Application of flavonoid glycoside in tea tree in preparation of medicine for inhibiting gastric cancer cell diffusion

Abstract

The invention discloses application of flavonoid glycoside in tea trees in preparation of drugs for inhibiting gastric cancer cell spread in the technical field of medicines, and the flavonoid glycoside in the tea trees is prepared by the following steps: taking fresh leaves of tea trees as raw materials, firstly performing alcohol extraction to obtain an alcohol extract, then performing extraction and vacuum concentration to obtain a concentrated solution, and finally performing vacuum concentration to obtain the flavonoid glycoside in the tea trees. And adsorbing by using macroporous adsorption resin, small-pore adsorption resin and size exclusion chromatographic resin in sequence to obtain the tea extract rich in flavonoid glycoside. The tea leaf extract rich in flavonoid glycoside has the activity of inhibiting growth of gastric cancer cells, the effect of resisting proliferation activity of the gastric cancer cells is improved to a certain extent by compounding the tea leaf extract rich in flavonoid glycoside with irinotecan, and the synergistic effect is achieved; therefore, the tea leaf extract rich in flavonoid glycoside can be used as an auxiliary synergistic component to be applied to preparation of drugs for inhibiting gastric cancer cell diffusion.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of flavonoid glycosides from tea trees in the preparation of drugs that inhibit the spread of gastric cancer cells. Background Technology

[0002] Tea plants contain numerous secondary metabolites, including flavonoids, phenolic acids, lignin, and plant hormones. Among these, flavonoids are characteristic substances constituting the quality and functional components of tea, and are important compounds contributing to the health benefits of tea. Flavonoid glycosides, obtained through glycosylation modification of flavonoids, possess various biological activities. For example, they exhibit significant free radical scavenging capabilities, inhibiting ROS generation, mitigating cellular oxidative damage, contributing to anti-aging and cell protection, regulating the expression of inflammatory factors (such as TNF-α and IL-6), and enhancing the body's immune response, making them suitable for chronic inflammation and immune regulation-related functions.

[0003] Flavonoid glycosides in tea mainly include flavonol glycosides, flavonoid glycosides, and anthocyanin glycosides, often existing in the form of O-glycosides or C-glycosides. Common aglycones include quercetin and kaempferol. Myricetin, apigenin, and other aglycones are linked to monosaccharides such as glucose, rhamnose, and galactose through glycosidic bonds to form various glycosidic forms, such as quercetin-3-O-glucoside, kaempferol-3-O-galactoside, and myricetin-3-O-rhamnoside. Flavonoid glycosides can be extracted from tea trees or produced through biosynthesis.

[0004] Flavonoid glycosides in tea plants are not only diverse and structurally complex, but also exhibit multiple biological activities, including antioxidant, antibacterial, anti-inflammatory, insecticidal, and sensory modulation. With the development of omics technologies and synthetic biology, their application potential in functional foods, natural medicines, plant protection, and cosmetics will continue to be explored. Based on this, this invention proposes the application of flavonoid glycosides from tea plants in the preparation of drugs that inhibit the spread of gastric cancer cells. Summary of the Invention

[0005] The purpose of this invention is to address the existing technical problems by proposing the application of flavonoid glycosides from tea in the preparation of drugs that inhibit the spread of gastric cancer cells.

[0006] The present invention achieves the above objectives through the following technical solutions: This invention provides the application of flavonoid glycosides from tea in the preparation of drugs that inhibit the spread of gastric cancer cells.

[0007] As a further optimization scheme of the present application, the flavonoid glycoside in the tea tree refers to a tea leaf extract rich in flavonoid glycoside obtained by using fresh tea leaves as raw materials, first obtaining an alcohol extract by alcohol extraction, then obtaining a concentrated solution by extraction and reduced pressure concentration, and then sequentially using macroporous adsorption resin, small-pore adsorption resin and size exclusion chromatography resin adsorption.

[0008] As a further optimization scheme of the present application, the application route is that the tea leaf extract rich in flavonoid glycoside is added as an auxiliary synergistic component together with a topoisomerase inhibitor into a pharmaceutically acceptable excipient to form a clinically available drug dosage form.

[0009] As a further optimization scheme of the present application, the topoisomerase inhibitor includes irinotecan, which belongs to a semi-synthetic water-soluble camptothecin derivative. Irinotecan and its metabolite SN38 are both DNA topoisomerase I inhibitors. The complex formed by irinotecan, Topo I and DNA can cause DNA single-strand breakage, prevent DNA replication and inhibit RNA synthesis, and is specific to the S phase of the cell cycle. Studies have shown that irinotecan has broad-spectrum anti-tumor activity on multiple tumor cell lines in vitro (including murine leukemia, human gastric cancer, lung cancer, breast cancer, etc.) and various experimental tumor models in vivo (Co4 colon cancer, S 15 and Sc 16 gastric cancer, Mx 1 breast cancer, etc.).

[0010] As a further optimization scheme of the present application, the drug dosage form is one or more of tablets, capsules, lozenges, injections, suspensions, suppositories and ointments.

[0011] As a further optimization scheme of the present application, the excipient is a carrier or an excipient.

[0012] As a further optimization scheme of the present application, the excipient is one or more of carbon microcrystalline cellulose, starch, lactose, cross-linked polyvinylpyrrolidone and hydroxypropyl cellulose.

[0013] As a further optimization scheme of the present application, the dose of the tea leaf extract rich in flavonoid glycoside in the medicine is 25-100 mg / kg / d.

[0014] The present application has the following beneficial effects: The present application uses fresh tea leaves as raw materials, first obtains an alcohol extract by alcohol extraction, then obtains a concentrated solution by extraction and reduced pressure concentration, and then sequentially uses macroporous adsorption resin, small-pore adsorption resin and size exclusion chromatography resin adsorption to obtain a tea leaf extract rich in flavonoid glycoside. MTT detection of cell growth inhibition rate test proves that the tea leaf extract rich in flavonoid glycoside has obvious inhibitory effect on the growth of human gastric cancer cell line SGC-7901 cells and has activity of inhibiting the growth of gastric cancer cells. The experiment of subcutaneous tumor-bearing in nude mice proves that the tea extract rich in flavonoid glycosides has anti-proliferation activity on gastric cancer cells in a concentration-dependent manner. In addition, the anti-proliferation activity of the tea extract rich in flavonoid glycosides combined with irinotecan is higher than that of irinotecan alone, which indicates that the tea extract rich in flavonoid glycosides alone has certain anti-proliferation activity on gastric cancer cells, and the anti-proliferation activity of the tea extract rich in flavonoid glycosides combined with irinotecan is improved to a certain extent, achieving a synergistic effect. DETAILED DESCRIPTION

[0015] The following further describes the present application, and it is necessary to point out here that the following detailed description is only used to further illustrate the present application, and cannot be understood as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0016] In the following examples, the preparation methods used are conventional methods known to those skilled in the art, such as the methods for preparing tablet, capsule, lozenge, injection, suspension, suppository, ointment and other dosage forms, and the carrier or excipient materials used, wherein the excipient is carbon microcrystalline cellulose, starch, lactose, cross-linked polyvinylpyrrolidone, hydroxypropyl cellulose; the carrier is diluent, disintegrant, binder or lubricant, and if not specifically stated, they are all commercially available products.

[0017] Example 1, extraction and preparation of tea extract rich in flavonoid glycosides The extraction and preparation method of the tea extract rich in flavonoid glycosides is as follows: (1) Fresh leaves of Qiancha 1 (Camellia sinensis (L.) O. Kuntze. var. sinensis cultivar Qiancha 1) are picked, and the Qiancha 1 is planted in the tea tree resource nursery of Guizhou Tea Research Institute.

[0018] (2) The fresh leaves are dried, and 60 °C hot reflux extraction is performed 3 times using a 70 % volume fraction ethanol aqueous solution, the extract is combined, and the ethanol is removed by concentration under reduced pressure to obtain an alcohol extract; (3) The alcohol extract is stirred with water to form a suspension, the suspension is extracted 3 times with an equal volume of ethyl acetate, the extracted suspension is filtered, and part of the water is removed by concentration under reduced pressure to obtain a concentrated solution; (4) AB 8 type macroporous adsorption resin is used to adsorb the concentrated solution, and 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % and 100 % volume fraction ethanol aqueous solutions are used for desorption in sequence to obtain a fraction containing flavonoid glycoside compounds; (5) The fraction is then adsorbed by MCI small pore adsorption resin CHP20 / P120, and desorbed by methanol aqueous solution with volume fraction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% successively, to obtain a secondary fraction containing flavonoid glycoside compounds; (6) The secondary fraction is then adsorbed by Toyopearl HW-40 resin, and desorbed by methanol aqueous solution with volume fraction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% successively, to obtain a tea extract rich in flavonoid glycosides.

[0019] Example 2, MTT detection of cell growth inhibition rate Human gastric cancer cell line SGC-7901 cells (from the Cell Bank of Shanghai Life Science Research Institute of Chinese Academy of Sciences) are cultured in RPMI-1640 medium (pH 7.2) containing 10% heat-inactivated calf serum, 1x10 5 U / L penicillin and 1x10 5 U / L streptomycin, and cultured at 37°C. After 24 h of cell culture, the tea extract rich in flavonoid glycosides is treated.

[0020] Logarithmic growth phase cells are inoculated at 5000 cells / well in a 96-well plate. After 24 h of inoculation, culture medium containing different concentrations of tea extract rich in flavonoid glycosides is added, with final concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 μg / ml, and the control group is added with fresh culture medium. Each concentration and the control group are set with 6 replicate wells.

[0021] 20 μl of 5 g / L MTT solution is added 4 h before the end of the experiment, and after 4 h of continued culture, the supernatant is discarded, 150 μl of DMSO is added, and after 10 min of shaking, the A value (absorbance) at 492 nm of each well is determined by enzyme-linked immunoassay. According to the A 492 value of the control group (denoted as A1) and the A 492 value of the experimental group (denoted as A2), the cell growth inhibition rate is calculated.

[0022] ; The results are shown in Table 1.

[0023] Table 1, cell growth inhibition rate statistical table ; As can be seen from Table 1, the tea extract rich in flavonoid glycosides has a significant inhibitory effect on the growth of human gastric cancer cell line SGC-7901 cells. In the same action time, with the increase of the drug concentration, the cell inhibition rate increases, showing a concentration-dependent manner. When the concentration of the tea extract rich in flavonoid glycosides is 0.6 μg / ml, the growth inhibition rate of SGC-7901 cells reaches the maximum, which is (88.44±0.5)%, and the change is not significant with the increase of the concentration.

[0024] Example 2, subcutaneous tumor-bearing experiment of nude mice The human gastric cancer cell line SGC 7901 was cultured in an RPMI-1640 medium (containing 10% FBS) at 37 °C in a 5% CO2 incubator until it was about 80% full, then it was digested with trypsin EDTA and resuspended with a phosphate buffer to prepare a suspension of 5×10 7 The cell suspension was placed on ice. 0.1 mL of the cell suspension was injected into the subcutaneous part of the back of each of the 4-5-week-old immunodeficient nude mice. After 12 days of implantation, the mice were randomly divided into 7 groups, with 5 mice in each group.

[0025] The following grouping was used for drug administration: Drug administration group A: 25 mg / kg of the tea extract rich in flavonoid glycosides was administered.

[0026] Drug administration group B: 50 mg / kg of the tea extract rich in flavonoid glycosides was administered.

[0027] Drug administration group C: 75 mg / kg of the tea extract rich in flavonoid glycosides was administered.

[0028] Drug administration group D: 100 mg / kg of the tea extract rich in flavonoid glycosides was administered Drug administration group E: 50 mg / kg of irinotecan was administered.

[0029] Drug administration group F: 50 mg / kg of irinotecan + 25 mg / kg of the tea extract rich in flavonoid glycosides was administered.

[0030] After being suspended in normal saline, the drug was administered by gavage once a day, and the administration was continued for 4 weeks. The blank control group was given an equal amount of normal saline. The long diameter and short diameter of the tumor were measured every 2 days using a digital caliper, and the tumor volume and specific tumor volume were calculated according to the following formula.

[0031] Tumor volume (TV) = tumor long diameter (mm) x tumor short diameter (mm) / 2 2 (mm 2 ) / 2 Relative tumor volume (RTV) = tumor volume on the day of measurement / tumor volume on the first day of administration The results were statistically analyzed, and the results were recorded in the form of χ±SD, as shown in Table 2.

[0032] Table 2, statistical results of the test ; As can be seen from Table 2, compared with the blank control group, the tea extract rich in flavonoid glycosides has an anti-gastric cancer cell proliferation activity, which is concentration-dependent, and as the administration dose increases, the anti-gastric cancer cell proliferation activity also increases, and when the dose exceeds 75 mg / kg, the anti-gastric cancer cell proliferation activity does not increase, and the anti-gastric cancer cell proliferation activity is still less than that of 50 mg / kg irinotecan.

[0033] Comparing administration group E and administration group F, under the same dose, after the tea extract rich in flavonoid glycosides is compounded with irinotecan, the anti-gastric cancer cell proliferation activity is improved to a certain extent compared with the effect of using irinotecan alone, which shows that the tea extract rich in flavonoid glycosides alone has a certain anti-gastric cancer cell proliferation activity, but when it is compounded with irinotecan, the anti-gastric cancer cell proliferation activity is improved to a certain extent, and a synergistic effect is achieved.

[0034] Example 3, preparation of a drug for inhibiting gastric cancer cell diffusion The present embodiment provides a specific preparation method of a tablet for inhibiting gastric cancer cell diffusion, and the specific preparation steps are as follows: taking a single tablet, 75 mg of active ingredients (50 mg / kg of irinotecan and 25 mg / kg of tea extract rich in flavonoid glycosides) are mixed with 10 mg of microcrystalline cellulose, 20 mg of starch and 30 mg of lactose, the mixture is moistened with 75% ethanol and sieved and granulated, and after drying, 4 mg of cross-linked polyvinylpyrrolidone and 2 mg of magnesium stearate are mixed uniformly, and the tablet is obtained by pressing.

[0035] The above embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. Application of flavonoid glycosides from tea in the preparation of drugs that inhibit the spread of gastric cancer cells.

2. The application according to claim 1, characterized in that, The flavonoid glycosides in tea refer to tea extracts rich in flavonoid glycosides obtained by first extracting fresh tea leaves with alcohol, then extracting and concentrating under reduced pressure to obtain a concentrated solution, and then adsorbing the solution sequentially using macroporous adsorption resin, microporous adsorption resin and size exclusion chromatography resin.

3. The application according to claim 2, characterized in that, The application method is as follows: tea extract rich in flavonoid glycosides is added together with topoisomerase inhibitors as pharmaceutically acceptable excipients to prepare a clinically usable drug dosage form.

4. The application according to claim 3, characterized in that, The topoisomerase inhibitors include irinotecan.

5. The application according to claim 3, characterized in that, The drug dosage form is one or more of the following: tablets, capsules, lozenges, injections, suspensions, suppositories, and ointments.

6. The application according to claim 3, characterized in that, The excipients are carriers or excipients.

7. The application according to claim 6, characterized in that, The excipient is one or more of carbon microcrystalline cellulose, starch, lactose, crospovidone, and hydroxypropyl cellulose.

8. The application according to any one of claims 1-7, characterized in that, The dosage of the tea extract rich in flavonoid glycosides in the drug is 25-100 mg / kg / day.