LU501046B1

LU501046B1 - Method for solubilizing hydrophobic polyphenol with hydrophilic polyphenols

Info

Publication number: LU501046B1
Application number: LU501046A
Authority: LU
Inventors: Haibo Pan; Huan Cheng; Xingqian Ye; Ziqi Qian; Xinlei Xu; Jianle Chen; Shiguo Chen
Original assignee: Univ Zhejiang; Zhejiang Univ Zhongyuan Institute
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2022-06-20

Abstract

The present disclosure discloses a method for solubilizing a hydrophobic polyphenol with hydrophilic polyphenols. The method comprises the following steps: (1) dissolving the hydrophilic polyphenols in pure water to prepare a hydrophilic polyphenol aqueous solution; (2) optionally selecting one of the following modes: in a first mode, dissolving the hydrophobic polyphenol in an organic solvent to prepare an HPs solution; adding the HPs solution into the hydrophilic polyphenol aqueous solution, and uniformly mixing to obtain an HPs aqueous solution; in a second mode, directly adding the hydrophobic polyphenol into the hydrophilic polyphenol aqueous solution, uniformly stirring and filtering, to obtain the HPs aqueous solution. According to the present disclosure, the hydrophilic polyphenols are used as a solubilizing factor, and the hydrophilic polyphenol aqueous solution with a certain concentration and the HPs are simply mixed so that the effects of solubilizing and protecting the HPs can be achieved, the complex preparation of a composite carrier is not needed, and the establishment of an HPs solubilizing system is greatly simplified.

Description

BL-5342 METHOD FOR SOLUBILIZING HYDROPHOBIC POLYPHENOL WITH LUS01046

HYDROPHILIC POLYPHENOLS TECHNICAL FIELD

[01] The present disclosure belongs to the field of food biotechnologies, in particular to a method for improving the aqueous solubility of hydrophobic polyphenols by using hydrophilic polyphenols.

BACKGROUND ART

[02] Many hydrophobic polyphenols (HPs) are widely used in the development and production of functional foods due to their unique physiological effects, such as enhancement on vascular toughness of hesperidin and antitumor efficacy of curcumin. However, the HPs are poorly water-soluble and sensitive to oxygen, heat, pH and other factors. As a result, the Hps are hardly digested and absorbed in human body and easily degraded during processing and storage, and has a low bioavailability in human body, which seriously affects their health efficacy.

SUMMARY

[03] The problem to be solved by the present disclosure is to provide a method for solubilizing hydrophobic polyphenols (HPs) with hydrophilic polyphenols. The HPs aqueous solution prepared by the method has a high stability and a long storage period, and can be widely used in various foods and beverages.

[04] In order to solve the above problem, the present disclosure provides a method for solubilizing hydrophobic polyphenols (HPs) with hydrophilic polyphenols, comprising steps of:

[05] (1) dissolving the hydrophilic polyphenols (pure hydrophilic polyphenols or products rich in the hydrophilic polyphenols) in pure water to prepare a hydrophilic polyphenol aqueous solution with a concentration of 0.5 to 5.0 g/L; and

[06] (2) optionally selecting one of the following modes:

[07] in the first mode,

[08] dissolving the HPs (pure HPs or products rich in the HPs) in an organic solvent to prepare an HPs solution with a concentration of 2.5 to 10 g/L;

[09] adding the HPs solution into the hydrophilic polyphenol aqueous solution, and uniformly mixing to obtain an HPs aqueous solution (a stable HPs aqueous solution), where a volume ratio of the HPs solution to the hydrophilic polyphenol aqueous solution is 0.5 to 2:100;

[10] in the second mode,

[11] directly adding 10 to 400 mg of the HPs into 1 L of the hydrophilic polyphenol aqueous solution, uniformly stirring (stirring at 200 rpm for 24 h) and filtering, to obtain the HPs aqueous solution (the stable HPs aqueous solution).

[12] As an improvement of the method for solubilizing the HPs with the hydrophilic polyphenols of the present disclosure:

[13] the organic solvent in the first mode is ethanol (pure ethanol) or dimethylformamide. 1

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[14] As a further improvement of the method for solubilizing the HPs with the LU501046 hydrophilic polyphenols:

[15] the hydrophilic polyphenols are catechins, proanthocyanidins and anthocyanins with a purity = 85%.

[16] That is, they are pure hydrophilic polyphenols or products rich in the hydrophilic polyphenols; a pure product of catechins is, for example, epigallocatechin gallate (EGCG).

[17] As a further improvement of the method for solubilizing the HPs with the hydrophilic polyphenols:

[18] the Hps are pure HPs or products rich in the HPs;

[19] the pure HPs are curcumin, hesperidin and resveratrol, and

[20] the products rich in the HPs are high-purity turmeric extract and orange peel extract.

[21] The present disclosure can solve the problems of poor water solubility and stability of the HPs in the prior art, and the difficulty of quality control and large-scale preparation of the existing solubilization system.

[22] The strong antioxidant activity of the carrier is the key to maintain the stability of the HPs. The present takes strong antioxidant hydrophilic polyphenols as an independent solubilization factor without the preparation of a composite carrier, improves the water solubility of the HPs and plays a protective effect. In addition, the quality control and large-scale preparation technology of the hydrophilic polyphenols (such as catechins and proanthocyanidins) adopted by the present disclosure has become mature, and the adopted hydrophilic polyphenols are commercially available. Therefore, the problems of great difficulty in quality control and immature large-scale preparation technology of polyphenol composite carriers can be effectively solved. Therefore, exploring and developing a method to improve the water solubility of the HPs with the hydrophilic polyphenols as independent solubilizing factors is of great significance for their applications in the food and pharmaceutical industries.

[23] The present disclosure has the following advantages: according to the present disclosure, the hydrophilic polyphenols are used as a solubilizing factor, and the hydrophilic polyphenol aqueous solution with a certain concentration and the HPs are simply mixed so that the effects of solubilizing and protecting the HPs can be achieved, the complex preparation of a composite carrier is not needed, and the establishment of an HPs solubilizing system is greatly simplified. In addition, the quality control and large-scale preparation technology of many hydrophilic polyphenols (such as catechins and proanthocyanidins) adopted by the invention has become mature, which can effectively solve the problems of great difficulty in quality control of polyphenol composite carriers and significantly improve the quality stability of the HPs solubilization system.

BRIEF DESCRIPTION OF THE DRAWINGS

[24] Fig. 1 is a comparison between the products of examples 1, 2, 3, 4 and 5 and their controls after 30 days of storage;

[25] in the pictures corresponding to each example, the left picture is the control 2

BL-5342 (pure water), and the right picture is the product. LU501046

[26] Fig. 2 is a comparison between the product of Example 6 and its control after 30 days of storage (an upper picture) and 96 hours of standing (a lower picture).

[27] Fig. 3 is a graph showing changes of curcumin during storage of the curcumin or turmeric extract aqueous solution for 30 days in Examples 1, 3, 4 and 6 (the curcumin content during storage is calculated with reference to the initial curcumin concentration).

[28] Fig. 4 is a graph showing changes of hesperidin during storage of the aqueous solution of hesperidin or orange peel extract in Examples 2 and 5 for 30 days (the hesperidin content during storage is calculated with reference to the initial hesperidin concentration).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[29] In the following solutions:

[30] Example 1

[31] 1 g of epigallocatechin gallate (EGCG) with a purity of 98% was weighted and placed in a 2 L container, into which 800 mL of pure water was added with stirring uniformly. Then, the container was heated to 35 °C, and the stirring was continued until the EGCG was dissolved to give a total volume of 1 L with addition of pure water to prepare an EGCG solution.

[32] 50 mg of curcumin with a purity of 98% was weighted and placed in a 50 mL container, into which 18 mL of pure ethanol was added with stirring for dissolution to give a total volume to 20 mL, resulting in a curcumin solution.

[33] The above 20 mL of curcumin solution was slowly added to 1 L of the EGCG solution and uniformly mixed with stirring at 200 rpm, standing for 10 min to obtain a stable curcumin aqueous solution.

[34] Pure water was used as a control, that is, an ethanol solution (20 mL) containing 50 mg of curcumin was added to 1 L of pure water, stirred uniformly, and then allowed to stand for 10 min.

[35] Example 2

[36] 2.5 g of myrica rubra proanthocyanidin with a purity of 85% was weighted and placed in a 2 L container, into which 800 mL of pure water was added with stirring uniformly. Then, the container was heated to 35 °C, and the stirring was continued until the myrica rubra proanthocyanidin was dissolved to give a total volume of 1 L with addition of pure water to prepare a myrica rubra proanthocyanidin solution.

[37] 100 mg of hesperidin with a purity of 98% was weighted and placed in a 50 mL container, into which 8 mL of dimethylformamide was added with stirring for dissolution to give a total volume to 10 mL, resulting in a hesperidin solution.

[38] The above 10 mL of hesperidin solution was slowly added to 1 L of the myrica rubra proanthocyanidin aqueous solution and uniformly mixed with stirring at 3rpm, standing for 10 min to obtain a stable myrica rubra proanthocyanidin aqueous solution.

[39] Pure water was used as a control, that is, a dimethylformamide solution (10 mL) containing 100 mg of hesperidin was added to 1 L of pure water, stirred uniformly, and then allowed to stand for 10 min.

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[40] Example 3 LU501046

[41] 2.0 g of tea polyphenol with a purity of 95% was weighted and placed in a 2 L container, into which 800 mL of pure water was added with stirring uniformly. Then, the container was heated to 35 °C, and the stirring was continued until the tea polyphenols was dissolved, followed with filtering to remove insoluble impurities, to give a total volume of 1 L with addition of pure water to prepare a tea polyphenol solution.

[42] 200 mg of turmeric extract with a purity of 95% was weighted and placed in a 50 mL container, into which 18 mL of pure ethanol was added with stirring for dissolution followed with filtering to remove insoluble impurities, to give a total volume to 20 mL, resulting in a turmeric extract solution.

[43] The above 20 mL of turmeric extract was slowly added to 1 L of the tea polyphenol and uniformly mixed with stirring at 200 rpm, standing for 10 min to obtain a stable turmeric extract.

[44] Pure water was used as a control, that is, an ethanol solution (20 mL) containing 200 mg of turmeric extract was added to 1 L of pure water, stirred uniformly, and then allowed to stand for 10 min.

[45] Example 4

[46] 3.0 g of grape seed proanthocyanidin was weighted and placed in a 2 L container, into which 800 mL of pure water was added with stirring uniformly. Then, the container was heated to 35 °C, and the stirring was continued until the grape seed proanthocyanidin was dissolved followed with filtering to remove remove insoluble impurities to give a total volume of 1 L with addition of pure water to prepare a grape seed proanthocyanidin.

[47] 100 mg of curcumin with a purity of 98% was weighted and placed in a 50 mL container, into which 18 mL of pure ethanol was added with stirring for dissolution to give a total volume to 20 mL, resulting in a curcumin solution.

[48] The above 20 mL of curcumin solution was slowly added to 1 L of the EGCG solution and uniformly mixed with stirring at 200 rpm, standing for 10 min to obtain a stable curcumin aqueous solution.

[49] Pure water was used as a control, that is, an ethanol solution (20 mL) containing 100 mg of grape seed proanthocyanidin was added to 1 L of pure water, stirred uniformly, and then allowed to stand for 10 min.

[50] Example 5

[51] 1.0g of EGCG with a purity of 98% was weighted and placed in a 2L container, into which 800ml of pure water was added with stirring uniformly. Then, the container was heated to 35 °C, and the stirring was continued until the was dissolved to give a total volume of 1L with addition of pure water to prepare an EGCG solution.

[52] 200 mg of orange peel extract with a purity of 95% was weighted and placed in a 50 mL container, into which 18 mL of dimethylformamide was added with stirring for dissolution followed with filtering to remove insoluble impurities, to give a total volume to 20 mL, resulting in an orange peel extract.

[53] The above 20 mL orange peel extract was slowly added to 1 L of the EGCG solution and uniformly mixed with stirring at 300 rpm, standing for 10 min to obtain an orange peel extract.

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[54] Pure water was used as a control, that is, an ethanol solution (20 mL) containing LU501046 200 mg of orange peel extract was added to 1 L of pure water, stirred uniformly, and then allowed to stand for 10 min.

[55] Example 6

[56] 1.0g of EGCG with a purity of 98% was weighted and placed in a 2L container, into which 800ml of pure water was added with stirring uniformly. Then, the container was heated to 35 °C, and the stirring was continued until the was dissolved to give a total volume of 1L with addition of pure water to prepare an EGCG solution.

[57] 100 mg of curcumin with a purity of 98% was weighted and added to the EGCG aqueous solution, with stirring at 200 rpm for 24 h, filtering to remove undissolved curcumin (75.4942 87 mg) to obtain a stable curcumin aqueous solution.

[58] Pure water was taken as a control, that is, 100 mg of curcumin was added to 1 L of pure water, stirred for 24 h, and filtered to remove undissolved curcumin.

[59] The comparison between the products of Examples 1 to 5 and their controls after 30 days of storage was as shown in Fig. 1. According to Fig. 1, it could be seen that the solubility of curcumin, hesperidin, turmeric extract and orange peel extract in the EGCG tea polyphenol, myrica rubra proanthocyanidin and grape seed proanthocyanidin aqueous solution was significantly improved, with a high physical stability and on insoluble precipitation after storage for 30 days.

[60] The comparison between the product of example 6 and its control after storage for 30 days and standing for 96 hours was described in Fig. 2. According to figure 2, the solubility of the curcumin powder in the EGCG aqueous solution was significantly improved, but it was insoluble in pure water, and the formed curcumin solution had a high physical stability with no insoluble precipitation after 30 days of storage.

[61] Experiment 1: HPs stability determination:

[62] For Examples 1, 3, 4 and 6: the prepared curcumin or turmeric extract aqueous solution was stored at room temperature (25+2°C) for 30 days, 1 mL of the curcumin or turmeric extract aqueous solution was sampled every 6 days, into which 1 mL of pure ethanol was added to dissolve the curcumin and filter with a 0.45um filter membrane for high performance liquid chromatography (HPLC) analysis. HPLC detection system: Waters e2695; detector: Waters 2489 UV-Vis detector, chromatographic column: Shimazu XDB-C18 chromatographic column (250 mmx4.6 mm, 5.0 um); mobile phase:

0.1% formic acid/water (phase A), 0.1% formic acid/acetonitrile (phase B); elution gradient: 0 to 20 min, 45 to 0% B; column temperature: 30°C; flow rate: 1.0 mL/min; detection wavelength: 425 nm; and injection volume: 10 ul. A standard curve of the Curcumin covered a range of 0.5 to 50 pg/ml (50% ethanol aqueous solution).

[63] The results obtained were as shown in Fig. 3. According to Fig. 3, it could be seen that the curcumin content of the curcumin and the turmeric extract aqueous solution prepared by the present disclosure did not change significantly during storage for 30 days, with a high stability.

[64] Note: the curcumin content was calculated according to the formula: C = (A + 46654)/39415, R? = 0.9997, in which A was a corresponding peak area of the curcumin in the HPLC spectrum, and C was the concentration of the curcumin (unit: pg/mL).

[65] Experiment 2:

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[66] For Examples 2 and 5: the prepared hesperidin or orange peel extract aqueous LU501046 solution was stored at room temperature (25+2°C) for 30 days, 1 mL of the curcumin or turmeric extract aqueous solution was sampled every 6 days, into which 1 mL of pure ethanol was added to dissolve the hesperidin and filter a 0.45um filter membrane for high performance liquid chromatography (HPLC) analysis. HPLC detection system: Waters e2695; detector: Waters 2489 UV-Vis detector, chromatographic column: Shimazu XDB-C18 chromatographic column (250 mmx4.6 mm, 5.0 um); mobile phase:

0.1% phosphoric acid/water (phase A), pure methanol (phase B); (phase B); elution gradient: 50% B; column temperature: 30°C; flow rate: 1.0 mL/min; detection wavelength: 280 nm; and injection volume: 10 uL. A standard curve of the hesperidin covered a range of 0.5 to 50 ug/ml (50% ethanol aqueous solution).

[67] The results obtained were as shown in Fig. 4. According to Fig. 4, it could be seen that the curcumin content of the hesperidin and orange peel extract aqueous solution prepared by the present disclosure did not change significantly during storage for 30 days, with a high stability.

[68] Note: the hesperidin content was calculated according to the formula: C = (A + +855)/43566, R2 = 0.9994, in which A was a corresponding peak area of the hesperidin in the HPLC spectrum, and C was the concentration of the hesperidin (unit: pg/mL).

[69] Comparative Example 1: the EGCG was changed to gallic acid with an unchanged amount, and the rest was the same as in Example 1.

[70] According to the above method, after 30 days of storage, obvious precipitation occurred, and the curcumin content was about 10.41%.

[71] Comparative Example 2-1: the amount of the myrica rubra proanthocyanidin was changed from 2.5 g to 0.4 g, and the rest was the same as in Example 2.

[72] According to the above method, after 30 days of storage, obvious precipitation occurred, and the curcumin content was about 21.74%.

[73] Comparative Example 2-2: the amount of the hesperidin content was changed from 100 mg to 400 mg, and the rest was the same as in Example 2.

[74] According to the above method, after 30 days of storage, obvious precipitation occurred, and the hesperidin content was 35.11%.

[75] Comparative Example 3-1: the amount of the tea polyphenol was changed from

2.0 gto 0.4 g, and the rest was the same as in Example 3.

[76] According to the above method, after 30 days of storage, obvious precipitation occurred, but the supernatant was still yellow and light in color, and the curcumin content was 45.37+1.56%.

[77] Comparative Example 3-2: the amount of the tea polyphenol was changed from

2.0 gto 10.0 g, and the rest was the same as in Example 3.

[78] According to the above method, there was no obvious precipitation after 30 days of storage. The curcumin content was 97.81 + 2.17%, which did not change significantly, but the amount of the tea polyphenol increased significantly, so it was not recommended.

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Claims

BL-5342 WHAT IS CLAIMED IS: LUS01046

1. A method for solubilizing hydrophobic polyphenols (HPs) with hydrophilic polyphenols, comprising steps of: (1) dissolving the hydrophilic polyphenols in pure water to prepare a hydrophilic polyphenol aqueous solution with a concentration of 0.5 to 5.0 g/L; and (2) optionally selecting one of the following modes: in the first mode, dissolving the HPs in an organic solvent to prepare an HPs solution with a concentration of 2.5 to 10 g/L; adding the HPs solution into the hydrophilic polyphenol aqueous solution, and uniformly mixing to obtain an HPs aqueous solution, wherein a volume ratio of the HPs solution to the hydrophilic polyphenol aqueous solution is 0.5 to 2:100; in the second mode, directly adding 10 to 400 mg of the HPs into 1 L of the hydrophilic polyphenol aqueous solution, uniformly stirring and filtering, to obtain the HPs aqueous solution.

2. The method for solubilizing the HPs with the hydrophilic polyphenols according to claim 1, wherein the organic solvent in the first mode is ethanol or dimethylformamide.

3. A method for solubilizing the HPs with the hydrophilic polyphenols according to claim 1 or 2, wherein the hydrophilic polyphenols are catechins, proanthocyanidins and anthocyanins with a purity = 85%.

4. A method for solubilizing the HPs with the hydrophilic polyphenols according to claim 1 or 2, wherein The HPs is pure HPs or products rich in the HPs; wherein the pure HPs are curcumin, hesperidin and resveratrol, and the products rich in the HPs are high-purity turmeric extract and orange peel extract.

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