Application of high flavonoid glycoside green tea in preparation of medicine for blocking colon cancer cell diffusion

Abstract

The invention discloses an application of high flavonoid glycoside green tea in preparation of a medicine for blocking colon cancer cell diffusion in the technical field of medicines, and the application way is as follows: fresh green tea leaves are taken as raw materials, and a flavonoid glycoside-containing green tea extract is obtained through extraction; and adding pharmaceutically acceptable auxiliary materials into the green tea extract containing the flavonoid glycoside serving as an auxiliary component and a platinum medicine to prepare a clinically available medicine dosage form. The green tea extract containing the flavonoid glycoside has a certain inhibition effect on the growth of human colorectal cancer cell line HCT 116 cells, and achieves a synergistic effect on blocking the proliferation and diffusion of the HCT 116 cells when being compounded with a platinum drug such as oxaliplatin, so that the green tea extract containing the flavonoid glycoside can be used as an auxiliary synergistic component to be applied to preparation of drugs for blocking the diffusion of the colon cancer cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of high-flavonoid glycoside green tea in the preparation of drugs to block the spread of colon cancer cells. Background Technology

[0002] Green tea is one of the main types of tea in China. It refers to tea made from unfermented new leaves or buds of tea trees through steps such as fixing, shaping, and drying. As an unfermented tea, it retains the natural substances of fresh leaves. Most of the natural substances such as tea polyphenols, flavonoids, chlorophyll, amino acids, and vitamins are retained. These natural nutrients have special effects on anti-aging, sterilization, anti-inflammation, lipid reduction and weight loss, and anti-cancer.

[0003] Green tea flavonoids are an important component of green tea extracts. Traditional extraction methods include alcohol extraction, hot water extraction, alkaline extraction, and enzymatic extraction. Green tea flavonoids possess strong biological activity. Besides the common anti-inflammatory, fat-reducing, antioxidant, antibacterial, and coronary artery-dilating effects of green tea, studies have shown that they can also slow the progression of liver fibrosis and even potentially reverse early-stage cirrhosis. However, there are relatively few reports on their application in blocking the spread of colon cancer cells. In-depth research on green tea flavonoids can expand their application prospects in the pharmaceutical, health, food, and cosmetic industries. Based on this, this invention proposes the application of high-flavonoid-glycoside green tea in the preparation of drugs to block the spread of colon cancer cells. Summary of the Invention

[0004] The purpose of this invention is to address existing technical problems by proposing the application of high-flavonoid glycoside green tea in the preparation of drugs to block the spread of colon cancer cells.

[0005] The present invention achieves the above objectives through the following technical solutions: This invention provides the application of high-flavonoid glycoside green tea in the preparation of drugs to block the spread of colon cancer cells.

[0006] As a further optimization of the present invention, the application method is as follows: using fresh green tea leaves as raw material, extracting green tea extract containing flavonoid glycosides, and then using the green tea extract containing flavonoid glycosides as an auxiliary component and platinum-based drugs together with pharmaceutically acceptable excipients to prepare a clinically usable drug dosage form.

[0007] As a further optimization of the present invention, the platinum-based drug is oxaliplatin.

[0008] As a further optimization of the present invention, the extraction method includes the following steps: (1) Using fresh green tea leaves as raw material, the alcohol extract is obtained by first extracting with alcohol and then by any one of the following auxiliary extraction methods: ultrasonic extraction, microwave extraction, or compound enzyme extraction. (2) The alcohol extract was extracted and concentrated under reduced pressure to obtain a concentrated solution; (3) The concentrated solution was subjected to three-stage adsorption using macroporous adsorption resin, microporous adsorption resin and size exclusion chromatography resin to obtain a green tea extract containing flavonoid glycosides.

[0009] As a further optimization of the present invention, the compound enzyme is cellulase at a dosage of 1% of the dry matter weight of the fresh leaves and pectinase at a dosage of 0.5% of the dry matter weight of the fresh leaves.

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

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

[0012] As a further optimization of the present invention, the excipient is one or more of carbon microcrystalline cellulose, starch, lactose, crospovidone, and hydroxypropyl cellulose.

[0013] The beneficial effects of this invention are as follows: This invention uses fresh green tea leaves as raw material. First, an alcohol extract is obtained through extraction combined with ultrasonic, microwave, or compound enzyme-assisted extraction methods. Then, a concentrated extract is obtained through extraction and vacuum concentration. Finally, a green tea extract containing flavonoid glycosides is obtained through tertiary resin adsorption. MTT assays showed that this extract inhibited the growth of human colorectal cancer cell line HCT 116. Subcutaneous tumor-bearing experiments in nude mice demonstrated that the green tea extract containing flavonoid glycosides had a certain inhibitory activity on the proliferation and spread of HCT 116 cells. Furthermore, when combined with oxaliplatin, a platinum-based drug, a synergistic effect was achieved in inhibiting the proliferation and spread of HCT 116 cells. Detailed Implementation

[0014] The present application will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0015] In the following examples, the preparation methods used are conventional methods known to those skilled in the art unless otherwise specified. For example, the methods for preparing dosage forms such as tablets, capsules, lozenges, injections, and suspensions are all conventional methods. The carriers or excipients used are, unless otherwise specified, commercially available products, including carbon microcrystalline cellulose, starch, lactose, crospovidone, and hydroxypropyl cellulose; and diluents, disintegrants, binders, or lubricants.

[0016] Example 1: Extraction and preparation of green tea extract containing flavonoid glycosides The extraction and preparation method of green tea extract containing flavonoid glycosides is as follows: (1) Pick fresh leaves of Qiancha No. 1 (Camellia sinensis (L.) O. Kuntze. var. sinensis cultivarQiancha 1), which is planted in the tea tree resource nursery of Guizhou Tea Research Institute.

[0017] (2) Extraction of flavonoid glycosides from green tea Fresh leaves were dried and extracted three times by hot reflux at 60 °C using a 70% ethanol aqueous solution. The extracts were combined and concentrated under reduced pressure to remove the ethanol, yielding the alcohol extract. (3) Add water to the alcohol extract and stir until a suspension is formed. Extract the suspension three times with an equal volume of ethyl acetate. Filter the extracted suspension and concentrate under reduced pressure to remove some water to obtain a concentrated solution. (4) Tertiary resin adsorption (4-1) The concentrated liquid was adsorbed using AB 8 type macroporous adsorption resin, and desorbed sequentially with ethanol aqueous solutions with volume fractions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% to obtain a fraction containing flavonoid glycoside compounds. (4-2) The fraction is then adsorbed by MCI small-pore adsorption resin CHP20 / P120, and desorbed sequentially by methanol aqueous solution with volume fractions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% to obtain a secondary fraction containing flavonoid glycoside compounds. (4-3) The secondary fraction was then adsorbed by Toyopearl HW-40 resin and desorbed sequentially by methanol aqueous solutions with volume fractions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% to obtain a green tea extract containing flavonoid glycosides.

[0018] Comparative Example 1 The difference from Example 1 is that ultrasonic-assisted extraction is added during the hot reflux extraction in step (2), and the ultrasonic power is controlled to be 550 W.

[0019] Comparative Example 2 The difference from Example 1 is that microwave-assisted extraction is added during the hot reflux extraction in step (2), and the microwave power is controlled to be 24 KW.

[0020] Comparative Example 3 The difference from Example 1 is that a compound enzyme extraction is added before step (2). The amount of cellulase is 1% of the dry matter weight of the fresh leaves, and the amount of pectinase is 0.5% of the dry matter weight of the fresh leaves. The mixture is placed at a constant temperature of 60 °C for 30 min for enzymatic hydrolysis. Then, it is extracted three times by hot reflux at 60 °C with a volume fraction of 70% ethanol aqueous solution. The extracts are combined and concentrated under reduced pressure to remove ethanol, thus obtaining the alcohol extract.

[0021] Comparative Example 4 The difference from Example 1 is that step (4) uses two-stage resin adsorption. First, the concentrated liquid is adsorbed using AB 8 type macroporous adsorption resin, and then desorbed with ethanol aqueous solution with volume fractions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% in sequence to obtain a fraction containing flavonoid glycosides. Then, the fraction is adsorbed by Toyopearl HW-40 resin, and desorbed with methanol aqueous solution with volume fractions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% in sequence to obtain a green tea extract containing flavonoid glycosides.

[0022] The green tea extracts containing flavonoid glycosides obtained in Example 1 and Comparative Examples 1-4 are designated as TQW-1, TQW-2, TQW-3, TQW-4 and TQW-5.

[0023] Test Example 1: MTT assay for cell growth inhibition rate Human colorectal cancer cell line HCT 116 (from the cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was revived from cryopreservation. Cells in the logarithmic growth phase were passaged and transferred to a 37 ℃ incubator with 5% CO2 for incubation. When the cells reached about 80% confluence, the adherent cells were passaged. Following the principle of one-to-three passage, one-third of the cell suspension was aspirated into the culture flask, and then 6-8 mL of complete culture medium (a mixture of DMEM and FBS at a volume ratio of 9:1) was added. The flask was gently shaken to distribute the cells evenly at the bottom of the culture dish, and the cells were then cultured in the incubator.

[0024] Logarithmic growth phase cells were fed at 1×10 5 Each well was seeded with different concentrations of TQW-1 to TQW-5. After 24 h of incubation, 20 μl of 5 g / L MTT solution was added to each well. After another 24 h of incubation, the supernatant was discarded, and 150 μl of DMSO was added. The mixture was then shaken in the dark for 10 min. The absorbance (A value) of each well at 570 nm was measured using an ELISA reader. The absorbance was compared with that of the control group. 570 Value (denoted as A1), experimental group A570 The value (denoted as A2) is used to calculate the cell growth inhibition rate (%).

[0025] ; Calculate IC based on cell growth inhibition rate (%) 50 The values ​​are shown in Table 1.

[0026] Table 1. Results of Cytotoxicity Assay ; As shown in Table 1, TQW-2, TQW-3, and TQW-4 were more sensitive to HCT 116 cells than TQW-1 and TQW-5. This indicates that in the preparation of green tea extracts containing flavonoid glycosides, combining ultrasonic, microwave, and compound enzyme-assisted extraction with alcohol extraction can effectively enhance the inhibitory effect of the extracts on HCT 116 cells. Therefore, TQW-2 to TQW-4 were further applied to subcutaneous tumor-bearing experiments in nude mice.

[0027] Test Example 2: Subcutaneous Tumor Bearing Experiment in Nude Mice Human colorectal cancer cell line HCT 116 cells were cultured in RPMI-1640 medium (containing 10% FBS) at 37 ℃ in a 5% CO2 incubator until approximately 80% confluence. Cells were then digested with trypsin-EDTA and resuspended in phosphate buffer to prepare 1×10⁻⁶ cells / mL. 8 A cell suspension of 0.1 mL / mL was placed on ice. 0.1 mL of the cell suspension was injected subcutaneously into the ventral and dorsal sides of 4-5 week old immunodeficient nude mice. Starting 12 days post-transplantation, the mice were randomly divided into 8 groups of 5 mice each.

[0028] The following experimental groups were administered the medication: Experimental group A: Administered 1 mL of TQW-2 at a concentration of 500 μg / mL; Experimental group B: Administered 1 mL of TQW-3 at a concentration of 500 μg / mL; Experimental group C: Administered 1 mL of TQW-4 at a concentration of 500 μg / mL; Experimental group D: 1 mL of TQW-2 at a concentration of 500 μg / mL and 15 μmol / L oxaliplatin was administered, with a volume ratio of 1:1. Experimental group E: 1 mL of TQW-3 at a concentration of 500 μg / mL and 15 μmol / L oxaliplatin was administered, with a volume ratio of 1:1. Experimental group F: 1 mL of TQW-4 at a concentration of 500 μg / mL and oxaliplatin at a concentration of 15 μmol / L were administered, with a volume ratio of 1:1. Control group: Administered 1 mL of oxaliplatin at a concentration of 15 μmol / L; The drugs used in each group were suspended in physiological saline and administered by gavage once daily for 4 consecutive weeks. The control group received an equal volume of physiological saline. Every 2 days, the long and short diameters of the tumor were measured using a digital caliper, and the tumor volume and specific tumor volume were calculated according to the following formula.

[0029] Tumor volume (TV) = Tumor long diameter (mm) × Tumor short diameter 2 (mm 2 ) / 2 Tumor volume ratio (RTV) = tumor volume on the measurement day / tumor volume on the first day of drug administration. The measurement results were statistically analyzed and recorded in the form of χ±SD, as shown in Table 2.

[0030] Table 2. Statistical Analysis of Experimental Results ; Table 2 shows that, compared to the blank control group, TQW-2 to TQW-4 exhibited certain inhibitory activity against the proliferation and spread of HCT 116 cells. Oxaliplatin, a platinum-based drug, has been widely used in clinical anticancer drugs, especially for the treatment of colon cancer. These drugs can block the growth and proliferation of cancer cells by inducing DNA damage within the cells and inhibiting transcription and translation. Table 2 also shows that it possesses excellent inhibitory activity against the proliferation and spread of HCT 116 cells. Compared with the experimental group DF, the combined use of TQW-2 to TQW-4 with oxaliplatin showed a certain degree of improvement in the activity of inhibiting the proliferation and spread of HCT 116 cells compared with the individual use of TQW-2 to TQW-4. In particular, the combined use of TQW-4 with oxaliplatin showed a certain degree of improvement in the activity of inhibiting the proliferation and spread of HCT 116 cells compared with the use of oxaliplatin alone. This indicates that the combination of the two achieved a synergistic effect in inhibiting the proliferation and spread of HCT 116 cells.

[0031] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of high-flavonoid glycoside green tea in the preparation of drugs to block the spread of colon cancer cells.

2. The application according to claim 1, characterized in that, The application method is as follows: using fresh green tea leaves as raw material, extracting green tea extract containing flavonoid glycosides, and then using the green tea extract containing flavonoid glycosides as an auxiliary component and platinum-based drugs together with pharmaceutically acceptable excipients to prepare a clinically usable drug dosage form.

3. The application according to claim 2, characterized in that, The platinum-based drug in question is oxaliplatin.

4. The application according to claim 2, characterized in that, The extraction method includes the following steps: (1) Using fresh green tea leaves as raw material, the alcohol extract is obtained by first extracting with alcohol and then by any one of the following auxiliary extraction methods: ultrasonic extraction, microwave extraction, or compound enzyme extraction. (2) The alcohol extract was extracted and concentrated under reduced pressure to obtain a concentrated solution; (3) The concentrated solution was subjected to three-stage adsorption using macroporous adsorption resin, microporous adsorption resin and size exclusion chromatography resin to obtain a green tea extract containing flavonoid glycosides.

5. The application according to claim 4, characterized in that, The compound enzyme consists of cellulase at a dosage of 1% of the dry weight of fresh leaves and pectinase at a dosage of 0.5% of the dry weight of fresh leaves.

6. The application according to claim 2, characterized in that, The drug dosage form is one or more of tablets, capsules, lozenges, injections, or suspensions.

7. The application according to claim 2, characterized in that, The excipients are carriers or excipients.

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