A polysaccharide-assisted tea extract and its preparation method and application

Abstract

The present invention relates to a polysaccharide-assisted tea extract and a preparation method and application thereof. The present invention soaks fresh tea leaves with aloe polysaccharide solution, filters the obtained tea leaves, and then dries them to obtain dry tea leaves. The filtrate obtained by filtration is then used to soak the dry tea leaves again and then filtered. The tea leaves are soaked in pure water and treated with high temperature and high pressure to obtain tea liquid. The tea liquid is concentrated, hydrolyzed with pure water, and treated with high temperature and high pressure to obtain hydrolyzed tea liquid. The filtrate after filtration of the hydrolyzed tea liquid is mixed with the filtrate of the previous step, filtered, concentrated, and dried to obtain the obtained product. The ethanol aqueous solution can dissolve the soluble substances of the aloe polysaccharide, and the insoluble impurities are removed in the subsequent filtering step. The present invention utilizes polysaccharide to assist in extracting effective substances from tea leaves, which can greatly increase the content of EGCG therein, and improve the anti-cancer activity of the tea extract through the synergistic effect of aloe polysaccharide and the active substances in the tea extract.

Description

Technical Field

[0001] The invention relates to the field of plant extracts, and in particular to a polysaccharide-assisted tea extract and a preparation method and application thereof. Background Art

[0002] Polyphenols are the main active ingredients in tea, and they exhibit biological activities including antioxidant, anti-cancer, and anti-aging, making them one of the important sources of polyphenols.

[0003] Although scientific research has shown the remarkable biological activity of polyphenols, large pharmaceutical companies have not adopted these substances into drugs. The main reason is that polyphenols have low bioavailability, low stability, low water solubility, and short biological half-life, which lead to various problems in clinical trials.

[0004] A large number of reports have discussed various tea extraction techniques, including solvent extraction adsorption, microwave solvent extraction, supercritical liquid combined adsorption extraction, etc. However, these extraction methods are based on harmful organic solvents, and the content of the effective substance epigallocatechin gallate (EGCG) in the obtained tea extract is not ideal. Therefore, it is extremely important to obtain a safe and healthy tea extraction method that can increase the content of effective substances in tea extracts. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a polysaccharide-assisted tea extract to increase the content of EGCG in the tea extract, making it possible to use the tea extract as a medicine.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The polysaccharide-assisted tea extract of the present invention is extracted by using aloe extract-aloe polysaccharide as an extraction aid. Aloe polysaccharide is first dissolved and purified with an ethanol aqueous solution to obtain an aloe polysaccharide solution, fresh tea leaves are soaked with the aloe polysaccharide solution, the tea leaves obtained by filtering are dried to obtain dry tea leaves, and then the filtrate obtained by filtering is used to soak the dry tea leaves again and then filtered, the tea leaves are soaked in pure water and treated with high temperature and high pressure to obtain tea liquid, the tea liquid is concentrated, hydrolyzed with pure water, and treated with high temperature and high pressure to obtain hydrolyzed tea liquid, and the filtrate after the hydrolyzed tea liquid is filtered is mixed with the filtrate of the previous step, filtered, concentrated and dried to obtain. The ethanol aqueous solution can dissolve the soluble substances of the aloe polysaccharide, and insoluble impurities are removed in the subsequent filtering step.

[0008] A polysaccharide-assisted tea extract of the present invention is prepared by the following method:

[0009] S1. Mix aloe polysaccharide, 95% edible alcohol by volume, and pure water in a volume ratio of 1:10:20, remove the precipitate, and take the supernatant to obtain an aloe polysaccharide solution;

[0010] S2, soaking fresh tea leaves with the aloe polysaccharide solution, and filtering to obtain aloe polysaccharide tea leaves A and filtrate A after soaking; during soaking, fresh tea leaves and aloe polysaccharide tea solution are added in a mass volume ratio of 5:1;

[0011] S3, sun-drying the aloe polysaccharide tea leaves A to obtain dry tea leaves;

[0012] S4, soaking the dry tea leaves again with the filtrate A obtained in step S2, and then filtering to obtain aloe polysaccharide tea leaves B and filtrate B;

[0013] S5, adding pure water to the aloe polysaccharide tea leaves B, treating under high temperature and high pressure conditions of 121° C. and 0.1-0.15 MPa for 20-40 min to obtain tea liquid A;

[0014] S6, concentrating the tea liquid A to obtain concentrated tea liquid B;

[0015] S7, adding pure water to the tea liquid B, treating under high temperature and high pressure conditions of 121° C. and 0.1-0.15 MPa for 20-40 min to obtain hydrolyzed tea liquid C;

[0016] S8, filtering the tea liquid C to remove the residue to obtain tea liquid D, and then mixing the tea liquid D with the filtrate B in step S4 and concentrating them to obtain a polysaccharide tea extract.

[0017] Tea leaves are susceptible to high temperature, light, humidity, and pH under traditional production processes and storage conditions, and a series of complex chemical reactions occur in the chemical components contained therein, and EGCG is particularly prone to auto-oxidation during storage, and the active ingredient is easily reduced. Therefore, in step S2, the aloe polysaccharide solution soaks fresh tea leaves so that more EGCG in the fresh tea leaves can be retained in the aloe polysaccharide solution. The aloe polysaccharide solution used in step S4 is the aloe polysaccharide solution obtained by filtering after soaking fresh tea leaves in step S2, and finally the aloe polysaccharide solution rich in EGCG is mixed with tea liquid to obtain a product. Step S3 is made into dry tea leaves for easy transportation to the factory for subsequent processing or storage.

[0018] The advantages of the present invention are:

[0019] The present invention utilizes polysaccharide to assist in extracting effective substances from tea, which can greatly increase the content of EGCG therein, and improves the anti-cancer activity of the tea extract through the synergistic effect of aloe polysaccharide and active substances in the tea extract; the tea extract obtained by the method of the present invention can withstand a high temperature of 121°C compared with the ultrasonic water extraction method, so the biological activity of the obtained tea extract is more stable and does not contain substances that are harmful to consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the results of measuring the EGCG content of the polysaccharide tea extract of an embodiment of the present invention;

[0021] Figure 2 This is a graph showing the results of measuring the EGCG content of the ultrasonic tea extract of the comparative example of the present invention;

[0022] Figure 3 The figure is a bar graph showing the apoptosis rate of NCI-H 1975 cells by the tea extracts of the examples of the present invention and the comparative examples;

[0023] Figure 4 The figure is a bar graph showing the apoptosis rate of NCI-H 460 cells by the tea extracts of the examples of the present invention and the comparative example. DETAILED DESCRIPTION

[0024] In order to make the technical personnel in the field understand the present invention more clearly and intuitively, the present invention will be further described below in conjunction with the accompanying drawings. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0025] Preparation of Aloe Polysaccharide

[0026] Extraction of polysaccharides from Aloe vera:

[0027] Peel the aloe vera and take out the aloe vera gel, grind the aloe vera gel into a gel using a gel grinder, stir for 24 hours at a temperature of 15-17°C to liquefy it, filter it after liquefaction, and concentrate the filtered aloe vera liquid at a temperature of 65-90°C to one twentieth of the volume of the original aloe vera liquid to obtain aloe polysaccharide.

[0028] Example

[0029] A method for preparing a polysaccharide-assisted tea extract comprises the following steps:

[0030] S1. Prepare an aloe polysaccharide solution by using 1 L aloe polysaccharide, 10 L 95% edible alcohol by volume, and 20 L pure water;

[0031] S2, soaking about 21L of aloe polysaccharide solution and 4.2kg of fresh Pu'er tea leaves for 48 hours, filtering with a 1000um filter bag to obtain aloe polysaccharide tea leaves A and filtrate A;

[0032] S3, dehydrating the aloe polysaccharide tea leaves A in the sun to obtain dry tea leaves;

[0033] S4, soaking the dry tea leaves again with the filtrate A obtained in step S2, and filtering to obtain aloe polysaccharide tea leaves B and filtrate B;

[0034] S5, mixing aloe polysaccharide tea leaves B and pure water in a weight-to-volume ratio of 1:5, and then treating under high temperature and high pressure conditions of 121° C. and 0.1-0.15 MPa for 30 minutes to obtain tea liquid A;

[0035] S6. Place the tea liquid A on a rotary evaporator, set the temperature to 80° C., and the negative pressure to -0.1 MPa, and concentrate it to 1 / 20 of the original volume to obtain concentrated tea liquid B;

[0036] S7, tea liquid B and pure water are mixed in a weight volume ratio of 1:5, and treated under high temperature and high pressure conditions of 121° C. and 0.1-0.15 MPa for 30 minutes to obtain hydrolyzed tea liquid C;

[0037] S8. Filter the tea liquid C using a 1000 μm filter bag to remove the residue to obtain tea liquid D. Then mix the tea liquid D with the filtrate B in step S4 and use a rotary evaporator with the temperature set to 80° C. and the negative pressure set to -0.1 MPa to concentrate the mixture to 1 / 20 of the original volume to obtain a polysaccharide tea extract.

[0038] The specific steps of reverse phase liquid chromatography for detecting EGCG content are as follows: take about 1g of polysaccharide tea extract and add it to a volumetric flask, dissolve it with an appropriate amount of water and make it up to 1000mL, then filter it with a 0.45μm water-based microporous filter membrane and perform liquid chromatography to detect and calculate the EGCG content in the sample. Figure 1 As shown, after determination, the content of EGC in the diluted polysaccharide tea extract sample was 0.55 mg / mL, the content of EGCG was 2.766 mg / mL (2766 mg / L), and the content of EC was 1.030 mg / mL.

[0039] Comparative Example

[0040] Traditional sun-dried Pu'er tea is extracted with ultrasonic water to obtain ultrasonic tea extract.

[0041] The material-liquid ratio is 1:20. For 100 grams of traditional sun-dried Pu'er tea, the required amount of water is 2000 grams.

[0042] Ultrasonic extraction conditions: ultrasonic power 500 W, ultrasonic time 20 min, extraction temperature 35 ° C, extraction time 2 hours, to obtain ultrasonic tea extract.

[0043] Take 50.0 mg of ultrasonic tea extract and add it to a volumetric flask, add appropriate amount of water to make up to 50 mL, and filter it with a 0.45 μm water-based microporous filter membrane, and then use liquid chromatography to detect and calculate the EGCG content in the sample, such as Figure 2 As shown, after determination, the content of EGC in the ultrasonic tea extract sample was 1.113 mg / mL, the content of EGCG was 1.663 mg / mL (1633 mg / L), and the content of EC was 1.214 mg / mL.

[0044] Functional testing of tea extracts

[0045] 1. NCI-H 1975 cell (human lung adenocarcinoma cell) apoptosis assay

[0046] 1. According to the EGCG concentration test results in the example, the polysaccharide tea extract was diluted with pure water. First, pure water was added to dilute the polysaccharide tea extract aqueous solution with an EGCG concentration of 15.6 mg / L, and then the polysaccharide tea extract aqueous solution with an EGCG concentration of 7.8 mg / L and 3.9 mg / L was further diluted. After adding pure water during preparation, the volumetric flask was sealed and shaken thoroughly.

[0047] Similarly, according to the EGCG concentration test results of the comparative example, the ultrasonic tea extract was diluted with pure water to prepare ultrasonic tea extract aqueous solutions with EGCG concentrations of 15.6 mg / L, 7.8 mg / L, and 3.9 mg / L.

[0048] 2. Take logarithmically growing NCI-H 1975 cells (human lung adenocarcinoma cells) and dispense them into 96-well plates, with 0.1 mL per well (containing about 0.75~12×10 3 Cells in the cell plate were routinely cultured in a culture medium, and the culture medium selected was RPMI 1640 culture medium (GIBCO catalog number: 11875093) supplemented with 10% fetal bovine serum. After culturing for 24 hours at 37°C and 5% CO2, aqueous solutions of tea extracts with different EGCG concentrations in the examples and comparative examples were added to the cells in different wells, respectively. Three parallel wells were set for each concentration. After drug treatment for 24 hours, the apoptosis rate was determined by flow cytometry. The results are shown in Tables 1 and 2. Figure 3 shown.

[0049] Table 1

[0050]

[0051] Effects of ultrasonic tea extract aqueous solution and polysaccharide tea extract aqueous solution on NCI-H 1975 (human lung adenocarcinoma cells): According to the data, the polysaccharide tea extract aqueous solution of the embodiment can cause more apoptosis of NCI-H 1975 cells relative to the ultrasonic tea extract aqueous solution. At EGCG concentrations of 7.8 mg / L and 15.6 mg / L, the polysaccharide tea extract aqueous solution can cause significant apoptosis of NCI-H 1975 cells relative to the ultrasonic tea extract aqueous solution. It can be seen that a low concentration of polysaccharide tea extract aqueous solution can exert the apoptotic effect of a high concentration of ultrasonic tea extract aqueous solution. At the same EGCG concentration, the aloe polysaccharide contained in the present invention can greatly promote the apoptosis of cells.

[0052] II. Apoptosis test of NCI-H 460 cells (human large cell lung cancer cells)

[0053] 1. According to the EGCG concentration test results of the example, the polysaccharide tea extract was diluted with pure water. Polysaccharide tea extract aqueous solutions with EGCG concentrations of 50 mg / L, 25 mg / L, and 12.5 mg / L were prepared successively.

[0054] At the same time, according to the EGCG concentration test results of the comparative example, the ultrasonic tea extract was diluted with pure water to prepare ultrasonic tea extract aqueous solutions with EGCG concentrations of 50 mg / L, 25 mg / L, and 12.5 mg / L.

[0055] 2. Take logarithmically growing NCI-H 460 cells and dispense them into 96-well plates, with 0.1 mL per well (about 1~5×10 4 Cells in the cell plate were routinely cultured in a culture medium, and the culture medium selected was RPMI-1640 culture medium supplemented with 10% fetal bovine serum. After culturing for 24 hours at 37°C and 5% CO2, aqueous solutions of tea extracts with different EGCG concentrations in the examples and comparative examples were added to the cells in different wells, respectively. Three parallel wells were set for each concentration. After 24 hours of drug treatment, the apoptosis rate was determined by flow cytometry. The results are shown in Tables 2 and 3. Figure 4 shown.

[0056] Table 2

[0057]

[0058] Effects of ultrasonic tea extract aqueous solution and polysaccharide tea extract aqueous solution on NCI-H 460 cells (human large cell lung cancer cells): Data show that the polysaccharide tea extract aqueous solution of the embodiment can cause more apoptosis in NCI-H460 cells relative to the ultrasonic tea extract aqueous solution. When the EGCG concentration is 25 mg / L and 50 mg / L, the polysaccharide tea extract aqueous solution can cause significant apoptosis in NCI-H 460 cells relative to the ultrasonic tea extract aqueous solution. It can be seen that a low concentration of polysaccharide tea extract aqueous solution can exert the apoptotic effect of a high concentration of ultrasonic tea extract aqueous solution. At the same EGCG concentration, the aloe polysaccharide contained in the present invention can greatly promote the apoptosis of cells.

[0059] In conclusion, in the tea extract obtained by aloe polysaccharide-assisted extraction, aloe polysaccharide and EGCG can synergistically promote the apoptosis of lung cancer cells.

[0060] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the embodiments herein, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a polysaccharide-assisted tea extract, characterized in that: The steps include: S1. Mix aloe polysaccharide, 95% by volume ethanol aqueous solution and pure water, remove the precipitate and take the supernatant to obtain an aloe polysaccharide solution; S2, soaking fresh tea leaves with the aloe polysaccharide solution, and filtering to obtain aloe polysaccharide tea leaves A and filtrate A after the soaking; S3, sun-drying the aloe polysaccharide tea leaves A to obtain dry tea leaves; S4, soaking the dry tea leaves again with the filtrate A obtained in step S2, and filtering to obtain aloe polysaccharide tea leaves B and filtrate B; S5, adding pure water to the aloe polysaccharide tea leaves B, and subjecting the mixture to high temperature and high pressure treatment to obtain tea liquid A; S6, concentrating the tea liquid A to obtain concentrated tea liquid B; S7, adding pure water to the tea liquid B, and subjecting it to high temperature and high pressure treatment to obtain hydrolyzed tea liquid C; S8, filtering the tea liquid C to remove the residue to obtain tea liquid D, and then mixing the tea liquid D with the filtrate B in step S4 and concentrating them to obtain a polysaccharide tea extract.

2. The method for preparing a polysaccharide-assisted tea extract according to claim 1, characterized in that: In step S1, aloe polysaccharide, 95% by volume ethanol aqueous solution, and pure water are mixed in a volume ratio of 1:10:

20.

3. The method for preparing a polysaccharide-assisted tea extract according to claim 1, characterized in that: In step S5 and / or S7, the high temperature and high pressure conditions are 121° C., pressure 0.1-0.15 MPa, and the processing time is 20-40 min.

4. The method for preparing a polysaccharide-assisted tea extract according to claim 1, characterized in that: In step S6 and / or S8, the volume needs to be concentrated to 1 / 20 of the original volume.

5. The method for preparing a polysaccharide-assisted tea extract according to claim 1, characterized in that: In step S5, aloe polysaccharide tea leaves B and pure water are mixed in a weight-to-volume ratio of 1:

5.

6. The method for preparing a polysaccharide-assisted tea extract according to claim 1, characterized in that: In step S7, the tea liquid B and pure water are mixed in a weight-to-volume ratio of 1:

5.

7. A polysaccharide tea extract obtained by the method for preparing a polysaccharide-assisted tea extract according to any one of claims 1 to 6.

8. Use of the polysaccharide tea extract obtained by the method for preparing a polysaccharide-assisted tea extract according to any one of claims 1 to 6 in preparing a drug for preventing and treating lung cancer.

9. A drug, characterized in that A polysaccharide tea extract prepared by the method for preparing a polysaccharide-assisted tea extract according to any one of claims 1 to 6.

10. The use of aloe polysaccharide as an extraction aid in increasing the EGCG content in tea extract, characterized in that: The tea extract is a tea extract obtained by the method for preparing a polysaccharide-assisted tea extract according to any one of claims 1 to 6.

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