CN106177187B - Tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects - Google Patents
Tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects Download PDFInfo
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Abstract
The invention discloses a tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects, which comprises the following components in part by weight: 0.5-2 parts of tea polyphenol and 1 part of tea polysaccharide, wherein the purity of the tea polyphenol is more than 90%; the tea polysaccharide has molecular weight of 80-100 kDa. The composition not only enhances the targeting property of the tea polyphenol, reduces the toxicity, improves the solubility of the tea polyphenol in body fluid, improves the bioavailability of the tea polyphenol, gives full play to the antioxidant activity of the tea polyphenol, eliminates free radicals in vivo, but also combines the immune function of tea polysaccharide, and achieves the obvious effect of resisting liver cancer by the synergy of the tea polyphenol and the tea polysaccharide. The composition has reduced adverse side effects, and the tea polyphenols and tea polysaccharide are derived from the same raw material, so as to improve utilization rate of the raw material, and the solvent can be recycled. In addition, the tea polysaccharide with the molecular weight of 80-100kDa is obtained by adopting an ultrafiltration technology, and has the characteristics of high efficiency, low cost, short production period, mild conditions and the like.
Description
Technical Field
The invention relates to a tea polyphenol and tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects and a preparation method thereof, belonging to the technical application field of medical and health care products.
Background
Liver cancer is a common cancer, and has extremely high morbidity and mortality. Surgical treatment and radiochemical therapy are the main means for treating liver cancer at present. However, the prognosis of the operation treatment is poor, and the disease is easy to relapse; the chemical medicine brings great toxic and side effects due to the killing effect on normal cells, and brings great pain to patients. Therefore, scientists have made efforts to find more effective and less toxic anticancer agents from natural products. Research shows that the improvement of antioxidant level and immunity level is two key means for treating tumor. Free radicals attack organs or tissues in vivo to cause cancer, and long-term low immunity is one of important causes of cancer generation, so that the search for anti-cancer drugs which have the effects of resisting oxidation and improving the immunity level is very necessary.
Tea polyphenol, also known as tea tannin, is a general name of polyhydroxy phenolic compounds contained in tea leaves and comprises catechin, flavonol, theaflavin, theabrownin and the like, wherein the catechin accounts for 65-80% of the total amount of the polyphenol. Research shows that catechin has pharmacological effects of increasing the antioxidant activity of blood, clearing free radicals in body, etc. The tea polyphenol has strong antioxidant activity and can be used as a potential medicament for treating cancers. However, tea polyphenols have low water solubility, are easily rapidly decomposed and metabolized by the upper digestive tract and liver, and are rapidly excreted through the urinary system, so that the biological activity of tea polyphenols is not maximized. In addition, although tea polyphenol has certain anticancer activity, it can penetrate all types of cells indiscriminately due to the characteristics of small molecules, thereby causing certain toxic and side effects.
Tea polysaccharide exerts a powerful immunoregulatory function in cancer treatment, but the curative effect of single and short-term administration is not significant.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a tea polyphenol tea polysaccharide composition with synergistic attenuation and liver cancer resistance.
The second purpose of the invention is to provide a preparation method of the tea polyphenol and tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects.
The technical scheme of the invention is summarized as follows:
a tea polyphenol tea polysaccharide composition with synergistic, attenuated and anti-liver cancer effects comprises the following components in parts by weight: 0.5-2 parts of tea polyphenol and 1 part of tea polysaccharide, wherein the purity of the tea polyphenol is more than 90%; the tea polysaccharide has molecular weight of 80-100 kDa.
A preparation method of a tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects comprises the following steps:
(1) preparing tea polyphenol: taking tea leaves, crushing the tea leaves, and adding 10-20 times by mass of ethanol water solution with volume concentration of 75% -99.7%; standing for 24-72 h, and performing vacuum filtration to obtain filtrate and tea residues; extracting the filtrate for 3-7 times by using ethyl acetate of which the amount is 1-3 times that of the filtrate, standing for layering, combining upper layers, and concentrating the combined solution under reduced pressure to dryness to obtain crude tea polyphenol; dissolving tea crude polyphenol in ethanol, adding the solution onto a treated macroporous adsorption resin column, eluting the column with water until the eluent is colorless, eluting the column with 3-5 column volumes of NaOH aqueous solution with the mass concentration of 0.2-0.4% to remove pigment, eluting the column with 10-12 column volumes of ethanol aqueous solution with the volume concentration of 50-90%, collecting the eluent, concentrating and drying to obtain the tea polyphenol with the purity of more than 90%;
(2) preparing tea polysaccharide: adding 10-20 mass times of deionized water into the tea residue obtained in the step (1), carrying out hot leaching for 2-3 times at 70-90 ℃, carrying out suction filtration to obtain a leaching solution, concentrating the leaching solution to 10-20% of the volume of the leaching solution to obtain a concentrated solution, adding an ethanol aqueous solution with the volume concentration of 95-99.7% which is 2.5-4 volume times of the concentrated solution, standing for 12-24 h, centrifuging at 3000-5000 rpm for 3-10 min, dissolving the precipitate in water, sequentially carrying out ultrafiltration on the precipitate by hollow cellulose membranes with the molecular weights of 100kDa and 80kDa to obtain a component with the molecular weight of 80kDa-100kDa, concentrating and drying to obtain tea polysaccharide;
(3) and uniformly mixing the tea polyphenol and the tea polysaccharide according to the mass ratio of 0.5-2: 1 to obtain the tea polyphenol and tea polysaccharide composition with the synergistic, attenuated and anti-liver cancer effects.
The model of the macroporous adsorption resin is preferably as follows: AB-8, D-101, DM-130, HPD-100 or H103.
A medicinal preparation containing the composition is prepared from the tea polyphenol tea polysaccharide composition with the effects of enhancing the efficacy, reducing the toxicity and resisting liver cancer and pharmaceutically acceptable auxiliary materials.
The invention has the advantages that:
(1) the tea polysaccharide is used as a low-toxicity drug carrier and functional materials and is combined with the tea polyphenol, so that the targeting property of the tea polyphenol is enhanced, the toxicity is reduced, the solubility of the tea polyphenol in body fluid is improved, the bioavailability of the tea polyphenol is improved, the antioxidation activity of the tea polyphenol is fully exerted, free radicals in a body are eliminated, the immune effect of the tea polysaccharide is combined, and the tea polysaccharide and the tea polyphenol are synergistic, so that the remarkable anti-liver cancer effect is achieved.
(2) After the combined administration of the tea polyphenol and the tea polysaccharide, the toxic and side effects are reduced, the adverse reaction is reduced, and the living quality of tumor-bearing mice is improved.
(3) The tea polyphenol and tea polysaccharide are derived from the same raw material, two components are comprehensively extracted for combined use, the utilization rate of the raw material is greatly improved, and the adopted solvent is pollution-free and can be recycled. In addition, the tea polysaccharide with the molecular weight of 80-100kDa is obtained by adopting an ultrafiltration technology, the defects of complicated column chromatography operation, unfavorable industrial mass production and the like are overcome, and the tea polysaccharide has the characteristics of high efficiency, low cost, short production period, mild conditions and the like, and provides a new method for industrial application of active compounds.
Drawings
FIG. 1 is a graph of tumor volume as a function of days observed for groups of tumor-bearing mice after administration of the composition of example 1;
FIG. 2 is a photograph of tumors in groups of tumor-bearing mice after administration of the composition of example 1;
FIG. 3 is a graph of the water and food efficiency of groups of tumor-bearing mice after administration of the composition of example 1;
FIG. 4 is a pathological section of tumors of various groups of tumor-bearing mice after administration of the composition of example 1.
The specific implementation mode is as follows:
the present invention will be further described with reference to the following examples, but the present invention is not limited thereto, and any changes made to the technical solution of the present invention should fall within the scope defined by the claims of the present invention without departing from the spirit and scope of the present invention.
The macroporous adsorption resin is treated by adopting a conventional method, namely, ethanol water solution with volume concentration of 95% is used for soaking, then is treated by acid and alkali, and finally is used after being washed to be neutral.
Example 1
A preparation method of a tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects comprises the following steps:
(1) preparing tea polyphenol: taking tea leaves, crushing the tea leaves, and adding 15 mass times of ethanol water solution with volume concentration of 80%; standing for 48h, and performing vacuum filtration to obtain filtrate and tea residue; extracting the filtrate with 3 times of ethyl acetate for 5 times, standing for layering, mixing the upper layers, and concentrating the combined solution under reduced pressure to dryness to obtain crude tea polyphenols; dissolving tea crude polyphenol with ethanol, adding the dissolved tea crude polyphenol onto a treated AB-8 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1:12, eluting with water until an eluent is colorless, then eluting with 4 column volumes of NaOH aqueous solution with the mass concentration of 0.25% to remove pigments, finally eluting with 11 column volumes of ethanol aqueous solution with the volume concentration of 75%, collecting the eluent, concentrating and drying to obtain the tea polyphenol with the purity of 94.87%;
(2) preparing tea polysaccharide: adding 17 mass times of deionized water into the tea residue obtained in the step (1), carrying out hot leaching for 2 times at 80 ℃, carrying out suction filtration to obtain a leaching solution, concentrating the leaching solution to 15% of the volume of the leaching solution to obtain a concentrated solution, adding 3 volume times of ethanol aqueous solution with the volume concentration of 95% of the concentrated solution, standing for 24h, centrifuging at 4000rpm for 6min, dissolving the precipitate in water, sequentially carrying out ultrafiltration by hollow cellulose membranes with the molecular weights of 100kDa and 80kDa to obtain a component with the molecular weight of 80kDa-100kDa, concentrating and drying to obtain tea polysaccharide;
(3) and uniformly mixing the tea polyphenol and the tea polysaccharide according to the mass ratio of 1:1 to obtain the tea polyphenol and tea polysaccharide composition with the synergistic, attenuated and anti-liver cancer effects.
Example 2
A preparation method of a tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects comprises the following steps:
(1) preparing tea polyphenol: taking tea leaves, crushing the tea leaves, and adding 10 mass times of 75% ethanol aqueous solution; standing for 72h, and performing vacuum filtration to obtain filtrate and tea residue; extracting the filtrate with 1 time of ethyl acetate for 7 times, standing for layering, mixing the upper layers, and concentrating the combined solution under reduced pressure to dryness to obtain crude tea polyphenols; dissolving tea crude polyphenol with ethanol, adding the solution onto a treated D-101 type macroporous adsorption resin column with the height ratio of the resin column diameter of 1:10, eluting with water until the eluent is colorless, then eluting with 3 column volumes of NaOH aqueous solution with the mass concentration of 0.2% to remove pigment, finally eluting with 10 column volumes of ethanol aqueous solution with the volume concentration of 50%, collecting the eluent, concentrating and drying to obtain the tea polyphenol with the purity of 93.63%;
(2) preparing tea polysaccharide: adding 10 mass times of deionized water into the tea residue obtained in the step (1), carrying out hot-extraction for 2 times at 70 ℃, carrying out suction filtration to obtain an extract, concentrating the extract to 18% of the volume of the extract to obtain a concentrated solution, adding an ethanol aqueous solution with the volume concentration of 95% which is 2.5 volume times of the concentrated solution, standing for 24h, centrifuging at 3000rpm for 3min, dissolving the precipitate in water, sequentially carrying out ultrafiltration by hollow cellulose membranes with the molecular weights of 100kDa and 80kDa to obtain a component with the molecular weight of 80kDa-100kDa, and carrying out concentration and drying to obtain tea polysaccharide;
(3) and uniformly mixing the tea polyphenol and the tea polysaccharide according to the mass ratio of 0.5:1 to obtain the tea polyphenol and tea polysaccharide composition with the synergistic, attenuated and anti-liver cancer effects.
Example 3
A preparation method of a tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects comprises the following steps:
(1) preparing tea polyphenol: taking tea leaves, crushing the tea leaves, and adding 20 times of ethanol water solution with volume concentration of 99.7 percent by mass; standing for 24h, and performing vacuum filtration to obtain filtrate and tea residue; extracting the filtrate with 3 times of ethyl acetate for 3 times, standing for layering, mixing the upper layers, and concentrating the mixed solution under reduced pressure to dryness to obtain crude tea polyphenols; dissolving tea crude polyphenol with ethanol, adding the solution onto a treated DM-130 type macroporous adsorption resin column with the height ratio of the resin column diameter of 1:15, eluting with water until the eluent is colorless, then eluting with 5 column volumes of NaOH aqueous solution with the mass concentration of 0.4% to remove pigment, finally eluting with 12 column volumes of ethanol aqueous solution with the volume concentration of 90%, collecting the eluent, concentrating and drying to obtain tea polyphenol with the purity of 94.07%;
(2) preparing tea polysaccharide: adding deionized water of which the mass is 20 times of that of the tea residue obtained in the step (1), hot-extracting for 3 times at 90 ℃, carrying out suction filtration to obtain a leaching solution, concentrating the leaching solution to 20% of the volume of the leaching solution to obtain a concentrated solution, adding an ethanol aqueous solution of which the volume concentration is 99.7% of that of the concentrated solution of which the volume is 4 times of that of the concentrated solution, standing for 12 hours, centrifuging at 5000rpm for 10 minutes, dissolving precipitates in water, sequentially carrying out ultrafiltration on the precipitates by hollow cellulose membranes of which the molecular weights are 100kDa and 80kDa to obtain components of which the molecular weights are 80kDa-100kDa, concentrating and drying to obtain tea;
(3) and uniformly mixing the tea polyphenol and the tea polysaccharide according to the mass ratio of 1.5:1 to obtain the tea polyphenol and tea polysaccharide composition with the synergistic, attenuated and anti-liver cancer effects.
Example 4
A preparation method of a tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects comprises the following steps:
(1) preparing tea polyphenol: taking tea leaves, crushing the tea leaves, and adding 17 times by mass of an ethanol water solution with the volume concentration of 85%; standing for 48h, and performing vacuum filtration to obtain filtrate and tea residue; extracting the filtrate with 2 times of ethyl acetate for 5 times, standing for layering, mixing the upper layers, and concentrating the combined solution under reduced pressure to dryness to obtain crude tea polyphenols; dissolving tea crude polyphenol with ethanol, adding the solution onto a processed HPD-100 type macroporous adsorption resin column with the column diameter height ratio of 1:13, eluting with water until the eluent is colorless, then eluting with 4 column volumes of NaOH aqueous solution with the mass concentration of 0.3% to remove pigment, finally eluting with 10 column volumes of ethanol aqueous solution with the volume concentration of 70%, collecting the eluent, concentrating and drying to obtain tea polyphenol with the purity of 92.18%;
the macroporous adsorption resin with the model number of H103 is used for replacing the HPD-100 type macroporous adsorption resin in the embodiment, and the purity of the prepared tea polyphenol is similar to that in the embodiment.
(2) Preparing tea polysaccharide: adding 17 mass times of deionized water into the tea residue obtained in the step (1), hot-extracting for 3 times at 85 ℃, performing suction filtration to obtain an extract, concentrating the extract to 10% of the volume of the extract to obtain a concentrated solution, adding an ethanol aqueous solution with the volume concentration of 95% which is 3 volume times of the concentrated solution, standing for 24h, centrifuging at 3500rpm for 8min, dissolving the precipitate in water, sequentially performing ultrafiltration by hollow cellulose membranes with the molecular weights of 100kDa and 80kDa to obtain a component with the molecular weight of 80kDa-100kDa, concentrating and drying to obtain tea polysaccharide;
(3) and uniformly mixing the tea polyphenol and the tea polysaccharide according to the mass ratio of 2:1 to obtain the tea polyphenol and tea polysaccharide composition with the synergistic, attenuated and anti-liver cancer effects.
Example 5
The effect of the composition on the survival rate of the liver cancer cell SMMC 7721.
Purpose of the experiment:
detecting the influence of the composition on the survival rate of the liver cancer cell SMMC 7721.
Experimental materials:
1. the test substance: tea polyphenols, tea polysaccharides, and the compositions obtained in examples 1 to 4, each test substance was accurately weighed, dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 0.15mg/mL, stored at-20 ℃, and diluted with dimethyl sulfoxide to solutions with concentrations of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.15mg/mL when used.
2. Reagent: DMEM medium: gibco (usa), 10% fetal bovine serum: gibco (usa); PBS buffer: solarbio (beijing); MTT: solarbio (beijing); the remaining reagents were commercially available.
3. Cell lines: human SMMC7721 liver cancer cell: purchased from Shanghai cell banks.
4. The instrument comprises the following steps: CO 22Incubator (BPN-80 CW): Shanghai-Hengchang scientific instruments, Inc.; clean bench (SW-CJ-1 FD): suzhou clarification plant, Inc.; inverted microscope (BDS-200): chongqing ott optical instruments, Inc. Microplate reader (DNM-9602): beijing Prian medical devices, Inc.
Experimental methods and results:
the experimental method comprises the following steps:
the MTT method detects the influence of the test object on the inhibition rate of SMMC7721 liver cancer cells.
The cells were cultured in a medium containing fetal bovine serum, 100U/mL penicillin, and 100mg/mL streptomycin at 37 ℃ with 5% CO2Culturing under the conditions of (1). After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating the cells into a 96-well cell culture plate according to the volume of 100 mu L per well, wherein the cell density is 6000-8000 cells per well. 37 ℃ and 5% CO2Adding tea polyphenols and tea polysaccharide composition with different concentrations after culturing in incubator at 37 deg.C and 5% CO2Incubate in incubator for 72 h. The medium was discarded, washed once with PBS, and then 100. mu.L of MTT containing 0.5mg/mL, 37 ℃ C., 5% CO was added to each well2Incubate in incubator for 4 h. The medium in the wells of the 96-well plate was aspirated and then 100. mu.L of dimethyl sulfoxide was added to each well. The detection is carried out by a microplate reader, and the detection wavelength is 490 nm.
Cell inhibition (%) is [ 1- (test group OD value/control group OD value) ] X100%
In order to determine whether the anti-liver cancer effect of the tea polyphenol and tea polysaccharide composition is synergistic effect or additive effect, a Combination Index (CI) is calculated by adopting a medium effect principle in Chou-Talady. CI is D1/DX1+ D2/DX 2. D1 and D2 are concentrations at which the inhibition of the combination of drug 1 and drug 2 is 50%. DX1 and DX2 were used at concentrations at which inhibition rates of drug 1 and drug 2 alone were 50%. According to the theory of Chou-Talady, if CI is 1, the combined effect of the medicines has additive effect; CI <1, and the combined effect of the medicaments has synergistic effect; CI is greater than 1, and the combined effect of the medicines has synergistic effect.
The experimental results are as follows:
TABLE 1 IC of inhibitory rate of the test substance obtained by the present invention on SMMC7721 hepatoma cells50The value is obtained.
The experimental result shows that the tea polyphenol and tea polysaccharide composition prepared according to the embodiment has good inhibition effect on the growth of liver cancer cells. The combination index is far less than 1, which indicates that the composition has synergistic anti-liver cancer curative effect.
Example 6
The composition has an inhibiting effect on mouse liver cancer H22 transplanted tumors.
Purpose of the experiment:
the effect of the composition on the growth of transplanted tumors in mice was examined.
Experimental materials:
1. the test substance: the composition obtained in example 1. Accurately weighing the composition, dissolving with normal saline to obtain stock solution with concentration of 15mg/mL, storing at-20 deg.C, and diluting with normal saline to obtain solutions with low dose, medium dose and high dose of 3, 6 dose and 12mg/mL dose; cyclophosphamide: jiangsu Shengdi medicine, Inc. (batch No. 15101225); 0.9% sodium chloride injection (normal saline): hebei Tian Chengyo GmbH (batch No. A15100801);
2. reagent: DMEM medium: gibco (usa), 10% fetal bovine serum: gibco (usa); PBS buffer: solarbio (beijing); MTT: solarbio (beijing); the remaining reagents were commercially available.
3. Animals and tumor strains: the male Kunming mouse has the weight of 20 +/-2 g and total 40 mice, and the animal certification SCXK (Jing) 2014-. Murine H22 sarcoma cells: purchased from Shanghai cell banks.
4. The instrument comprises the following steps: CO 22Incubator (BPN-80 CW): Shanghai-Hengchang scientific instruments, Inc.; clean bench (SW-CJ-1 FD): suzhou clarification plant, Inc.; inverted microscope (BDS-200): chongqing ott optical instruments, Inc. Ultraviolet spectrophotometer (UV-9200): beijing Rayleigh Analyzer Mill. Microplate reader (DNM-9602): beijing Prian medical devices, Inc.
Experimental methods and results:
1. inhibition of H22 solid tumors by test substances
Unfreezing frozen H22 mouse liver cancer cells in a 37 ℃ constant-temperature water bath, centrifuging, collecting the cells, washing the cells twice by using PBS buffer solution, resuspending the cells by using the PBS solution, transferring the cells to an ICR mouse abdominal cavity for more than 3 generations, extracting tumor liquid under an aseptic condition after ascites grows out, diluting the tumor liquid and normal saline according to the volume ratio of 1:1, and fully mixing to obtain cell suspension. After staining with trypan blue, the cells were counted on a hemocytometer in which the number of viable cells was more than 95%, and the concentration of the cell suspension was adjusted to 1X 10 with physiological saline7Tumor cells/mL. Then 0.2mL of the cell suspension with the adjusted concentration is inoculated in the right anterior limb armpit subcutaneous of the mouse, and a solid tumor animal model is constructed to obtain the H22 tumor-bearing mouse.
The day after inoculation, 8 subjects were randomized into groups, test group (three doses low, medium and high), model group (normal saline) and positive control group (25 mg/kg). The mice were gavaged once a day at a dose of 0.01mL/g (dose volume/mouse body weight) for 14 consecutive days, during which the maximum diameter a and the minimum diameter b of the tumor were measured with a vernier caliper every 2 to 3 days, according to V of 1/2 × a × b2And calculating the tumor volume and drawing a mouse tumor growth curve. The experimental results are shown in table 2 and fig. 1; the body weight of each mouse was weighed on day 15, the eyeball was removed to collect blood, the cervical spine was removed to sacrifice the mouse, and the tumor, spleen and thymus were removed and weighed, and the tumor inhibition rate, spleen index and thymus index were calculated as follows. Tumors were photographed.The results are shown in tables 3-4, and the photographs of tumors are shown in FIG. 2.
Tumor inhibition (%) was (1-administration group average tumor weight/model group average tumor weight) × 100%
Spleen index (mg/g) ═ spleen weight/body weight
Thymus index (mg/g) ═ weight of thymus/body weight
TABLE 2 experiment of tumor volume during administration in groups of mice
Example 1 the tumor volumes of the mice of each group during administration are shown in table 2, and fig. 1 is a graph showing the change of the tumor volumes of the mice of each group of example 1 with the number of observation days, and the results show that the composition of the present invention can slow down the growth rate of transplanted tumors in mice and has an effect of inhibiting the growth of tumors.
Experiments prove that the compositions of examples 2, 3 and 4 can slow down the growth rate of transplanted tumors of mice and have an inhibiting effect on the growth of the tumors.
TABLE 3 tumor weight and tumor inhibition in mice of each group
Note:*P<0.05，**P<0.01，***P<0.001vs model set;#P<0.05，##P<0.01，###P<0.001vs positive control drug group
As can be seen from the experimental data shown in table 3, compared with the model group, the composition of example 1 has a significant inhibitory effect (P <0.001) on the solid tumor of mouse H22, and has concentration dependence, and the inhibitory effect is enhanced with the increase of the dosage; the tumor inhibition effect of the medium and high dose groups is close to that of a positive control drug, no significant difference exists, and P is greater than 0.05.
Experiments prove that the compositions of examples 2, 3 and 4 have obvious inhibition effect on mouse H22 solid tumor.
TABLE 4 spleen and thymus indices of mice from each group were tested
Note:*P<0.05，**P<0.01，***P<0.001vs model set;#P<0.05，##P<0.01，###P<0.001vs positive control drug group
The experimental results in table 4 show that the middle and high doses of the composition of example 1 can significantly improve spleen index and thymus index of tumor-bearing mice, and improve the innate immunity of the mice.
Experiments prove that the spleen index and the thymus index of tumor-bearing mice can be obviously improved by the medium and high doses of the compositions of the examples 2, 3 and 4, and the inherent immunity of the mice is improved.
Influence of the drug of the invention on the survival quality of tumor-bearing mice
Experimental methods and results:
the body weight, food intake and water intake of the mice were measured daily. The water and food efficiency of the mice was calculated. Feeding efficiency ═ (increased body weight/food intake) × 100%; water uptake efficiency (weight gain/water uptake) × 100%. The results of the experiment are shown in FIG. 3.
As can be seen from FIG. 3, compared with the positive control drug, the composition of example 1 can significantly improve the water-food efficiency (P <0.001) of tumor-bearing mice, and greatly improve the quality of life of the tumor-bearing mice.
Experiments prove that the compositions of the examples 2, 3 and 4 can obviously improve the water and food efficiency of tumor-bearing mice and greatly improve the living quality of the tumor-bearing mice.
The influence of the drug of the invention on related enzymes in the liver of tumor-bearing mice
Experimental methods and results:
the activities of hydrogen peroxide (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminase (AST) and the content of Malondialdehyde (MDA) in the liver tissue homogenate were measured. The determination kit is purchased from Nanjing institute of bioengineering (with the serial numbers of A007-1, A005, A001-3, C009-2, C010-2 and A003-1). The amounts of relevant enzymes in the livers of the mice in each group are shown in Table 5.
TABLE 5 experiment of liver content of relevant enzymes in groups of mice
Note:aP<0.05，bP<0.01，cP<0.001vs model set
As is clear from the results in Table 5, the composition of example 1 of the present invention was found to increase the activities of the antioxidants CAT, GSH-Px and SOD in the liver and to decrease the level of MDA in the liver, and to have concentration dependency. In addition, the composition of example 1 can significantly reduce the activity of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in liver, reduce liver toxicity, and reduce the generation of liver inflammation. Compared with the model group, the positive drug group mice have increased transaminase in the liver, which indicates that the chemotherapy drug cyclophosphamide has greater toxicity to the liver.
Experiments prove that the compositions of examples 2, 3 and 4 improve the activity of antioxidant enzymes CAT, GSH-Px and SOD in liver, reduce the level of MDA in liver and have concentration dependence. Can obviously reduce the activity of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in liver, reduce hepatotoxicity and reduce the generation of liver inflammation. Compared with the model group, the positive drug group mice have increased transaminase in the liver, which indicates that the chemotherapy drug cyclophosphamide has greater toxicity to the liver.
Effect of the composition of the present invention on the level of immune factors in the serum of tumor-bearing mice
Experimental methods and results:
blood is taken by an eyeball taking method the next day after each group of mice are stopped, the blood serum is centrifugally separated after standing, the content of Vascular Endothelial Growth Factor (VEGF), interleukin (IF-2) and tumor necrosis factor (TNF- α) in the blood serum of tumor-bearing mice is detected by an ELIAS A method, and the result is shown in Table 6.
TABLE 6 content of vascular endothelial growth factor, interleukin 2 and tumor necrosis factor α in serum of mice of each group
Note:*P<0.05，**P<0.01，***P<0.001vs model set;#P<0.05，##P<0.01，###P<0.001vs positive control drug group
As shown in Table 6, the composition of example 1 can reduce the expression of tumor vascular endothelial growth factor, increase the expression of interleukin 2 and tumor necrosis factor α, and improve the immunity of the organism.
Experiments prove that the compositions of examples 2, 3 and 4 can reduce the expression of tumor vascular endothelial growth factor, improve the expression of interleukin 2 and tumor necrosis factor α, and improve the immunity of the organism.
EXAMPLE 1 pathological section of tumor in tumor-bearing mice is shown in FIG. 4(A-E represent, respectively, model group, positive control group, low dose group, medium dose group, and high dose group)
The tumor cells of the administration group are not as active as those of the model group, part of the tumor cells are closely arranged and part of the tumor cells are loose, the high-dose group shows multiple-focus and large-sheet tumor tissue necrosis, a large part of coagulative necrosis can be seen, and blood vessels and fibrous tissue hyperplasia can be seen around the tumor; a small amount of inflammatory cell infiltration.
Claims (4)
1. A preparation method of a tea polyphenol and tea polysaccharide composition with synergistic, attenuated and anti-liver cancer effects is characterized by comprising the following steps:
(1) preparing tea polyphenol: taking tea leaves, crushing the tea leaves, and adding 10-20 times by mass of ethanol water solution with volume concentration of 75% -99.7%; standing for 24-72 h, and performing vacuum filtration to obtain filtrate and tea residues; extracting the filtrate for 3-7 times by using ethyl acetate of which the amount is 1-3 times that of the filtrate, standing for layering, combining upper layers, and concentrating the combined solution under reduced pressure to dryness to obtain crude tea polyphenol; dissolving tea crude polyphenol in ethanol, adding the solution onto a treated macroporous adsorption resin column, eluting the column with water until the eluent is colorless, eluting the column with 3-5 column volumes of NaOH aqueous solution with the mass concentration of 0.2-0.4% to remove pigment, eluting the column with 10-12 column volumes of ethanol aqueous solution with the volume concentration of 50-90%, collecting the eluent, concentrating and drying to obtain the tea polyphenol with the purity of more than 90%;
(2) preparing tea polysaccharide: adding 10-20 mass times of deionized water into the tea residue obtained in the step (1), carrying out hot leaching for 2-3 times at 70-90 ℃, carrying out suction filtration to obtain a leaching solution, concentrating the leaching solution to 10-20% of the volume of the leaching solution to obtain a concentrated solution, adding an ethanol aqueous solution with the volume concentration of 95-99.7% which is 2.5-4 volume times of the concentrated solution, standing for 12-24 h, centrifuging at 3000-5000 rpm for 3-10 min, dissolving the precipitate in water, sequentially carrying out ultrafiltration on the precipitate by hollow cellulose membranes with the molecular weights of 100kDa and 80kDa to obtain a component with the molecular weight of 80kDa-100kDa, concentrating and drying to obtain tea polysaccharide;
(3) and uniformly mixing the tea polyphenol and the tea polysaccharide according to the mass ratio of 0.5-2: 1 to obtain the tea polyphenol and tea polysaccharide composition with the synergistic, attenuated and anti-liver cancer effects.
2. The method according to claim 1, wherein the macroporous adsorbent resin is of the type: AB-8, D-101, DM-130, HPD-100 or H103.
3. A tea polyphenol tea polysaccharide composition with synergistic attenuation and anti-liver cancer effects prepared by the method of claim 1 or 2.
4. A pharmaceutical preparation comprising the composition of claim 3, which is characterized in that the tea polyphenol tea polysaccharide composition with the effects of synergy, attenuation and anti-liver cancer is prepared with pharmaceutically acceptable auxiliary materials.
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| CN1348817A (en) * | 2001-11-05 | 2002-05-15 | 黄宝生 | Production process of natural anticarcinogen |
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| CN1348817A (en) * | 2001-11-05 | 2002-05-15 | 黄宝生 | Production process of natural anticarcinogen |
| CN1837201A (en) * | 2006-02-21 | 2006-09-27 | 张守政 | Process for extracting tea polyphenol, theanine, tea polysaccharide and tea pigment from tea |
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