CN105153092B

CN105153092B - A kind of mercaptamine base of condensed tannin replaces the preparation method of catabolite

Info

Publication number: CN105153092B
Application number: CN201510685495.0A
Authority: CN
Inventors: 孙磊; 乔善义; 刘晓晴; 李姝靓
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2015-10-21
Filing date: 2015-10-21
Publication date: 2019-03-08
Anticipated expiration: 2035-10-21
Also published as: CN105153092A

Abstract

The invention belongs to natural medicine fields, the mercaptamine base for being related to a kind of condensed tannin replaces the preparation method of catabolite, more particularly to a kind of separation or the method for preparing hydroxyflavan compounds mercaptamine base substituent, include the following steps: that sample is carried out C18 normal pressure pillar layer separation by (1), the C18 normal pressure pillar layer separation is primary or multiple.Separation of the invention or the method for preparing hydroxyflavan compounds mercaptamine base substituent are easy to operate, and yield is high, and product purity is high.It has a good application prospect.

Description

Preparation method of sulfhydryl ethylamine substituted degradation product of condensed tannin

Technical Field

The invention belongs to the field of natural medicines, and relates to a preparation method of a thiol ethylamine substituted degradation product of condensed tannin.

Background

Condensed tannins are condensates of hydroxyflavan compounds connected by carbon-carbon bonds (Yaohao, Nature pharmaceutical chemistry, Beijing: people health Press, 1999:247.), the research of condensed tannin structural units generally adopts an acid-catalyzed degradation method, the connecting bonds between flavan-3-ol units are easy to break under proper acidic conditions, the terminal structural units are released, and the carbocation formed at the C-ring 4 position in the extension units is easy to be captured by proper nucleophilic reagent to form nucleophilic reagent additive products (Delphiine calimien, Sylvain Guyot, Sonia Ciollin. use olylysis hyphenated RP-HPLC-ESI (-) -MS/MS for the analysis of flavan origin fresh lager beers [ J ]. 1018. 2008,110: 1012.). Based on the chemical characteristics, the phloroglucinol degradation method and the mercaptan degradation method taking benzyl mercaptan as a main nucleophilic reagent become the classical analysis means of the structural analysis of various condensed tannins and are applied up to now. But benzyl mercaptan is toxic and smells bad, and phloroglucinol degradation efficiency is low.

For the research of the condensed tannin structural units, the inventor adopts a method of sulfhydrolysis of a cysteamine hydrochloride reagent combined with reversed phase HPLC analysis, firstly obtains the information of the constituent structural units of the condensed tannin position, further establishes an external standard working curve of each structural unit, carries out HPLC analysis by taking the degradation products of the condensed tannin position as the substances to be detected, and obtains the structural information of the condensed tannin position such as average molecular weight, average polymerization degree, galloylation rate and the like through calculation. In order to carry out the structural research, a reference substance for condensing each degradation product of tannin is needed, and the degradation product comprises hydroxy flavane compounds which form a condensation tannin structural unit, such as catechin, epicatechin, catechin gallate and epicatechin gallate, and the like, and also comprises mercaptoethylamino substitutes corresponding to each compound. The compounds of each structural unit for forming the condensed tannin have commodities, and the corresponding mercaptoethylamine substitutes have no corresponding commodities, so the condensed tannin self-prepared.

According to the literature reports (J.L. Torr and R.Bobet, New Flavoanol Derivatives from gradient (vitas vinifera) bypass products, Antiodant Aminoethyl-Flavan-3-ol conjugates from a Polymeric Waste Fraction Used as a Source of Flavanols, J.Agric.food chem.2001,49, 4627-containing 4634), the former researchers Used cation exchange to separate the hydroxy flavanoid mercaptoethylamino substituent, utilized the charge property of the hydroxy flavanoid mercaptoethylamino substituent to separate it from other substances, and then further purified by reversed phase semi-preparative high performance liquid chromatography, but this method not only is complicated to operate, but also requires the use of a large amount of inorganic salts, which causes great difficulty in the purification of the compound, and has long and low yield, which seriously hampers the progress of the research.

There is a need for a novel method for preparing mercaptoethylamino substitute of hydroxyflavan compound with relatively simple operation.

Disclosure of Invention

The inventor obtains a separation or preparation method of a hydroxy flavane compound mercaptoethylamino substitute through intensive research and creative labor. The inventor surprisingly finds that the method for preparing the mercaptoethylamino substitute of the hydroxyflavan compound has less steps and simple and convenient operation. The following invention is thus provided:

one aspect of the invention relates to a method for separating mercaptoethylamino substitute of hydroxyflavan compound, which comprises the following steps:

(1) the sample is subjected to C18 atmospheric column chromatography, which is C18 atmospheric column chromatography in one or more (e.g., 2, 3, 4, or 5) times.

The sample may be a liquid sample or may be a solid such as a powdered sample, which is dissolved by a suitable solvent such as methanol or the like.

The method according to any one of the present invention, wherein the sample contains a hydroxy flavanoid thioethylamino substitute;

preferably, the sample is or comprises a or b as follows:

a. degradation products of the condensed tannin by a thiolytic reagent; or

b. Degradation products of condensed tannin-containing plant extract (such as condensed tannin-containing grape seed extract or condensed tannin-containing wild buckwheat rhizome extract such as wimenine) with thiolytic reagent;

preferably, the thiolytic reagent described in a or b above is cysteamine hydrochloride.

In a preferred embodiment of the invention, the above-described degradation is carried out under water bath conditions; preferably, the temperature is 50-80 ℃ water bath temperature, more preferably 60-70 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, or any two of the above specific temperature value formed by the temperature range; and/or preferably, the time of the water bath is 5-50min, more preferably 10-30min, 10-25min, 10-20min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, or a time range formed by any two of the specific time values. In a particularly preferred embodiment of the invention, the water is bathed for 15min at 65 ℃. The preferable temperature and/or time of the water bath is favorable for full degradation and is also favorable for improving the yield of the mercaptoethylamino substitute of the hydroxyflavan compound.

The degrading reagent benzyl mercaptan used in the prior art is toxic and smelly, the degradation efficiency of phloroglucinol is low, and the tasteless and nontoxic cysteamine hydrochloride is adopted to replace benzyl mercaptan and phloroglucinol as nucleophilic reagents to degrade condensed tannin. The structural formula of the cysteamine hydrochloride is H₂N-H₂C-H₂C-SH.Hcl。

The grape seed extract containing condensed tannins can be prepared by the method in the prior art. Preferably, the extract is extracted by 50-95% ethanol and then purified by macroporous adsorption resin.

The Fagopyrum dibotrys extract containing condensed tannin can be extracted with 50-95% ethanol, and purified by macroporous adsorbent resin. Weimeining is an ethanol extract of plant wild buckwheat rhizome, the main component of which is condensed tannin, and the Wemeining is extracted by adopting 95 percent ethanol and purified by macroporous absorption resin.

The process according to any of the invention, wherein the condensed tannin in a or b is represented by formula A below (wherein the arrow indicates the attachment of the next building block at this site),

wherein,

R₁selected from H and OH;

R₂selected from the group consisting of OH and formula II below,

the method according to any one of the invention, wherein the mercaptoethylamino substitute of the hydroxyflavan compound is shown as the following formula I,

wherein,

R₁selected from H and OH;

R₂selected from H, OH and formula II;

R₃selected from H, OH and formula II;

and R is₂And R₃Different;

the method according to any one of the present invention, wherein,

the mercaptoethylamino substitute of the hydroxyflavan compound is the following compound:

table 1: the invention relates to partial specific hydroxy flavane compound mercaptoethylamino substitute

In the present invention, the above compound 1 is also simply referred to as a peak 1 compound, the compound 2 is also simply referred to as a peak 2 compound, and the compound 3 is also simply referred to as a peak 3 compound.

The method of any one of the invention, wherein the C18 atmospheric column chromatography in the step (1) satisfies any one, two, three or four of the following items 1) to 4):

1) the mobile phase is methanol aqueous solution, the concentration is 2% -12%, preferably, the concentration is 3% -12%, 4% -12%, 5% -12%, 2% -10%, 4% -11%, 2%, 3%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5% or 12%, or the numerical range formed by any two of the above values;

2) the mobile phase also contains formic acid or acetic acid, and the concentration of the formic acid or the acetic acid is 0.01 to 1 percent; preferably, the concentration is 0.05% to 0.5%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.25%, 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or 1%, or a range of any two of the foregoing;

3) the volume of each flow part is 1-15 mL; preferably, 2-10mL, 3-8mL, 4-6mL, 2, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, or 10mL, or a volume range of any two of the above specific volume values;

4) filling a column by adopting a wet method, and loading a sample by adopting the wet method; after loading the column, washing with methanol until free of impurities (e.g. by HPLC), and gradually transitioning the mobile phase to the starting ratio; preferably, the column volume is 10-1000mL, 50-500mL, 100-500mL, 200-200 mL, 250mL, 300mL, 400mL, 500mL, or a volume range consisting of any two of the specific volume values.

The method according to any one of the present invention, wherein in the step (1), the elution fractions are detected by HPLC, the fractions having the same or similar purity are combined, and methanol is removed; preferably, the methanol-depleted product is also freeze-dried.

Methanol removal can be accomplished by methods known to those skilled in the art, such as concentration under reduced pressure. It is also known to those skilled in the art that formic acid or acetic acid is also removed during this process.

The process according to any of the invention, wherein the conditions of HPLC are as follows:

a chromatographic column: thermo Syoronis C18 chromatography column (4.6 mm. times.250 mm, 5 μm); column temperature: 30 ℃; flow rate: 1 mL/min; sample introduction amount: 20 mu L of the solution; detection wavelength: 280 nm;

mobile phase: gradient elution with 0.2% formic acid (A) and 0.2% formic acid acetonitrile (B) as mobile phase, the elution procedure is shown in Table 2 below,

table 2: gradient elution procedure

Time (min)	A(％)	B(％)
			0	93	7
15	86	14
			22	84	16
28	83	17
			43	60	40
46	20	80
			55	20	80

In a preferred embodiment of the present invention, when isolating peak 1 compound, after 1C 18 atmospheric column chromatography, the product is again subjected to C18 atmospheric column chromatography using 5% methanol in water (containing 0.2% formic acid) as the mobile phase, and 5mL portions are combined (e.g., as measured by the above HPLC assay conditions), concentrated under reduced pressure to remove methanol, and freeze-dried to obtain a 95% or greater purity catechin mercaptoethylamino substitute.

In a preferred embodiment of the present invention, when isolating peak 3 compound, after 1C 18 atmospheric column chromatography, the product is again subjected to C18 atmospheric column chromatography using 10% methanol in water (containing 0.2% formic acid) as the mobile phase, and each 5mL of the mobile phase is fractionated, the fractions of similar purity are combined (e.g., as determined by the above HPLC assay conditions), concentrated under reduced pressure to remove methanol, and then lyophilized to obtain an epigallocatechin gallate mercaptoethylamino substitute with a purity of 95% or more.

The method according to any one of the present invention, further comprising a step (2),

(2) carrying out semi-preparative HPLC separation on the product in the step (1);

preferably, the conditions for semi-preparative HPLC are as follows:

a chromatographic column: YMC-Pack ODS-A column (10 mm. times.250 mm, 5 μm); column temperature: 30 ℃; flow rate: 1 mL/min; sample introduction amount: 1 mL; detection wavelength: 280 nm;

mobile phase: elution was carried out in a gradient with 0.2% formic acid (A) and 0.2% formic acid methanol (B) as mobile phases, according to the following procedure in Table 3.

Table 3: gradient elution procedure

Time (min)	A(％)	B(％)
			0	97	3
8	80	20
			25	75	25
35	60	40
			45	20	80
60	97	3

In a preferred embodiment of the invention, when isolating the compound having peak 2, the product is subjected to semi-preparative HPLC after 1C 18 atmospheric column chromatography. Preferably, the preparation is carried out according to the semi-preparative HPLC chromatographic conditions described above. Fractions were collected according to peak appearance: fractions of the first 33min are collected together as front impurity fractions, fractions of 33 min-43 min are collected together as product fractions, and fractions of the second impurity are collected together after 43 min. Mixing the impurity fractions, concentrating, introducing sample once again according to the above method, detecting the product fraction according to the above HPLC condition, concentrating under reduced pressure to remove methanol, and freeze drying to obtain the above 95% purity catechin mercaptoethylamine substitute.

Another aspect of the present invention relates to a method for preparing mercaptoethylamino substitutes of hydroxyflavan compounds, comprising the method for isolating mercaptoethylamino substitutes of hydroxyflavan compounds as described in any one of the above.

The invention belongs to a C18 atmospheric pressure column, which is a reversed phase chromatographic column with octadecylsilane chemically bonded silica as a filler. Octadecylsilane chemically bonded silica (ODS or C18) is a porous silica with complete spherical and smooth surface, has very high surface bonding coverage, and is obtained by bonding octadecylsilane on silica by a chemical reaction method. Since the bonded stationary phase surface of the C18 column is octadecyl with very low polarity, the eluent is mostly prepared from strong polar solvent such as water, methanol, acetonitrile, or buffer solution of inorganic salt. In elution, solutes are separated according to their hydrophobicity, and solutes having higher polarity and lower hydrophobicity are less likely to bind to nonpolar immobilization, and are eluted first. Since C18 is a long chain alkyl bonded phase with higher carbon content and better hydrophobicity, it is often used for separation analysis of medium polar compounds and high polar compounds dissolved in water.

Without being limited by theory, the normal pressure column chromatography separation operation is slightly troublesome, but the sample is not lost; and the semi-preparation liquid phase is simple and convenient to operate, and more samples can be lost. Since 3 compounds have a greater amount of peak 2 only, further purification of peak 2 only employs a semi-preparative liquid phase procedure.

The percentage concentrations of methanol or formic acid in the present invention are, if not otherwise specified, volume percentage concentrations.

Advantageous effects

The method for separating or preparing the mercaptoethylamino substitute of the hydroxyflavan compound has simple and convenient operation. The invention also has the advantages of higher yield, higher product purity and the like. Has good application prospect.

Drawings

FIG. 1: HPLC profile of condensed tannin site (bulk drug of wilkinin) thiolysis product.

FIG. 2: HPLC of the 3 obtained compounds with purity of 95% or more was prepared. Fig. 2A shows peak No. 1, fig. 2B shows peak No. 2, and fig. 2C shows peak No. 3.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. Wherein, the reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be obtained commercially.

Instruments and reagents:

LC-20A high performance liquid chromatograph, Shimadzu corporation, Japan (for HPLC analysis of the thionated product of example 2); HPLC-MS system, Agilent, usa (equipped with model 1200 high performance liquid chromatograph and model 6330 ion trap mass spectrometer for HPLC-MS analysis of the thiolytes of example 2); KQ2200E model ultrasonic cleaner, kunshan ultrasonic instruments ltd; Sartorius-BS 124S electronic balance, beijing sidoris balance ltd; N-1001D-WA rotary evaporator, Eyela, Japan; HW SY-K4 model intelligent electric heating constant temperature water bath, Changfeng instruments and meters company in Beijing city. Hitachi L-2000 high performance liquid chromatograph, Hitachi, Japan (for semi-preparative HPLC separation in example 4); JMN-ECA-400 superconducting NMR instrument, Japan Electron.

Weimaining raw material medicine (batch number: 12003), provided by Beijing Hua Yi pharmaceutical industry; the water is the Wahaha purified water; methanol and acetonitrile were chromatographically pure, Fisher corporation, usa; formic acid is analytically pure, chemical reagent of national drug group, ltd. C18 filler (specification: 120A, 50 μm), Japan YMC.

Example 1: thiolation and HPLC analysis of condensed tannin sites (Wilmining drug substance)

Dissolving 200.0mg of a Weimeining raw material medicine in 20.0mL of methanol, adding 21.6mL (containing 1.6mL of concentrated hydrochloric acid) of 100mg/mL cysteamine hydrochloride solution, putting the mixture into a 250mL distillation flask, carrying out water bath at 65 ℃ for 15min, cooling, adding 160.0mL of water for quenching, and concentrating to about 3.0mL to obtain a thiolation solution.

Example 2: HPLC analysis of sulfur decomposition product and qualitative identification of chromatographic peak

The sample used was the thiolated solution prepared in example 1.

HPLC conditions: a chromatographic column: thermo Syoronis C18 chromatography column (4.6 mm. times.250 mm, 5 μm); column temperature: 30 ℃; flow rate: 1 mL/min; sample introduction amount: 20 mu L of the solution; detection wavelength: 280 nm; mobile phase: elution was carried out in a gradient with 0.2% formic acid (A) and 0.2% formic acid acetonitrile (B) as mobile phases, the elution procedure being shown in Table 2 below.

Table 2: gradient elution procedure for HPLC analysis of thiolytes

HPLC-MS analysis conditions:

HPLC conditions: a chromatographic column: agilent TC-C18(2) chromatography column (250 mm. times.4.6 mm, 5 μm); column temperature: 25 ℃; flow rate: 1 mL/min; the sample volume is 20 mu L; mobile phase: the elution was carried out in a gradient manner using 0.2% formic acid (A) and 0.2% formic acid acetonitrile (B) as mobile phases, and the procedure was as shown in Table 2.

MS conditions: the ion source is ESI, negative ion scanning mode, atomizing gas pressure is 40.0psi, dry gas flow is 10.0L/min, ion source temperature is 350 ℃, compound stability is 100%, and trap depth is 100%.

The analysis results are shown in FIG. 1.

The three larger sulfydryl ethylamine substitutes are respectively named as a peak 1 (presumed to be catechin sulfydryl ethylamine substitutes), a peak 2 (presumed to be epicatechin sulfydryl ethylamine substitutes) and a peak 3 (presumed to be epicatechin gallate sulfydryl ethylamine substitutes) by combining the HPLC-MS analysis result and the peak appearance sequence reported by the literature.

Example 3: c18 atmospheric column chromatographic separation of condensed tannin position sulfhydrolysis product

C18 normal pressure column adopts wet method to pack column and wet method to load sample. After loading the column (column volume 250mL) it was washed free of impurities (HPLC assay) with methanol and the mobile phase was gradually switched to the starting ratio.

Taking 3mL (prepared according to example 1) of the Weimaining bulk drug thiolate solution for C18 normal pressure column chromatography, taking 10% methanol water (containing 0.2% formic acid) as a mobile phase, taking each 5mL as a flow portion, combining the parts with similar purity of each compound according to the detection condition of HPLC in example 2, and combining the principles as follows: fractions with a purity of 95% or more were pooled together, 95% or less were pooled together and purified again by HPLC normalization. After removing methanol by concentration under reduced pressure, freeze-drying was carried out to obtain 4 samples: the sample for normal pressure column chromatography of peak 1C 18, the sample for normal pressure column chromatography of peak 2C 18 (purity 95% or more) 1, the sample for normal pressure column chromatography of peak 2C 18 (purity 95% or less) and the sample for normal pressure column chromatography of peak 3C 18.

And (3) performing C18 normal pressure column chromatography again on the No. 1 peak C18 normal pressure column chromatography separation sample, wherein the mobile phase is 5% methanol water (containing 0.2% formic acid), each 5mL of the mobile phase is used as one flow, the HPLC detection conditions are detected according to the example 2, the parts with similar purity are combined, the methanol is removed by decompression concentration, and the freeze drying is carried out, so that the No. 1 peak compound (supposed to be the catechin mercaptoethylamino substitute) with the purity of more than 95% (determined by an HPLC normalization method) is obtained.

And (3) carrying out C18 normal pressure column chromatography again on the No. 3 peak C18 normal pressure column chromatography separation sample, wherein the mobile phase is 10% methanol water (containing 0.2% formic acid), each 5mL of the mobile phase is used as one flow, the HPLC detection conditions are detected according to the example 2, the parts with similar purity are combined, the methanol is removed by decompression concentration, and then the mixture is frozen and dried to obtain the No. 3 peak compound (supposedly, the epigallocatechin gallate mercaptoethylamino substitute) with the purity of more than 95%.

Example 4: semi-preparative HPLC separation of Peak 2 Compound

Experimental samples: peak 2C 18 obtained in example 3 above was used to isolate sample 2 (purity 95% or less) by atmospheric column chromatography.

Semi-preparative HPLC conditions: a chromatographic column: YMC-Pack ODS-A column (10 mm. times.250 mm, 5 μm); column temperature: 30 ℃; flow rate: 1 mL/min; sample introduction amount: 1 mL; detection wavelength: 280 nm; the elution was carried out in a gradient with 0.2% formic acid (A) and 0.2% formic acid methanol (B) as mobile phases, the elution being carried out according to the following Table 3.

Table 3: semi-preparative HPLC gradient elution procedure

Time (min)	A(％)	B(％)
			0	97	3
8	80	20
			25	75	25
35	60	40
			45	20	80
60	97	3

And (3) carrying out semi-preparative HPLC separation and purification on the sample of the No. 2 peak: and C18 normal pressure column chromatography separation No. 2 peak sample solution is injected with sample, 1 mL/time, and the preparation is carried out according to the semi-preparative HPLC chromatographic conditions. Fractions from peak 2 samples were collected as peak: fractions of the first 33min are collected together as front impurity fractions, fractions of 33 min-43 min are collected together as product fractions, and fractions of the second impurity are collected together after 43 min. And (3) merging and concentrating the impurity fractions before and after the impurity fractions are subjected to sample injection once according to the method, detecting the product fraction according to the HPLC condition in the embodiment 2, concentrating under reduced pressure to remove methanol, and freeze-drying to obtain the compound of No. 2 peak with the purity of more than 95% (supposedly, the compound is the epicatechin mercaptoethylamino substitute).

The HPLC charts of 3 compounds with purity of 95% or more prepared in this example are shown in FIGS. 2A, 2B, and 2C.

Example 5: 3 compounds with purity of more than 95 percent¹Determination of H-NMR spectra

The samples used were the peak 1 compound, the peak 3 compound obtained in example 3, the peak 2 compound obtained in example 4, or the peak 2C 18 obtained in example 3, and sample 1 (purity 95% or more) was separated by atmospheric column chromatography.

Each test compound was precisely weighed at 3mg, dissolved in DMSO-d6, and then measured.

Peak 1 compound:¹H-NMR spectrum (400MHz, DMSO-d6) delta: 2.93-2.62 (4H, m, -CH2-CH2-), delta: 3.86-3.82 (1H, m,4-H), delta: 4.03(1H, s,3-H), delta: 4.64(1H, d, J ═ 9.64Hz,2-H), delta: 5.47(1H, s,6-H), delta: 5.78(1H, s,8-H), delta: 6.65-6.54 (3H, m, J ═ 8.08Hz,2 ' -H,5 ' -H,6 ' -H), combined with literature data (J.L.Torpedo and R.Bobeth 2001, New Flavovatives yield from Graves (vitamins of great variety) and B.B.Oxidata. and C.1. the compound of formula 1, see the previous compound of amino group of formulas, see the formula of formula 1, 35, family of formula, family II, family III.

Peak 2 compound:¹H-NMR spectrum (400MHz, DMSO-d6) delta: 3.09-2.72 (4H, m, -CH2-CH2-), delta: 3.82(2H, ss,3-H,4-H), Δ: 5.02(1H, s,2-H), Δ: 5.68(1H, d, J ═ 2.24Hz,6-H), δ: 5.89(1H, d, J ═ 2.24Hz,8-H), δ: 6.88 to 6.65(2H, m,5 '-H, 6' -H), δ: 6.91(1H, s, 2' -H). Combine literature data (J.L.Torres and R.Bobet, New Flavonol Derivatives from Grape (vitas vinifera) Byproducts.Antionodal Aminoethyl thiohi-Flavan-3-ol Conjug)ates from a polymeric Waste Fraction Used as a Source of Flavanols, J.Agric.food chem.2001,49, 4627-one 4634), compound 2 was identified as 4 β - (2-aminoethylthio) epicatechin, the structure of which is shown in Compound 2 above.

Peak 3 compound:¹H-NMR spectrum (400MHz, DMSO-d6) delta: 3.08-2.86 (4H, m, -CH2-CH2-), delta: 3.93(1H, s,4-H), delta: 5.07(1H, d, J ═ 4.72Hz,3-H), delta: 5.29(1H, s,2-H), delta: 5.78(1H, d, J ═ 2.04Hz,6-H), delta: 5.90(1H, s,8-H), delta: 6.74-6.62 (4H, m,6 ' -H,2 ' -H,5 ' -H,6 ' -H), delta: 6.88(1H, s,2 ' -H), combined literature data (J.L.Botors and R.B.B.R.V.F.V.A. and N.F.E. beta.P.E. Catechu.E.C.A. combined literature data (J.L.P.E. Botorum and R.E.E. C., N.E. C. see Table 3, U.E. 5, N.E. C. 7, N.E. C. 3, N. 5, N. C. H, N.E. 5, N. H, N. 4, N. H, N. 4, N. H, N. 5, N. H, N. 4, N. H.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for separating mercaptoethylamino substitute of hydroxyflavan compound comprises the following steps:

(1) subjecting the sample to C18 atmospheric column chromatography, the C18 atmospheric column chromatography being one or more times; the mobile phase of the C18 normal pressure column chromatographic separation is methanol water solution with the concentration of 2-12 percent, and the mobile phase also contains formic acid or acetic acid with the concentration of 0.1-1 percent;

in the step (1), the elution fractions are detected by HPLC, and the fractions with the same or similar purity are combined to remove methanol;

wherein, the HPLC conditions are as follows:

a chromatographic column: thermo Syncronis C18 chromatography column; column temperature: 30 ℃; flow rate: 1 mL/min; sample introduction amount: 20 mu L of the solution; detection wavelength: 280 nm;

mobile phase: gradient elution is carried out by taking 0.2 percent formic acid water (A) and 0.2 percent formic acid acetonitrile (B) as mobile phases; the elution procedure is as in table 2 below,

TABLE 2

The separation method further comprises a step (2),

wherein, the conditions of the semi-preparative HPLC are as follows:

a chromatographic column: a YMC-Pack ODS-A chromatography column; column temperature: 30 ℃; flow rate: 1 mL/min; sample introduction amount: 1 mL; detection wavelength: 280 nm;

mobile phase: gradient elution with 0.2% formic acid water (A) and 0.2% formic acid methanol (B) as mobile phase;

elution procedure for semi-preparative HPLC is shown in table 3 below,

TABLE 3

Time (min) A(％) B(％) 0 97 3 8 80 20 25 75 25 35 60 40 45 20 80 60 97 3

；

Wherein, the mercaptoethylamino substitute of the hydroxyflavan compound is shown as the following formula I,

wherein,

R₁selected from H and OH;

R₂selected from H, OH and formula II;

R₃selected from H, OH and formula II;

and R is₂And R₃Different;

and, the sample is a or b as follows:

a. degradation products of the condensed tannin by a thiolytic reagent;

b. degradation products of condensed tannin containing plant extracts by thiolytic agents.

2. The method of claim 1, wherein the C18 atmospheric column chromatography is performed 2, 3, 4, or 5 times.

3. The method of claim 1 or 2, wherein the sample contains a hydroxy flavanoid thioethylamino substitution.

4. The method according to claim 1 or 2, wherein the condensed tannin-containing plant extract is a condensed tannin-containing grape seed extract or a condensed tannin-containing wild buckwheat rhizome extract.

5. The method according to claim 1 or 2, wherein the condensed tannin containing plant extract is wiener.

6. The process of claim 1 or 2 wherein the thiolytic reagent of a or b is cysteamine hydrochloride.

7. The method according to claim 1 or 2, wherein the degradation is carried out in a water bath at 50-80 ℃ for 5-50 min.

8. The method according to claim 1 or 2, wherein the condensed tannin in a or b is represented by the following formula A,

wherein,

R₁selected from H and OH;

R₂selected from the group consisting of OH and formula II below,

9. the method according to claim 1 or 2, wherein the hydroxy flavanoid mercaptoethylamino substitution is selected from the group consisting of:

4 β - (2-aminoethylthio) catechin,

4 β - (2-aminoethylthio) epicatechin,

4 β - (2-aminoethylthio) epicatechin gallate,

4 β - (2-aminoethylthio) catechin gallate,

4 β - (2-aminoethylthio) gallocatechin,

4 β - (2-aminoethylthio) epigallocatechin,

4 β - (2-aminoethylthio) gallocatechin gallate, and

4 β - (2-aminoethylthio) epigallocatechin gallate.

10. The method according to claim 1, wherein the C18 atmospheric column chromatography in step (1) satisfies any one, two or three of the following 1) to 3):

1) the concentration of the methanol water solution is 3% -12%;

2) the volume of each flow part is 1-15 mL;

3) filling a column by adopting a wet method, and loading a sample by adopting the wet method; after column packing, washing with methanol until no impurities exist, and gradually transitioning the mobile phase to the initial ratio.

11. The method according to claim 10, wherein the concentration of the aqueous methanol solution in item 1) is 4% to 12%.

12. The method according to claim 10, wherein the concentration of the aqueous methanol solution in item 1) is 5% to 12%.

13. The method according to claim 1, wherein the concentration of the aqueous methanol solution in step (1) is 2% to 10%.

14. The method according to claim 10, wherein the concentration of the aqueous methanol solution in item 1) is 4% to 11%.

15. The method according to claim 1, wherein the concentration of the aqueous methanol solution in step (1) is 2%, 3%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5% or 12%.

16. The method of claim 1, wherein the concentration of formic acid or acetic acid in the mobile phase of the C18 atmospheric column chromatography in step (1) is 0.1% -0.5%.

17. The method of claim 1, wherein the concentration of formic acid or acetic acid in the mobile phase of the C18 atmospheric column chromatography in step (1) is 0.1% -0.4%.

18. The method of claim 1, wherein the concentration of formic acid or acetic acid in the mobile phase of the C18 atmospheric column chromatography in step (1) is 0.1% -0.3%.

19. The method of claim 1, wherein the concentration of formic acid or acetic acid in the mobile phase of the C18 atmospheric column chromatography in step (1) is 0.15% -0.25%.

20. The method of claim 1, wherein in step (1), the concentration of formic acid or acetic acid in the mobile phase of the C18 atmospheric column chromatography is 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or 1%.

21. The method of claim 10, wherein the volume of each fraction in item 2) is 2-10 mL.

22. The method of claim 10, wherein the volume of each fraction in item 2) is 3-8 mL.

23. The method of claim 10, wherein the volume of each fraction in item 2) is 4-6 mL.

24. The method of claim 10, wherein the volume of each fraction in item 2) is 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, or 10 mL.

25. The method according to claim 10, wherein in item 3), the packed column volume is 10 to 1000 mL.

26. The method according to claim 1 or 2, wherein in the step (1), the product from which methanol is removed is further subjected to freeze-drying.

27. A method for preparing mercaptoethylamino substitutes of hydroxyflavan compounds, comprising the method for isolating mercaptoethylamino substitutes of hydroxyflavan compounds as claimed in any one of claims 1 to 26.