阅读说明：本技术 一种改善茶多酚茶多糖溶解性的方法及所制备复合物与应用 (Method for improving tea polyphenol and tea polysaccharide solubility, prepared compound and application ) 是由 字成庭 盛军 王宣军 杨柳 张宁 吴以龙 孙秀丽 谢引荣 于 2021-09-29 设计创作，主要内容包括：本发明涉及一种改善茶多酚茶多糖溶解性的方法及所制备复合物与应用,属于植物化学技术领域,本发明首次从普洱熟茶中分离纯化得到一种茶多糖,该化合物结构新颖,具有生物活性,尤其是较好的降糖活性,为进一步研究药用茶多糖提供重要的启示,可用于降糖药物的研发制备。本发明还提供了茶多酚改善高聚茶多糖溶解性的方法,通过多糖-多酚的相互作用,使高聚茶多糖与多酚类物质在水溶液中自组装形成稳定的大分子复合物,以显著提高高聚茶多糖的溶解性。本发明所提供的茶多糖的增溶方法工艺简单,操作方便,实际应用效果优异,通过茶多糖与茶多酚复配形成的复合物具有显著的协同降糖作用。(The invention relates to a method for improving the solubility of tea polyphenol and tea polysaccharide, a prepared compound and application, and belongs to the technical field of phytochemistry. The invention also provides a method for improving the solubility of the high polymer tea polysaccharide by the tea polyphenol, which enables the high polymer tea polysaccharide and the polyphenol substance to self-assemble in the water solution to form a stable macromolecular compound through the interaction of the polysaccharide and the polyphenol so as to obviously improve the solubility of the high polymer tea polysaccharide. The solubilization method of tea polysaccharide provided by the invention has the advantages of simple process, convenient operation and excellent practical application effect, and the compound formed by compounding the tea polysaccharide and the tea polyphenol has a remarkable synergistic hypoglycemic effect.)

1. A tea polysaccharide, which is characterized in that: has the following structure:

the compound is named as PTPs, and the molecular formula of the compound is C₄₁H₈₂O₄₀(ii) a The polysaccharide has a molecular weight of 4.59 KDa, the monosaccharide is composed of rhamnose, arabinose = 1:7, and the skeleton of PTPs is composed of 6 → 2) -alpha-L-Arap- (3 → is a main chain and 1 repeating unit of beta-L-Rhap- (3 → 1) -alpha-L-Arap.

2. The tea polysaccharide of claim 1, wherein: the tea polysaccharide is a new polysaccharide separated from Pu' er tea leaves of Yunnan big leaf species.

3. A method for improving the solubility of tea polysaccharide-tea polyphenol is characterized in that: the tea polysaccharide and polyphenol are built into a self-assembly compound, so that the solubility of the tea polysaccharide is improved.

4. A method of improving the solubility of tea polysaccharides-tea polyphenols according to claim 3 wherein: adding tea polyphenols into tea polysaccharide water solution, mixing and stirring.

5. A method of improving the solubility of tea polysaccharides-tea polyphenols according to claim 3 or 4, wherein: the tea polysaccharide is high-polymer tea polysaccharide or the tea polysaccharide of claim 1; the tea polyphenols comprise one or more of gallocatechin (EC), gallocatechin gallate (ECG), Epigallocatechin (EGC), and epigallocatechin gallate (EGCG).

6. A method of improving the solubility of tea polysaccharides-tea polyphenols according to any of claims 3 to 5, wherein: the molar mass ratio of the tea polysaccharide to the tea polyphenol substances is 1: 1.

7. A tea polysaccharide-polyphenol complex characterized by: the molar mass ratio of the tea polysaccharide to the tea polyphenol substances is 1: 1.

8. The tea polysaccharide-polyphenol complex of claim 8, wherein: the tea polysaccharide is high-polymer tea polysaccharide or the tea polysaccharide of claim 1; the tea polyphenols comprise one or more of gallocatechin (EC), gallocatechin gallate (ECG), Epigallocatechin (EGC), and epigallocatechin gallate (EGCG).

9. Use of a tea polysaccharide-polyphenol complex as claimed in claim 7 or 8 in the preparation of a carbohydrate lowering food or medicament.

Technical Field

The invention relates to the technical field of dissolution of phytochemical functional components, and in particular relates to a method for extracting and purifying tea polysaccharide and improving the solubility of tea polyphenol tea polysaccharide.

Background

The Pu' er tea is a special tea prepared from Yunnan special big leaf tea. Pu' er tea contains a large amount of tea polysaccharide, and is one of main flavor substances of tea soup. The tea polysaccharide content in tea is about 6%, the composition is complex, the tea polysaccharide belongs to heteropolysaccharide complex, and the tea polysaccharide is composed of substances such as saccharides, proteins and pectin, and is a compound polysaccharide with biological activity.

Modern researches show that tea polysaccharide has the effects of reducing blood fat, losing weight, lowering blood pressure, regulating immunity, resisting cancer and the like. The polysaccharides in tea include neutral polysaccharides and acidic polysaccharides, and are often glycoprotein complexes closely bound to proteins. At present, animal in vivo model tests prove that the tea polysaccharide has the function of reducing blood sugar, and the tea polysaccharide is mainly used for removing free radicals and regulating key enzyme activity in the carbohydrate metabolism process to regulate the blood sugar level, slow down the emptying speed of the stomach and reduce the absorption of glucose. Therefore, the research of tea polysaccharide is also attracting more and more attention. However, the water solubility of the extracted Pu' er tea polysaccharide, especially the high-polymer tea polysaccharide, is poor, and the blood sugar reducing effect of the tea polysaccharide is further influenced. With the growing concern over their health, there has been a trend to research and develop natural hypoglycemic agents to replace chemically synthesized hypoglycemic agents. In conclusion, how to overcome the technical problem of difficult dissolution of high-polymer tea polysaccharide is a key technical problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In order to overcome the problems in the background art, the invention provides a method for extracting, purifying and solubilizing high-polymer tea polysaccharide, which extracts tea polysaccharide from Pu' er tea, mixes the tea polysaccharide with EGCG and establishes a self-assembly compound. Through the inhibition activity experiment of alpha-glucosidase, the PTPs-EGCG compound is found to be improved by 1.1 times compared with EGCG, and the PTPs-EGCG compound is improved by 7.8 times compared with PTPs, so that the compound has a better hypoglycemic effect.

The following technical scheme is used for realizing the aim of the invention:

the tea polysaccharide provided by the invention is a new polysaccharide separated from Pu' er tea of Yunnan large-leaf species, belongs to a homogeneous polysaccharide, the polysaccharide has a molecular weight of about 4.59 KDa, monosaccharide is composed of rhamnose, arabinose = 1:7, the skeleton of PTPs is composed of 6 → 2) -alpha-L-Arap- (3 → is a main chain and 1 repeating unit of beta-L-Rhap- (3 → 1) -alpha-L-Arap, and the polysaccharide has the following structure:

the compound is named as PTPs, and the molecular formula of the compound is C₄₁H₈₂O₄₀。

The preparation method of the tea polysaccharide specifically comprises the following steps:

1) drying Pu her tea in shade, pulverizing, and sieving (40 mesh) to obtain raw material;

2) putting the step 1) into a hot water extractor, adding ultrapure water, extracting for 2 hours at 85 ℃, filtering insoluble substances to obtain a water extract, and evaporating and concentrating;

3) precipitating the concentrated solution obtained in the step 2) by using 95% ethanol, wherein the final concentration is 70% under the condition of vigorous stirring; standing overnight at room temperature, centrifuging (4000 r/min, 15 min, 0 ℃) to obtain a precipitate; redissolving the precipitate with hot water (80 ℃) and cooling to room temperature;

4) washing the solution obtained in the step 3) with Sevag reagent (n-butanol/chloroform 1:4, V/V) to remove protein; finally, crude tea polysaccharide is obtained by concentration and freeze drying;

5) purifying the crude tea polysaccharide obtained in the step 4) by using a DEAE-52 column, washing the column by using pure water, collecting eluent and concentrating; removing impurities such as monosaccharide and oligosaccharide with dialysis bag, and lyophilizing to obtain white floccule;

6) further purifying the white floccule obtained in the step 5) by using a Sephadex G-100 column, eluting by using a constant flow pump and purified water, collecting the eluent, and freeze-drying to obtain the purified tea polysaccharide.

Preferably, in step 2), the mass ratio of the raw material to water is 1: 15.

Preferably, in step 5), the DEAE-52 column is selected to be 2.9 cm × 50 cm, and the dialysis bag is 7000 Da.

Preferably, in step 6), the DEAE-52 column is selected to be 2.9 cm × 100 cm.

The invention also provides a method for improving the solubility of tea polysaccharide by using tea polyphenol, which comprises the following steps: the tea polysaccharide and polyphenol are built into a self-assembly compound, so that the solubility of the tea polysaccharide is improved. The method specifically comprises the following steps: adding tea polyphenols into tea polysaccharide water solution, mixing and stirring.

Further, the tea polysaccharide is high-polymer tea polysaccharide or tea polysaccharide with the structure; the tea polyphenols comprise one or more of gallocatechin (EC), gallocatechin gallate (ECG), Epigallocatechin (EGC), and epigallocatechin gallate (EGCG).

Further, the molar mass ratio of the tea polysaccharide to the tea polyphenol substances is 1: 1.

The dissolution time of tea polysaccharides in aqueous solution at 20 ℃ can be increased from 214 s to 150 s.

The invention also provides a tea polysaccharide-polyphenol compound, wherein the molar mass ratio of the tea polysaccharide to the tea polyphenol is 1: 1.

Further, the tea polysaccharide is high-polymer tea polysaccharide or tea polysaccharide with the structure; the tea polyphenols comprise one or more of gallocatechin (EC), gallocatechin gallate (ECG), Epigallocatechin (EGC), and epigallocatechin gallate (EGCG).

The invention also provides the application of the tea polysaccharide-polyphenol compound in preparing food or medicines for reducing blood sugar and lipid.

The invention has the beneficial effects that:

(1) the invention separates and purifies the Pu-Er ripe tea for the first time to obtain the tea polysaccharide, the compound has novel structure and better hypoglycemic effect, provides important inspiration for further researching medicinal tea polysaccharide, and can be used for researching, developing and preparing hypoglycemic drugs.

(2) According to the tea polysaccharide solubilization method provided by the invention, the polyphenol substances are added, and the high-polymer tea polysaccharide and the polyphenol substances form a stable compound in an aqueous solution under the condition that the molar mass ratio of the tea polysaccharide to the tea polyphenol substances is 1:1 through the interaction of polysaccharide and polyphenol substances, so that the dissolution time is prolonged. The dissolution time of tea polysaccharides in aqueous solution at 20 ℃ can be increased from 214 s to 150 s.

(3) The tea polysaccharide-polyphenol compound provided by the invention can be applied to the preparation of hypoglycemic foods or medicines.

Drawings

FIG. 1 is a structural formula of tea polysaccharide PTPs.

FIG. 2 is a pure HPLC chromatogram of PTPs.

FIG. 3 is a PMP-HPLC chromatogram of pure PTPs.

FIG. 4 is a pure IR spectrum of PTPs.

FIG. 5 is a pure UV spectrum of PTPs.

FIG. 6 is an NMR spectrum of pure PTPs.

FIG. 7 is an ITC plot of 800. mu.M PTPs titrated to 800. mu.M EGCG at 25 ℃.

FIG. 8 shows transmission electron micrographs of pure PTPs (a) and PTPs-EGCG (b) dissolved in water.

FIG. 9 shows that pure PTPs are miscible with EGCG to increase solubility; (a) zeta potential of EGCG, PTPs and PTPs-EGCG, (b) solubility of PTPs and PTPs-EGCG.

FIG. 10 shows that pure PTPs are miscible with EGCG to increase the dispersibility of the complex.

FIG. 11 is a test of the inhibitory activity of PTPs-EGCG on alpha-glucosidase.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.

Example 1: preparation method of tea polysaccharide PTPs

The preparation method of tea polysaccharide PTPs comprises the following steps: crushing 1 kg of big-leaf Pu' er tea dried in the shade, and sieving by a 40-mesh sieve to be used as a raw material; placing into a hot water extractor, extracting the obtained raw material with 8 times (mass ratio) of ultrapure water at 85 deg.C for 2 hr, repeatedly extracting for three times, filtering insoluble substances to obtain water extract, and concentrating by evaporation to obtain 500 mL of concentrated solution. The resulting concentrate was precipitated with 95% ethanol and the final concentration was 70% under vigorous stirring. Standing overnight at room temperature, and centrifuging (4000 r/min, 15 min, 0 ℃) to obtain a precipitate. The precipitate was redissolved with hot water (80 ℃) and cooled to room temperature. The resulting solution was washed several times with Sevag reagent (n-butanol/chloroform 1:4, V/V) to remove the protein. Finally, crude tea polysaccharide is obtained by concentration and freeze drying. The crude tea polysaccharide is purified by using DEAE-52 column (2.9 cm. times.50 cm), washing the column with pure water, collecting eluate, and concentrating. Dialyzing (7000 Da) with dialysis bag to remove impurities such as monosaccharide and oligosaccharide, and freeze drying to obtain white floccule. The resulting white floc was further purified using a Sephadex G-100 column (2.9 cm. times.100 cm), eluted with a constant flow pump and purified water at a flow rate of 0.6 mL/min, and the eluate (3 mL/tube) was automatically collected and lyophilized to give purified tea polysaccharide PTPs.

The structure of purified tea polysaccharide PTPs is shown in FIG. 1. Pure HPLC profiles of PTPs are shown in FIG. 2. The PMP-HPLC profile of pure PTPs is shown in FIG. 3. The IR spectrum of pure PTPs is shown in FIG. 4. The pure UV spectra of PTPs are shown in FIG. 5. FIG. 6 shows the NMR spectra of pure PTPs.

Example 2: test for improving high-polymer tea polysaccharide by using tea polyphenol

Pure tea polysaccharide PTPs obtained by the method of example 1 and tea polyphenol EGCG are used as experimental materials, and the tea polyphenol is extracted by the laboratory.

Preparing 1 mM purified tea polysaccharide aqueous solution and PTPs-EGCG aqueous solution (molar ratio is 1:1, see figure 7), and observing the existence form of the purified tea polysaccharide aqueous solution and the PTPs-EGCG aqueous solution in the aqueous solution by adopting a transmission electron microscope.

As shown in FIG. 8, it can be seen from FIG. 8 (a) that the purified tea polysaccharide has poor dissolution effect in water and is not uniformly dispersed; the characteristic of difficult water solubility causes that the glucose-reducing lipid-lowering. As shown in FIG. 8 (b), it can be seen from FIG. 8 (b) that the PTPs-EGCG formed stable nanoparticles and were uniformly dispersed when the PTPs and EGCG were prepared at a molar ratio of 1: 1.

As shown in FIG. 9, the potential values of the PTPs-EGCG solutions were lower than those of the PTPs solutions, the potential of the 1 mg/mL TPs solution was-17.8 mV, the potential of the 1 mg/mL EGCG solution was-33.4 mV, and the potential of the 1 mg/mL TPs-EGCG mixture was-29.7 mV. When EGCG is added into PTPs, the potential is increased, the higher the absolute value (positive and negative) of the potential is, the more stable the system is, and the solubility is improved.

As a result, as shown in FIG. 10, the particle size of EGCG was 110.29 nm; the particle size of the PTPs was 241.53 nm; when EGCG was added to PTPs, the particle size of the PTPs-EGCG complex was reduced to 134.17 nm, indicating that EGCG was effective in inhibiting autopolymerization of PTPs to form PTPs-EGCG complexes.

Example 3: PTPs-EGCG Activity test for alpha-glucosidase inhibition

The inhibition activity experiment of the alpha-glucosidase is carried out by taking pNPG as a substrate, acarbose as a positive control and EGCG, PTPs and PTPs-EGCG as an experimental group. 50 μ L of PBS, 50 μ L of alpha-glucosidase (0.5U/mL) and 50 μ L of EGCG, PTPs and PTPs-EGCG and acarbose (5, 10, 20, 40, 80 μ M) at different concentrations (0, 12.5, 25, 50, 100, 200 μ M) were incubated at 37 ℃ for 15 min. Then, 50. mu.L of pNPG solution (6 mM) was added thereto, and the mixture was incubated at 37 ℃ for 15 min. Finally adding 50 mu L of Na₂CO₃(0.2M) the reaction was stopped and the absorbance was measured at 405 nm. The experiment was repeated 3 times. The inhibitory activity of α -glucosidase is formulated as follows:

as a result, as shown in FIG. 11, under in vitro conditions, the ratio of PTPs to EGCG (molar ratio, μ M) was higher than that of EGCG at 12.5:12.5, 25:25, 50:50, and 100: 100. However, the inhibition activity of α -glucosidase was poor when PTPs were used alone. The PTPs-EGCG can effectively inhibit the activity of alpha-glucosidase.

The high-polymer tea polysaccharide is obtained by separating, purifying and identifying Pu' er tea leaves for the first time, has a novel structure, has various biological activities, particularly has a better blood sugar reducing effect, provides important inspiration for further research and preparation of blood sugar reducing lipid food medicines, and can be used for research and development and preparation of blood sugar reducing medicines. The preparation method is simple, the preparation cost is low, and the application scene is wide.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.