Compositions for ameliorating adverse effects of chemotherapy and methods of making, using and products thereof

Abstract

The application discloses a composition for improving adverse reactions of chemotherapy and a preparation method, application and product thereof, and particularly relates to the technical field of drugs.The composition of the application comprises raw materials including turtles, soft-shelled turtles, deer antlers, sea cucumbers, sand ginger, dried orange peel and hawthorn; the composition is obtained by enzymolysis of alkaline protease and flavor protease, can 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 composition of the application is used in the preparation of related products for improving adverse reactions of chemotherapy, and the prepared products are easy to be absorbed and have a remarkable improvement effect on adverse reactions 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 compositions for preventing and treating chemotherapy damage are less studied, and are limited to the individual effects of multiple components, without considering the mutual synergistic effect of the components, and the effect of the conventional functional components on improving the adverse reactions of chemotherapy is limited, and the effect of reducing the tumor volume and improving bone marrow suppression needs to be improved. SUMMARY

[0007] The application provides a composition for improving adverse reactions of chemotherapy and a preparation method, application and product thereof. In the composition, soft-shelled turtles, Chinese paddlefish, deer antlers, sea cucumbers, amomum villosum, dried orange peel and hawthorn are used in cooperation, and are subjected to enzymolysis by using alkaline protease and flavor protease, so that 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 has a significant effect on improving adverse reactions of chemotherapy.

[0008] To achieve the above-mentioned application purposes, the technical solutions of the application are as follows.

[0009] Firstly, the application provides a composition for improving adverse reactions of chemotherapy, and raw materials thereof include soft-shelled turtles, Chinese paddlefish, deer antlers, sea cucumbers, amomum villosum, dried orange peel and hawthorn.

[0010] Preferably, the raw materials of the composition include, in terms of weight parts, 30-40 parts of soft-shelled turtles, 30-40 parts of Chinese paddlefish, 4-8 parts of deer antlers, 30-40 parts of sea cucumbers, 0.5-2 parts of amomum villosum, 1-3 parts of dried orange peel and 2-4 parts of hawthorn.

[0011] Further preferably, the raw materials of the composition include, in terms of weight parts, 35 parts of soft-shelled turtles, 35 parts of Chinese paddlefish, 6 parts of deer antlers, 35 parts of sea cucumbers, 1 part of amomum villosum, 2 parts of dried orange peel and 2 parts of hawthorn.

[0012] Then, the application provides a preparation method of the above-mentioned composition, including the following steps.

[0013] (1) soft-shelled turtles, Chinese paddlefish and deer antlers are decocted with water, filtered to obtain decocted liquid 1, and the residue is heated with water and subjected to enzymolysis by using alkaline protease and flavor protease respectively to obtain enzymolysis liquid A;

[0014] (2) sea cucumbers are heated with water and subjected to enzymolysis by using alkaline protease and flavor protease respectively to obtain enzymolysis liquid B;

[0015] (3) amomum villosum, dried orange peel and hawthorn are decocted with water to obtain decocted liquid 2, and the decocted liquid 1, the decocted liquid 2 and the enzymolysis liquid A and the enzymolysis liquid B are mixed to obtain the composition.

[0016] Preferably, before decocting with water in step (1), the raw materials need to be crushed.

[0017] Preferably, in step (1), the decoction is performed with water for 3-5 hours; further preferably for 4 hours.

[0018] Preferably, in step (1), the decoction is performed with water at a solid-liquid ratio of 1 g:8-15 mL; further preferably at a solid-liquid ratio of 1 g:10 mL.

[0019] Preferably, in step (1), the heating is performed with water to a temperature of 60-65°C at a solid-liquid ratio of 1 g:6-10 mL; further preferably at a temperature of 60°C at a solid-liquid ratio of 1 g:8 mL.

[0020] 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 hours; in the second step, the flavor protease is used for enzymolysis for 20-60 minutes.

[0021] Preferably, in step (1), the enzymolysis is performed in two steps: in the first step, the alkaline protease is used for enzymolysis for 3 hours; in the second step, the flavor protease is used for enzymolysis for 30 minutes.

[0022] Further preferably, in step (1), the temperature in the first step of enzymolysis is 60-65°C, and the temperature in the second step of enzymolysis is 50-55°C.

[0023] More preferably, in step (1), the temperature in the first step of enzymolysis is 60°C, and the temperature in the second step of enzymolysis is 55°C.

[0024] Preferably, in step (1), the alkaline protease is added in 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.

[0025] Further preferably, in step (1), the alkaline protease is added in an amount of 2% of the net weight of the raw materials of turtle, soft-shelled turtle and deer antler.

[0026] Preferably, in step (1), the flavor protease is added in 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.

[0027] Further preferably, in step (1), the flavor protease is added in an amount of 0.5% of the net weight of the raw materials of turtle, soft-shelled turtle and deer antler.

[0028] Preferably, in step (1), after the enzymolysis, the enzyme is inactivated at a temperature of 100-121°C.

[0029] Preferably, in step (1), the enzymolysis liquid A can be concentrated.

[0030] Preferably, in step (2), before heating with water, the raw material needs to be crushed.

[0031] Preferably, in step (2), the heating with water is heated to 60-65℃, and the solid-liquid ratio is 1g:6-10mL; further preferably, 60℃, and the solid-liquid ratio is 1g:8mL.

[0032] Preferably, in step (2), the enzymolysis is twice: the first enzymolysis is carried out for 2-5h using alkaline protease; the second enzymolysis is carried out for 20-60min using flavor protease.

[0033] Further preferably, in step (2), the enzymolysis is twice: the first enzymolysis is carried out for 3h using alkaline protease; the second enzymolysis is carried out for 30min using flavor protease.

[0034] Further preferably, in step (2), the temperature of the first enzymolysis is 60-65℃, and the temperature of the second enzymolysis is 50-55℃.

[0035] More preferably, in step (2), the temperature of the first enzymolysis is 60℃, and the temperature of the second enzymolysis is 55℃.

[0036] Preferably, in step (2), the addition amount of alkaline protease is 1%-3% of the net weight of raw material sea cucumber; the net weight is the weight of the sea cucumber after soaking.

[0037] Further preferably, in step (2), the addition amount of alkaline protease is 2% of the net weight of raw material sea cucumber.

[0038] Preferably, in step (2), the addition amount of flavor protease is 0.3%-0.8% of the net weight of raw material sea cucumber.

[0039] Further preferably, in step (2), the addition amount of flavor protease is 0.5% of the net weight of raw material sea cucumber.

[0040] Preferably, in step (2), after enzymolysis, the enzymolysis needs to be inactivated, and the inactivation temperature is 100-121℃.

[0041] Preferably, in step (2), the enzymolysis liquid B can be concentrated.

[0042] Preferably, in step (3), the number of water decoction is 1-4 times, and each decoction is 0.5-2h.

[0043] Further preferably, in step (3), the number of water decoction is 2 times, and each decoction is 1h.

[0044] Preferably, in step (3), the decocting is carried out with water at a solid-to-liquid ratio of 1 g:8-15 mL; more preferably, 1 g:10 mL.

[0045] Preferably, in step (3), the mixing is not limited to mixing after concentration or mixing after drying.

[0046] Further, the present application provides use of the above-mentioned composition in the preparation of a medicament for improving adverse reactions of chemotherapy.

[0047] Finally, the present application provides a product for improving adverse reactions of chemotherapy, comprising the following component: the above-mentioned composition.

[0048] Preferably, the improvement of adverse reactions of chemotherapy includes: improvement of myelosuppression, reduction of tumor volume in tumor-bearing animals, increase of bone marrow DNA content in chemotherapy animals, and increase of white blood cell count and red blood cell count in peripheral blood of chemotherapy animals.

[0049] Preferably, the product can further comprise: a sweetener, a flavoring agent.

[0050] Preferably, the product is a medicament.

[0051] More 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.

[0052] 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, solutions and emulsions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, emulsifying agents, 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. Optionally coated tablets can be employed.

[0053] 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 included to prevent the growth of microorganisms.

[0054] More 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.

[0055] The beneficial effects of the present application are:

[0056] 1、The composition of the present application, turtle, crab, deer antler, sea cucumber, sand ginger, dried orange peel and hawthorn 7 ingredients interact with each other, by using alkaline protease and flavor protease for enzymolysis, the composition obtained can significantly reduce the tumor volume of tumor-bearing animals, increase the bone marrow DNA content of chemotherapy animals, significantly increase the white blood cell count and red blood cell count in the peripheral blood of chemotherapy animals.

[0057] 2、The composition of the present application, 7 ingredients are indispensable, under the condition of specific weight ratio, mutual cooperation, bring the effect of improving the adverse reactions of chemotherapy better than the conventional efficacy ingredients.

[0058] 3、The composition of the present application, turtle, crab, deer antler, kidney yang, benefit blood, strong bones, is the monarch drug; sea cucumber, kidney and essence, nourishing blood and dryness, enhancing the yin and essence of turtle, crab and deer antler, is the ministerial medicine; sand ginger, dampness, spleen and stomach, prevent monarch drug and ministerial medicine from being greasy and stomach; dried orange peel, regulating qi and spleen, dampness and phlegm, assisting sand ginger to dissolve the sticky nature, dredging the middle jiao qi machine, promoting the absorption of tonifying medicine, realizing "tonifying without stagnation", is the auxiliary medicine; hawthorn, food accumulation, auxiliary sand ginger, dried orange peel, digestion and prevention of abdominal distension and food accumulation, is the service medicine; Seven ingredients synergistically improve the adverse reactions of chemotherapy. DETAILED DESCRIPTION

[0059] In order to make the technical means, creative characteristics, purposes and effects realized by the present application easy to understand, the following specific embodiments are further illustrated, but the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application. In the following examples, if not otherwise specified, the operation method used is the conventional operation method, and the equipment used is the conventional equipment, and the equipment materials used in each example are the same.

[0060] In the specific embodiments of the present application, the raw materials used are obtained through conventional commercial channels, and the products of different manufacturers do not have a significant effect on the effect.

[0061] Basic embodiment

[0062] A composition for improving the adverse reactions of chemotherapy, the raw materials according to weight parts include: turtle 30-40 parts, crab 30-40 parts, deer antler 4-8 parts, sea cucumber 30-40 parts, sand ginger 0.5-2 parts, dried orange peel 1-3 parts and hawthorn 2-4 parts.

[0063] The preparation method of the composition comprises the following steps:

[0064] (1) Turtle, turtle and deer antler are stirred and boiled with 8-15 times the amount of water for 3-5 h, filtered, to obtain decoction liquid 1; 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 antler) 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 antler) 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;

[0065] (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;

[0066] (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.

[0067] Example 1

[0068] A composition for improving the adverse reactions of chemotherapy, the raw materials are turtle 35 parts, turtle 35 parts, deer antler 6 parts, sea cucumber 35 parts, sand ginger 1 part, dried tangerine or orange peel 2 parts and hawthorn 2 parts by weight.

[0069] The preparation method of the composition is as follows:

[0070] (1) Turtle, turtle and deer antler are stirred and boiled with 10 times the amount of water for 4 h, filtered, to obtain decoction liquid 1; the residue is heated with 8 times the amount of water to 60℃, and 2% of the net weight (turtle, turtle and deer antler) of alkaline protease is used for enzymolysis for 3 h; cooled to 55℃, and 0.5% of the net weight (turtle, turtle and deer antler) 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;

[0071] (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;

[0072] (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.

[0073] Example 2

[0074] A composition for improving adverse reactions of chemotherapy, raw materials according to weight parts are: turtle 30 parts, soft-shelled turtle 40 parts, deer horn 8 parts, sea cucumber 30 parts, amomum villosum 2 parts, dried tangerine or orange peel 3 parts and hawthorn 2 parts.

[0075] The preparation method of the composition is:

[0076] (1) The turtle, soft-shelled turtle and deer horn are stirred and crushed, and are decocted with 8 times the amount of water for 5 h, 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 horn for 2 h; cooled to 50℃, and is subjected to enzymatic hydrolysis with 0.3% of flavor protease based on the net weight of the turtle, soft-shelled turtle and deer horn for 60 min, 100℃ for enzyme inactivation, and the enzymatic hydrolysis liquid is concentrated to obtain enzymatic hydrolysis liquid A;

[0077] (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; cooled to 50℃, and is subjected to enzymatic hydrolysis with 0.3% of flavor protease based on the net weight of the sea cucumber for 60 min, 100℃ for enzyme inactivation, and the enzymatic hydrolysis liquid is concentrated to obtain enzymatic hydrolysis liquid B;

[0078] (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; mixed with the decoction liquid 1, enzymatic hydrolysis liquid A and enzymatic hydrolysis liquid B to obtain the composition.

[0079] Example 3

[0080] A composition for improving adverse reactions of chemotherapy, raw materials according to weight parts are: turtle 40 parts, soft-shelled turtle 30 parts, deer horn 4 parts, sea cucumber 40 parts, amomum villosum 0.5 parts, dried tangerine or orange peel 1 part and hawthorn 4 parts.

[0081] The preparation method of the composition is:

[0082] (1) The turtle, soft-shelled turtle and deer horn are stirred and crushed, and are decocted with 15 times the amount of water for 3 h, 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 horn for 5 h; cooled to 55℃, and is subjected to enzymatic hydrolysis with 0.8% of flavor protease based on the net weight of the turtle, soft-shelled turtle and deer horn for 20 min, 100℃ for enzyme inactivation, and the enzymatic hydrolysis liquid is concentrated to obtain enzymatic hydrolysis liquid A;

[0083] (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; cooled to 55℃, and is subjected to enzymatic hydrolysis with 0.8% of flavor protease based on the net weight of the sea cucumber for 20 min, 100℃ for enzyme inactivation, and the enzymatic hydrolysis liquid is concentrated to obtain enzymatic hydrolysis liquid B;

[0084] (3) Taking amomum villosum, dried tangerine or orange peel and hawthorn, decocting once with 15 times of water for 2 hours to obtain decocting liquid 2; mixing with decocting liquid 1, enzyme hydrolysis liquid A and enzyme hydrolysis liquid B to obtain the composition.

[0085] Comparative Example 1

[0086] Different from Example 1, the raw material of soft-shelled turtle is replaced by tortoise shell (the shell of soft-shelled turtle), and the raw material of paddlefish is replaced by turtle shell.

[0087] Comparative Example 2

[0088] Different from Example 1, the raw material of deer horn is replaced by donkey-hide gelatin (Dong'e donkey-hide gelatin).

[0089] Comparative Example 3

[0090] Different from Example 1, the raw material of sea cucumber is replaced by American ginseng.

[0091] Comparative Example 4

[0092] Different from Example 1, the hawthorn is replaced by roasted barley.

[0093] Comparative Example 5

[0094] Different from Example 1, the weight ratio of each component in the composition is different.

[0095] A composition for improving adverse reactions of chemotherapy, the raw materials are as follows in terms of weight parts: soft-shelled turtle 50 parts, paddlefish 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.

[0096] Evaluation of the effect of assisting in improving adverse reactions of chemotherapy

[0097] 1. Materials

[0098] 1.1. Drugs and reagents:

[0099] The compositions prepared in each example and comparative example are turtle and turtle oral liquid;

[0100] 5-fluorouracil (5-FU), purchased from Shanghai Xudong Haipu Pharmaceutical Co., Ltd.;

[0101] Whey protein, Beijing Combit Sports Technology Co., Ltd.;

[0102] DNA extraction kit, Beijing TransGene BioTech Co., Ltd.;

[0103] Leukocyte counting hemolysis reagent: Nanchang Kewei Reagent Co., Ltd.;

[0104] DMEM high-sugar culture medium, Gibico Company.

[0105] Preparation method of turtle and soft-shelled turtle oral liquid:

[0106] (1) Take 35 g of turtles and 35 g of soft-shelled turtles, wash and crush, add 10 times the amount of water and decoct for 4 hours, filter the soup 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 (turtles and soft-shelled turtles) and enzymatically hydrolyze for 3 hours, cool to 55℃, then add 0.5% of flavor protease and enzymatically hydrolyze for 0.5 hours, heat to 100℃ to inactivate the enzymes, and concentrate to obtain an enzymatic hydrolysate;

[0107] (2) Take 1 g of amomum villosum, 2 g of dried tangerine or orange peel, and 2 g of hawthorn, add water and decoct for 2 times, each time for 1 hour, concentrate the decocting liquid, combine with the enzymatic hydrolysate of (1), sterilize, and obtain turtle and soft-shelled turtle oral liquid.

[0108] 1.2 Cell strain:

[0109] Human gastric cancer cell line BGC-823 was purchased from the Cell Bank of Chinese Academy of Medical Sciences, and was subcultured and stored by the Pharmacology Group of Jiangxi College of Traditional Chinese Medicine.

[0110] 1.3 Experimental animals:

[0111] Balb / C Nu / Nu nude mice, male, 18-22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with license number 0290346.

[0112] 1.4 Main instruments:

[0113] NanoVue microprotein nucleic acid instrument, GE Company; AL204 electronic balance, Mettler-Toledo Instrument (Shanghai) Co., Ltd.

[0114] XT-200I type blood cell analyzer, Japan Sysmex Corporation;

[0115] Digital vernier caliper, Cangzhou Jilu Test Instrument Co., Ltd.

[0116] 2 Experimental method

[0117] 2.1 Cell culture

[0118] 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℃, 5% CO2, and saturated humidity. When the cells grew to the logarithmic growth phase, the cells were digested with trypsin solution.

[0119] 2.2 Establishment of human gastric cancer nude mouse xenograft model

[0120] ​Tumor culture: Male 4-6 week old BALB / C Nu / Nu nude mice were inoculated with BGC-823 human gastric cancer cells 1 x 10 7 / per mouse intradermally on the back, and when the tumor volume reached 1 cm 3 in diameter, part of the tumor mass was removed under sterile conditions.

[0121] Tumor-bearing animals: The tumor mass was cut into small pieces of about 1 mm 3 in diameter, and was inoculated into the back of other experimental nude mice (male 4-6 week old BALB / C Nu / Nu nude mice) subcutaneously using a trocar to form subcutaneous transplanted tumors, thereby obtaining tumor-transplanted mice (tumor-bearing animals).

[0122] 2.3 Grouping and administration

[0123] The above tumor-transplanted mice were randomly divided into 14 groups of 10 mice each 48 hours after inoculation, and the grouping is as follows:

[0124] A, blank control group, administered with distilled water by gavage.

[0125] B, 5-FU group, administered with 5-FU at a dose of 30 mg / kg BW by intraperitoneal injection, once a week.

[0126] C, 5-FU + whey protein group, the dose and administration method of 5-FU are the same as those in group B; whey protein is administered by gavage at a dose of 8.125 g / Kg BW (80% purity, actual dose 6.5 g / Kg BW), once a day.

[0127] D, 5-FU + Biejia oral liquid group, the dose and administration method of 5-FU are the same as those in group B; Biejia oral liquid is administered by gavage at a dose of 45.5 g crude drug / kg BW, once a day (equivalent to 5 times the clinical dose).

[0128] E, 5-FU + low-dose composition of Example 1 group, the dose and administration method of 5-FU are the same as those in group B; the composition of Example 1 is administered by gavage at a dose of 11.25 g crude drug / kg BW, once a day (equivalent to 1 times the clinical dose).

[0129] F, 5-FU + medium-dose composition of Example 1 group, the dose and administration method of 5-FU are the same as those in group B; the composition of Example 1 is administered by gavage at a dose of 56.25 g crude drug / kg BW, once a day (equivalent to 5 times the clinical dose).

[0130] G, 5-FU + high-dose composition of Example 1 group, the dose and administration method of 5-FU are the same as those in group B; the composition of Example 1 is administered by gavage at a dose of 112.5 g crude drug / kg BW, once a day (equivalent to 10 times the clinical dose).

[0131] H, 5-FU + Example 2 composition dose group, 5-FU dose and administration method same as group B; Example 2 composition was administered by gavage, the dose was 56.25 g crude drug / kg BW, once a day (equivalent to 5 times the amount of clinical).

[0132] I, 5-FU + Example 3 composition dose group, 5-FU dose and administration method same as group B; Example 3 composition was administered by gavage, the dose was 56.25 g crude drug / kg BW, once a day (equivalent to 5 times the amount of clinical).

[0133] J, 5-FU + Comparative Example 1 composition dose group, 5-FU dose and administration method same as group B; Comparative Example 1 composition was administered by gavage, the dose was 56.25 g crude drug / kg BW, once a day (equivalent to 5 times the amount of clinical).

[0134] K, 5-FU + Comparative Example 2 composition dose group, 5-FU dose and administration method same as group B; Comparative Example 2 composition was administered by gavage, the dose was 56.25 g crude drug / kg BW, once a day (equivalent to 5 times the amount of clinical).

[0135] L, 5-FU + Comparative Example 3 composition dose group, 5-FU dose and administration method same as group B; Comparative Example 3 composition was administered by gavage, the dose was 56.25 g crude drug / kg BW, once a day (equivalent to 5 times the amount of clinical).

[0136] M, 5-FU + Comparative Example 4 composition dose group, 5-FU dose and administration method same as group B; Comparative Example 4 composition was administered by gavage, the dose was 56.25 g crude drug / kg BW, once a day (equivalent to 5 times the amount of clinical).

[0137] N, 5-FU + Comparative Example 5 composition dose group, 5-FU dose and administration method same as group B; Comparative Example 5 composition was administered by gavage, the dose was 56.25 g crude drug / kg BW, once a day (equivalent to 5 times the amount of clinical).

[0138] The above animals were administered from the day of grouping, 5-FU once a week, and the rest of the samples were administered by gavage once a day for 35 consecutive days. The tumor volume of tumor-bearing animals was measured twice a week, and the blood was collected from the orbital venous plexus 24 hours after the last administration. The peripheral blood cells of mice were detected by automatic blood cell analyzer, and the white blood cell count in blood was determined. The animals were sacrificed, the tumor tissue was stripped and weighed, and the tumor inhibition rate was calculated.

[0139] 2.4 Determination of bone marrow DNA content

[0140] The complete femur was stripped out, and a certain amount of Hank's solution was sucked by a 1 mL syringe to flush out all the bone marrow cells in the femur. Finally, the supernatant was discarded after centrifugation at 1000 x g for 5 minutes. After washing with a certain amount of PBS solution and centrifugation, 180 μL of PBS solution was added, and the cells were mixed thoroughly. Then, the DNA in the bone marrow cells was extracted by using an EasyPure™ DNA Extraction Kit (TransGene BioTech, Beijing), and finally, the sample was accurately diluted to 300 μL with a lysis buffer for determination of the DNA content.

[0141] 2.5 Observation of the growth of bone marrow cells

[0142] The effect of the test sample on the proliferation of bone marrow cells was detected by using the method of bone marrow smear. The sternum was taken, and the basic bone marrow liquid was mixed with the calf serum on one end of the glass sheet. The conventional smear was naturally dried and then fixed in methanol for 5-10 minutes. The fixed smear was put into Giemsa staining solution and dyed for 10-15 minutes. Then, the smear was immediately washed with phosphate buffer, dried, and read.

[0143] 3. Results

[0144] 3.1 Effect of the test sample on the body weight of animals

[0145] The body weight results are shown in Table 1.

[0146] Table 1: Effect of the test sample on the body weight of the chemotherapy animals

[0147]

[0148] Notes: ## 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; & Compared with the 5-FU-Example 1 composition dose group, p < 0.05.

[0149] In each of the above groups, except for the blank group, 5-FU was injected intraperitoneally once a week, and the dose was 30 mg / Kg BW.

[0150] 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 the tumor-bearing animals, but the body weight of the test animals significantly increases after being given the turtle oral liquid, and the difference is statistically significant (p < 0.05); the whey protein control group does not have statistical significance. The body weight of the test animals increases more after being given the composition of the example, and the difference is extremely significant compared with the 5-FU group, p < 0.01.

[0151] The weight of the animals in the dosage group of Example 1 composition increased significantly, while the weight of the animals in each of the other comparative groups did not increase as much as the weight of the animals in the dosage group of the Example composition.

[0152] 3.2 Effect of the test samples on the tumor volume of the chemotherapy animals

[0153] Table 2 shows the effect of each test sample on the tumor volume of the tumor-bearing animals. As can be seen from Table 2, 5-FU can significantly reduce the tumor volume of the tumor-bearing animals, and the tumor volume of the animals in each of the groups receiving the test samples in combination with 5-FU was significantly reduced compared with the blank control group, with statistical significance (p<0.05).

[0154] Table 2 Effect of the test samples on the tumor volume of the tumor-bearing animals

[0155]

[0156] Notes: ## 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.

[0157] Among the above groups, except for the blank control group, each group was injected intraperitoneally with 5-FU once a week at a dose of 30 mg / Kg BW. The dosage group of the composition of Example had a significant difference compared with the 5-FU group, the high-dose group of Example 1 had a very significant difference compared with the 5-FU group, and was able to significantly reduce the tumor volume; the other groups had no significant difference compared with the 5-FU group.

[0158] 3.3 Effect of the test samples on the bone marrow DNA content of the chemotherapy animals

[0159] The mice in each group were sacrificed, and the femur was taken out, 2 mL of Hanks solution was sucked out using a syringe (6.5 gauge needle), and all the bone marrow cells in the femur were flushed out, so that the cell suspension passed through a 4-gauge needle syringe, and the cells were fully dispersed in the suspension. The OD value was measured at 260 nm using a UV spectrophotometer, and the results of the bone marrow DNA content OD value of the mice in each group are shown in Table 3.

[0160] Table 3 Effect of the test samples on the bone marrow DNA content of the tumor-bearing animals

[0161]

[0162] Notes: ## 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. In the above groups, except the blank group, 5-FU was injected intraperitoneally once a week, and the dosage was 30 mg / Kg BW.

[0163] Table 3 shows the effect of each test sample on the bone marrow DNA content of the chemotherapy animals. As shown in the table, 5-FU can significantly reduce the bone marrow DNA content of the tumor-bearing animals, and produce a significant bone marrow suppression effect (p < 0.01). After the high and medium doses of the composition of Example 1 were combined with 5-FU for treatment, the bone marrow DNA content was significantly higher than that of the 5-FU alone group (p < 0.01), and after the low dose of Example 1 and the medium doses of Example 2 and Example 3 were combined with 5-FU for treatment, the bone marrow DNA content was significantly higher than that of the 5-FU alone group (p < 0.05), indicating that the composition of the present application can significantly improve the bone marrow suppression effect caused by the chemotherapy drug. The bone marrow DNA content of the animals in the combined administration groups of the remaining test samples, the comparative examples and 5-FU had no significant difference compared with the 5-FU alone group (p > 0.05).

[0164] 3.4 Effect of test samples on the peripheral blood cell count of chemotherapy animals

[0165] 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 animals.

[0166] 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 animals.

[0167]

[0168] In Table 4, the data in each vertical column are compared: ## p < 0.01 compared with the blank control group; # p < 0.05 compared with the blank control group; p < 0.05 compared with the 5-FU group; p < 0.01 compared with the 5-FU group.

[0169] In the above groups, except the blank group, 5-FU was injected intraperitoneally once a week, and the dosage was 30 mg / Kg BW.

[0170] As shown in Table 4, 5-FU can significantly reduce the white blood cell count of the chemotherapy animals, and the white blood cell count of the animals in the 5-FU group (chemotherapy group) was significantly decreased compared with the blank control group (p < 0.01). After the test animals were administered the composition of the present application, the peripheral white blood cell count was significantly increased, and the peripheral white blood cell count of the animals in the other combined treatment groups had no significant change compared with the 5-FU alone group (p > 0.05).

[0171] As can be seen from Table 4, the red blood cell count of the 5-FU group is significantly reduced, and obvious bone marrow suppression is produced (p<0.01). Except for the high-dose composition of Example 1 group, the red blood cell count of the animals in the rest of the combined treatment groups has no obvious change. After the high-dose composition of Example 1 is used in combination with 5-FU, the red blood cell count is significantly higher than that of the 5-FU group, and the difference is statistically significant (p<0.05).

[0172] 4 Summary

[0173] The present application uses the method of subcutaneously transplanting human gastric cancer BGC-823 cells into nude mice to establish a human gastric cancer nude mouse transplantation model, and understands the auxiliary effects of the test samples on the chemotherapy of gastric cancer by combining 5-FU with turtle and soft-shelled turtle oral liquid and the composition respectively, and compares the chemotherapy auxiliary effect characteristics of the test samples from the aspects of the efficacy and toxic side effects of chemotherapy.

[0174] (1) The present application uses tumor volume as an index for evaluating the efficacy of the combined treatment, and evaluates the influence of the above samples on the efficacy of chemotherapy. The results show that the composition of the present application in combination with 5-FU has obvious anti-tumor effect.

[0175] (2) The present application uses the indexes of animal weight, bone marrow DNA content and peripheral blood cell count as the toxic side effect indexes of the chemotherapeutic drugs, and investigates the influence of the above test samples on the toxic side effects of the chemotherapeutic drugs. The results show 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 have this effect, which shows that the chemotherapy protection effect of the composition of the present application is not a non-specific effect of protein.

[0176] (3) The present application also comparatively studies the chemotherapy auxiliary effects of the composition and turtle and soft-shelled turtle oral liquid. The results show that the same dose of the composition and turtle and soft-shelled 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 significantly higher than that of turtle and soft-shelled turtle oral liquid. In addition, the composition can also significantly increase the bone marrow DNA content of the chemotherapy animals, the white blood cell and red blood cell counts in the peripheral blood, while turtle and soft-shelled turtle oral liquid has no such 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 turtle and soft-shelled turtle oral liquid.

[0177] (4) As can be seen from the comparative results of the examples and the comparative examples, the composition of the present application has better effects of reducing the tumor volume of the tumor-bearing animals, increasing the bone marrow DNA content of the chemotherapy animals, and increasing the white blood cell count and red blood cell count in the peripheral blood of the chemotherapy animals than the comparative examples, and the bone marrow suppression protection effect is significantly improved.

[0178] 5 Conclusion

[0179] (1) The composition of the embodiment of the present application has obvious chemotherapy auxiliary effect, and has protective effect on bone marrow suppression of the chemotherapy drugs, and the turtle and turtle oral liquid does not have this effect.

[0180] (2) The composition of the embodiment of the present application has no obvious influence on the curative effect of the chemotherapy drugs, and the turtle and turtle oral liquid also does not have a chemotherapy synergistic effect.

[0181] 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 ameliorating adverse reactions of chemotherapy, characterized by comprising, The raw materials include turtle 30-40 parts, soft-shelled turtle 30-40 parts, deer horn 4-8 parts, sea cucumber 30-40 parts, amomum villosum 0.5-2 parts, dried tangerine or orange peel 1-3 parts and hawthorn 2-4 parts by weight; (1) The turtle, soft-shelled turtle and deer horn are decocted with water, filtered to obtain decocting liquid 1; the filter residue is heated with water and subjected to enzymatic hydrolysis with alkaline protease and flavor protease respectively to obtain enzymatic hydrolysis liquid A; (2) The sea cucumber is heated with water and subjected to enzymatic hydrolysis with alkaline protease and flavor protease respectively to obtain enzymatic hydrolysis liquid B; (3) The amomum villosum, dried tangerine or orange peel and hawthorn are decocted with water to obtain decocting liquid 2; the decocting liquid 1, decocting liquid 2 and enzymatic hydrolysis liquid A and B are mixed to obtain the composition.

2. The composition of claim 1, wherein, The raw materials include turtle 35 parts, soft-shelled turtle 35 parts, deer horn 6 parts, sea cucumber 35 parts, amomum villosum 1 part, dried tangerine or orange peel 2 parts and hawthorn 2 parts by weight.

3. Process for the preparation of a composition according to any one of claims 1-2, characterized in that, The steps include: (1) The turtle, soft-shelled turtle and deer horn are decocted with water, filtered to obtain decocting liquid 1; the filter residue is heated with water and subjected to enzymatic hydrolysis with alkaline protease and flavor protease respectively to obtain enzymatic hydrolysis liquid A; (2) The sea cucumber is heated with water and subjected to enzymatic hydrolysis with alkaline protease and flavor protease respectively to obtain enzymatic hydrolysis liquid B; (3) The amomum villosum, dried tangerine or orange peel and hawthorn are decocted with water to obtain decocting liquid 2; the decocting liquid 1, decocting liquid 2 and enzymatic hydrolysis liquid A and B are mixed to obtain the composition.

4. The production method according to claim 3, characterized by, In step (1), the decocting is performed for 3-5 h, and the solid-liquid ratio of decocting is 1 g:8-15 mL; In step (1), the heating is performed to 60-65 ℃, and the solid-liquid ratio is 1 g:6-10 mL.

5. The preparation method according to claim 3, characterized in that, In step (1), the enzymatic hydrolysis is performed twice: the first enzymatic hydrolysis is performed for 2-5 h with alkaline protease; the second enzymatic hydrolysis is performed for 20-60 min with flavor protease; The temperature of the first enzymatic hydrolysis is 60-65 ℃, and the temperature of the second enzymatic hydrolysis is 50-55 ℃; In step (1), the addition amount of alkaline protease is 1%-3% of the net weight of the turtle, soft-shelled turtle and deer horn; the addition amount of flavor protease is 0.3%-0.8% of the net weight of the turtle, soft-shelled turtle and deer horn.

6. The preparation method according to claim 3, characterized in that, In step (2), the heating is performed to 60-65 ℃, and the solid-liquid ratio is 1 g:6-10 mL; In step (2), the enzymatic hydrolysis is performed twice: the first enzymatic hydrolysis is performed for 2-5 h with alkaline protease; the second enzymatic hydrolysis is performed for 20-60 min with flavor protease; 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 addition amount of alkaline protease is 1%-3% of the net weight of the sea cucumber; the addition amount of flavor protease is 0.3%-0.8% of the net weight of the sea cucumber.

7. The preparation method according to claim 3, characterized in that, In step (3), the decocting is performed for 1-4 times, and each time is for 0.5-2 h; the solid-liquid ratio of decocting is 1 g:8-15 mL.

8. Use of the composition according to any one of claims 1 to 2 or the composition prepared according to any one of claims 3 to 7 in the manufacture of a medicament for ameliorating adverse effects of chemotherapy; wherein the medicament for ameliorating adverse effects of chemotherapy is a medicament for ameliorating myelosuppression.

9. A medicament having an effect of ameliorating adverse reactions of chemotherapy, characterized by comprising the compound according to claim 1. A medicament for ameliorating adverse effects of chemotherapy comprising the composition according to any one of claims 1 to 2 or the composition prepared according to any one of claims 3 to 7.

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