Tea polysaccharide-polyphenol compound as well as extraction method and application thereof

Abstract

The invention belongs to the technical field of plant extraction, and particularly relates to a tea polysaccharide-polyphenol compound as well as an extraction method and application thereof. According to the extraction method provided by the invention, a tea powder solution is treated by adopting a pulsed electric field to strengthen extraction, the field intensity of the pulsed electric field is 5.3 kV / cm, the flow velocity is 1.25 mL / s, pulse circulation is performed for 4 times, each time is 5 minutes, and then the tea polysaccharide-polyphenol compound is obtained through the steps of removing protein, pigment and the like. The extraction method disclosed by the invention has the advantages of being green, low in solvent toxicity, safe to operate, short in time and low in energy consumption, and lays a foundation for broadening the research and development of the tea polysaccharide-polyphenol compound in the aspects of food, medicine and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and specifically relates to a tea polysaccharide-polyphenol complex, an extraction method thereof, and an application thereof. Background Art

[0002] A polysaccharide-polyphenol complex is a complex formed by two bioactive components, polysaccharide and polyphenol, through covalent or non-covalent binding. It is reported that naturally occurring polysaccharide-polyphenol complexes have antithrombotic, antioxidant, radiation protection, antiplatelet, antitussive, and bronchodilatory effects. In recent years, some medicinal plants of the Compositae and Rosaceae families have been proven to be valuable resources of natural polysaccharide-polyphenol complexes, such as Melastoma candidum, Echinacea purpurea, Fragaria vesca leaves, Chamomilla recutita, Sanguisorba officinalis, etc. Since ancient times, tea has been used as a health product or medicine to prevent and treat various diseases and is a representative of medicine and food homology. The functions of its main components, tea polysaccharide and tea polyphenol, such as antidiabetic, antioxidant, antitumor, and enhancing immune regulation ability, have also been widely studied. However, there is no report on the research of tea polysaccharide-polyphenol complexes. Therefore, it is of great significance to develop an extraction method for tea polysaccharide-polyphenol complexes.

[0003] In view of the limited yield and complex structure of natural polysaccharide-polyphenol complexes, there are also studies on synthesizing polysaccharide-polyphenol complexes by chemical synthesis methods to increase their yield. However, the safety of the synthesized polysaccharide-polyphenol complexes remains to be evaluated. Currently, the main extraction methods for natural polysaccharide-polyphenol complexes are hot alkali extraction method and organic solvent extraction method, and these extraction methods have cumbersome steps, long time consumption, and use a large amount of organic reagents. Summary of the Invention

[0004] Based on the above technical problems, the purpose of the present invention is to provide a tea polysaccharide-polyphenol complex, an extraction method thereof, and an application thereof.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The present invention provides an extraction method for a tea polysaccharide-polyphenol complex, comprising the following steps: S1. Take dry green tea leaves, grind them into powder, and mix them with water to obtain a tea powder solution; S2. After treating the tea powder solution with pulsed electric field, perform extraction, centrifugation, and concentration to obtain a first concentrated solution. Remove proteins from the first concentrated solution to obtain a second concentrated solution. Remove pigments from the second concentrated solution to obtain a third concentrated solution. Perform alcohol precipitation and centrifugation on the third concentrated solution, collect the precipitate, remove the excess ethanol in the precipitate, and then dry it to obtain the tea polysaccharide-polyphenol complex; The conditions for the pulsed electric field treatment are: the field strength is 5.3 kV / cm, the flow rate is 1.25 mL / s, the pulse cycle is 4 times, and each time is 5 min.

[0006] The extraction method of the tea polysaccharide-polyphenol complex provided by the present invention is enhanced by pulsed electric field treatment, and the extraction efficiency and product quality are significantly improved. The tea polysaccharide-polyphenol complex mainly exists in the cell wall, and the pulsed electric field mainly causes electroporation of the cell wall through high voltage, thereby accelerating the exudation of substances in the cell wall and increasing the extraction efficiency. After pulsed electric field treatment, the cell wall is damaged, which promotes the extraction of the tea polysaccharide-polyphenol complex. Specifically, polyphenols are soluble in ethanol, while tea polysaccharides are insoluble in ethanol. Therefore, in the alcohol precipitation step, the free polyphenols will dissolve in the supernatant, and the precipitate is tea polysaccharides and bound polyphenols. The method for extracting the tea polysaccharide-polyphenol complex collects the precipitate. Therefore, the final precipitate contains both tea polysaccharides and a certain amount of bound polyphenols. The precipitate is detected by an ultraviolet spectrophotometer, which proves that the tea polysaccharide-polyphenol complex is extracted in the precipitate. The extraction method of the present invention reduces the use of organic solvents and has the advantages of simple operation steps and short time.

[0007] Further, the material-liquid ratio in the leaching is 1 g: 30 mL, and the leaching conditions are 90 °C and 4.5 h.

[0008] Further, the centrifugation conditions are all 9000 rpm / min for 15 min.

[0009] Further, the Sevage method is used to remove proteins from the first concentrated solution. Specifically, the first concentrated solution is mixed with a chloroform-n-butanol mixture with 3 times its volume, shaken and then allowed to stand for stratification, and the upper clear liquid is retained, which is the second concentrated solution. The volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixture is 4:1.

[0010] Further, removing pigments from the second concentrated solution is specifically: adding 5% (by volume fraction) of H 2 O 2 to the second concentrated solution, and keeping it at 50 °C for 2 h for decolorization to remove pigments in the second concentrated solution.

[0011] Further, alcohol precipitation of the third concentrated solution is specifically: mixing the third concentrated solution with 4 times its volume of ethanol and allowing it to stand at 4 °C for 12 h.

[0012] The present invention also provides a tea polysaccharide-polyphenol complex prepared by the above method.

[0013] The present invention also provides the application of the tea polysaccharide-polyphenol complex in the preparation of antioxidant drugs.

[0014] The present invention has the following beneficial effects: The present invention significantly improves the extraction efficiency and product quality of tea polysaccharide-polyphenol complexes by introducing pulsed electric field enhanced extraction technology. In addition, the process conditions are more green and efficient, with a short pulsed electric field treatment time, low energy consumption, water as the main solvent, reducing the use of organic reagents, and the operation process is also more simplified. The present invention conforms to the concept of green chemistry, has a lower cost, and the high-purity and high-activity complexes have broad application prospects in the fields of food, health products and medicine. Description of the Drawings

[0015] Figure 1 It is the ultraviolet absorption spectrum diagram of the tea polysaccharide-polyphenol complex extracted in Example 1.

[0016] Figure 2 It is the percentage diagram of polyphenol and total sugar contents of the tea polysaccharide-polyphenol complexes extracted under different electric field intensities. Among them, lowercase letters represent the inter-group difference analysis. The same letters indicate no significant difference P > 0.05, different letters indicate significant differences P < 0.05.

[0017] Figure 3 It is the percentage diagram of polyphenol and total sugar contents of the tea polysaccharide-polyphenol complexes extracted under different flow rates. Among them, lowercase letters represent the inter-group difference analysis. The same letters indicate no significant difference P > 0.05, different letters indicate significant differences P < 0.05.

[0018] Figure 4 It is the percentage diagram of polyphenol and total sugar contents of the tea polysaccharide-polyphenol complexes extracted under different cycle numbers. Among them, lowercase letters represent the inter-group difference analysis. The same letters indicate no significant difference P > 0.05, different letters indicate significant differences P < 0.05.

[0019] Figure 5 It is the DPPH free radical scavenging rate diagram. Detailed Description of the Invention

[0020] The present invention will be described in detail below with specific examples, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0021] Example 1: Extraction and identification of tea polysaccharide-polyphenol complexes.

[0022] 1. Experimental method.

[0023] (1) Extraction of tea polysaccharide-polyphenol complexes.

[0024] The dry green tea leaves are ground into powder and mixed with water at a mass-to-volume ratio of 1 g: 30 mL to obtain a tea powder solution.

[0025] The tea powder solution is placed in a pulsed electric field for treatment. The pulsed electric field strength is 5.3 kV / cm, the flow rate is 1.25 mL / s, the pulse cycle is 4 times, and each time is 5 min.

[0026] The product treated by the pulsed electric field is extracted at 90 °C for 4.5 h, centrifuged at 9000 rpm / min for 15 min, and concentrated to obtain a first concentrated solution.

[0027] The first concentrated solution is mixed with a chloroform-n-butanol mixture with 3 times its volume, shaken for 20 min, then transferred to a separating funnel and allowed to stand for stratification to separate the protein. The above operation is repeated until there is no flocculent precipitate in the lower layer, and the supernatant is taken out, which is the second concentrated solution.

[0028] Add H 2 O 2 accounting for 5% of its volume to the second concentrated solution, and keep it at 50 °C for 2 h for decolorization to remove the pigments in the solution to obtain a third concentrated solution.

[0029] The third concentrated solution is mixed with ethanol with 4 times its volume, allowed to stand at 4 °C for 12 h for alcohol precipitation, centrifuged, and the precipitate is collected, dissolved in a small amount of ultrapure water, and the ethanol is removed by rotary evaporation to obtain an extract. The extract is poured into a petri dish, frozen in a -80 °C refrigerator, and freeze-dried in a vacuum freeze dryer for 72 h to obtain a tea polysaccharide-polyphenol complex.

[0030] (2) Identification of the tea polysaccharide-polyphenol complex.

[0031] Prepare solutions of gallic acid, tea polysaccharide and tea polysaccharide-polyphenol complex with a mass concentration of 1 mg / mL, where the tea polysaccharide-polyphenol complex is obtained by extraction in the above steps. Use an ultraviolet spectrophotometer to scan in the wavelength range of 190 nm to 490 nm.

[0032] 2. Experimental results.

[0033] The experimental results are as Figure 1 shown: The carboxyl groups in the polysaccharide part and the carboxyl and carbonyl groups derived from the polyphenol part will produce absorption peaks at a wavelength of about 212 nm. The absorption peaks of gallic acid at 218 nm and 263 nm belong to the π-system of the benzene ring. The absence of an absorption peak of tea polysaccharide at 265 nm indicates that this method can fully remove free phenols, while the absorption peak produced by the polysaccharide-polyphenol complex at 265 nm indicates the successful extraction of the tea polysaccharide-polyphenol complex.

[0034] Verification Example 1: Influence of pulsed electric field conditions on the extraction effect of the tea polysaccharide-polyphenol complex.

[0035] 1. Experimental method.

[0036] (1) Optimization of pulsed electric field conditions.

[0037] To prove the effects of pulsed electric field on the contents of polysaccharides and polyphenols, the following test treatments were carried out: The influencing factors set included: pulsed electric field intensities of 3.3 kV / cm, 4 kV / cm, 4.6 kV / cm, 5.3 kV / cm, and 6 kV / cm respectively; cycle numbers of 1, 2, 3, 4, and 5 times respectively; flow rates of 1.25 mL / s, 1.67 mL / s, 2 mL / s, 2.5 mL / s, and 2.86 mL / s respectively. When the pulsed electric field intensity was the variable, the cycle number was 3 times and the flow rate was 1.25 mL / s; when the flow rate was the variable, the field intensity was 4.6 kV / cm and the cycle number was 3; when the cycle number was the variable, the field intensity was 4.6 kV / cm and the flow rate was 1.25 mL / s.

[0038] The remaining steps were the same as those in Example 1.

[0039] (2) Detection of the contents of polyphenols and total sugars in the tea polysaccharide-polyphenol complex.

[0040] Determination of polyphenol content: The Folin-Ciocalteu method was used. Using gallic acid as the standard, a standard curve y = 0.0071x + 0.0434, R 2 = 0.9989 was drawn. 20 μL of a sample solution with a concentration of 0.5 mg / mL was taken and mixed with 100 μL of a Folin-Ciocalteu reagent with a volume fraction of 10%. After reacting for 4 min, 80 μL of a Na 2 CO 3 solution with a mass concentration of 7.5% was added. It was placed in the dark at room temperature for 2 h, and the absorbance value was measured at 765 nm. The polyphenol content of the sample was calculated according to the following formula, and the result was expressed as the mass fraction of polyphenols contained in the sample per unit mass.

[0041] TP = [(C × n × V) / m] × 10 -3 × 100.

[0042] In the formula: TP is the polyphenol content, in %; C is the polyphenol mass concentration of the measured sample solution, in μg / mL; n is the dilution factor; V is the volume of the measured sample solution, in mL; m is the sample mass, in mg.

[0043] Determination of total sugar content: The phenol-sulfuric acid method was used. Using glucose as the standard, a standard curve y = 4.5229x + 0.1135, R 2= 0.9996. Take 100 μL of the sample solution with a concentration of 0.2 mg / mL and mix it with 100 μL of the phenol solution with a mass concentration of 5%. Then add 500 μL of concentrated sulfuric acid with a mass fraction of 98%. React at room temperature for 30 min, measure the absorbance at 490 nm, and calculate the total sugar content according to the following formula. The result is expressed as the mass fraction of the total sugar contained in the sample per unit mass.

[0044] TS = [(C × n × V) / m] × 100.

[0045] In the formula: TS is the total sugar content, %; C is the total sugar mass concentration of the measured sample solution, with the unit of mg / mL; n is the dilution factor; V is the sample solution volume, with the unit of mL; m is the sample mass, with the unit of mg.

[0046] 2. Experimental results.

[0047] The experimental results are as Figure 2 , Figure 3 and Figure 4 shown. The electric field strength, flow rate, and number of cycles have a great influence on the total sugar content and polyphenol content of the tea polysaccharide-polyphenol complex. Among them, the best effect is obtained when the pulsed electric field strength is 5.3 kV / cm, the best effect is obtained when the flow rate is 1.25 mL / s, and the highest polysaccharide content and polyphenol content are obtained under the condition of pulsed electric field treatment with 4 pulses, each for 5 min. The optimal extraction conditions are a pulsed electric field strength of 5.3 kV / cm, a flow rate of 1.25 mL / s, 4 pulses, and each for 5 min.

[0048] Verification Example 2: Comparison of the extraction effects of the extraction method in Example 1 and the traditional method.

[0049] 1. Experimental method.

[0050] (1) Extraction of tea polysaccharide-polyphenol complex.

[0051] Traditional method for extracting tea polysaccharide-polyphenol complex: Pressurized liquid extraction was carried out using a semi-continuous system. Among them, the extraction solvent was water, the extraction temperature was 200 °C, the flow rate was 5 mL / min, and the extract was vacuum filtered and evaporated to dryness. The dried part was dissolved in 250 mL of deionized water, and extracted twice with a mixture of water:n-hexane at a volume ratio of 1:1 at 69 °C for 6 h each time. Then, the aqueous phase was extracted twice with 250 mL of diethyl ether at 34 °C for 6 h each time. The separated aqueous phase was evaporated to a paste product. The paste product was dissolved in 2 L of methanol at room temperature, filtered under reduced pressure in a glass funnel, and dried at room temperature to obtain a residue. Finally, the residue was dissolved in distilled water and dialyzed thoroughly for 5 days and then freeze-dried to obtain the polysaccharide-polyphenol complex. The method is derived from "Ho T C, Kiddane A T, Sivagnanam S P, et al. Green extraction of polyphenolic-polysaccharide conjugates from Pseuderanthemum palatiferum Radlk: Chemical profile and anticoagulant activity. International Journal of Biological Macromolecules, 2020, 157".

[0052] Weigh and calculate the yield of the tea polysaccharide-polyphenol complex extracted in Example 1 and the above traditional method. The calculation formula is as follows.

[0053] Yield = (m 1 / m 2 ) × 100.

[0054] In the formula, m 1 is the mass of the powder after freeze-drying, in g; m 2 is the mass of the tea powder, in g.

[0055] Protein content: Using bovine serum albumin as the standard product, it was determined by the Coomassie brilliant blue method. According to the regression equation y = 0.92x + 0.6783, R 2 = 0.9985, the measured amount was calculated; Uronic acid content: Using galacturonic acid as the standard product, it was determined by the m-hydroxybiphenyl method. According to the regression equation y = 7.4727x + 0.0024, R 2 = 0.9995, the measured amount was calculated.

[0056] 3. Comparison results.

[0057] The comparison results are shown in Table 1. The yields of tea polysaccharide-polyphenol complexes, total sugar contents, and uronic acid contents extracted by the extraction method of Example 1 are significantly increased compared with the existing traditional methods, which are 8.1%, 35.7%, and 25.71% respectively, with an increase of 1.26%, 8.6%, and 8.94%, and the protein content is relatively low, which is 2.07%.

[0058] Table 1: Analysis Table of Extracted Components Example 2: Determination of the antioxidant function of tea polysaccharide-polyphenol complexes.

[0059] 1. Experimental method.

[0060] DPPH scavenging rate: Prepare solutions of tea polysaccharide and the tea polysaccharide-polyphenol complex extracted in Example 1 with a concentration of 1 mg / mL, and dilute them into a series of concentrations: 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL. Then, take 100 μL of each solution and mix it with 100 μL of a 0.1 mM DPPH solution in a 96-well plate as the reaction group, react in the dark for 30 min, measure the absorbance at 517 nm with an enzyme-labeled instrument, use distilled water instead of the sample as the blank group, and use absolute ethanol instead of DPPH as the control group. The formula for the DPPH radical scavenging rate is as follows.

[0061] DPPH radical scavenging rate = [1 - (A 1 - A 2 ) / A 0 ] × 100.

[0062] In the formula: A 0 is the absorbance of the blank group; A 1 is the absorbance of the reaction group; A 2 is the absorbance of the control group.

[0063] 2. Experimental results.

[0064] The experimental results are as Figure 5As shown, when the mass concentration is 50 μg / mL, the DPPH radical scavenging rate of the tea polysaccharide-polyphenol mixture is 76.8%, and that of the tea polysaccharide is 51%. When the mass concentration is 100 μg / mL, the DPPH radical scavenging rate of the tea polysaccharide-polyphenol mixture is 86%, and that of the tea polysaccharide is 75%. When the mass concentration is 150 μg / mL, the DPPH radical scavenging rate of the tea polysaccharide-polyphenol mixture is 87.7%, and that of the tea polysaccharide is 78.9%. When the mass concentration is 200 μg / mL, the DPPH radical scavenging rate of the tea polysaccharide-polyphenol mixture is 90%, and that of the tea polysaccharide is 80.5%. In summary, the DPPH radical scavenging rate of the tea polysaccharide-polyphenol complex is much higher than that of the tea polysaccharide.

[0065] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made by those skilled in the art once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

Claims

1. A method for extracting tea polysaccharide-polyphenol complex, characterized in that: The following steps are involved: S1, grinding green tea leaves into powder and mixing with water in a mass volume ratio of 1 g:30 mL to obtain a tea powder solution; S2, treating the tea powder solution with a pulsed electric field, extracting, centrifuging and concentrating to obtain a first concentrated solution, removing protein from the first concentrated solution to obtain a second concentrated solution, removing pigment from the second concentrated solution to obtain a third concentrated solution, subjecting the third concentrated solution to alcohol precipitation, centrifuging and collecting the precipitate, removing excess ethanol from the precipitate, and then drying to obtain the tea polysaccharide-polyphenol complex; The pulse electric field treatment conditions are: field intensity of 5.3 kV / cm, flow rate of 1.25 mL / s, 4 pulse cycles, each for 5 minutes.

2. The method for extracting tea polysaccharide-polyphenol complex according to claim 1, characterized in that: The extraction conditions are 90° C., 4.5 h.

3. The method for extracting tea polysaccharide-polyphenol complex according to claim 1, characterized in that: The centrifugation conditions are all 9000 rpm / min, 15 min.

4. The method for extracting tea polysaccharide-polyphenol complex according to claim 1, characterized in that: The first concentrated solution was deproteinized by the Sevage method, specifically, the first concentrated solution was mixed with a chloroform-n-butanol mixture of 3 times its volume, shaken and then allowed to stand for stratification, and the upper clear liquid was retained, i.e., the second concentrated solution, wherein the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixture was 4:

1.

5. The method for extracting tea polysaccharide-polyphenol complex according to claim 1, characterized in that: The pigment in the second concentrated solution is removed by adding 5% by volume of H2O2 to the second concentrated solution, and decolorizing the solution at 50°C for 2 hours to remove the pigment in the second concentrated solution.

6. The method for extracting tea polysaccharide-polyphenol complex according to claim 1, characterized in that: The alcohol precipitation of the third concentrated solution is specifically as follows: the third concentrated solution is mixed with 4 times the volume of ethanol and allowed to stand at 4° C. for 12 hours.

7. A tea polysaccharide-polyphenol complex, characterized in that: The extract is obtained by extraction using the extraction method described in any one of claims 1 to 6.

8. Use of the tea polysaccharide-polyphenol complex according to claim 7 in the preparation of antioxidant drugs.

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