude product;

[0051] S3, add 120kg ethanol to the crude product and stir for 18min, let stand for 3.5h, collect the precipitate, vacuum dry, add 180kg ethanol and stir for 12min, let stand for 9h, collect the precipitate, vacuum dry, send it into a 3000Da dialysis bag and dialyze it with running water for 71h, and vacuum dry it to obtain the purified material;

[0052] S4, add 14kg dopamine hydrochloride and 7kg 2,2,6,6-tetramethylpiperidinoxide to 120kg 25% ethanol aqueous solution, stir at 1800r / min for 80min, add 7kg 1,3,5-trimethylbenzene, continue stirring for 8 minutes, adjust the pH value of the system to 10-12, continue stirring for 100 minutes, centrifuge, remove the supernatant, freeze-dry, wash twice with deionized water and anhydrous ethanol alternately, freeze-dry, then mix with 12kg purified material and 45kg polyethylene glycol 400 and dissolve in an appropriate amount of chloroform, ultrasonicate at room temperature for 15 minutes, the ultrasonic frequency is 9kHz, add 1.3kg hydrogenated soybean lecithin, 9kg lecithin, 7kg cholesterol and mix, rotary evaporate, add pH=7.1-7.5 phosphate buffer solution for hydration for 30 minutes, ultrasonicate for 4.5 minutes, the ultrasonic power is 310w; pass through 0.45μm and 0.22μm microporous filter membranes in turn for whole particles, place in a cillin bottle, fill with nitrogen and seal for storage.

[0053] Example 4

[0054] A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity comprises the following steps:

[0055] S1. Add 7kg of deionized water to 18kg of Grifola frondosa fruiting bodies and grind for 18min at a grinding speed of 2000r / min and a grinding pressure of 0.12MPa. Add to 35kg of deionized water and stir at a speed of 1700r / min for 15min. Reduce to -35°C, keep warm for 80min, raise to 37°C, ultrasonically treat for 80min at an ultrasonic frequency of 11kHz, stand for 15min, reduce to -57°C, keep warm for 80min, return to room temperature, and filter to obtain an extract. a ;

[0056] S2, add 90kg water to the residue obtained in S1, ultrasonically treat for 80min, the ultrasonic frequency is 12kHz, and filter to obtain the extract b ; Combined extracts a With extract b , sent into a UV box for treatment for 1.5 min under stirring, wherein the UV lamp power in the UV box is 14w, the distance is 20±0.5cm, concentrated to 45% of the original volume, added 150kg of ethanol and stirred for 25min, allowed to stand for 7h, filtered, the precipitate was collected, and vacuum dried to obtain a crude product;

[0057] S3, add 180kg ethanol to the crude product and stir for 12min, let stand for 4.5h, collect the precipitate, vacuum dry, add 120kg ethanol and stir for 18min, let stand for 7h, collect the precipitate, vacuum dry, send it into a 3000Da dialysis bag and dialyze it with running water for 73h, and vacuum dry it to obtain a purified material;

[0058] S4, add 12kg dopamine hydrochloride and 9kg 2,2,6,6-tetramethylpiperidinoxide to 80kg 35% ethanol aqueous solution, stir at 1200r / min for 100min, add 3kg 1,3,5-trimethylbenzene, continue stirring for 12 minutes, adjust the pH value of the system to 10-12, continue stirring for 80 minutes, centrifuge, remove the supernatant, freeze-dry, wash twice with deionized water and anhydrous ethanol alternately, freeze-dry, then mix with 18kg purified material and 35kg polyethylene glycol 400 and dissolve in an appropriate amount of chloroform, ultrasonicate at room temperature for 25 minutes, the ultrasonic frequency is 6kHz, add 1.7kg hydrogenated soybean lecithin, 7kg lecithin, 9kg cholesterol and mix, rotary evaporate, add pH=7.1-7.5 phosphate buffer solution for hydration for 30 minutes, ultrasonicate for 5.5 minutes, the ultrasonic power is 280w; pass through 0.45μm and 0.22μm microporous filter membranes in turn for whole particles, place in a cillin bottle, fill with nitrogen and seal for storage.

[0059] Example 5

[0060] A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity comprises the following steps:

[0061] S1. Add 8 kg of deionized water to 15 kg of Grifola frondosa fruiting bodies and grind for 15 min at a grinding speed of 3000 r / min and a grinding pressure of 0.1 MPa. Add to 40 kg of deionized water and stir at a speed of 1500 r / min for 20 min. Reduce to -30 ° C., keep warm for 90 min, raise to 35 ° C., ultrasonically treat for 90 min at an ultrasonic frequency of 9 kHz, stand for 20 min, reduce to -55 ° C., keep warm for 90 min, return to room temperature, and filter to obtain an extract. a ;

[0062] S2. Add 80 kg of water to the residue obtained in S1 and perform ultrasonic treatment for 90 min at an ultrasonic frequency of 10.5 kHz. Filter and obtain an extract. b ; Combined extracts a With extract b , sent into a UV box for treatment for 1.5 min under stirring, wherein the UV lamp power in the UV box is 15w, the distance is 20±0.5cm, concentrated to 40% of the original volume, added 200kg ethanol and stirred for 20min, allowed to stand for 8h, filtered, the precipitate was collected, and vacuum dried to obtain a crude product;

[0063] S3, add 150kg ethanol to the crude product and stir for 15min, let stand for 4h, collect the precipitate, vacuum dry, add 150kg ethanol and stir for 15min, let stand for 8h, collect the precipitate, vacuum dry, send it into a 3000Da dialysis bag and dialyze it with running water for 72h, and vacuum dry it to obtain a purified material;

[0064] S4, add 13kg dopamine hydrochloride and 8kg 2,2,6,6-tetramethylpiperidinoxide to 100kg 30% ethanol aqueous solution, stir at 1500r / min for 90min, add 5kg 1,3,5-trimethylbenzene, continue stirring for 10 minutes, adjust the pH value of the system to 10-12, continue stirring for 90 minutes, centrifuge, remove the supernatant, freeze-dry, wash twice with deionized water and anhydrous ethanol alternately, freeze-dry, then mix with 15kg purified material and 40kg polyethylene glycol 400 and dissolve in an appropriate amount of chloroform, ultrasonicate at room temperature for 20 minutes, the ultrasonic frequency is 7.5kHz, add 1.5kg hydrogenated soybean lecithin, 8kg lecithin, 8kg cholesterol and mix, rotary evaporate, add pH=7.1-7.5 phosphate buffer solution for hydration for 30 minutes, ultrasonicate for 5 minutes, the ultrasonic power is 300w; pass through 0.45μm and 0.22μm microporous filter membranes in turn for whole particles, place in a cillin bottle, fill with nitrogen and seal for storage.

[0065] The stability of the glycine frondosa polysaccharide-protein complex obtained in this example was tested for a long term with reference to the Chinese Pharmacopoeia 2010. The complex was placed in a sealed container at 4±2°C (with an internal environment filled with N 2 ), stored under light-shielding conditions for 12 months. Samples were taken at the 0th, 3rd, 6th, 9th and 12th months, and the appearance, particle size, leakage rate, oxidation index and stability coefficient were evaluated.

[0066] The appearance of the Grifola frondosa polysaccharide-protein complex obtained in this example was always in a state of no flocculation, no precipitation, and uniform dispersion within 12 months. Figure 1 As shown, its various indicators were stable within 12 months, proving that its properties are stable and has long-term stability.

[0067] Comparative Example 1

[0068] A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity comprises the following steps:

[0069] S1. Add 8 kg of deionized water to 15 kg of Grifola frondosa fruiting bodies and grind for 15 min at a grinding speed of 3000 r / min and a grinding pressure of 0.1 MPa. Add to 40 kg of deionized water and stir at a speed of 1500 r / min for 20 min. Reduce to -30 ° C., keep warm for 90 min, raise to 35 ° C., ultrasonically treat for 90 min at an ultrasonic frequency of 9 kHz, stand for 20 min, reduce to -55 ° C., keep warm for 90 min, return to room temperature, and filter to obtain an extract. a ;

[0070] S2. Add 80 kg of water to the residue obtained in S1 and perform ultrasonic treatment for 90 min at an ultrasonic frequency of 10.5 kHz. Filter and obtain an extract. b ; Combined extracts a With extract b , concentrated to 40% of the original volume, added 200 kg of ethanol and stirred for 20 min, allowed to stand for 8 h, filtered, collected the precipitate, and vacuum dried to obtain a crude product;

[0071] S3, add 150kg ethanol to the crude product and stir for 15min, let stand for 4h, collect the precipitate, vacuum dry, add 150kg ethanol and stir for 15min, let stand for 8h, collect the precipitate, vacuum dry, send it into a 3000Da dialysis bag and dialyze it with running water for 72h, and vacuum dry it to obtain a purified material;

[0072] S4, add 13kg dopamine hydrochloride and 8kg 2,2,6,6-tetramethylpiperidinoxide to 100kg 30% ethanol aqueous solution, stir at 1500r / min for 90min, add 5kg 1,3,5-trimethylbenzene, continue stirring for 10 minutes, adjust the pH value of the system to 10-12, continue stirring for 90 minutes, centrifuge, remove the supernatant, freeze-dry, wash twice with deionized water and anhydrous ethanol alternately, freeze-dry, then mix with 15kg purified material and 40kg polyethylene glycol 400 and dissolve in an appropriate amount of chloroform, ultrasonicate at room temperature for 20 minutes, the ultrasonic frequency is 7.5kHz, add 1.5kg hydrogenated soybean lecithin, 8kg lecithin, 8kg cholesterol and mix, rotary evaporate, add pH=7.1-7.5 phosphate buffer solution for hydration for 30 minutes, ultrasonicate for 5 minutes, the ultrasonic power is 300w; pass through 0.45μm and 0.22μm microporous filter membranes in turn for whole particles, place in a cillin bottle, fill with nitrogen and seal for storage.

[0073] Comparative Example 2

[0074] A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity comprises the following steps:

[0075] S1. Add 8 kg of deionized water to 15 kg of Grifola frondosa fruiting bodies and grind for 15 min at a grinding speed of 3000 r / min and a grinding pressure of 0.1 MPa. Add to 40 kg of deionized water and stir at a speed of 1500 r / min for 20 min. Reduce to -30 ° C., keep warm for 90 min, raise to 35 ° C., ultrasonically treat for 90 min at an ultrasonic frequency of 9 kHz, stand for 20 min, reduce to -55 ° C., keep warm for 90 min, return to room temperature, and filter to obtain an extract. a ;

[0076] S2. Add 80 kg of water to the residue obtained in S1 and perform ultrasonic treatment for 90 min at an ultrasonic frequency of 10.5 kHz. Filter and obtain an extract. b ; Combined extracts a With extract b , sent into a UV box for treatment for 1.5 min under stirring, wherein the UV lamp power in the UV box is 15w, the distance is 20±0.5cm, concentrated to 40% of the original volume, added 200kg ethanol and stirred for 20min, allowed to stand for 8h, filtered, the precipitate was collected, and vacuum dried to obtain a crude product;

[0077] S3, add 150kg ethanol to the crude product and stir for 15min, let stand for 4h, collect the precipitate, vacuum dry, add 150kg ethanol and stir for 15min, let stand for 8h, collect the precipitate, vacuum dry, send it into a 3000Da dialysis bag and dialyze it with running water for 72h, and vacuum dry it to obtain a purified material;

[0078] S4. Mix 15kg of purified material and 40kg of polyethylene glycol 400 and dissolve them in an appropriate amount of chloroform. Ultrasonicate at room temperature for 20 minutes at an ultrasonic frequency of 7.5kHz. Add 1.5kg of hydrogenated soybean lecithin, 8kg of lecithin and 8kg of cholesterol, mix them, evaporate them, add pH=7.1-7.5 phosphate buffer to hydrate for 30 minutes, and ultrasonicate for 5 minutes at an ultrasonic power of 300w. Pass through 0.45μm and 0.22μm microporous filter membranes in turn to size the particles, place them in a vial, fill them with nitrogen and seal them for storage.

[0079] The polysaccharide content (measured by phenol-sulfuric acid method with glucose as standard) and protein content (measured by Coomassie brilliant blue method with calf serum albumin as standard) of the purified materials obtained in Example 5 and Comparative Examples 1-2 were measured.

[0080]

[0081] It is confirmed that the purified material obtained by extracting Grifola frondosa fruiting bodies using the method of the present invention is a polysaccharide-protein complex or a polysaccharide-glycoprotein complex. There is no significant difference in polysaccharide content and protein content between the purified materials obtained in Example 5 and Comparative Examples 1-2 (P<0.05).

[0082] Human gastric cancer cell line SGC-7901 and human laryngeal cancer cell line Hep-2 were used as test subjects, and the Grifola frondosa polysaccharide-protein complex obtained in Example 5 and Comparative Examples 1-2 was used for in vitro testing, as follows:

[0083] Human gastric cancer cell line SGC-7901 and human laryngeal cancer cell line Hep-2 were routinely cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator with saturated humidity; the cells were digested and passaged using 0.25% trypsin.

[0084] The cells in the logarithmic growth phase were digested with 0.25% trypsin, collected, counted, and the cell density was adjusted to 4 × 10 using RMPI1640 culture medium containing 10% fetal bovine serum. 4 / mL, add the cell suspension into a 96-well plate, add 100 μL into each well. At the same time, add only RMPI1640 culture medium containing 10% fetal bovine serum into 3 wells as blank wells for zero adjustment. Place the 96-well plate with cells at 37°C and 5% CO 2 , and cultured overnight in a cell culture incubator with saturated humidity. After the cells adhered to the wall, the polysaccharide-protein complexes of Grifola frondosa in Example 5 and Comparative Examples 1-2 were added to each group, respectively, so that the concentration of the purified material in each group reached 50 mg / L, and a negative control well was set. The culture was continued for 48 hours, and then 10 μL of 5g / L MTT was added to each well and cultured for 4 hours. The culture solution was discarded, and 150 μL of DMSO was added to each well and shaken for 10 minutes until the crystals were completely dissolved. The A490 light absorption value was detected by a microplate reader.

[0085] Tumor cell growth inhibition rate = (1-OD value of drug group ÷ OD value of control group) × 100%

[0086] like Figure 2 As shown, the inhibitory effect of the Grifola frondosa polysaccharide-protein complex obtained in Example 5 on tumor cell growth in vitro was better than that of Comparative Example 1 (P < 0.05), but worse than that of Comparative Example 2 (P < 0.05).

[0087] The applicant believes that this is because Comparative Example 2 only uses hydrogenated soybean lecithin, lecithin, and cholesterol to liposome-coat the purified material, causing it to rupture rapidly during in vitro culture, resulting in the purified material directly entering the tumor cell line culture environment, making the tumor cell growth inhibition effect of the Grifola frondosa polysaccharide-protein complex obtained in Comparative Example 2 the best; while Comparative Example 1 and Example 5 both use substances such as dopamine hydrochloride for reaction to further enhance its sustained release effect, resulting in unsatisfactory in vitro tumor cell growth inhibition effects of the two groups.

[0088] The human gastric cancer cell line SGC-7901 and the human laryngeal cancer cell line Hep-2 were used as test subjects for in vivo mouse experiments. They were cultured to the logarithmic growth phase. BALB / C female mice (6-8 weeks old, weighing 20±2g) were selected and fed adaptively for one week to prepare for the experiment. The above-mentioned tumor cell lines in the logarithmic growth phase were digested and counted, and the cell density was adjusted to 5×10 5 cells / mL, implanted subcutaneously in mice. After confirming the successful modeling, the mice were randomly divided into a model group and three intervention groups (Example 5 group, Comparative Example 1 group, Comparative Example 2 group), with five replicates in each group, three mice in each replicate, and a negative control group was set up at the same time.

[0089] On the second day after grouping, the intervention group was given oral gavage, the dose of the intervention group was 100 mg / kg / day, and the model group was given the same volume of normal saline, and the drug was administered for 8 consecutive weeks. On the second day after the last administration, the mice were killed by cervical dislocation, the tumor was completely removed and weighed, and the tumor inhibition rate was calculated; and the blood of the mice implanted with the human gastric cancer cell line SGC-7901 was collected by sodium heparin tube, and the CD3 / CD45 and CD8 / CD4 were calculated by flow cytometry within 48 hours.

[0090] Tumor inhibition rate (%) = (average tumor weight of model group - average tumor weight of intervention group) / average tumor weight of model group × 100%

[0091] like Figure 3 As shown, both intervention groups could inhibit tumor growth in mice, and the inhibitory effect of Example 5 group was better than that of Comparative Example 1 group and Comparative Example 2 group (P < 0.05).

[0092] Since cancer patients have reduced immunity, lymphocyte proliferation and activation are inhibited. Cytotoxic T cells (CD8+), as the main effector immune cells, are suppressed in tumors, so enhancing and activating CD8+ T cells is the key to tumor immunotherapy.

[0093] like Figure 4 As shown, compared with the negative control group, the total T cells (CD3+) of the model group mice were significantly reduced, and the ratio of CD8+ / CD4+ decreased, while the CD3+T cells of the intervention group increased, and the ratio of CD8+ / CD4+ increased, proving that the intervention with the Grifola frondosa polysaccharide-protein complex obtained in Example 5 and Comparative Examples 1-2 can improve the immune function of tumor mice. Among the intervention groups, the Example 5 group was superior to the Comparative Examples 1-2 group (P<0.05).

[0094] The etoposide synergistic experiment was continued according to the above in vivo mouse experimental steps. After confirming the successful modeling, the mice were randomly assigned to a model group, an etoposide group, and three intervention groups (Example 5 group, Comparative Example 1 group, Comparative Example 2 group), with five replicates for each group of mice, three mice in each replicate, and a negative control group was set up at the same time.

[0095] On the second day after grouping, the etoposide group and the intervention group were given drugs by oral gavage. The etoposide group was given etoposide 20 mg / kg by gavage for 1 week, and then continued after 3 weeks of stopping; the intervention group was given Grifola frondosa polysaccharide-protein complex 100 mg / kg by gavage every day, and etoposide 20 mg / kg by gavage for 1 week, and then continued after 3 weeks of stopping. The model group was given the same volume of normal saline.

[0096] The experiment lasted for 8 weeks. The mice were killed by cervical dislocation, the tumors were completely removed and weighed, and the tumor inhibition rate was calculated.

[0097] like Figure 5 As shown, both the etoposide group and the intervention group can inhibit tumor growth in mice, and the intervention group adopts a combined drug administration method, so that its inhibitory effect is better than the etoposide group (P < 0.05). Among the intervention groups, the inhibitory effect of Example 5 group is better than that of Comparative Example 1 group and Comparative Example 2 group (P < 0.05).

[0098] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a Grifola frondosa polysaccharide-protein complex with anti-tumor activity, characterized in that: The steps include: S1. Add deionized water to the fruiting bodies of Grifola frondosa, grind for 10-20 min, add to deionized water, stir for 10-30 min, cool to -20--40°C, keep warm for 1-2 h, raise to 30-40°C, ultrasonically treat for 1-2 h, let stand for 10-30 min, cool to -50--60°C, keep warm for 1-2 h, return to room temperature, and filter to obtain extract a; S2, add water to the filter residue obtained in S1, ultrasonically treat for 1-2h, and filter to obtain extract b; combine extract a and extract b, and treat them in a UV box under stirring for 1-2min, the UV power is 13-17w, and the treatment distance of the UV lamp is 20±0.5cm; concentrate, add ethanol and stir for 10-30min, let stand for 5-10h, filter, collect the precipitate, and vacuum dry to obtain a crude product; S3, add ethanol to the crude product and stir for 10-20 min, let stand for 3-5 h, collect the precipitate, vacuum dry, add ethanol and stir for 10-20 min, let stand for 5-10 h, collect the precipitate, vacuum dry, dialyze with a 3000Da dialysis bag for 70-80 h, and vacuum dry to obtain a purified material; S4. Add dopamine hydrochloride and 2,2,6,6-tetramethylpiperidinoxide to an ethanol aqueous solution and stir for 1-2 hours. Add 1,3,5-trimethylbenzene and continue stirring for 5-15 minutes. Adjust the pH value of the system to 10-12 and continue stirring for 1-2 hours. Centrifuge, remove the supernatant, freeze-dry, wash, freeze-dry, mix with purified material and polyethylene glycol 400, add to chloroform, ultrasonicate at room temperature for 10-30 minutes, add hydrogenated soybean lecithin, lecithin and cholesterol, mix, rotary evaporate, add phosphate buffer to hydrate, ultrasonicate, and use a microporous filter membrane to granulate.

2. The method for preparing the Grifola frondosa polysaccharide-protein complex with anti-tumor activity according to claim 1, characterized in that: In S1, the grinding pressure is 0.05-0.15 MPa, and the grinding speed is 1000-5000 r / min.

3. The method for preparing the Grifola frondosa polysaccharide-protein complex with anti-tumor activity according to claim 1, characterized in that: In S1, the ultrasound frequency is 5-12kHz.

4. The method for preparing the Grifola frondosa polysaccharide-protein complex with anti-tumor activity according to claim 1, characterized in that: In S2, the ultrasound frequency is 8-15kHz.

5. The method for preparing the Grifola frondosa polysaccharide-protein complex with anti-tumor activity according to claim 1, characterized in that: S2 was concentrated to 30-50% of the original volume.

6. The method for preparing the Grifola frondosa polysaccharide-protein complex with anti-tumor activity according to claim 1, characterized in that: In S4, the mass ratio of dopamine hydrochloride, 2,2,6,6-tetramethylpiperidinoxide, 1,3,5-trimethylbenzene, purified material, polyethylene glycol 400, hydrogenated soybean lecithin, lecithin, and cholesterol is 10-15: 5-10: 1-10: 10-20: 30-50: 1-2: 5-10: 5-10.

7. The method for preparing the Grifola frondosa polysaccharide-protein complex with anti-tumor activity according to claim 1, characterized in that: In S4, the mass fraction of the ethanol aqueous solution is 20-40%, and the pH value of the phosphate buffer is 7.1-7.

5.

8. A Grifola frondosa polysaccharide-protein complex with anti-tumor activity, characterized in that: The polysaccharide-protein complex with anti-tumor activity is prepared by the method for preparing the polysaccharide-protein complex with anti-tumor activity according to any one of claims 1 to 7.

9. Use of the Grifola frondosa polysaccharide-protein complex with anti-tumor activity as claimed in claim 8 in the preparation of a drug for treating gastric cancer and / or laryngeal cancer.

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