CN112841643A

CN112841643A - Method for solubilizing hydrophobic polyphenol by using hydrophilic polyphenol

Info

Publication number: CN112841643A
Application number: CN202011620846.7A
Authority: CN
Inventors: 叶兴乾; 潘海波; 陈士国; 程焕; 陈健乐; 徐新雷; 钱子琪
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-05-28
Also published as: WO2022142423A1

Abstract

The invention discloses a method for solubilizing hydrophobic polyphenol by hydrophilic polyphenol, which comprises the following steps: 1) dissolving hydrophilic polyphenol in pure water to prepare a hydrophilic polyphenol water solution; 2) optionally one of the following: dissolving hydrophobic polyphenol in an organic solvent to prepare HPs solution; adding the HPs solution into the hydrophilic polyphenol aqueous solution, and uniformly mixing to obtain HPs aqueous solution; and secondly, directly adding the hydrophobic polyphenol into the hydrophilic polyphenol water solution, uniformly stirring, and filtering to obtain HPs water solution. The invention takes hydrophilic polyphenol as a solubilizing factor, can play roles of solubilizing and protecting HPs by simply mixing hydrophilic polyphenol aqueous solution with certain concentration and HPs, does not need to carry out complicated composite carrier preparation, and greatly simplifies the establishment of a HPs solubilizing system.

Description

Method for solubilizing hydrophobic polyphenol by using hydrophilic polyphenol

Technical Field

The invention belongs to the technical field of food biology, and particularly relates to a method for improving water solubility of hydrophobic polyphenol by using hydrophilic polyphenol.

Background

Many Hydrophobic Polyphenols (HPs) have been widely used in the development and production of functional foods due to their unique physiological effects, such as the vascular toughness-enhancing effect of hesperidin and the anti-tumor effect of curcumin. However, HPs has poor water solubility and is sensitive to oxygen, heat, pH and other factors, so that it is difficult to digest and absorb by human body, and is easily degraded in the processing and storage processes, and the bioavailability of human body is low, thus seriously affecting its health efficacy.

Delivery systems with food-borne ingredients (proteins, polysaccharides, lipids, etc.) as carriers are often used for embedding HPs, not only to achieve the effect of solubilization, but also to protect and deliver HPs, to improve its bioavailability, to exert its health effects. Common delivery systems include food-borne ingredients such as proteins, polysaccharides and lipids, and nanoparticles, emulsions, liposomes and the like formed from HPs, which are favored by researchers due to their low toxicity, degradability and biocompatibility; it can effectively improve HPs water solubility, but has limited protection effect, and HPs stability is still poor in the processing and storage process. In order to improve stability, hydrophilic polyphenols as antioxidants are often combined physically or chemically with food-borne ingredients to form composite carriers, which improve their antioxidant capacity. The food-derived ingredients and the water-soluble polyphenol are combined through physical effects such as electrostatic combination, hydrogen bond effect and pi-pi effect, but the physical combination strength is influenced by environmental factors such as ionic strength, pH and temperature. The indexes of ionic strength, pH, temperature and the like in a food system are wide in range and large in change, the stability of the food-derived component-polyphenol composite carrier is seriously influenced, and the actual application range of the food-derived component-polyphenol composite carrier is limited. Compared with physical action, the covalent bond is not easily affected by factors such as temperature, ionic strength and the like. The chemical grafting is connected with the traditional food-borne ingredients and the water-soluble polyphenol by covalent bonds, and the formed polyphenol composite carrier has high stability. The chemical grafting method mainly comprises a free radical induction method, a chemical coupling method and a polyphenol oxidase catalysis method; the two methods of free radical initiation and chemical coupling have high reaction speed, but many reaction sites, many byproducts and difficult control of product quality; the reaction site of the polyphenol oxidase catalysis method is special, the byproducts are few, but the reaction rate is extremely slow, the large-scale preparation is difficult to realize, and the oxidation resistance is reduced due to the oxidation of the water-soluble polyphenol. The practical application of the polyphenol composite carrier is limited by the problems of large quality control difficulty and immature large-scale preparation technology.

Disclosure of Invention

The problem to be solved by the invention is to provide a method for solubilizing Hydrophobic Polyphenol (HPs) by hydrophilic polyphenol, wherein HPs aqueous solution prepared by the method has high stability and long storage period, and can be widely applied to various foods and beverages.

In order to solve the above technical problems, the present invention provides a method for solubilizing a hydrophobic polyphenol with a hydrophilic polyphenol, comprising the steps of:

1) dissolving hydrophilic polyphenol (a pure hydrophilic polyphenol product or a product rich in hydrophilic polyphenol) in pure water to prepare a hydrophilic polyphenol water solution with the concentration of 0.5-5.0 g/L;

2) optionally one of the following:

in a first way,

Dissolving hydrophobic polyphenol (HPs pure product or HPs-enriched product) in organic solvent to obtain HPs solution with concentration of 2.5-10 g/L;

adding HPs solution into hydrophilic polyphenol water solution, and mixing uniformly to obtain HPs water solution (stable HPs water solution); HPs solution: the hydrophilic polyphenol water solution is 0.5-2: 100 in volume ratio;

the second way,

Directly adding 10-400 mg of Hydrophobic Polyphenol (HPs) into 1L of hydrophilic polyphenol aqueous solution, uniformly stirring (stirring at 200rpm for 24 hours), and filtering to obtain HPs aqueous solution (stable HPs aqueous solution).

As an improvement of the method of solubilizing hydrophobic polyphenols with hydrophilic polyphenols of the present invention:

the organic solvent in the first mode is ethanol (pure ethanol) or dimethylformamide.

As a further improvement of the method of solubilizing hydrophobic polyphenols with hydrophilic polyphenols of the present invention:

the hydrophilic polyphenol is catechin, proanthocyanidin, and anthocyanin with purity of not less than 85%.

Namely, the product is a pure hydrophilic polyphenol product or a product rich in hydrophilic polyphenol; the purified product of catechins is, for example, epigallocatechin gallate (EGCG).

the Hydrophobic Polyphenol (HPs) is HPs pure product or HPs enriched product;

the HPs pure product is curcumin, hesperidin, resveratrol,

the HPs-enriched product is high-purity (purity is more than or equal to 90%) Curcuma rhizome extract and pericarpium Citri Tangerinae extract.

The invention can solve the problems of poor water solubility and stability of HPs, great difficulty in quality control and large-scale preparation of the existing solubilization system and the like in the prior art.

The strong oxidation resistance of the carrier is critical to maintaining HPs stable. The invention takes the hydrophilic polyphenol with strong oxidation resistance as an independent solubilizing factor, does not need to prepare a composite carrier, and improves HPs water solubility and plays a role in protection. In addition, the quality control and large-scale preparation technology of the hydrophilic polyphenol (such as catechin and proanthocyanidin) adopted by the invention is mature, and the hydrophilic polyphenol can be easily obtained in a commercially available mode, so that the problems of high quality control difficulty and immature large-scale preparation technology of the polyphenol composite carrier are effectively solved. Therefore, the exploration and development of the method for improving the water solubility of HPs by taking the hydrophilic polyphenol as the independent solubilizing factor are of great significance for the application of the method in the food and medicine industries.

The invention has the following technical advantages: the invention takes hydrophilic polyphenol as a solubilizing factor, can play roles of solubilizing and protecting HPs by simply mixing hydrophilic polyphenol aqueous solution with certain concentration and HPs, does not need to carry out complicated composite carrier preparation, and greatly simplifies the establishment of a HPs solubilizing system. In addition, a plurality of water-soluble polyphenols (such as catechins and proanthocyanidins) have mature quality control and large-scale preparation technologies, so that the problem of great difficulty in quality control of the polyphenol composite carrier is effectively solved, and the quality stability of the HPs solubilization system is obviously improved.

Drawings

FIG. 1 is a comparison of the products of examples 1, 2, 3, 4, 5 and their controls after 30 days of storage;

in the corresponding picture of each example, the left image is the control (pure water) and the right image is the product.

FIG. 2 is a comparison of the product of example 6 and its control preparation after storage for 30 days (top panel) and after standing for 96 hours (bottom panel).

Fig. 3 is a graph showing the change in curcumin during storage for 30 days in examples 1, 3, 4 and 6 of curcumin or an aqueous solution of turmeric extract (curcumin content during storage was calculated with reference to the initial curcumin concentration).

Fig. 4 is a graph of the change in hesperidin during storage of the aqueous solutions of hesperidin or of an extract of citrus peel in examples 2 and 5 for 30 days (the hesperidin content during storage was calculated with the initial hesperidin concentration as a reference).

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of making the objects and technical solutions of the present invention clearer, but the present invention is not limited thereto.

In the following scheme, the method comprises the following steps,

example 1:

weighing 1g of epigallocatechin gallate (EGCG) with the purity of 98 percent, placing the EGCG in a 2L container, adding 800mL of pure water, stirring uniformly, heating to 35 ℃, continuing stirring until the EGCG is dissolved, adding the pure water to a constant volume of 1L, and preparing the EGCG solution.

Weighing 50mg of 98% curcumin with purity, placing the curcumin in a 50mL container, adding 18mL of pure ethanol, stirring for dissolving, and metering the volume to 20mL to prepare a curcumin solution.

Slowly adding the 20mL curcumin solution into 1L EGCG solution, stirring at 200rpm, mixing, and standing for 10 min to obtain stable curcumin solution.

With pure water as a control, an ethanol solution (20mL) containing 50mg of curcumin was added to 1L of pure water, uniformly stirred, and then allowed to stand for 10 minutes.

Example 2:

weighing 2.5g of myrica rubra leaf proanthocyanidin with purity of 85 percent, placing the myrica rubra leaf proanthocyanidin into a 2L container, adding 800mL of pure water, stirring uniformly, heating to 35 ℃, continuing stirring until the pure water is dissolved, filtering to remove insoluble impurities, adding pure water to a constant volume of 1L, and preparing the myrica rubra leaf proanthocyanidin solution.

Weighing 100mg of hesperidin with the purity of 98 percent, putting the hesperidin into a 50mL container, adding 8mL of dimethylformamide, stirring to dissolve, and fixing the volume to 10mL to prepare a hesperidin solution.

Slowly adding the above 10mL of hesperidin solution into 1L of myrica rubra leaf proanthocyanidin aqueous solution, stirring at 300rpm, mixing, and standing for 10 min to obtain stable hesperidin aqueous solution.

Pure water was used as a control, i.e., a dimethylformamide solution (10mL) containing 100mg of hesperidin was added to 1L of pure water, stirred uniformly and then allowed to stand for 10 minutes.

Example 3:

weighing 2.0g of tea polyphenol with purity of 95 percent, placing the tea polyphenol into a 2L container, adding 800mL of pure water, stirring uniformly, heating to 35 ℃, continuing stirring until the mixture is dissolved, filtering to remove insoluble impurities, adding pure water to a constant volume of 1L, and preparing a tea polyphenol solution.

Weighing 200mg of turmeric extract with purity of 95% in a 50mL container, adding 18mL of pure ethanol, stirring for dissolving, filtering to remove insoluble impurities, and diluting to 20mL to obtain turmeric extract solution.

Slowly adding 20mL of Curcuma rhizome extract solution into 1L of tea polyphenols solution while stirring at 200rpm, and standing for 10 min to obtain stable Curcuma rhizome extract water solution.

With purified water as a control, an ethanol solution (20mL) containing 200mg of curcumin extract was added to 1L of purified water, stirred uniformly and then allowed to stand for 10 minutes.

Example 4:

weighing 3.0g of grape seed proanthocyanidin with the purity of 95 percent, placing the grape seed proanthocyanidin in a 2L container, adding 800mL of pure water, stirring uniformly, heating to 35 ℃, continuing stirring until the mixture is dissolved, filtering to remove insoluble impurities, adding the pure water to a constant volume of 1L, and preparing the grape seed proanthocyanidin solution.

Weighing 100mg of 98% curcumin with purity, placing the curcumin in a 50mL container, adding 18mL of pure ethanol, stirring for dissolving, and metering to 20mL to prepare a curcumin solution.

Slowly adding 20mL of Curcuma rhizome solution into 1L of grape seed proanthocyanidin solution while stirring at 200rpm, and standing for 10 min to obtain stable curcumin water solution.

With pure water as a control, namely, an ethanol solution (20mL) containing 100mg of curcumin was added to 1L of pure water, uniformly stirred and then allowed to stand for 10 minutes.

Example 5:

weighing 1.0g of 98% EGCG with purity, placing in a 2L container, adding 800mL of pure water, stirring, heating to 35 deg.C, stirring to dissolve, adding pure water to constant volume to 1L, and making into EGCG solution.

Weighing 200mg of orange peel extract with the purity of 95 percent, placing the orange peel extract into a 50mL container, adding 18mL of dimethylformamide, stirring for dissolving, filtering to remove insoluble impurities, and fixing the volume to 20mL to prepare an orange peel extract solution.

Slowly adding 20mL of the orange peel extract solution into 1L of EGCG solution, stirring and mixing uniformly at 300rpm, and standing for 10 min to obtain a stable orange peel extract aqueous solution.

With pure water as a control, an ethanol solution (20mL) containing 200mg of orange peel extract was added to 1L of pure water, stirred uniformly and then allowed to stand for 10 minutes.

Example 6:

100mg of curcumin with purity of 98 percent is weighed and added into the EGCG aqueous solution, the mixture is stirred for 24 hours at 200rpm, and undissolved curcumin (75.49 +/-2.87 mg) is removed by filtration, thus obtaining the stable curcumin aqueous solution.

With purified water as a control, 100mg of curcumin was added to 1L of purified water, stirred for 24 hours, and filtered to remove undissolved curcumin.

Comparison of the products of examples 1-5 and their controls after 30 days storage, as described in FIG. 1; from fig. 1, it can be seen that: the solubility of curcumin, hesperidin, Curcuma rhizome extract and pericarpium Citri Tangerinae extract in aqueous solution of EGCG, tea polyphenols, bayberry leaf proanthocyanidin and grape seed proanthocyanidin is remarkably improved, and the physical stability is high, and no insoluble precipitate is formed after storage for 30 days.

Comparison of the product of example 6 and its control after 30 days of storage and 96 hours of standing, as depicted in FIG. 2; from fig. 2, it can be seen that: the curcumin powder has remarkably improved solubility in EGCG aqueous solution, but is insoluble in pure water, and the formed curcumin solution has high physical stability, and no insoluble precipitate is formed after storage for 30 days.

Experiment 1: HPs stability determination:

for examples 1, 3, 4 and 6: storing the prepared curcumin or Curcuma rhizome extract water solution at room temperature (25 + -2 deg.C) for 30 days, sampling 1mL every 6 days, adding 1mL pure ethanol to dissolve curcumin, filtering with 0.45 μm filter membrane, and analyzing with High Performance Liquid Chromatography (HPLC). HPLC detection system: waters e 2695; a detector: waters 2489 uv-vis detectors; a chromatographic column: shimadzu XDB-C18 column (250 mm. times.4.6 mm,5.0 um); mobile phase: 0.1% formic acid/water (phase a), 0.1% formic acid/acetonitrile (phase B); elution gradient: 0-20 min, 45-60% B; column temperature: 30 ℃; flow rate: 1.0 mL/min; detection wavelength: 425 nm; sample introduction amount: 10 μ L. Curcumin standard curve range: 0.5-50. mu.g/mL (50% aqueous ethanol).

The results are shown in FIG. 3, and it can be seen from FIG. 3 that: the curcumin and the turmeric extract aqueous solution prepared by the invention have no obvious change in curcumin content in the process of storing for 30 days, and have high stability.

Description of the drawings: the calculation formula of the curcumin content is as follows: c ═ A +46654)/39415, R²0.9997. Wherein A is the corresponding peak area of curcumin in an HPLC (high performance liquid chromatography) spectrum, and C is the concentration of curcumin (unit is mu g/mL).

Experiment 2:

for examples 2 and 5: storing the prepared hesperidin or orange peel extract water solution at room temperature (25 + -2 deg.C) for 30 days, sampling 1mL every 6 days, adding 1mL of pure ethanol to dissolve hesperidin, and filtering with 0.45 μm filter membrane for High Performance Liquid Chromatography (HPLC) analysis. HPLC detection system: waters e 2695; a detector: waters 2489 uv-vis detectors; a chromatographic column: shimadzu XDB-C18 column (250 mm. times.4.6 mm,5.0 um); mobile phase: 0.1% phosphoric acid/water (phase a), pure methanol (phase B); isocratic elution: 50% of B; column temperature: 30 ℃; flow rate: 1.0 mL/min; detection wavelength: 280 nm; sample introduction amount: 10 μ L. Standard curve range of hesperidin: 0.5-50. mu.g/mL (50% aqueous ethanol).

The results are shown in FIG. 4, and it can be seen from FIG. 4 that: the hesperidin content and the hesperidin content of the aqueous solution of the orange peel extract prepared by the method are not obviously changed in the process of storing for 30 days, and the stability is high.

Description of the drawings: the calculation formula of the hesperidin content is as follows: c ═ a +855)/43566, R²0.9994. Wherein A is the corresponding peak area of curcumin in an HPLC (high performance liquid chromatography) spectrum, and C is the hesperidin concentration (unit is mu g/mL).

Comparative example 1, EGCG was changed to gallic acid, and the amount of EGCG was kept constant, and the rest was the same as example 1.

The detection is carried out according to the method, after 30 days of storage, obvious precipitation occurs, and the curcumin content is about 10.41 percent.

In comparative example 2-1, the amount of proanthocyanidin in the leaves of Myrica rubra was changed from 2.5g to 0.4g, and the rest was the same as in example 2.

The detection is carried out according to the method, after 30 days of storage, obvious precipitation phenomenon appears, and the content of hesperidin is about 21.74%.

Comparative example 2-2, the amount of hesperidin was changed from 100mg to 400mg, and the rest was the same as example 2.

The detection is carried out according to the method, after 30 days of storage, obvious precipitation phenomenon appears, and the content of hesperidin is 35.11%.

Comparative example 3-1, the amount of tea polyphenol was changed from 2.0g to 0.4g, and the rest was the same as in example 3.

The detection is carried out according to the method, after 30 days of storage, obvious precipitation phenomenon appears, but the supernatant is still yellow and has lighter color, and the curcumin content is 45.37 +/-1.56%.

Comparative example 3-2, the amount of tea polyphenol was changed from 2.0g to 10.0g, and the rest was the same as in example 3.

The detection is carried out according to the method, after 30 days of storage, no obvious precipitation phenomenon occurs, the curcumin content is 97.81 +/-2.17%, no obvious change occurs, but the dosage of the tea polyphenol is obviously increased, so that the use is not recommended.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims

1. A method for solubilizing hydrophobic polyphenols with hydrophilic polyphenols, characterized by comprising the steps of:

1) dissolving hydrophilic polyphenol in pure water to prepare a hydrophilic polyphenol water solution with the concentration of 0.5-5.0 g/L;

2) optionally one of the following:

in a first way,

Dissolving hydrophobic polyphenol in organic solvent to obtain HPs solution with concentration of 2.5-10 g/L;

adding the HPs solution into the hydrophilic polyphenol aqueous solution, and uniformly mixing to obtain HPs aqueous solution; HPs solution: the hydrophilic polyphenol water solution is 0.5-2: 100 in volume ratio;

the second way,

Directly adding 10-400 mg of hydrophobic polyphenol into 1L of hydrophilic polyphenol aqueous solution, uniformly stirring, and filtering to obtain HPs aqueous solution.

2. The method of solubilizing hydrophobic polyphenols as claimed in claim 1, wherein:

the organic solvent in the first mode is ethanol or dimethylformamide.

3. The method of solubilizing hydrophobic polyphenols with hydrophilic polyphenols according to claim 1 or 2, characterized in that:

4. The method of solubilizing hydrophobic polyphenols with hydrophilic polyphenols according to claim 1 or 2, characterized in that:

the hydrophobic polyphenol is HPs pure product or HPs-enriched product;

the HPs pure product is curcumin, hesperidin, resveratrol,

the HPs-enriched product is high-purity Curcuma rhizome extract and pericarpium Citri Tangerinae extract.