LU501076B1

LU501076B1 - Method for analyzing structure of proanthocyanidin by combination of hydrophilic interaction liquid chromatography (hilic) and reversed-phase liquid chromatography (rplc)

Info

Publication number: LU501076B1
Application number: LU501076A
Authority: LU
Inventors: Huan Cheng; Shiguo Chen; Xingqian Ye; Xinlei Xu; Yi Wang; Jianle Chen; Haibo Pan; Ziqi Qian
Original assignee: Univ Zhejiang; Zhejiang Univ Zhongyuan Institute
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2022-06-22

Abstract

The present disclosure discloses a method for analyzing a structure of proanthocyanidin by a combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC).

Description

BL-5344 METHOD FOR ANALYZING STRUCTURE OF PROANTHOCYANIDIN BY HUS01076

COMBINATION OF HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY (HILIC) AND REVERSED-PHASE LIQUID CHROMATOGRAPHY (RPLC) TECHNICAL FIELD

[01] The present disclosure relates to a method for rapidly analyzing a structure of proanthocyanidin by a combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC).

BACKGROUND ART

[02] At present, anthocyanidin formation involved will affect an accuracy of analysis results in known technical solutions for proanthocyanidin structure analysis. Moreover, purification of the proanthocyanidin is time-consuming and low-efficiency.

SUMMARY

[03] To solve the above technical problems, the present disclosure provides a method for analyzing a structure of proanthocyanidin by a combination of HILIC and RPLC, including the following steps:

[04] 1) analysis of a flavan-3-ol monomer:

[05] dissolving 5.0 mg of a to-be-tested sample containing proanthocyanidin in 1.0 mL of a 97% acetonitrile aqueous solution to obtain a to-be-tested sample solution; filtering the to-be-tested sample solution through a 0.22 um filter membrane, and analyzing the flavan-3-ol monomer in the to-be-tested sample by HILIC;

[06] 2) acid degradation of the proanthocyanidin:

[07] dissolving 100 mg of phloroglucinol and 20 mg of ascorbic acid in 2 mL of a 0.1 mol/L methanol hydrochloride solution to obtain an acid degradation reaction solution; and

[08] dissolving 10.0 mg of the to-be-tested sample containing proanthocyanidin in the acid degradation reaction solution, conducting reaction at 50°C for 20 min to obtain an acid degradation product, and filtering the acid degradation product through a 0.22 um filter membrane to obtain a to-be-analyzed acid degradation product;

[09] 3) analysis of the acid degradation product:

[10] analyzing the to-be-analyzed acid degradation product obtained in step 2) by RPLC (that is, analyzing the acid degradation product of the sample), and detecting a flavan-3-ol- phloroglucinol additive product and the flavan-3-ol in the to-be-analyzed acid degradation product at 280 nm; and 1

BL-5344

[11] 4) data analysis: HUS01076

[12] analyzing a type and a content of a terminal unit of the proanthocyanidin according to a type and a content difference of the flavan-3-ol analyzed by the RPLC and the HILIC, analyzing a type and a content of an extension unit of the proanthocyanidin according to the flavan-3-ol- phloroglucinol additive product analyzed by the RPLC, and calculating an average degree of polymerization of the proanthocyanidin.

[13] The present disclosure has the following technical advantages.

[14] 1. In the present disclosure, the type and concentration of the flavan-3-ol are analyzed in a sample solution rich in proanthocyanidin, and compared with the type and concentration of the flavan-3-ol in the acid degradation reaction solution, to determine the type and content of the terminal unit of proanthocyanidin in the sample; this scheme replaces a step of directly removing the flavan-3-ol in the sample in traditional methods, thereby greatly improving an analysis rate of the terminal unit of the proanthocyanidin.

[15] 2. Side reactions of anthocyanidin formation are analyzed during the acid degradation to further improve the accuracy of structure analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

[16] FIG. 1 shows a chromatogram of HILIC analysis on a flavan-3-ol monomer in a flavan-3- ol standard product and a grape seed extract solution; where proanthocyanidin Bl, as proanthocyanidin with the minimum molecular weight in grape seeds, is used to demarcate a boundary between the proanthocyanidin and the flavan-3-ol monomer;

[17] FIG. 2 shows a chromatogram of a by-product cyanidin produced by acid degradation of the proanthocyanidin and a chromatogram of standard cyanidin, by RPLC analysis of the method of the present disclosure; and

[18] FIG. 3 shows a chromatogram of the flavan-3-ol-phloroglucinol and the flavan-3-ol analyzed by RPLC using the method of the present disclosure and the traditional method; where

[19] 1: catechin-phloroglucinol, 2: epicatechin-phloroglucinol, 3: catechin, 4: epicatechin gallate-phloroglucinol, 5: epicatechin, and 6: epicatechin gallate.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[20] 1. HILIC analysis

[21] A concentration of each flavan-3-ol in a grape seed extract solution is calculated using (epi)catechin and gallates thereof as standard products.

[22] 1) HILIC analysis conditions

[23] A liquid chromatography uses a Waters €2695 chromatograph; a detector uses a Waters 2

BL-5344 2489 ultraviolet-visible light detector with a detection wavelength of 280 nm; and à 201076 chromatographic column uses a Luna Hilic column, with a column temperature of 30°C, an injection volume of 10 uL and an elution rate of 0.35 mL/min.

[24] A mobile phase includes acetonitrile (A) containing 0.5% acetic acid and water (B) containing 0.5% acetic acid, where A is composed of 0.5% acetic acid and 99.5% acetonitrile, B is composed of 0.5% acetic acid and 99.5% water, and % refers to % (volume).

[25] An elution gradient is: 0-30 min, 3-13% B.

[26] 2) A type and a concentration of flavan-3-ol is analyzed in a sample solution using a flavan-3-ol standard as reference. A standard solution of flavan-3-ol (flavan-3-ol standard product) with a concentration of 100 ug/mL is prepared (using a 97% acetonitrile aqueous solution as a solvent and diluent), and subjected to gradient dilution to 50.0, 25.0, 10.0, 5.0, 2.5 and 1.0 ug/mL; with these diluents, detection is conducted according to the above method to obtain a corresponding relationship between a peak area and a concentration of the flavan-3-ol in the diluents, and curve equations are established as follows:

[27] (epi)catechin: Y=15.5X+4.7, R° =0.996;

[28] (epi)gallocatechin: Y=3.9X+2.1, R° =0.993;

[29] (epi)catechin gallate: Y=38.7X-0.3, R? =0.998; and

[30] (epi)gallocatechin gallate: Y=27.4X-21.6, R? =0.993; where

[31] Y is a peak area of the flavan-3-ol (mAU*min), X is a corresponding concentration of the flavan-3-ol (ug/mL).

[32] 15.5 min corresponds to (epi)catechin; 19.0 min corresponds to (epi)gallocatechin; 22.8 min corresponds to (epi)catechin gallate; and 27.1 min corresponds to (epi)gallocatechin gallate.

[33] 2. RPLC analysis

[34] 1) RPLC analysis conditions

[35] A liquid chromatography uses a Waters €2695 chromatograph; a detector uses a Waters 2489 ultraviolet-visible light detector with detection wavelengths of 280 nm and 550 nm; and a chromatographic column uses an Eclipse XDB-C18 chromatographic column (250 mm x 4.6 mm,

5.0 um; Agilent), with a column temperature of 30°C, an injection volume of 10 uL and an elution rate of 1.0 mL/min.

[36] A mobile phase is water containing 1% acetic acid (A) and methanol containing 1% acetic acid (B). A is composed of 1% acetic acid and 99% water, B is composed of 1% acetic acid and 99% methanol, and % refers to % (volume).

[37] An elution gradient is: 0-10 min, 5% B;

[38] 10-30 min, 5-20% B; and

[39] 30-55 min, 20-40% B.

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BL-5344

[40] 2) a type and a concentration of an additive product, flavan-3-ol and anthocyanin in an 201076 acid degradation product are analyzed using a flavan-3-ol-phloroglucinol additive product, the flavan-3-ol and an anthocyanidin standard product as reference.

[41] (1) A standard solution is prepared for the flavan-3-ol-phloroglucinol additive product with a concentration of 100 ng/mL (using methanol as a solvent and diluent); gradient dilution is conducted to 50.0, 25.0, 10.0, 5.0, 2.5 and 1.0 pg/mL; with these diluents, detection is conducted according to the above method to obtain a corresponding relationship between a peak area and a concentration of the flavan-3-ol-phloroglucinol additive product in the diluents. Curve equations are established as follows:

[42] catechin-phloroglucinol additive product: Y=14.1X+4.0, R? =0.991;

[43] epicatechin-phloroglucinol additive product: Y=13.8X+4.3, R° =0.996;

[44] catechin gallate-phloroglucinol additive product: Y=32.6X+1.3, R° =0.991;

[45] epicatechin gallate-phloroglucinol additive product: Y=33.8X-0.2, R° =0.995;

[46] gallocatechin-phloroglucinol additive product: Y=3.4X+3.7, R° =0.997;

[47] epigallocatechin-phloroglucinol additive product: Y=3.6X+2.4, R° =0.992;

[48] gallocatechin gallate-phloroglucinol additive product: Y=26.5X-32.1, R* =0.992; and

[49] epigallocatechin gallate-phloroglucinol additive product: Y=29.6X-13.7, R? =0.996; where

[50] Y is a peak area of the flavan-3-ol-phloroglucinol additive product (mAU*min), X is a corresponding concentration of the flavan-3-ol-phloroglucinol additive product (ug/mL).

[51] 14.4 min corresponds to the catechin-phloroglucinol additive product,

[52] 17.4 min corresponds to the epicatechin-phloroglucinol additive product,

[53] 32.7 min corresponds to the catechin gallate-phloroglucinol additive product,

[54] 28.5 min corresponds to the epicatechin gallate-phloroglucinol additive product,

[55] 7.8 min corresponds to the gallocatechin-phloroglucinol additive product,

[56] 12.9 min corresponds to the epigallocatechin-phloroglucinol additive product,

[57] 25.3 min corresponds to the gallocatechin gallate-phloroglucinol additive product, and

[58] 23.2 min corresponds to the epigallocatechin gallate-phloroglucinol additive product.

[59] (2) A standard solution is prepared for the flavan-3-ol with a concentration of 100 pg/mL (using methanol as a solvent and diluent); gradient dilution is conducted to 50.0, 25.0, 10.0, 5.0,

2.5 and 1.0 pg/mL; with these diluents, detection is conducted according to the above method to obtain a corresponding relationship between a peak area and a concentration of the flavan-3-ol in the diluents, and curve equations are established as follows:

[60] catechin: Y=16.4X+2.4, R° =0.992;

[61] epicatechin: Y=15.2X+4.3, R° =0.991; 4

BL-5344

[62] gallocatechin: Y=3.7X+4.3, R° =0.997; HUS01076

[63] epigallocatechin: Y=3.9X+2.7, R° =0.992;

[64] catechin gallate: Y=36.9X+6.4, R° = 0.991;

[65] epicatechin gallate: Y=38.4X+2.7, R° =0.994;

[66] gallocatechin gallate: Y=29.1X-33.4, R? =0.999; and

[67] epigallocatechin gallate: Y=32.5X-17.3, R? =0.993; where

[68] Y is a peak area of the flavan-3-ol (mAU*min), X is a corresponding concentration of the flavan-3-ol (ug/mL).

[69] Note:

[70] 27.1 min corresponds to the catechin,

[71] 37.0 min corresponds to the epicatechin,

[72] 15.5 min corresponds to the gallocatechin,

[73] 20.6 min corresponds to the epigallocatechin,

[74] 45.4 min corresponds to the catechin gallate,

[75] 43.7 min corresponds to the epicatechin gallate,

[76] 41.5 min corresponds to the gallocatechin gallate, and [771 39.2 min corresponds to the epigallocatechin gallate.

[78] (3) A standard solution is prepared for the anthocyanin with a concentration of 100 pg/mL (using methanol as a solvent and diluent); gradient dilution is conducted to 50.0, 25.0, 10.0, 5.0,

2.5 and 1.0 pg/mL; with these diluents, detection is conducted according to the above method to obtain a corresponding relationship between a peak area and a concentration of the anthocyanin in the diluents. Curve equations are established as follows:

[79] cyanidin: Y=61.5X-32.8, R° =0.999; and

[80] delphinidin: Y=66.3X-43.1, R° =0.995; where

[81] Y is a peak area of the anthocyanin (mAU*min), X is a corresponding concentration of the anthocyanin (ug/mL).

[82] Note:

[83] 48.2 min corresponds to the cyanidin; and

[84] 35.9 min corresponds to the delphinidin.

[85] Example1

[86] A method for analyzing a structure of proanthocyanidin by a combination of HILIC and RPLC was provided, where a to-be-tested sample containing proanthocyanidin was a grape seed extract, and the method included the following steps sequentially:

[87] 1) Analysis of a flavan-3-ol monomer:

[88] (epi)catechin and gallates thereof were analyzed by HILIC in a solution of the to-be-tested

BL-5344 sample, specifically as follows: HUS01076

[89] 5.0 mg of the grape seed extract was accurately weighed and dissolved in 1.0 mL of a 97% acetonitrile aqueous solution to obtain the solution of the to-be-tested sample; the solution was filtered with a 0.22 um filter membrane, detected by the "1.1). HILIC analysis conditions", and an obtained peak area was substituted into the formula obtained in "1.2)" to obtain concentrations of the four components in the solution.

[90] A chromatogram of the HILIC analysis was shown in FIG. 1.

[91] 2) Acid degradation of the proanthocyanidin:

[92] 100 mg of phloroglucinol and 20 mg of ascorbic acid were weighed and dissolved in 2 mL of a 0.1 mol/L methanol hydrochloride solution to obtain an acid degradation reaction solution; and

[93] 10.0 mg of the grape seed extract was dissolved in the acid degradation reaction solution, reaction was conducted at 50°C for 20 min to obtain an acid degradation product, and the acid degradation product was filtered through a 0.22 um filter membrane to obtain a to-be-analyzed acid degradation product (capable of being stored at -20°C).

[94] 3) Analysis of the acid degradation product:

[95] The acid degradation product of the grape seed proanthocyanidin sample was subjected to RPLC analysis; a flavan-3-ol-phloroglucinol additive product and the flavan-3-ol were detected in the acid degradation product to be analyzed at 280 nm, and anthocyanidin (as an acid degradation by-product) was detected in the acid degradation product to be analyzed at 550 nm; and a chromatogram of the RPLC analysis was shown in FIG. 2 and FIG. 3.

[96] After testing, the ingredients include: (epi)catechin, (epi)gallocatechin and gallates thereof, phloroglucinol additive product and cyanidin.

[97] The acid degradation product to be analyzed obtained in step 2) was detected in accordance with the "2.1). RPLC analysis conditions", and a peak area obtained was correspondingly substituted into a formula obtained in the "2.2)", to obtain concentrations of corresponding components of the acid degradation product to be analyzed.

[98] 4) Data analysis:

[99] A difference between the concentration of each flavan-3-ol analyzed by the RPLC and the HILIC was a terminal unit concentration of the grape seed proanthocyanidin, a sum of the concentration of each flavan-3-ol-phloroglucinol and the anthocyanin analyzed by the RPLC was a terminal unit concentration of the grape seed proanthocyanidin; and an extension unit mole number/a terminal unit mole number + 1 was an average degree of polymerization of the grape seed proanthocyanidin.

[100] The following formula was used: 6

BL-5344

[101] Average degree of polymerization = Pr en EE +1 9501076

[102] Actual detection results were as follows:

[103] step 1)

[104] In the grape seed extract solution, the (epi)catechin and epicatechin gallate thereof have concentrations of 0.13 umol/mL and 0.01 umol/mL, respectively.

[105] step 2)

[106] In a reaction solution after the acid degradation, the catechin, the epicatechin and the epicatechin gallate have concentrations of 0.20 umol/mL, 0.16 umol/ML and 0.08 jumol/mL, respectively;

[107] the cyanidin has a concentration of 0.17 umol/mL; and

[108] the catechin-phloroglucinol, the epicatechin-phloroglucinol and the epicatechin gallate- phloroglucinol have concentrations of 0.13 umol/mL, 0.76 pmol/mL and 0.16 umol/mL, respectively.

[109] Therefore, a specific calculation formula of step 4) was as follows:

[110] Average degree of polymerization = Catechin—phloroglucinol +epicatechin-phloroglucinol +epicatechin gallate—phloroglucinol+cyanidin |; _ 5 97 (Catechin+epicatechin+epicatechin gallate)rpzc-((epi)catechin+epicatechin gallate) pic

[111] In the method of the present disclosure:

[112] 1) The concentrations of the (epi)catechin and the epicatechin gallate in the reaction solution after the acid degradation are higher than those of the (epi)catechin and gallates thereof in the grape seed extract solution. This indicates that structural units of the proanthocyanidin in the grape seed extract are catechin, epicatechin and epicatechin gallate.

[113] 2) The proanthocyanidin in the grape seed extract has an average degree of polymerization of 5.07.

[114] Comparative Example (traditional method)

[115] 1) Purification of proanthocyanidin: 1.0 g of grape seed extract was accurately weighed and dissolved in 4.0 mL of a 50% methanol aqueous solution to obtain a sample, and the sample was loaded on a Sephadex LH20 column equilibrated with a 50% methanol aqueous solution; elution was conducted with a 50% methanol aqueous solution for 5 column volumes, an eluent 1 was collected, and elution was conducted with a 70% acetone aqueous solution for 3 column volumes, and an eluent 2 was collected; the eluent 2 was dried to obtain a purified grape seed proanthocyanidin.

[116] 2) Acid degradation of the proanthocyanidin: the "to-be-tested sample containing proanthocyanidin" in Example 1 was replaced with the purified grape seed proanthocyanidin obtained in step 1), with a dosage remaining unchanged, where other operations were the same as step 2 in Example 1).

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BL-5344

[117] 3) Analysis of an acid degradation product: the operations were the same as step 3 hr 201076 Example 1).

[118] That is, the acid degradation products of the grape seed proanthocyanidin were subjected to RPLC analysis, including (epi)catechin and gallates thereof and the phloroglucinol additive product. A chromatogram of the RPLC analysis was shown in FIG. 3.

[119] Actual detection results were as follows:

[120] In a reaction solution after the acid degradation, the catechin, the epicatechin and the epicatechin gallate have concentrations of 0.15 umol/mL, 0.11 umol/ML and 0.06 jumol/mL, respectively;

[121] the catechin-phloroglucinol, the epicatechin-phloroglucinol and the epicatechin gallate- phloroglucinol have concentrations of 0.14 umol/mL, 0.80 pmol/mL and 0.17 pumol/mL, respectively.

[122] 4) Data analysis:

[123] Each flavan-3-ol detected by the RPLC was a terminal unit of the grape seed proanthocyanidin; each flavan-3-ol-phloroglucinol detected by the RPLC was an extension unit of the grape seed proanthocyanidin; and an extension unit mole number/a terminal unit mole number + 1 was an average degree of polymerization of the grape seed proanthocyanidin.

[124] Average degree of polymerization = Catechin—phloroglucinol+epicatechin-phloroglucinol+epicatechin gallate-phloroglucinol+cyanidin | 4 _ 4 47 Catechin+epicatechin+epicatechin gallate

[125] In the traditional method:

[126] 1) The concentrations of the (epi)catechin and the epicatechin gallate in the reaction solution after the acid degradation are higher than those of the (epi)catechin and gallates thereof in the grape seed extract solution. This indicates that structural units of the proanthocyanidin in the grape seed extract are catechin, epicatechin and epicatechin gallate.

[127] 2) The proanthocyanidin in the grape seed extract has an average degree of polymerization of 4.47.

8

Claims

BL-5344 WHAT IS CLAIMED IS: LU501076

1. A method for analyzing a structure of proanthocyanidin by a combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), comprising the following steps: 1) analysis of a flavan-3-ol monomer: dissolving 5.0 mg of a to-be-tested sample containing proanthocyanidin in 1.0 mL of a 97% acetonitrile aqueous solution to obtain a to-be-tested sample solution; filtering the to-be-tested sample solution through a 0.22 um filter membrane, and analyzing the flavan-3-ol monomer in the to-be-tested sample by HILIC; 2) acid degradation of the proanthocyanidin: dissolving 100 mg of phloroglucinol and 20 mg of ascorbic acid in 2 mL of a 0.1 mol/L methanol hydrochloride solution to obtain an acid degradation reaction solution; and dissolving 10.0 mg of the to-be-tested sample containing proanthocyanidin in the acid degradation reaction solution, conducting reaction at 50°C for 20 min to obtain an acid degradation product, and filtering the acid degradation product through a 0.22 um filter membrane to obtain a to-be-analyzed acid degradation product; 3) analysis of the acid degradation product: analyzing the to-be-analyzed acid degradation product obtained in step 2) by RPLC, and detecting a flavan-3-ol-phloroglucinol additive product and the flavan-3-ol in the to-be-analyzed acid degradation product at 280 nm; and 4) data analysis: analyzing a type and a content of a terminal unit of the proanthocyanidin according to a type and a content difference of the flavan-3-ol analyzed by the RPLC and the HILIC, analyzing a type and a content of an extension unit of the proanthocyanidin according to the flavan-3-ol- phloroglucinol additive product analyzed by the RPLC, and calculating an average degree of polymerization of the proanthocyanidin.

2. The method for analyzing a structure of proanthocyanidin by a combination of HILIC and RPLC according to claim 1, wherein step 3) further comprises: detecting anthocyanin of the to-be-analyzed acid degradation product at 550 nm; step 4) comprises: analyzing the type and the content of the extension unit of the proanthocyanidin according to the flavan-3-ol-phloroglucinol additive product and the anthocyanin analyzed by the RPLC; 9

BL-5344 the anthocyanin comprises: cyanidin and delphinidin; LU501076 in step 4), a calculation formula for the average degree of polymerization of the proanthocyanidin is as follows: . . __ Flavan-3-ol-phloroglucinol additive product ; Average degree of polymerization = Flavan-3-0)rpue — (Flavan3-oDume + 1; and in step 4), a calculation formula for the average degree of polymerization of the proanthocyanidin is as follows: . . __ Flavan-3-ol-phloroglucinol additive product Average degree of polymerization = Flavan-3-0)rpue — (Flavan3-oDume +1.