Donkey-hide gelatin product with tumor inhibition effect as well as preparation method and application of donkey-hide gelatin product

Abstract

The invention provides a donkey-hide gelatin product with a tumor inhibiting effect and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The colla corii asini product with the tumor inhibition effect comprises colla corii asini, angelica sinensis, codonopsis pilosula, astragalus membranaceus, oldenlandia diffusa, callistephus chinensis, dandelion roots, rhizoma phragmitis, liquidambar formosana hance, Chinese yam and liquorice. All the medicines are combined to achieve a synergistic effect, and the traditional Chinese medicine composition has the effects of inhibiting tumors, improving anemia, enhancing immunity and nourishing bodies. The product disclosed by the invention is combined with chemotherapeutic drugs, namely paclitaxel and adriamycin, so that the tumor can be remarkably inhibited, and the side effects of anemia, low immunity and the like caused by the chemotherapeutic drugs can be relieved. Experimental results show that the activity of breast cancer cells and ovarian cancer cells can be remarkably inhibited by combining traditional Chinese medicines with chemotherapeutic drugs. Therefore, the product disclosed by the invention can be used as a candidate medicine for preventing / treating breast cancer and ovarian cancer.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a donkey-hide gelatin product having tumor-inhibiting efficacy, and a preparation method and application thereof. Background Art

[0002] A tumor is a new growth formed by the proliferation of local tissue cells under the action of various tumorigenic factors, and it often manifests as a local abnormal mass. Tumor cells grow vigorously and out of control, and are relatively autonomous. They can still stably propagate after the initial factors that cause neoplastic proliferation disappear. Tumors are divided into two categories: benign tumors and malignant tumors. Benign tumors grow slowly, have no or weak invasiveness, do not spread, and have little harm. Malignant tumors grow rapidly, are highly invasive, can spread, and have serious harm, and are called cancer. The occurrence mechanism of tumors is complex, involving multiple factors such as genetics, environment, and lifestyle habits. Among them, breast cancer and ovarian cancer are malignant tumors with a higher incidence in women.

[0003] At present, the drugs used in clinical treatment of breast cancer and ovarian cancer are mainly Western medicines. For example, docetaxel, carboplatin, paclitaxel, doxorubicin and other drugs are used as the main chemotherapy regimens for breast cancer and ovarian cancer. However, clinical findings show that the chemotherapy efficacy of the above drugs has certain limitations, and patients often suffer from serious side effects due to chemotherapy drugs, such as anemia, low immunity, gastrointestinal discomfort, liver and kidney damage, which in turn affects the overall treatment effect of breast cancer and ovarian cancer. Therefore, it is urgent to invent a drug that can not only inhibit tumors but also alleviate the side effects of chemotherapy drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a donkey-hide gelatin product with tumor inhibition effect and its preparation method and application. The product of the present invention has proper compatibility and can not only inhibit tumors but also alleviate the side effects caused by chemotherapy drugs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a donkey-hide gelatin product with tumor inhibition effect, comprising the following raw materials in parts by weight: 20-30 parts of donkey-hide gelatin, 15-25 parts of angelica, 15-25 parts of codonopsis, 15-25 parts of astragalus, 10-20 parts of oldenlandia diffusa, 10-20 parts of aster, 10-20 parts of dandelion root, 10-20 parts of reed root, 5-10 parts of liquidambar, 5-10 parts of yam, and 5-10 parts of liquorice; The weight ratio of the angelica, codonopsis and astragalus is 1:1:1; The weight ratio of the oldenlandia diffusa, aster, dandelion root and reed root is 1:1:1:1; The weight ratio of the Rhizoma Lulufen, Rhizoma Dioscoreae and Radix Glycyrrhizae is 1:1:1.

[0006] The present invention also provides a method for preparing the above-mentioned donkey-hide gelatin product, comprising: mixing raw materials except donkey-hide gelatin and then breaking the wall to obtain a broken wall composition, mixing it with water, performing reduced pressure ultrasonic extraction, filtering, and concentrating under reduced pressure to obtain a concentrated paste; heating the donkey-hide gelatin and water until they are melted, mixing them with the concentrated paste, and cooling them to obtain the donkey-hide gelatin product.

[0007] More preferably, the wall breaking comprises: crushing the raw materials except donkey-hide gelatin, maintaining the pressure at 50-200 MPa for 1-5 min, and releasing the pressure instantly.

[0008] More preferably, the water used in the preparation method is deuterium-depleted small molecular cluster water.

[0009] Preferably, the solid-liquid ratio of the reduced pressure ultrasonic extraction is 1:10-15 g / mL, the pressure is 0.03-0.07 MPa, the frequency is 20-30 kHz, the power is 200-300 W, the temperature is 40-50° C., the time is 20-30 min, and the number of times is 1-3 times.

[0010] Preferably, the reduced pressure concentration has a rotation speed of 80-120 r / min, a pressure of 0.02-0.07 MPa, a temperature of 45-55° C., and is concentrated to a relative density of 1.12-1.17.

[0011] Preferably, the weight ratio of the donkey-hide gelatin to water is 1:1-3.

[0012] The present invention also provides the use of the donkey-hide gelatin product in the preparation of tumor-inhibiting drugs.

[0013] The present invention also provides the use of the above-mentioned donkey-hide gelatin product in combination with chemotherapy drugs in the preparation of tumor-inhibiting drugs, wherein the chemotherapy drugs are paclitaxel and doxorubicin.

[0014] Preferably, the tumors include breast cancer and ovarian cancer.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a donkey-hide gelatin product with tumor inhibition effect, comprising the following raw materials in parts by weight: donkey-hide gelatin, angelica, codonopsis, astragalus, oldenlandia diffusa, aster, dandelion root, reed root, rhizome of liquidambar, yam and liquorice. The donkey-hide gelatin of the present invention nourishes blood and is the main medicine; angelica, codonopsis and astragalus nourish qi and promote blood circulation, enhance the blood nourishing effect of donkey-hide gelatin, and are the ministerial medicine; oldenlandia diffusa, aster, dandelion root and reed root clear away heat and detoxify, can relieve the internal heat caused by the main medicine and ministerial medicine, and are adjuvant medicines; rhizome of liquidambar, yam and liquorice harmonize various medicines, and are the guiding medicines. Each Chinese medicine in the product of the present invention has anti-tumor activity and the effect of enhancing human resistance, and can alleviate the problems of low immunity, leukopenia, anemia and the like caused by chemotherapy drugs. The various medicines of the present invention are used together, have a synergistic effect, and have the effects of inhibiting tumors, improving anemia, enhancing immunity and nourishing the body. The product of the present invention is used in combination with paclitaxel and doxorubicin, a chemotherapy drug, to significantly inhibit tumors and alleviate the side effects of anemia, low immunity and the like caused by chemotherapy drugs. The experimental results show that the combination of traditional Chinese medicine and chemotherapy drugs in the present invention can significantly inhibit the activity of breast cancer cells and ovarian cancer cells. Therefore, the product of the present invention can be used as a candidate drug for the prevention / treatment of breast cancer and ovarian cancer. DETAILED DESCRIPTION

[0016] The invention provides a donkey-hide gelatin product with tumor inhibition effect, comprising the following raw materials in parts by weight: 20-30 parts of donkey-hide gelatin, 15-25 parts of angelica, 15-25 parts of codonopsis, 15-25 parts of astragalus, 10-20 parts of oldenlandia diffusa, 10-20 parts of aster, 10-20 parts of dandelion root, 10-20 parts of reed root, 5-10 parts of liquidambar, 5-10 parts of yam, and 5-10 parts of liquorice; The weight ratio of the angelica, codonopsis and astragalus is 1:1:1; The weight ratio of the oldenlandia diffusa, aster, dandelion root and reed root is 1:1:1:1; The weight ratio of the Rhizoma Lulufen, Rhizoma Dioscoreae and Radix Glycyrrhizae is 1:1:1.

[0017] The donkey-hide gelatin of the present invention can nourish blood, reduce anemia symptoms caused by chemotherapy drugs, improve the physical condition of patients, and enhance the tolerance of tumor treatment; it can promote the generation and activity of immune cells and enhance the immune function of the body; it has an anti-fatigue effect and can help patients improve their physical endurance and recovery ability; it has antioxidant properties and can reduce the damage of free radicals to cells and prevent normal cells from becoming cancerous, and is a monarch drug.

[0018] The angelica of the present invention can replenish qi and promote blood circulation, can enhance the body's immune response, promote the proliferation and activation of immune cells, and enhance the patient's immunity; can inhibit the growth and division of tumors, and induce tumor cell apoptosis; has an anti-angiogenic effect, inhibits the supply of nutrients and oxygen to tumor tissues, and limits the growth and metastasis of tumors; has antioxidant properties, reduces the damage of oxidative stress to cells, and protects normal cells from oxidative damage, and is an adjuvant drug.

[0019] The dangshen of the invention can nourish the middle and replenish qi, strengthen the spleen and benefit the lungs, relieve fatigue, improve the physical condition of patients, and enhance tolerance to tumor treatment; can stimulate and regulate the immune system and enhance the body's resistance; has an antioxidant effect and inhibits the growth of tumor cells; and affects the proliferation, differentiation and apoptosis of tumor cells by regulating cell signaling pathways to exert an anti-tumor effect; and is an adjuvant drug.

[0020] The astragalus root of the present invention replenishes qi and nourishes blood, and can improve the symptoms of qi deficiency, fatigue, shortness of breath and the like in patients; the effective ingredients such as polysaccharides and triterpenoid compounds can enhance immunity and improve the body's ability to attack tumor cells; it has antioxidant and anti-free radical effects, can reduce the occurrence of gene mutations, and reduce the risk of tumor occurrence; it can inhibit the growth and division of tumor cells by regulating cell cycles, inducing tumor cell apoptosis and the like; it can inhibit the development of tumors by regulating factors such as cytokines and angiogenesis in the tumor microenvironment, and is an adjuvant drug.

[0021] The oldenlandia diffusa herb of the invention can clear away heat and detoxify, inhibit the proliferation of cancer cells and promote their apoptosis; it has an immunomodulatory effect, enhances the resistance of patients and is an adjuvant.

[0022] The aster of the present invention has the functions of clearing away heat and detoxicating, and is an adjuvant.

[0023] The dandelion root of the invention can clear away heat and detoxify, reduce swelling and resolve knots, and enhance the body's immunity; it can reduce the inflammatory reaction of the gastrointestinal tract, thereby alleviating the side effects caused by chemotherapy drugs, and is an adjuvant.

[0024] The reed root of the present invention can clear away heat and purge fire, promote the production of body fluid and quench thirst, and relieve restlessness, and is helpful to relieve vomiting caused by chemotherapy, improve the patient's appetite and digestive function; replenish body fluid in the body, and relieve dryness and heat discomfort; can have a certain regulating effect on the body's immune system, help improve the patient's immunity during tumor treatment, and enhance the body's ability to fight tumors and treat side effects, and is an adjuvant.

[0025] The Lulutong of the present invention dredges the meridians, helps to improve blood circulation, dredges the meridians, thereby alleviating the discomfort of limb numbness; regulates the immune state of the body and enhances the body's resistance; guides other drugs to the diseased area and plays the role of guiding the meridians, thus serving as an adjuvant drug.

[0026] The yam of the present invention can nourish the spleen and stomach, promote the production of body fluid and benefit the lungs, nourish the kidneys and astringe the essence, can regulate the spleen and stomach functions, help improve the digestive dysfunction caused by chemotherapy, increase appetite, relieve discomfort such as nausea and vomiting; help improve the body's immune function, and enhance the ability to resist the side effects of chemotherapy; can provide energy and nutritional support for the body, and help patients maintain a good physical condition during chemotherapy; reduce the mutual conflict between drugs, make the medicinal properties of the entire prescription more peaceful and coordinated, and serve as the drug.

[0027] The liquorice root of the invention nourishes the spleen and replenishes qi, helps to relieve spasm and inflammation of the gastrointestinal tract; has an immunomodulatory effect, helps to enhance the body's resistance, reduces the damage of chemotherapy to the immune system, harmonizes the properties and effects of different drugs, makes the overall therapeutic effect of the prescription more stable and coordinated, and is a drug.

[0028] The donkey-hide gelatin of the present invention nourishes blood and is the main drug; Angelica sinensis, Codonopsis pilosula and Astragalus membranaceus nourish qi and promote blood circulation, enhance the blood-nourishing and blood-nourishing effect of donkey-hide gelatin, and are the ministerial drugs; Hedyotis diffusa, Astragalus membranaceus, Taraxacum root and Phragmites australis clear away heat and detoxify, and can relieve the internal heat caused by the main drug and the ministerial drugs, and are the adjuvant drugs; Smilax glabra, Dioscorea opposita and Licorice harmonize the drugs and are the guiding drugs. Each Chinese medicine in the product of the present invention has anti-tumor activity and the effect of enhancing human resistance, and can alleviate the problem of low immunity caused by chemotherapy drugs. The combination of the drugs of the present invention has the effects of inhibiting tumors, improving anemia, enhancing immunity and nourishing the body.

[0029] The present invention also provides a method for preparing the above-mentioned donkey-hide gelatin product, comprising: mixing the raw materials except donkey-hide gelatin and then breaking the wall to obtain a broken wall composition, mixing it with water, performing reduced pressure ultrasonic extraction, filtering, and concentrating under reduced pressure to obtain a concentrated paste; heating the donkey-hide gelatin and water until they are melted, mixing them with the concentrated paste, and cooling them to obtain the traditional Chinese medicine components.

[0030] The wall breaking of the present invention preferably includes: crushing the raw materials except donkey-hide gelatin, maintaining them under 50-200Mpa for 1-5 minutes, and releasing the pressure instantly; the particle size of the crushed particles is preferably 100-200 mesh, more preferably 150 mesh, the pressure is more preferably 100Mpa, and the time is more preferably 3 minutes. The high pressure in the wall breaking method of the present invention kills the microorganisms in the raw materials of traditional Chinese medicine and reduces microbial contamination; it can rupture the cell walls of the raw materials of traditional Chinese medicine, thereby facilitating the release and dissolution of the active ingredients therein and improving the bioavailability of the drug.

[0031] The water used in the preparation method of the present invention is preferably low-deuterium small-molecule water. Low-deuterium small-molecule water can improve the extraction efficiency, help destroy the structure of Chinese medicine cells, have stronger penetration ability, can more effectively enter the interior of Chinese medicine tissues, fully contact with active ingredients, accelerate the release of active ingredients, reduce extraction time and cost, thereby improving the extraction efficiency, dissolving more active ingredients; it has a positive effect on the stability and activity of Chinese medicine active ingredients, and helps to maintain the biological activity and efficacy of the extracted ingredients; the low-deuterium environment may interfere with the energy metabolism and biosynthesis process of tumor cells, thereby inhibiting the growth and proliferation of tumor cells; it can help enhance the body's immune function, so that the immune system can better monitor and kill tumor cells.

[0032] The material-liquid ratio of the reduced pressure ultrasonic extraction of the present invention is preferably 1:10-15g / mL, more preferably 1:12g / mL, the pressure is preferably 0.03-0.07MPa, more preferably 0.05MPa, the frequency is preferably 20-30kHz, more preferably 25kHz, the power is preferably 200-300W, more preferably 250W, the temperature is preferably 40-50°C, more preferably 45°C, the time is preferably 20-30min, more preferably 25min, and the number of times is preferably 1-3 times, more preferably 2 times. Under reduced pressure, the boiling point of the solvent is reduced, which promotes the dissolution of the effective ingredients; the cavitation effect and mechanical action of ultrasound can further accelerate cell disruption and component release, and the synergistic effect of the two greatly improves the extraction efficiency and shortens the extraction time; the reduced pressure condition reduces the extraction temperature, reduces the decomposition, deterioration or inactivation of heat-sensitive components due to high temperature, and helps to maintain the biological activity and quality of the extract.

[0033] The rotation speed of the reduced pressure concentration of the present invention is preferably 80-120r / min, more preferably 100r / min, the pressure is preferably 0.02-0.07Mpa, more preferably 0.08Mpa, the temperature is preferably 45-55°C, more preferably 50°C, and the concentration is to a relative density of 1.12-1.17, more preferably 1.15.

[0034] The weight ratio of donkey-hide gelatin to water in the present invention is preferably 1:1-3, more preferably 1:2.

[0035] The present invention also provides the use of the donkey-hide gelatin product in the preparation of tumor-inhibiting drugs.

[0036] The present invention also provides the use of the above-mentioned donkey-hide gelatin product in combination with chemotherapy drugs in the preparation of tumor-inhibiting drugs, wherein the chemotherapy drugs are paclitaxel and doxorubicin.

[0037] The tumors described in the present invention include breast cancer and ovarian cancer.

[0038] The dosage form of the product of the present invention is preferably an injection, capsule, tablet, oral preparation or microcapsule preparation.

[0039] The product of the present invention preferably comprises the donkey-hide gelatin prepared as described above and auxiliary materials; the auxiliary material components are substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio, and can be selected according to the dosage form, such as dispersants, granulating agents, surfactants, emulsifiers, disintegrants, adhesives, preservatives, lubricants, colorants, coating agents, sweeteners and flavoring agents.

[0040] The product of the present invention can be prepared by methods known in the art, including combining the donkey-hide gelatin peptide with auxiliary ingredients, and if necessary and / or desired, molding and / or packaging the product into desired single-dose or multi-dose units.

[0041] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0042] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1 Preparation of donkey-hide gelatin product with tumor inhibition effect (1) Cell wall breaking: accurately weigh 20 g of Chinese Angelica sinensis, 20 g of Codonopsis pilosula, 20 g of Astragalus membranaceus, 15 g of Hedyotis diffusa, 15 g of Astragalus membranaceus, 15 g of Taraxacum officinale root, 15 g of Phragmites australis, 8 g of Fructus Lycopodiellae, 8 g of Dioscorea opposita, and 8 g of Licorice, mix and grind the mixture through a 150-mesh sieve, maintain the mixture under 100 MPa for 3 min, and release the pressure instantly to obtain a cell wall breaking composition; (2) Vacuum ultrasonic extraction: the cell wall-breaking composition was mixed with 1728 mL of low-deuterium small molecular cluster water, and vacuum ultrasonic extraction was performed for 25 min at a pressure of 0.05 MPa, a frequency of 25 kHz, a power of 250 W, and a temperature of 45°C. After filtration, the obtained residue was extracted again according to the above extraction method, and the filtrate was combined and concentrated to a relative density of 1.15 at 100 r / min, 0.05 MPa, and 50°C to obtain a concentrated paste; (3) Preparation of the product: Heat 25 g of donkey-hide gelatin and 50 g of low-deuterium small molecular water until they are melted, mix with the above-mentioned concentrated paste, and after cooling, obtain the donkey-hide gelatin product.

[0044] Example 2 Preparation of donkey-hide gelatin product with tumor inhibition effect (1) Broken cell wall: accurately weigh 15 g of angelica sinensis, 15 g of codonopsis pilosula, 15 g of astragalus, 10 g of oldenlandia diffusa, 10 g of aster, 10 g of dandelion root, 10 g of reed root, 5 g of fennel, 5 g of yam, and 5 g of liquorice, mix and grind, pass through a 100-mesh sieve, maintain at 50 MPa for 5 min, and release the pressure instantly to obtain a broken cell wall composition; (2) Vacuum ultrasonic extraction: the cell wall-breaking composition was mixed with 1000 mL of low-deuterium small molecular cluster water, and vacuum ultrasonic extraction was performed for 30 min under the conditions of pressure 0.03 MPa, frequency 20 kHz, power 200 W, and temperature 40°C. After filtration, the obtained filter residue was extracted again according to the above extraction method, the filtrate was combined, and the filtrate was concentrated to a relative density of 1.12 under the conditions of 80 r / min, 0.05 MPa, and 45°C to obtain a concentrated paste; (3) Preparation of the product: 20 g of donkey-hide gelatin and 60 g of low-deuterium small molecular water are heated until melted, mixed with the above-mentioned concentrated paste, and cooled to obtain the donkey-hide gelatin product.

[0045] Example 3 Preparation of donkey-hide gelatin product with tumor inhibition effect (1) Cell wall breaking: accurately weigh 25 g of Chinese Angelica sinensis, 25 g of Codonopsis pilosula, 25 g of Astragalus membranaceus, 20 g of Hedyotis diffusa, 20 g of Astragalus membranaceus, 20 g of Taraxacum officinale root, 20 g of Phragmites australis, 10 g of Fructus liquidambarii, 10 g of Dioscorea opposita, and 10 g of Licorice, mix and grind, pass through a 200-mesh sieve, maintain at 200 MPa for 1 min, and release the pressure instantly to obtain a cell wall breaking composition; (2) Vacuum ultrasonic extraction: the cell wall-breaking composition was mixed with 2775 mL of low-deuterium small molecular cluster water, and vacuum ultrasonic extraction was performed for 20 min under the conditions of pressure 0.07 MPa, frequency 25 kHz, power 300 W, and temperature 50°C. After filtration, the obtained residue was extracted twice again according to the above extraction method, the filtrate was combined, and the filtrate was concentrated to a relative density of 1.17 under the conditions of 80 r / min, 0.05 MPa, and 45°C to obtain a concentrated paste; (3) Preparation of the product: Heat 30 g of donkey-hide gelatin and 30 g of low-deuterium small molecular water until they are melted, mix with the above-mentioned concentrated paste, and after cooling, obtain the donkey-hide gelatin product.

[0046] Comparative Example 1 The specific implementation is the same as that of Example 1, except that no donkey-hide gelatin is added.

[0047] Comparative Example 2 The specific implementation is the same as that of Example 1, except that Angelica sinensis, Codonopsis pilosula and Astragalus membranaceus are not added.

[0048] Comparative Example 3 The specific implementation is the same as that of Example 1, except that Hedyotis diffusa, Cuscuta australis, dandelion root and Phragmites australis are not added.

[0049] Comparative Example 4 The specific implementation is the same as that of Example 1, except that no Rhizoma Lulufen, Rhizoma Dioscoreae and Radix Glycyrrhizae were added.

[0050] Comparative Example 5 The specific implementation is the same as that of Example 1, except that the deuterium-depleted small molecular cluster water is replaced by ordinary pure water.

[0051] Comparative Example 6 The specific implementation method is the same as that of Example 1, except that (1) crushing: accurately weigh 20 g of Angelica sinensis, 20 g of Codonopsis pilosula, 20 g of Astragalus membranaceus, 15 g of Hedyotis diffusa, 15 g of Astragalus membranaceus, 15 g of Taraxacum officinale root, 15 g of Phragmites australis, 8 g of Fructus Lycii, 8 g of Dioscorea opposita, and 8 g of Licorice, mix them, and then crush them through a 150-mesh sieve to obtain a crushed composition.

[0052] Comparative Example 7 The specific implementation is the same as that of Example 1, except that (2) is normal pressure extraction, specifically: The broken wall composition was mixed with 1000 mL of low-deuterium small molecular cluster water, stirred at 500 rpm for 60 min at a temperature of 40°C, and then filtered. The obtained filter residue was extracted again according to the above-mentioned extraction method, and the filtrate was combined and concentrated to a relative density of 1.12 at 80 r / min, 0.05 MPa, and 45°C to obtain a concentrated paste.

[0053] Experimental Example 1 Cell experiments Breast cancer cells: human breast cancer MDA-MB-231 cells; Ovarian cancer cells: human ovarian cancer A2780 cells.

[0054] Breast cancer cells and ovarian cancer cells were cultured to the logarithmic phase, digested with trypsin, centrifuged at 800 rpm for 5 min, and complete culture medium was added to mix the cells to make a single cell suspension until the cell density reached 1.5×10000 / 100 μL. 100 μL / well of cell solution was added to a 96-well plate, with 3 replicates per group. The plates were placed in a constant temperature incubator at 37°C and 5% CO2 for overnight culture.

[0055] CCK8 method to detect the anti-tumor activity of the product The products of Examples 1-3 and Comparative Examples 1-7 were prepared with different drug concentrations (0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 8, 10, 20 mg / mL), 200 μL of DMEM medium was added to each well, 3 replicates were set for each group, and after 48 hours of culture, CCK8 solution was replaced, and after 2 hours of incubation, the absorbance was measured at 450 nm using an ELISA reader, and the cell viability IC50 value was calculated. The IC50 values ​​of the products of Examples 1-3 and Comparative Examples 1-7 for breast cancer cells and ovarian cancer cells are shown in Table 1.

[0056] Table 1 IC50 values ​​of the products of Examples 1-3 and Comparative Examples 1-7 against breast cancer cells and ovarian cancer cells (mg / mL)

[0057] As can be seen from the data in Table 1, compared with Comparative Examples 1-4, the IC50 values ​​of the products of the present invention against breast cancer cells and ovarian cancer cells are lower, indicating that the various drugs in the products of the present invention have a synergistic effect in the activity against breast cancer cells and ovarian cancer cells. Compared with Comparative Examples 5-7, the products obtained by the preparation method of the present invention have better anti-cancer cell activity. The product obtained by the technical solution of the present invention has certain anti-breast cancer cell and ovarian cancer cell activity.

[0058] Experimental Example 2 Animal experiments 1. Breast cancer (1) Establishment and drug administration of 4T1 mouse breast cancer model Twenty-five SPF female BALB / c mice aged 4-6 weeks and weighing 20-25 g were selected and divided into 5 groups: blank group, model group, chemotherapy group, Chinese medicine group and chemotherapy + Chinese medicine group.

[0059] Blank group: no treatment; Except for the blank group, breast cancer models were established in all other groups: 4T1 cells cultured to the logarithmic phase were configured with a density of 1×10 7 50 μL of cell suspension was inoculated into the fourth pair of mammary fat pads on the right side of the mouse and the mice were raised for 7 days after inoculation; The mice in the model group did not receive any treatment after modeling; Chemotherapy group: intraperitoneal administration of 5 mg kg / week -1 One injection of paclitaxel 5 mg kg -1 One day later, 5 mg kg -1 One injection of doxorubicin; Chinese medicine group: gavage the donkey-hide gelatin composition of Example 1 at 15 mg / kg per day; Chemotherapy + Chinese medicine group: 15 mg / kg of donkey-hide gelatin composition of Example 1 was administered intragastricly every day, and 5 mg / kg of donkey-hide gelatin was administered intraperitoneally every week-1 One injection of paclitaxel 5 mg kg -1 One day later, 5 mg kg -1 One injection of doxorubicin.

[0060] The dosing cycle is 4 weeks.

[0061] (2) Tumor inhibition rate The long and short diameters of the mouse tumors were measured with a vernier caliper, and the tumor inhibition rate was calculated. The results of the tumor inhibition rate after drug administration are shown in Table 2.

[0062] Tumor volume = 1 / 2 × (tumor long diameter × tumor short diameter) 2 ); Tumor inhibition rate = (average tumor volume of model group - tumor volume of experimental group) / average tumor volume of model group × 100%; the experimental groups were chemotherapy group, Chinese medicine group or chemotherapy + Chinese medicine group.

[0063] Table 2 Tumor inhibition rate of each group

[0064] From the experimental results in Table 2, it can be seen that the chemotherapy + Chinese medicine group can better inhibit breast tumors than the chemotherapy group and the Chinese medicine group.

[0065] (3) Immune function After 4 weeks of administration, blood from the orbital venous plexus of the blank group, model group, chemotherapy group, Chinese medicine group, and chemotherapy + Chinese medicine group was collected, and the lymphocyte subsets (CD3 + 、CD4 + 、CD8 + ) percentage and CD4 + / CD8 + , the specific results are shown in Table 3.

[0066] Table 3 Immune function indicators of each group

[0067] From the data in Table 3, we can see that the Chinese medicine + chemotherapy group can increase the number of lymphocyte subsets CD3 + 、CD4 + The percentage of CD4 + / CD8 + This indicates that the product of the present invention can alleviate the symptoms of low immunity caused by chemotherapy drugs.

[0068] 2. Ovarian cancer (1) Establishment and drug administration of ID 8 mouse ovarian cancer model Twenty-five SPF female BALB / c mice aged 4-6 weeks and weighing 20-25 g were selected and divided into 5 groups: blank group, model group, chemotherapy group, Chinese medicine group and chemotherapy + Chinese medicine group.

[0069] Blank group: no treatment; Except for the blank group, ovarian cancer models were established in all other groups: the density of ID 8 cells cultured to the logarithmic phase was 3×10 6 50 μL of cell suspension was inoculated subcutaneously in the right rib area of ​​the mouse until the tumor volume reached 100 mm 3 about; The mice in the model group did not receive any treatment after modeling; Chemotherapy group: intraperitoneal administration of 5 mg kg / week -1 One injection of paclitaxel 5 mg kg -1 One day later, 5 mg kg -1 One injection of doxorubicin; Chinese medicine group: gavage the donkey-hide gelatin composition of Example 1 at 15 mg / kg per day; Chemotherapy + Chinese medicine group: 15 mg / kg of donkey-hide gelatin composition of Example 1 was administered intragastricly every day, and 5 mg / kg of donkey-hide gelatin was administered intraperitoneally every week -1 One injection of paclitaxel 5 mg kg -1 One day later, 5 mg kg -1 One injection of doxorubicin.

[0070] The dosing cycle is 4 weeks.

[0071] (2) Tumor inhibition rate The long and short diameters of the mouse tumors were measured with a vernier caliper, and the tumor inhibition rate was calculated. The results of the tumor inhibition rates of each group after administration are shown in Table 4.

[0072] Tumor volume = 1 / 2 × (tumor long diameter × tumor short diameter 2 ); Tumor inhibition rate = (average tumor volume of model group - tumor volume of experimental group) / average tumor volume of model group × 100%; the experimental groups were chemotherapy group, Chinese medicine group or chemotherapy + Chinese medicine group.

[0073] Table 4 Tumor inhibition rate of each group

[0074] From the experimental results in Table 4, it can be seen that the chemotherapy + Chinese medicine group can better inhibit ovarian tumors than the chemotherapy group and the Chinese medicine group.

[0075] (3) Immune function After 4 weeks of administration, blood from the orbital venous plexus of the blank group, model group, chemotherapy group, Chinese medicine group, and chemotherapy + Chinese medicine group was collected, and the lymphocyte subsets (CD3 + 、CD4 + 、CD8 + ) percentage and CD4 + / CD8 + , the specific results are shown in Table 5.

[0076] Table 5 Immune function indicators before and after treatment

[0077] From the data in Table 5, we can see that the Chinese medicine + chemotherapy group can increase the lymphocyte subsets CD3 + 、CD4 + The percentage of CD4 + / CD8 + This indicates that the product of the present invention can alleviate the symptoms of low immunity caused by chemotherapy drugs.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A donkey-hide gelatin product with tumor-inhibiting effect, characterized in that: The invention comprises the following raw materials in parts by weight: 20-30 parts of donkey-hide gelatin, 15-25 parts of angelica, 15-25 parts of codonopsis, 15-25 parts of astragalus, 10-20 parts of oldenlandia diffusa, 10-20 parts of aster, 10-20 parts of dandelion root, 10-20 parts of reed root, 5-10 parts of fennel, 5-10 parts of yam and 5-10 parts of liquorice; The weight ratio of the angelica, codonopsis and astragalus is 1:1:1; The weight ratio of the oldenlandia diffusa, aster, dandelion root and reed root is 1:1:1:1; The weight ratio of the Rhizoma Lulufen, Rhizoma Dioscoreae and Radix Glycyrrhizae is 1:1:

1.

2. The method for preparing the donkey-hide gelatin product according to claim 1, characterized in that: include: The raw materials except donkey-hide gelatin are mixed and then broken to obtain a broken-wall composition, which is mixed with water and subjected to reduced-pressure ultrasonic extraction. The mixture is filtered and concentrated under reduced pressure to obtain a concentrated paste. The donkey-hide gelatin and water are heated until melted, mixed with the concentrated paste, and cooled to obtain the donkey-hide gelatin product.

3. The preparation method according to claim 2, characterized in that: The wall breaking comprises: crushing the raw materials except donkey-hide gelatin, maintaining the pressure at 50-200Mpa for 1-5min, and releasing the pressure instantly.

4. The preparation method according to claim 2, characterized in that: The water used in the preparation method is all deuterium-depleted small molecular cluster water.

5. The preparation method according to claim 2, characterized in that: The material-liquid ratio of the reduced pressure ultrasonic extraction is 1:10-15 g / mL, the pressure is 0.03-0.07 MPa, the frequency is 20-30 kHz, the power is 200-300 W, the temperature is 40-50° C., the time is 20-30 min, and the number of times is 1-3 times.

6. The preparation method according to claim 2, characterized in that: The reduced pressure concentration is carried out at a rotation speed of 80-120 r / min, a pressure of 0.02-0.07 MPa, a temperature of 45-55° C., and is concentrated to a relative density of 1.12-1.

17.

7. The preparation method according to claim 2, characterized in that: The weight ratio of the donkey-hide gelatin to water is 1:1-3.

8. Use of the donkey-hide gelatin product according to claim 1 in the preparation of tumor-inhibiting drugs.

9. The use of the donkey-hide gelatin product according to claim 1 in combination with chemotherapy drugs in the preparation of tumor-inhibiting drugs, characterized in that: The chemotherapy drugs are paclitaxel and doxorubicin.

10. The use according to claim 8 or 9, characterized in that: Such tumors include breast cancer and ovarian cancer.

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