阅读说明：本技术 一种从藤茶组织中分离纯化和鉴定花旗松素的方法 (A method of texifolin is isolated and purified and identified from vine tea tissue ) 是由 何美军 李宇 艾伦强 黄东海 张宇 廖璐婧 方诗琪 于 2019-09-17 设计创作，主要内容包括：本发明提供了一种从藤茶组织中分离纯化和鉴定花旗松素的方法,本发明通过利用硅胶柱色谱及半制备高效液相从藤茶组织中分离、纯化花旗松素活性化合物,利用LC-HR-ESI MS、1D和2D NMR波谱数据分析,鉴定其结构为花旗松素(4.6mg),通过HPLC检测分析花旗松素的纯度≥98％。本发明解决高效、经济地从藤茶老叶、残次叶中分离、纯化和鉴定花旗松素(1)单体化合物的方法,充分利用藤茶老叶、残次叶,避免藤茶资源浪费,创造巨大经济效益,有利于促进藤茶产业发展。(The present invention provides a kind of methods for isolating and purifying and identifying texifolin from vine tea tissue, the present invention using silica gel column chromatography and half preparation efficient liquid phase from vine tea tissue by being separated, purifying texifolin reactive compound, it is analyzed using LC-HR-ESI MS, 1D and 2D NMR spectra data, it identifies that its structure is texifolin (4.6mg), purity >=98% of texifolin is tested and analyzed by HPLC.The present invention solves the method for efficiently, economically separating from vine tea old leaf, inferior leaf, purifying and identifying texifolin (1) monomeric compound, make full use of vine tea old leaf, inferior leaf, the vine tea wasting of resources is avoided, great economic benefit is created, may advantageously facilitate vine tea industry development.)

1. a kind of method for isolating and purifying texifolin from vine tea tissue, it is characterised in that steps are as follows:

A. vine tea old leaf, inferior leaf, EtOH Sonicate coarse extraction Flavonoids are collected；

B. crude flavonoid powder medicinal extract is obtained by step a to separate using normal phase silica gel column chromatography, obtain the component containing targeted activity ingredient；

C. the component containing targeted activity ingredient is isolated and purified to obtain the monomer of reactive compound with half preparation efficient liquid phase；

D. according to the monomer structure using LC-HR-ESIMS, 1D and 2D NMR spectra data parsing target compound.

2. a kind of method for isolating and purifying texifolin from vine tea tissue described in accordance with the claim 1, it is characterised in that step General flavone is extracted medicinal extract to rapid b and 100-200 mesh purification on normal-phase silica gel mixed in equal amounts mixes sample, 5 times of quality 300-400 mesh purification on normal-phase silica gel dresses Column, number A column, with solvent petroleum ether, ethyl acetate, methanol elute, elution program according to: A-1, A-2 to A-7, wherein A column is washed De- program are as follows: elution volume: for 5 times of column volumes, 80% petroleum ether of A-1 elution fraction, 20% ethyl acetate；A-2 elution fraction 60% petroleum ether, 40% ethyl acetate；40% petroleum ether of A-3 elution fraction, 60% ethyl acetate；20% stone of A-4 elution fraction Oily ether, 80% ethyl acetate；100% ethyl acetate of A-5 elution fraction；80% ethyl acetate of A-6 elution fraction, 20% methanol； 50% ethyl acetate of A-7 elution fraction, 50% methanol；

The component A-6 containing targeted activity compound and 100-200 mesh purification on normal-phase silica gel mixed in equal amounts are mixed into sample, 5 times of quality 300-400 mesh purification on normal-phase silica gel fills column, number B column, with solvent chloroform, methanol elution, elution program according to B-1, B-2 to B-7, Wherein B column elution program are as follows: elution volume: for 5 times of column volumes, 100% chloroform of B-1 elution fraction；98% chlorine of B-2 elution fraction It is imitative, 2% methanol；96% chloroform of B-3 elution fraction, 4% methanol；94% chloroform of B-4 elution fraction, 6% methanol；B-5 elution group Divide 92% chloroform, 8% methanol；85% chloroform of B-6 elution fraction, 15% methanol；80% chloroform of B-7 elution fraction, 20% methanol；

Component B-3, B-4 and 300-400 mesh reverse phase silica gel mixed in equal amounts containing targeted activity compound is mixed into sample, 10 times of quality 300-400 mesh reverse phase silica gel fills column, number C column, with solvent acetone, water elution, elution program according to C-1, C-2 to C-8, Middle C column elution program are as follows: elution volume: for 5 times of column volumes, 5% acetone of C-1 elution fraction, 95% water；C-2 elution fraction 15% acetone, 85% water；20% acetone of C-3 elution fraction, 80% water；25% acetone of C-4 elution fraction, 75% water；C-5 elution 30% acetone of component, 70% water；35% acetone of C-6 elution fraction, 65% water；40% acetone of C-7 elution fraction, 60% water；C-8 45% acetone of elution fraction, 55% water.

3. a kind of method for isolating and purifying texifolin from vine tea tissue described in accordance with the claim 1, it is characterised in that step Rapid C by described by the component C-6 containing targeted activity compound, it is anti-with Shimadzu efficient liquid phase C18 with acetonitrile and water gradient elution Phase column separating purification: A phase: 85%H₂O, 15% acetonitrile, 0.1% acetic acid, B phase: 15%H₂O, 85% acetonitrile, 0.1% acetic acid, stream Speed is 2.5ml/min, and elution program is pressed: Time:0.01min, A phase 85%, B phase 15%；Time:23min, A phase 50%, B phase 50%；Time:28min, A phase 0%, B phase 100%；Time:27min, A phase 0%, B phase 100%；Time:27.1min, A phase 80%, B phase 20%；Time:30min, stop；The elution of Detection wavelength 290nm, 28%B equality, obtains compound pesudotsuga taxifolia Element: Rt=17.5min.

4. a kind of identification method of texifolin, it is characterised in that the MS of texifolin reactive compound structure,¹H H NMR spectroscopy,¹³C H NMR spectroscopy, the identification of DEPT135 H NMR spectroscopy: [M+H] of compound texifolin⁺305.1, it is accredited as taxifolin monomer compound.

Technical field

The invention belongs to vine tea the Study on Resources evaluation and exploration technology field, it is related to a kind of utilizing high performance liquid chromatography (HPLC) and liquid NMR spectrum (Liquid NMR) instrument isolates and purifies and identifies taxifolin monomer from vine tea tissue The method of compound, present invention relates particularly to a kind of methods for isolating and purifying and identifying texifolin from vine tea tissue.

Background technique

Vine tea is commonly called as certain kind of berries tea (Wulin tomb specialty), rattan mother-in-law's tea, is Vitaceae Ampelopsis ampelopsis grossdentata kind.Hubei Province, Hunan, River, Guizhou Province, Fujian are distributed, and are mainly distributed on the sandstone hillside fields of Chinese Wuling mountain area.Vine tea is the treasure omitted by Compendium of Material Medica Product are the native Se and flavones composition having been found that rich in the necessary 17 kinds of amino acid of human body, 14 kinds of microelements and polysaccharide etc. The highest wild plant of content is valuable dual-purpose of drug and food pure plant.

It is Vitaceae (Vitaceae), Ampelopsis (Ampelopsis Michx) plant present invention relates particularly to vine tea The dried leaf and stem of Ampelopsis grossedentata is processed into, and is the dual-purpose of drug and food for being distributed widely in the mountain area of China Drink.Some column rich contents, the flavonoids with important physiological function such as texifolin, poplar are separated and identified in vine tea Syphilis and texifolin isoreactivity ingredient.Texifolin contains in the present inventor's research discovery vine tea young tender stem tip (the Lichuan place of production) Amount is 14.76 ± 2.26mg/g, and being accumulated in vine tea young tender stem tip tissue for texifolin is up to 18%.Research shows that pesudotsuga taxifolia Element has anti-oxidant, anti-inflammatory, protection angiocarpy, protection liver, anti-senile dementia disease, a variety of physiological activity such as antibacterial and anticancer. But the vine tea young tender stem tip tissue of different producing area harvesting after, old leaf, inferior leaf flavones active component do not obtain abundant benefit With causing the waste of vine tea resource.

Dihydromyricetin extracts from a kind of bejuco of Vitaceae Ampelopsis more, and also useful honey raisin tree is extracted, Middle main active is flavone compound, and substance of this kind, which has, removes free radical, anti-oxidant, antithrombotic, antitumor, anti-inflammatory Etc. a variety of peculiar effects；And dihydromyricetin is a kind of more special flavone compound, except with flavone compound Outside general characteristic, also has and release alcoholism, prevention alcoholic liver, fatty liver, liver cell inhibited to deteriorate, reduce the disease incidence of liver cancer The effects of.It is liver protecting, the non-defective unit of Dealcoholic sobering-up.

Summary of the invention

It is an object of the invention to solve, efficiently, economically from vine tea old leaf, inferior leaf, separation is separated, purifies and is identified The method of texifolin (1) monomeric compound, makes full use of vine tea old leaf, inferior leaf, avoids the wasting of resources, to create economy Benefit.Present invention ultrasonic extraction general flavone from vine tea old leaf, inferior leaf with 60% ethanol solution utilizes silica gel column chromatography and half Efficient liquid phase separation is prepared, is analyzed using LC-HR-ESI MS, 1D and 2D NMR spectra data, identifies that its structure is pesudotsuga taxifolia Element.

A kind of method that texifolin is isolated and purified from vine tea tissue of the present invention, it is characterised in that steps are as follows:

A. vine tea old leaf, inferior leaf, EtOH Sonicate coarse extraction Flavonoids are collected；

B. crude flavonoid powder medicinal extract is obtained by step a to separate using normal phase silica gel column chromatography, obtain containing targeted activity ingredient Component；

C. the component containing targeted activity ingredient is isolated and purified to obtain the list of reactive compound with half preparation efficient liquid phase Body；

D. according to the monomer structure using LC-HR-ESI MS, 1D and 2D NMR spectra data parsing target compound.

A kind of method that texifolin is isolated and purified from vine tea tissue of the present invention, it is characterised in that step b is by general flavone It extracts medicinal extract and 100-200 mesh purification on normal-phase silica gel mixed in equal amounts and mixes sample, 5 times of quality 300-400 mesh purification on normal-phase silica gel fill columns, number A column, With solvent petroleum ether, ethyl acetate, methanol elution, elution program is according to A-1, A-2 to A-7, wherein A column elution program are as follows: Elution volume: for 5 times of column volumes, 80% petroleum ether of A-1 elution fraction, 20% ethyl acetate；60% petroleum of A-2 elution fraction Ether, 40% ethyl acetate；40% petroleum ether of A-3 elution fraction, 60% ethyl acetate；20% petroleum ether of A-4 elution fraction, 80% Ethyl acetate；100% ethyl acetate of A-5 elution fraction；80% ethyl acetate of A-6 elution fraction, 20% methanol；A-7 elution group Divide 50% ethyl acetate, 50% methanol；

The component A-6 containing targeted activity compound and 100-200 mesh purification on normal-phase silica gel mixed in equal amounts are mixed into sample, 5 times Quality 300-400 mesh purification on normal-phase silica gel fill column, number B column, with solvent chloroform, methanol elution, elution program according to: B-1, B-2 are extremely B-7, wherein B column elution program are as follows: elution volume: for 5 times of column volumes, 100% chloroform of B-1 elution fraction；B-2 elution fraction 98% chloroform, 2% methanol；96% chloroform of B-3 elution fraction, 4% methanol；94% chloroform of B-4 elution fraction, 6% methanol；B-5 92% chloroform of elution fraction, 8% methanol；85% chloroform of B-6 elution fraction, 15% methanol；80% chloroform of B-7 elution fraction, 20% methanol；

Component B-3, B-4 and 300-400 mesh reverse phase silica gel mixed in equal amounts containing targeted activity compound is mixed into sample, 10 times Quality 300-400 mesh reverse phase silica gel fills column, number C column, and with solvent acetone, water elution, elution program is according to C-1, C-2 to C- 8, wherein C column elution program are as follows: elution volume: for 5 times of column volumes, 5% acetone of C-1 elution fraction, 95% water；C-2 elution group Divide 15% acetone, 85% water；20% acetone of C-3 elution fraction, 80% water；25% acetone of C-4 elution fraction, 75% water；C-5 is washed De- 30% acetone of component, 70% water；35% acetone of C-6 elution fraction, 65% water；40% acetone of C-7 elution fraction, 60% water； 45% acetone of C-8 elution fraction, 55% water；

A kind of method that texifolin is isolated and purified from vine tea tissue of the present invention, it is characterised in that step C is incited somebody to action described Component C-6 containing targeted activity compound is pure with Shimadzu efficient liquid phase C18 reverse phase post separation with acetonitrile and water gradient elution Change: A phase: 85%H₂O, 15% acetonitrile, 0.1% acetic acid, B phase: 15%H₂O, 85% acetonitrile, 0.1% acetic acid, flow velocity 2.5ml/ Min, elution program are pressed: Time:0.01min, A phase 85%, B phase 15%；Time:23min, A phase 50%, B phase 50%；Time: 28min, A phase 0%, B phase 100%；Time:27min, A phase 0%, B phase 100%；Time:27.1min, A phase 80%, B phase 20%；Time:30min, stop；The elution of Detection wavelength 290nm, 28%B equality, obtains compound texifolin: Rt= 17.5min。

A kind of identification method of texifolin of the present invention, it is characterised in that the MS of texifolin reactive compound structure,¹H H NMR spectroscopy,¹³C H NMR spectroscopy, the identification of DEPT135 H NMR spectroscopy: [M+H] of compound texifolin⁺305.1, it is accredited as texifolin list Body compound.

¹H (400MHz) and¹³C (101MHz) NMR data

Note:^*With CD₃OD is test solvent；

The present invention is a kind of to be separated using silica gel column chromatography and half preparation efficient liquid phase, utilizes LC-HR-ESI MS, 1D and 2D The analysis of NMR spectra data identifies that its structure is the method for texifolin, and feature includes the following steps:

Step 1, vine tea vine tea old leaf, inferior leaf sample, EtOH Sonicate coarse extraction Flavonoids are collected；

Step 2, by step 1 obtain crude flavonoid powder medicinal extract using normal phase silica gel column chromatography separation, obtain containing targeted activity at The component divided；

Step 3, the component containing targeted activity ingredient is isolated and purified to obtain reactive compound with half preparation efficient liquid phase Monomer；

Step 4, according to the monomer knot using LC-HR-ESI MS, 1D and 2D NMR spectra data parsing target compound Structure.

General flavone is extracted medicinal extract and purification on normal-phase silica gel by one of the preferred embodiment as technical solution of the present invention (100-200 mesh) mixed in equal amounts mixes sample, and 5 times of quality purification on normal-phase silica gel (300-400 mesh) fill column (number A column), with solvent petroleum Ether, ethyl acetate, methanol elution, elution program is according to A-1, A-2 to A-7, wherein A column elution program are as follows: elution volume: being 5 Times column volume, 80% petroleum ether of A-1 elution fraction, 20% ethyl acetate；60% petroleum ether of A-2 elution fraction, 40% acetic acid second Ester；40% petroleum ether of A-3 elution fraction, 60% ethyl acetate；20% petroleum ether of A-4 elution fraction, 80% ethyl acetate；A-5 100% ethyl acetate of elution fraction；80% ethyl acetate of A-6 elution fraction, 20% methanol；50% acetic acid second of A-7 elution fraction Ester, 50% methanol；

One of preferred embodiment as technical solution of the present invention, by the component A- containing targeted activity compound 6 mix sample with purification on normal-phase silica gel (100-200 mesh) mixed in equal amounts, and 5 times of quality purification on normal-phase silica gel (300-400 mesh) fill column (number B column), It is eluted with solvent chloroform, methanol, wherein B column elution program are as follows: elution volume: for 5 times of column volumes, 100% chlorine of B-1 elution fraction It is imitative；98% chloroform of B-2 elution fraction, 2% methanol；96% chloroform of B-3 elution fraction, 4% methanol；94% chlorine of B-4 elution fraction It is imitative, 6% methanol；92% chloroform of B-5 elution fraction, 8% methanol；85% chloroform of B-6 elution fraction, 15% methanol；B-7 elution 80% chloroform of component, 20% methanol；

One of preferred embodiment as technical solution of the present invention, by the component B- containing targeted activity compound 3, B-4 and reverse phase silica gel (300-400 mesh) mixed in equal amounts mix sample, and 10 times of quality reverse phase silica gels (300-400 mesh) fill column (number C Column), with solvent acetone, water elution, elution program is according to C-1, C-2 to C-8, wherein C column elution program are as follows: elution volume: for 5 times of column volumes, 5% acetone of C-1 elution fraction, 95% water；15% acetone of C-2 elution fraction, 85% water；C-3 elution fraction 20% acetone, 80% water；25% acetone of C-4 elution fraction, 75% water；30% acetone of C-5 elution fraction, 70% water；C-6 elution 35% acetone of component, 65% water；40% acetone of C-7 elution fraction, 60% water；45% acetone of C-8 elution fraction, 55% water；

One of preferred embodiment as technical solution of the present invention, by the component C- containing targeted activity compound 6, with acetonitrile and water gradient elution, with Shimadzu efficient liquid phase C18 reverse phase column separating purification: A phase: 85% H₂O, 15% acetonitrile, 0.1% acetic acid, B phase: 15%H₂O, 85% acetonitrile, 0.1% acetic acid, flow velocity 2.5ml/min, elution program are pressed: Time: 0.01min, A phase 85%, B phase 15%；Time:23min, A phase 50%, B phase 50%；Time:28min, A phase 0%, B phase 100%；Time:27min, A phase 0%, B phase 100%；Time:27.1min, A phase 80%, B phase 20%；Time:30min, stop；The elution of Detection wavelength 290nm, 28%B equality, obtains compound texifolin: Rt=17.5min.

One of preferred embodiment as technical solution of the present invention is selected and utilizes LC-HR-ESI MS, Bruker AV400 superconduction (liquid) nuclear magnetic resonance chemical analyser (400/175MHz, TMS are internal standard) test target reactive compound structure MS、¹H spectrum,¹³C spectrum, DEPT135 spectrum are to identify structure.

It is of the present invention a kind of to separate from vine tea old leaf, the inferior leaf, purify and identification taxifolin monomer compound Method, at least having the following beneficial effects is or advantage.

1) present invention utilizes 60% ethanol solution ultrasonic extraction general flavone from vine tea old leaf, inferior leaf, utilizes silicagel column Chromatography and half preparation efficient liquid phase separation, are analyzed using LC-HR-ESI MS, 1D and 2D NMR spectra data, identify that its structure is Texifolin.Easy to operate, economic cost is low, and gained subject monomers compound purity is high (>=98%).

It is an object of the invention to solve, efficiently, economically from vine tea old leaf, inferior leaf, separation is separated, purifies and is identified The method of texifolin (1) monomeric compound, makes full use of vine tea old leaf, inferior leaf, avoids the wasting of resources, to create economy Benefit.

2) present invention solves efficient, economically separation, purifying and identification texifolin (1) from vine tea old leaf, inferior leaf The method of monomeric compound makes full use of vine tea old leaf, inferior leaf, avoids the wasting of resources, creates great economic benefit, is conducive to Promote vine tea industry development.

The present invention using silica gel column chromatography and half preparation efficient liquid phase from vine tea tissue by being separated, purifying texifolin Reactive compound is analyzed using LC-HR-ESI MS, 1D and 2D NMR spectra data, identifies that its structure is texifolin (4.6mg) tests and analyzes purity >=98% of texifolin by HPLC.The present invention solve efficiently, economically from vine tea old leaf, The method for separating in inferior leaf, purifying and identifying texifolin (1) monomeric compound, makes full use of vine tea old leaf, inferior leaf, keeps away Exempt from the vine tea wasting of resources, creates great economic benefit, may advantageously facilitate vine tea industry development.

Detailed description of the invention

Attached drawing 1 is the HPLC test map of taxifolin monomer compound；

Attached drawing 2 is texifolin structural formula；

Specific embodiment

Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described, Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all Belong to the scope of protection of the invention.