Composition for improving adverse reaction of chemotherapy and preparation method, application and product thereof

Abstract

The invention discloses a composition for improving adverse effects of chemotherapy as well as a preparation method, application and a product of the composition, and particularly relates to the technical field of medicines. The composition comprises the following raw materials: tortoise, soft-shelled turtle, antler, sea cucumber, fructus amomi, pericarpium citri reticulatae and hawthorn. Alkaline protease and flavourzyme are adopted for enzymolysis, and the obtained composition can remarkably reduce the tumor volume of tumor-bearing animals, increase the bone marrow DNA content of chemotherapy animals and remarkably increase the number of white blood cells and the number of red blood cells in peripheral blood of the chemotherapy animals. The composition disclosed by the invention is used for preparing related products for improving the adverse effects of chemotherapy, and the prepared products are easy to absorb and have a remarkable improvement effect on the adverse effects of chemotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to a composition for improving adverse reactions of chemotherapy, a preparation method, application and product thereof. BACKGROUND

[0002] Bone marrow is the main hematopoietic organ of the human body, responsible for generating red blood cells, white blood cells, platelets and other key blood cells, and the precursor cells of these blood cells have the characteristics of rapid division. Chemotherapy drugs (especially cytotoxic drugs) kill cancer cells by interfering with cell DNA synthesis, division or protein synthesis, but cannot accurately distinguish between cancer cells and normal rapidly dividing cells (such as bone marrow hematopoietic stem cells, gastrointestinal mucosa cells, hair follicle cells, etc.). When bone marrow hematopoietic stem cells are damaged, their proliferation and differentiation capacity decreases, leading to a decrease in the production of various blood cells, which is called "myelosuppression".

[0003] The core manifestation of myelosuppression is a decrease in the number of various blood cells in peripheral blood, such as leukopenia, thrombocytopenia, and anemia. After chemotherapy, bone marrow hematopoietic function can gradually recover, but some patients may have long-term effects. Long-term bone marrow reserve function decreases, especially after multiple chemotherapy or high-dose chemotherapy, the number of hematopoietic stem cells decreases, and the recovery speed slows down; a small number of drugs can damage the DNA of hematopoietic stem cells, increasing the risk of long-term leukemia (such as acute myeloid leukemia). Therefore, it is necessary to improve myelosuppression caused by chemotherapy drugs.

[0004] Chinese patent CN103845723A discloses a composition for preventing and treating radiation damage or chemical drug treatment damage and a preparation method thereof. The composition comprises the following raw materials: 10-2000 parts, soybean oligopeptide 1-100 parts, and marine fish oligopeptide 1-100 parts. Marine fish oligopeptide is a mixture of oligopeptides with a molecular weight of less than 1000 Dalton produced by enzymatic hydrolysis, acid hydrolysis or microbial fermentation using marine fish skin, fish bone or fish meat as the main raw material. The composition also includes ingredients such as wolfberry extract. The prepared composition has the effects of preventing and treating tumors and improving adverse reactions of radiotherapy or chemical drug therapy, and enhancing immunity.

[0005] Chinese patent CN102327390A discloses a traditional Chinese medicine formula for preventing and treating chemotherapy side effects and a preparation method of its pills. The formula is composed of the following ingredients in a certain weight ratio: donkey-hide gelatin, ginseng, cassia bark, Szechuan lovage rhizome, prepared rehmannia root, poria cocos, atractylodes, milkvetch root, angelica, white peony root, dodder seed, wolfberry fruit, Szechuan chenpi, chenpi, ganoderma, cistanche, deer horn, musk, stalactite, baijiatian, aconite, sea cucumber, licorice, deerhorn glue, and yellow rice wine. After taking the traditional Chinese medicine formula, the total effective rate of patients with chemotherapy side effects reaches 87.9%.

[0006] However, the existing research on the composition for preventing and treating the chemotherapy damage is less, and is limited to the single effect of multiple components, without considering the mutual synergistic effect of each component, the effect of improving the adverse reactions of chemotherapy brought by the conventional functional components is limited, and the effect of reducing the tumor volume and improving the bone marrow suppression needs to be improved. SUMMARY

[0007] The embodiment of the present application provides a composition for improving adverse reactions of chemotherapy and a preparation method, application and product thereof, in the composition of the present application, soft-shelled turtle, Chinese paddlefish, deer antler, sea cucumber, amomum villosum, dried orange peel and hawthorn are used in cooperation, and alkaline protease and flavor protease are used for enzymolysis, the obtained composition can improve bone marrow suppression, significantly reduce the tumor volume of tumor-bearing animals, increase the DNA content of bone marrow of chemotherapy animals, and significantly increase the white blood cell count and red blood cell count in the peripheral blood of chemotherapy animals. The prepared product is easy to be absorbed, and the improvement effect on the adverse reactions of chemotherapy is significant.

[0008] To achieve the above-mentioned purposes of the application, the technical solutions of the present application are as follows: Firstly, the present application provides a composition for improving adverse reactions of chemotherapy, the raw materials of which include soft-shelled turtle, Chinese paddlefish, deer antler, sea cucumber, amomum villosum, dried orange peel and hawthorn.

[0009] Preferably, the raw materials of the composition include soft-shelled turtle 30-40 parts, Chinese paddlefish 30-40 parts, deer antler 4-8 parts, sea cucumber 30-40 parts, amomum villosum 0.5-2 parts, dried orange peel 1-3 parts and hawthorn 2-4 parts by weight.

[0010] Further preferably, the raw materials of the composition include soft-shelled turtle 35 parts, Chinese paddlefish 35 parts, deer antler 6 parts, sea cucumber 35 parts, amomum villosum 1 part, dried orange peel 2 parts and hawthorn 2 parts by weight.

[0011] Then, the present application provides a preparation method of the above-mentioned composition, which comprises the following steps: (1) soft-shelled turtle, Chinese paddlefish and deer antler are decocted with water, filtered to obtain decocting liquid 1, and the residue is heated with water and subjected to enzymolysis with alkaline protease and flavor protease respectively to obtain enzymolysis liquid A; (2) sea cucumber is heated with water and subjected to enzymolysis with alkaline protease and flavor protease respectively to obtain enzymolysis liquid B; (3) amomum villosum, dried orange peel and hawthorn are decocted with water to obtain decocting liquid 2, and the decocting liquid 1, the decocting liquid 2 and the enzymolysis liquid A and B are mixed to obtain the composition.

[0012] Preferably, before decocting with water in step (1), the raw materials need to be crushed.

[0013] Preferably, in step (1), the decocting time is 3-5 h, and more preferably 4 h.

[0014] Preferably, in step (1), the decocting is performed with water at a solid-to-liquid ratio of 1 g:8-15 mL; further preferably, 1 g:10 mL.

[0015] Preferably, in step (1), the heating is performed with water at a temperature of 60-65℃ and a solid-to-liquid ratio of 1 g:6-10 mL; further preferably, 60℃ and a solid-to-liquid ratio of 1 g:8 mL.

[0016] Preferably, in step (1), the enzymolysis is performed in two steps: in the first step, the alkaline protease is used for enzymolysis for 2-5 h; in the second step, the flavor protease is used for enzymolysis for 20-60 min.

[0017] Preferably, in step (1), the enzymolysis is performed in two steps: in the first step, the alkaline protease is used for enzymolysis for 3 h; in the second step, the flavor protease is used for enzymolysis for 30 min.

[0018] Further preferably, in step (1), the temperature in the first step of enzymolysis is 60-65℃, and the temperature in the second step of enzymolysis is 50-55℃.

[0019] More preferably, in step (1), the temperature in the first step of enzymolysis is 60℃, and the temperature in the second step of enzymolysis is 55℃.

[0020] Preferably, in step (1), the alkaline protease is added at an amount of 1%-3% of the net weight of the raw materials of turtle, soft-shelled turtle and deer antler; the net weight is the weight of fresh turtle, soft-shelled turtle and conventional deer antler decoction pieces.

[0021] Further preferably, in step (1), the alkaline protease is added at an amount of 2% of the net weight of the raw materials of turtle, soft-shelled turtle and deer antler.

[0022] Preferably, in step (1), the flavor protease is added at an amount of 0.3%-0.8% of the net weight of the raw materials of turtle, soft-shelled turtle and deer antler; the net weight is the weight of fresh turtle, soft-shelled turtle and conventional deer antler decoction pieces.

[0023] Further preferably, in step (1), the flavor protease is added at an amount of 0.5% of the net weight of the raw materials of turtle, soft-shelled turtle and deer antler.

[0024] Preferably, in step (1), after the enzymolysis, the enzyme needs to be inactivated at a temperature of 100-121℃.

[0025] Preferably, in step (1), the enzymolysis liquid A can be concentrated.

[0026] Preferably, in step (2), before the water is heated, the raw materials need to be crushed.

[0027] Preferably, in step (2), the heating with water is heating to 60-65℃, and the solid-liquid ratio is 1g:6-10mL; further preferably 60℃, and the solid-liquid ratio is 1g:8mL.

[0028] Preferably, in step (2), the enzymolysis is two times of enzymolysis: the first enzymolysis is enzymolysis for 2-5h using alkaline protease; the second enzymolysis is enzymolysis for 20-60min using flavor protease.

[0029] Further preferably, in step (2), the enzymolysis is two times of enzymolysis: the first enzymolysis is enzymolysis for 3h using alkaline protease; the second enzymolysis is enzymolysis for 30min using flavor protease.

[0030] Further preferably, in step (2), the temperature of the first enzymolysis is 60-65℃, and the temperature of the second enzymolysis is 50-55℃.

[0031] More preferably, in step (2), the temperature of the first enzymolysis is 60℃, and the temperature of the second enzymolysis is 55℃.

[0032] Preferably, in step (2), the addition amount of the alkaline protease is 1%-3% of the net weight of the raw material sea cucumber; the net weight is the weight of the sea cucumber after soaking.

[0033] Further preferably, in step (2), the addition amount of the alkaline protease is 2% of the net weight of the raw material sea cucumber.

[0034] Preferably, in step (2), the addition amount of the flavor protease is 0.3%-0.8% of the net weight of the raw material sea cucumber.

[0035] Further preferably, in step (2), the addition amount of the flavor protease is 0.5% of the net weight of the raw material sea cucumber.

[0036] Preferably, in step (2), after the enzymolysis, enzyme inactivation is needed, and the enzyme inactivation temperature is 100-121℃.

[0037] Preferably, in step (2), the enzymolysis liquid B can be concentrated.

[0038] Preferably, in step (3), the water decoction is 1-4 times, and each time is decocted for 0.5-2h.

[0039] Further preferably, in step (3), the water decoction is 2 times, and each time is decocted for 1h.

[0040] Preferably, in step (3), the water decoction is 1g:8-15mL; further preferably 1g:10mL.

[0041] Preferably, in step (3), the mixing is not limited to mixing after concentration or mixing after drying.

[0042] Furthermore, the present application provides the use of the above-mentioned composition in the preparation of a medicament for improving adverse reactions of chemotherapy.

[0043] Finally, the present application provides a product for improving adverse reactions of chemotherapy, comprising the component: the above-mentioned composition.

[0044] Preferably, the improvement of adverse reactions of chemotherapy includes: improving bone marrow suppression, reducing tumor volume of tumor-bearing animals, increasing bone marrow DNA content of chemotherapy animals, and increasing white blood cell count and red blood cell count in peripheral blood of chemotherapy animals.

[0045] Preferably, the product can further comprise: a sweetener, a flavoring agent.

[0046] Preferably, the product is a medicament.

[0047] Further preferably, the medicament can further comprise: a pharmaceutical carrier. The pharmaceutical carrier can be solid, liquid or gas. Examples of solid carriers include lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0048] In preparing the compositions in oral dosage form, any convenient pharmaceutical media can be employed. For example, water, ethanol, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, emulsifiers, lubricants, binders, disintegrating agents can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are preferred oral dosage units. It is also possible to coat tablets to mask the unpleasant taste of the drug.

[0049] The pharmaceutical compositions of the present application can be prepared as aqueous solutions or suspensions. Suitable surfactants, such as hydroxypropylcellulose, can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof with oils. Additionally, preservatives can be added to prevent the growth of microorganisms.

[0050] Further preferably, the dosage form of the medicament is not limited to pills, capsules, granules, oral liquids, powders, tablets, lozenges, ointments and the like, and suitable pharmaceutical carriers in the art can be selected for different dosage forms.

[0051] The beneficial effects of the present application are: 1. In the composition of the present invention, seven components, namely turtle, soft-shelled turtle, deer antler, sea cucumber, cardamom, tangerine peel and hawthorn, interact with each other. By using alkaline protease and flavor protease for enzymatic hydrolysis, the resulting composition can significantly reduce the tumor volume of tumor-bearing animals, increase the bone marrow DNA content of chemotherapy animals, and significantly increase the number of white blood cells and red blood cells in the peripheral blood of chemotherapy animals.

[0052] 2. In the composition of the present invention, all seven components are indispensable. They work together under specific weight ratios to bring about a better effect on improving the adverse reactions of chemotherapy compared with conventional active ingredients.

[0053] 3. In the composition of the present invention, turtle, soft-shelled turtle, and deer antler warm and tonify kidney yang, benefit essence and blood, and strengthen tendons and bones, serving as the principal ingredient; sea cucumber tonifies the kidney and benefits essence, nourishes blood and moistens dryness, and enhances the yin-nourishing and essence-consolidating effects of turtle, soft-shelled turtle, and deer antler, serving as the assistant ingredient; amomum villosum resolves dampness and promotes qi circulation, invigorates the spleen and stomach, and prevents the principal and assistant ingredients from being too greasy and hindering the stomach; tangerine peel regulates qi and strengthens the spleen, dries dampness and resolves phlegm, helps amomum villosum to resolve its sticky nature, unblocks the qi mechanism of the middle jiao, promotes the absorption of tonifying medicines, and achieves "tonification without stagnation," serving as the adjuvant ingredient; hawthorn eliminates food stagnation and promotes digestion, assists amomum villosum and tangerine peel in eliminating greasiness, and prevents abdominal distension and food stagnation, serving as the guiding ingredient; the seven ingredients work synergistically to promote and improve adverse reactions of chemotherapy. Detailed Implementation

[0054] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0055] In the specific embodiments of the present invention, the raw materials used are obtained through conventional commercial channels unless otherwise specified, and products from different manufacturers do not have a significant impact on the effect.

[0056] Basic Implementation A composition for improving adverse reactions to chemotherapy, comprising the following ingredients by weight: 30-40 parts turtle, 30-40 parts soft-shelled turtle, 4-8 parts deer antler, 30-40 parts sea cucumber, 0.5-2 parts cardamom, 1-3 parts dried tangerine peel, and 2-4 parts hawthorn.

[0057] The method for preparing the composition includes the following steps: (1) Turtle, turtle and deer horn are stirred and boiled with 8-15 times the amount of water for 3-5 h, filtered, and decoction liquid 1 is obtained; the residue is heated with 6-10 times the amount of water to 60-65℃, and 1%-3% of the net weight (turtle, turtle and deer horn) of alkaline protease is used for enzymolysis for 2-5 h; cooled to 50-55℃, and 0.3%-0.8% of the net weight (turtle, turtle and deer horn) of flavor protease is used for enzymolysis for 20-60 min, 100-121℃ for enzyme inactivation, and the enzymolysis liquid is concentrated to obtain enzymolysis liquid A; (2) The soaked sea cucumber is stirred and boiled with 6-10 times the amount of water to 60-65℃, and 1%-3% of the net weight (sea cucumber) of alkaline protease is used for enzymolysis for 2-5 h; cooled to 50-55℃, and 0.3%-0.8% of the net weight (sea cucumber) of flavor protease is used for enzymolysis for 20-60 min, 100-121℃ for enzyme inactivation, and the enzymolysis liquid is concentrated to obtain enzymolysis liquid B; (3) Take sand ginger, dried tangerine or orange peel and hawthorn, and boil with 8-15 times the amount of water for 1-4 times, each time for 0.5-2 h, to obtain decoction liquid 2; the decoction liquid is concentrated and mixed with decoction liquid 1, enzymolysis liquid A and enzymolysis liquid B to obtain the composition.

[0058] Example 1 A composition for improving the adverse reactions of chemotherapy, the raw materials are turtle 35 parts, turtle 35 parts, deer horn 6 parts, sea cucumber 35 parts, sand ginger 1 part, dried tangerine or orange peel 2 parts and hawthorn 2 parts by weight.

[0059] The preparation method of the composition is as follows: (1) Turtle, turtle and deer horn are stirred and boiled with 10 times the amount of water for 4 h, filtered, and decoction liquid 1 is obtained; the residue is heated with 8 times the amount of water to 60℃, and 2% of the net weight (turtle, turtle and deer horn) of alkaline protease is used for enzymolysis for 3 h; cooled to 55℃, and 0.5% of the net weight (turtle, turtle and deer horn) of flavor protease is used for enzymolysis for 30 min, 100℃ for enzyme inactivation, and the enzymolysis liquid is concentrated to obtain enzymolysis liquid A; (2) The soaked sea cucumber is stirred and boiled with 8 times the amount of water to 60℃, and 2% of the net weight (sea cucumber) of alkaline protease is used for enzymolysis for 3 h; cooled to 55℃, and 0.5% of the net weight (sea cucumber) of flavor protease is used for enzymolysis for 30 min, 100℃ for enzyme inactivation, and the enzymolysis liquid is concentrated to obtain enzymolysis liquid B; (3) Take sand ginger, dried tangerine or orange peel and hawthorn, and boil with 10 times the amount of water for 2 times, each time for 1 h, to obtain decoction liquid 2; and mix with decoction liquid 1, enzymolysis liquid A and enzymolysis liquid B to obtain the composition.

[0060] Example 2 A composition for improving adverse reactions of chemotherapy, raw materials according to weight parts are: turtle 30 parts, soft-shelled turtle 40 parts, deer antler 8 parts, sea cucumber 30 parts, amomum villosum 2 parts, dried tangerine or orange peel 3 parts and hawthorn 2 parts.

[0061] The preparation method of the composition is: (1) The turtle, soft-shelled turtle and deer antler are stirred and crushed, and are decocted with 8 times the amount of water for 5 h, and filtered to obtain decoction liquid 1; the filter residue is heated to 65℃ with 6 times the amount of water, and is subjected to enzymatic hydrolysis with 3% of alkaline protease based on the net weight of the turtle, soft-shelled turtle and deer antler for 2 h; the temperature is lowered to 50℃, and then 0.3% of flavor protease based on the net weight of the turtle, soft-shelled turtle and deer antler is used for enzymatic hydrolysis for 60 min, and the enzyme is inactivated at 100℃, and the enzymatic hydrolysis liquid is concentrated to obtain enzymatic hydrolysis liquid A; (2) The sea cucumber after soaking is stirred and crushed, and is heated to 65℃ with 6 times the amount of water, and is subjected to enzymatic hydrolysis with 3% of alkaline protease based on the net weight of the sea cucumber for 2 h; the temperature is lowered to 50℃, and then 0.3% of flavor protease based on the net weight of the sea cucumber is used for enzymatic hydrolysis for 60 min, and the enzyme is inactivated at 100℃, and the enzymatic hydrolysis liquid is concentrated to obtain enzymatic hydrolysis liquid B; (3) The amomum villosum, dried tangerine or orange peel and hawthorn are taken, and are decocted with 8 times the amount of water for 4 times, each time for 0.5 h to obtain decoction liquid 2; the decoction liquid 2 is mixed with the decoction liquid 1, the enzymatic hydrolysis liquid A and the enzymatic hydrolysis liquid B to obtain the composition.

[0062] Example 3 A composition for improving adverse reactions of chemotherapy, raw materials according to weight parts are: turtle 40 parts, soft-shelled turtle 30 parts, deer antler 4 parts, sea cucumber 40 parts, amomum villosum 0.5 parts, dried tangerine or orange peel 1 part and hawthorn 4 parts.

[0063] The preparation method of the composition is: (1) The turtle, soft-shelled turtle and deer antler are stirred and crushed, and are decocted with 15 times the amount of water for 3 h, and filtered to obtain decoction liquid 1; the filter residue is heated to 60℃ with 10 times the amount of water, and is subjected to enzymatic hydrolysis with 1% of alkaline protease based on the net weight of the turtle, soft-shelled turtle and deer antler for 5 h; the temperature is lowered to 55℃, and then 0.8% of flavor protease based on the net weight of the turtle, soft-shelled turtle and deer antler is used for enzymatic hydrolysis for 20 min, and the enzyme is inactivated at 100℃, and the enzymatic hydrolysis liquid is concentrated to obtain enzymatic hydrolysis liquid A; (2) The sea cucumber after soaking is stirred and crushed, and is heated to 60℃ with 10 times the amount of water, and is subjected to enzymatic hydrolysis with 1% of alkaline protease based on the net weight of the sea cucumber for 5 h; the temperature is lowered to 55℃, and then 0.8% of flavor protease based on the net weight of the sea cucumber is used for enzymatic hydrolysis for 20 min, and the enzyme is inactivated at 100℃, and the enzymatic hydrolysis liquid is concentrated to obtain enzymatic hydrolysis liquid B; (3) The amomum villosum, dried tangerine or orange peel and hawthorn are taken, and are decocted with 15 times the amount of water for 1 time, and are decocted for 2 h to obtain decoction liquid 2; the decoction liquid 2 is mixed with the decoction liquid 1, the enzymatic hydrolysis liquid A and the enzymatic hydrolysis liquid B to obtain the composition.

[0064] Comparative Example 1 Different from Example 1, the raw material turtle is replaced by tortoise shell (the shell of turtle), and the raw material carp is replaced by soft-shelled turtle shell.

[0065] Comparative Example 2 Different from Example 1, the raw material deer horn is replaced by donkey-hide gelatin (Dong'e donkey-hide gelatin).

[0066] Comparative Example 3 Different from Example 1, the raw material sea cucumber is replaced by American ginseng.

[0067] Comparative Example 4 Different from Example 1, hawthorn is replaced by roasted wheat germ.

[0068] Comparative Example 5 Different from Example 1, the weight ratio of each component in the composition is different.

[0069] A composition for improving adverse reactions of chemotherapy, the raw materials are as follows in terms of weight parts: turtle 50 parts, carp 50 parts, deer horn 6 parts, sea cucumber 5 parts, amomum villosum 0.5 part, dried tangerine or orange peel 0.5 part, and hawthorn 4 parts. The rest is the same as Example 1.

[0070] Evaluation of the effect of assisting in improving adverse reactions of chemotherapy 1 Material 1.1 Drugs and reagents: The composition prepared in each example and comparative example is turtle-turtle oral liquid; 5-fluorouracil (5-FU), purchased from Shanghai Xudong Haipu Pharmaceutical Co., Ltd.; Whey protein, Beijing Combit Sports Technology Co., Ltd.; DNA extraction kit, Beijing TransGene BioTech Co., Ltd.; Leukocyte counting hemolysis reagent: Nanchang Kewei Reagent Co., Ltd.; DMEM high-sugar culture medium, Gibico Company.

[0071] Preparation method of turtle-turtle oral liquid: (1) Take 35 g of turtle and 35 g of carp, wash and crush, add 10 times the amount of water to decoct for 4 hours, filter the decoction and concentrate; add 8 times the amount of water to the residue, heat to 60℃, add 2% of alkaline protease based on the net weight (turtle and carp) for enzymolysis for 3 hours, cool to 55℃, then add 0.5% of flavor protease for enzymolysis for 0.5 hours, heat to 100℃ to inactivate the enzyme, and concentrate to obtain an enzymolysis liquid; (2) Take 1 g of amomum villosum, 2 g of dried tangerine or orange peel, and 2 g of hawthorn, decoct with water for 2 times, each time for 1 hour, concentrate the decoction, combine with the enzymolysis liquid of (1), and sterilize to obtain turtle-turtle oral liquid.

[0072] 1.2 Cell strain: Human gastric cancer cell line BGC-823 was purchased from China Medical Science Institute Cell Bank, and was subcultured and preserved by the Pharmacology Group of Jiangxi College of Traditional Chinese Medicine.

[0073] 1.3 Experimental animals: Balb / C Nu / Nu nude mice, male, 18-22g, purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd., license number 0290346.

[0074] 1.4 Main instruments: NanoVue microprotein nucleic acid instrument, GE Company; AL204 electronic balance, Mettler-Toledo Instrument (Shanghai) Co., Ltd. XT-200I type blood cell analyzer, Japan Sysmex Corporation; Digital vernier caliper, Cangzhou Jilu Test Instrument Co., Ltd.

[0075] 2 Experimental method 2.1 Cell culture BGC-823 cells were routinely cultured in DMEM medium containing 10% fetal bovine serum, 100 U / L penicillin, and 100 μg / mL streptomycin at 37°C, 5% CO2, and saturated humidity. When the cells grew to the logarithmic growth phase, the cells were digested with trypsin solution.

[0076] 2.2 Establishment of human gastric cancer nude mouse xenograft model Cultured tumors: Male 4-6 week old BALB / C Nu / Nu nude mice were inoculated with BGC-823 human gastric cancer cells 1×10 7 / per mouse on the back intradermally, and when the tumor volume grew to 1 cm 3 around, part of the tumor mass was removed under sterile conditions.

[0077] Tumor-bearing animals: The tumor mass was cut into small pieces of about 1 mm 3 , and was inoculated into the backs of other experimental nude mice (male 4-6 week old BALB / C Nu / Nu nude mice) subcutaneously with a trocar to form subcutaneous xenografts, obtaining tumor-bearing animals.

[0078] 2.3 Grouping and administration The above tumor-bearing mice were randomly divided into 14 groups 48h after inoculation, 10 mice in each group, and the grouping is as follows: A, blank control group, intragastrically administered with distilled water.

[0079] B, 5-FU group, intraperitoneally injected with 5-FU at a dose of 30 mg / kg BW, once a week.

[0080] C, 5-FU + whey protein group, 5-FU dose and administration method are the same as group B; whey protein is administered by gavage, 8.125 g / Kg BW (purity is 80%, the actual dose is 6.5 g / Kg BW), once a day.

[0081] D, 5-FU + Guqie oral liquid group, 5-FU dose and administration method are the same as group B; Guqie oral liquid is administered by gavage, the dose is 45.5 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0082] E, 5-FU + Example 1 composition low dose group, 5-FU dose and administration method are the same as group B; Example 1 composition is administered by gavage, the dose is 11.25 g crude drug / kg BW, once a day (equivalent to 1 times of clinical dose).

[0083] F, 5-FU + Example 1 composition medium dose group, 5-FU dose and administration method are the same as group B; Example 1 composition is administered by gavage, the dose is 56.25 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0084] G, 5-FU + Example 1 composition high dose group, 5-FU dose and administration method are the same as group B; Example 1 composition is administered by gavage, the dose is 112.5 g crude drug / kg BW, once a day (equivalent to 10 times of clinical dose).

[0085] H, 5-FU + Example 2 composition medium dose group, 5-FU dose and administration method are the same as group B; Example 2 composition is administered by gavage, the dose is 56.25 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0086] I, 5-FU + Example 3 composition medium dose group, 5-FU dose and administration method are the same as group B; Example 3 composition is administered by gavage, the dose is 56.25 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0087] J, 5-FU + Comparative Example 1 composition medium dose group, 5-FU dose and administration method are the same as group B; Comparative Example 1 composition is administered by gavage, the dose is 56.25 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0088] K, 5-FU + Comparative Example 2 composition medium dose group, 5-FU dose and administration method are the same as group B; Comparative Example 2 composition is administered by gavage, the dose is 56.25 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0089] L, 5-FU + the composition of Comparative Example 3, 5-FU dose and administration method are the same as group B; the composition of Comparative Example 3 is administered by gavage, the dose is 56.25 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0090] M, 5-FU + the composition of Comparative Example 4, 5-FU dose and administration method are the same as group B; the composition of Comparative Example 4 is administered by gavage, the dose is 56.25 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0091] N, 5-FU + the composition of Comparative Example 5, 5-FU dose and administration method are the same as group B; the composition of Comparative Example 5 is administered by gavage, the dose is 56.25 g crude drug / kg BW, once a day (equivalent to 5 times of clinical dose).

[0092] The animals are administered from the day of grouping, 5-FU once a week, and the rest of the samples are administered by gavage once a day for 35 consecutive days. The tumor volume of tumor-bearing animals is measured twice a week, and blood is collected from the orbital venous plexus 24 hours after the last administration. The white blood cell count in the blood is determined by automatic blood cell analyzer. The animals are sacrificed, the tumor tissue is peeled off, weighed and the tumor inhibition rate is calculated.

[0093] 2.4 Determination of bone marrow DNA content The complete femur is peeled off, a certain amount of Hank's solution is sucked with a 1 mL syringe to flush out all the bone marrow cells in the femur. Finally, centrifugation is performed at 1000xg for 5 minutes, and the supernatant is discarded. After washing with a certain amount of PBS solution and centrifugation, 180 μL of PBS solution is added, and the cells are suspended. Then, the DNA in the bone marrow cells is extracted by using EasyPure™ DNA Extraction Kit (TransGene BioTech, Beijing), and finally the sample is accurately diluted to 300 μL with lysis buffer for determination of DNA content.

[0094] 2.5 Observation of bone marrow cell growth The method of bone marrow smear is used to detect the effect of the test sample on the proliferation of bone marrow cells. The sternum is taken, the base bone marrow liquid is mixed with the small calf serum at one end of the glass sheet, the conventional smear is made, and after natural drying, it is placed in methanol for 5-10 min for fixation. The fixed smear is placed in Giemsa staining solution for 10-15 min. Immediately wash with phosphate buffer, dry and read the film.

[0095] 3. Results 3.1 Effect of test sample on animal body weight The body weight results are shown in Table 1.

[0096] Table 1 Effect of test sample on the body weight of chemotherapy animals

[0097] Note: ## p<0.01 compared with the blank control group; p<0.05 compared with the 5-FU group; p<0.01 compared with the 5-FU group; & p<0.05 compared with the dose group in the 5-FU-Example 1 composition.

[0098] In each of the above groups, except the blank group, 5-FU was injected intraperitoneally once a week, with a dose of 30 mg / Kg BW.

[0099] Table 1 shows the effect of each test sample on the body weight of the chemotherapy animals. As can be seen from the table, 5-FU can significantly reduce the body weight of tumor-bearing animals, but the body weight of the test animals significantly increased after being given the turtle oral liquid comparative composition, with statistical significance (p<0.05); while the whey protein control group had no statistical significance. The test animals had a greater increase in body weight after being given the composition of the example, and there was a very significant difference compared with the 5-FU group, p<0.01.

[0100] The animals in the dose group of the composition of Example 1 had a significant increase in body weight, while the body weight of the animals in each of the comparative groups did not increase as much as the dose group in the composition of Example.

[0101] 3.2 Effect of test samples on the tumor volume of chemotherapy animals Table 2 shows the effect of each test sample on the tumor volume of tumor-bearing animals. As can be seen from Table 2, 5-FU can significantly reduce the tumor volume of tumor-bearing animals, and the tumor volume of animals in each of the test sample and 5-FU combined administration groups was significantly reduced compared with the blank control group, with statistical significance (p<0.05).

[0102] Table 2 Effect of test samples on the tumor volume of tumor-bearing animals

[0103] Note: ## p<0.01 compared with the blank group; p<0.05 compared with the 5-FU group; p<0.01 compared with the 5-FU group.

[0104] Each group of the above, except the blank control group, was injected intraperitoneally with 5-FU once a week, and the dosage was 30 mg / Kg BW. The dosage of the composition of the example group had a significant difference compared with the 5-FU group, the high dosage of the example 1 group had a very significant difference compared with the 5-FU group, and the tumor volume was significantly reduced; the rest of the groups had no obvious difference compared with the 5-FU group.

[0105] 3.3 Effect of the test sample on the bone marrow DNA content of the chemotherapy animal The mice in each group were sacrificed, and femurs were taken, 2 mL of Hanks solution was sucked by a syringe (6.5 needle), and all the bone marrow cells in the femur were flushed out, so that the cell suspension passed through a 4 needle syringe, and the cells were fully dispersed in the suspension. The OD value was measured at 260 nm by a UV spectrophotometer, and the bone marrow DNA content OD value of the mice in each group was shown in Table 3.

[0106] Table 3 Effect of the test sample on the bone marrow DNA content of the tumor-bearing animal

[0107] Note: ## Compared with the blank control group, p<0.01; Compared with the 5-FU group, p<0.05; Compared with the 5-FU group, p<0.01. Each group of the above, except the blank group, was injected intraperitoneally with 5-FU once a week, and the dosage was 30 mg / Kg BW.

[0108] Table 3 shows the effect of each test sample on the bone marrow DNA content of the chemotherapy animal. As shown in the table, 5-FU can significantly reduce the bone marrow DNA content of the tumor-bearing animal, and has an obvious bone marrow inhibition effect (p<0.01). After the combination treatment of the medium and high doses of the composition of the example 1 and 5-FU, the bone marrow DNA content was very significantly improved compared with the 5-FU single administration group (p<0.01), and after the combination treatment of the low dose of the example 1, the medium dose of the example 2 and the medium dose of the example 3 and 5-FU, the bone marrow DNA content was significantly improved compared with the 5-FU single administration group (p<0.05), which indicated that the composition of the example of the application can significantly improve the bone marrow inhibition effect caused by the chemotherapy drug. The bone marrow DNA content of the animals in the combination administration group of the rest of the test samples, the comparative examples and 5-FU had no significant difference compared with the 5-FU single administration group (p>0.05).

[0109] 3.4 Effect of the test sample on the number of peripheral blood cells of the chemotherapy animal Table 4 shows the effect of each test sample on the white blood cells and red blood cells in the peripheral blood of the chemotherapy animal.

[0110] Table 4 Effect of the test sample on the white blood cells and red blood cells in the peripheral blood of the chemotherapy animal

[0111] In Table 4, the data in each vertical column are compared: ## Compared with the blank control group, p<0.01; # Compared with the blank control group, p<0.05; Compared with the 5-FU group, p<0.05; Compared with the 5-FU group, p<0.01.

[0112] In each group above, except the blank group, 5-FU was injected intraperitoneally once a week, and the dose was 30 mg / Kg BW.

[0113] As can be seen from Table 4, 5-FU can significantly reduce the number of white blood cells in the peripheral blood of the chemotherapy animals, and the number of white blood cells of the animals in the 5-FU group (chemotherapy group) is significantly decreased compared with the blank control group, p<0.01. After the test animals are administered the composition of the embodiment in combination, the number of peripheral white blood cells is significantly increased, and the number of peripheral white blood cells of the other combined treatment groups does not change significantly compared with the 5-FU alone treatment group (p>0.05).

[0114] As can be seen from Table 4, the red blood cell count of the 5-FU group is significantly decreased, and obvious bone marrow suppression is produced (p<0.01). Except the high-dose group of the composition of Example 1, the red blood cell count of the animals in the other combined treatment groups does not change significantly. After the high-dose composition of Example 1 is administered in combination with 5-FU, the number of red blood cells is significantly higher than that of the 5-FU group, and the difference is statistically significant, p<0.05.

[0115] 4 Summary In the present application, a human gastric cancer nude mouse transplantation model is established by the method of subcutaneously transplanting human gastric cancer BGC-823 cells in nude mice, and the auxiliary effects of the test samples on the chemotherapy of gastric cancer are understood by administering 5-FU in combination with turtle and soft-shelled turtle oral liquid and the composition, respectively, and the auxiliary effect characteristics of the test samples are compared from the aspects of the efficacy and the side effects of the chemotherapy.

[0116] (1) In the present application, the tumor volume is used as an index for evaluating the efficacy of the combined treatment, and the influence of the above samples on the efficacy of the chemotherapy is evaluated, and the results show that the anti-tumor effect of the composition of the embodiment of the present application in combination with 5-FU is obvious.

[0117] (2) The present application takes animal weight, bone marrow DNA content, and peripheral blood cell count as indexes of the toxic side effects of chemotherapeutic drugs, and investigates the influence of the above-mentioned test sample on the toxic side effects of chemotherapeutic drugs. It is found that the composition of the present application can significantly increase the weight of the chemotherapy animals, the bone marrow DNA content, and the white blood cell and red blood cell counts in the peripheral blood, and has obvious chemotherapy protection effect. The whey protein control group does not show this effect, indicating that the chemotherapy protection effect of the composition of the present application is not a non-specific effect of protein.

[0118] (3) The present application also comparatively studies the chemotherapy auxiliary effect of the composition and the turtle and turtle oral liquid. It is found that the same dose of the composition and the turtle and turtle oral liquid can significantly increase the weight of the chemotherapy animals, but the effect of the composition is more significant, and the weight is increased to be significantly higher than that of the turtle and turtle oral liquid. In addition, the composition can also significantly increase the bone marrow DNA content, white blood cell and red blood cell counts of the chemotherapy animals, while the turtle and turtle oral liquid does not have this effect. The above results suggest that the chemotherapy protection effect of the composition of the present application is obviously stronger than that of the same dose of the turtle and turtle oral liquid.

[0119] (4) From the comparative results of the examples and the comparative examples, it can be seen that the composition of the present application has better effects of reducing the tumor volume of tumor-bearing animals, increasing the bone marrow DNA content of chemotherapy animals, and increasing the white blood cell and red blood cell counts in the peripheral blood of chemotherapy animals than the comparative examples, and the bone marrow suppression protection effect is significantly improved.

[0120] 5 Conclusion (1) The composition of the present application has obvious chemotherapy auxiliary effect, and has protection effect on the bone marrow suppression of chemotherapeutic drugs, while the turtle and turtle oral liquid does not show this effect.

[0121] (2) The composition of the present application has no obvious influence on the curative effect of chemotherapeutic drugs, and the turtle and turtle oral liquid also does not show chemotherapy synergistic effect.

[0122] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A composition for improving adverse reactions to chemotherapy, characterized in that, Raw materials include: Turtle, soft-shelled turtle, deer antler, sea cucumber, cardamom, dried tangerine peel, and hawthorn.

2. The composition according to claim 1, characterized in that, The ingredients, by weight, include: 30-40 parts turtle, 30-40 parts soft-shelled turtle, 4-8 parts deer antler, 30-40 parts sea cucumber, 0.5-2 parts cardamom, 1-3 parts dried tangerine peel, and 2-4 parts hawthorn.

3. The composition according to claim 2, characterized in that, The ingredients, by weight, include: 35 parts turtle, 35 parts soft-shelled turtle, 6 parts deer antler, 35 parts sea cucumber, 1 part cardamom, 2 parts dried tangerine peel, and 2 parts hawthorn.

4. A method for preparing the composition according to any one of claims 1-3, characterized in that, Including the following steps: (1) Boil turtle, soft-shelled turtle and deer antler in water, filter to obtain decoction 1; heat the residue in water and enzymatically hydrolyze it with alkaline protease and flavor protease respectively to obtain hydrolysate A. (2) The sea cucumber was heated with water and then enzymatically hydrolyzed with alkaline protease and flavor protease respectively to obtain hydrolysate B; (3) Take Amomum villosum, dried tangerine peel and hawthorn, decoct them in water to obtain decoction 2; mix decoction 1, decoction 2 and enzymatic hydrolysate A and enzymatic hydrolysate B to obtain a composition.

5. The preparation method according to claim 4, characterized in that, In step (1), the water is used for decoction, the decoction time is 3-5 hours, and the solid-liquid ratio is 1g:8-15mL. In step (1), water is used to heat the water to 60-65°C, and the solid-liquid ratio is 1g:6-10mL.

6. The preparation method according to claim 4, characterized in that, In step (1), the enzymatic hydrolysis is performed twice: the first enzymatic hydrolysis is performed using alkaline protease for 2-5 hours; the second enzymatic hydrolysis is performed using flavor protease for 20-60 minutes. The temperature for the first enzymatic hydrolysis is 60-65℃, and the temperature for the second enzymatic hydrolysis is 50-55℃. In step (1), the amount of alkaline protease added is 1%-3% of the net weight of the raw materials turtle, soft-shelled turtle and deer antler; the amount of flavor protease added is 0.3%-0.8% of the net weight of the raw materials turtle, soft-shelled turtle and deer antler.

7. The preparation method according to claim 4, characterized in that, In step (2), the water is used for heating to 60-65°C, and the solid-liquid ratio is 1g:6-10mL; In step (2), the enzymatic hydrolysis is performed twice: the first enzymatic hydrolysis is performed using alkaline protease for 2-5 hours; the second enzymatic hydrolysis is performed using flavor protease for 20-60 minutes; the temperature of the first enzymatic hydrolysis is 60-65℃, and the temperature of the second enzymatic hydrolysis is 50-55℃. In step (2), the amount of alkaline protease added is 1%-3% of the net weight of the raw sea cucumber; the amount of flavor protease added is 0.3%-0.8% of the net weight of the raw sea cucumber.

8. The preparation method according to claim 4, characterized in that, In step (3), the number of times the water is boiled is 1-4 times, and each boiling is 0.5-2 hours; the solid-liquid ratio of the boiling is 1g:8-15mL.

9. The use of the composition according to any one of claims 1-3 or the composition prepared by any one of claims 4-8 in the preparation of a drug for improving adverse reactions to chemotherapy.

10. A product that improves the adverse reactions of chemotherapy, characterized in that, Includes the following components: the composition according to any one of claims 1-3 or the composition prepared by the preparation method according to any one of claims 4-8.

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