阅读说明：本技术 一种从藤茶组织中分离纯化和鉴定二氢杨梅素的方法 (A method of dihydromyricetin is isolated and purified and identified from vine tea tissue ) 是由 何美军 李宇 黄东海 艾伦强 方诗琪 张宇 廖璐婧 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二氢杨梅素的方法,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、纯化二氢杨梅素,利用LC-HR-ESI MS、1D和2D NMR波谱数据分析,鉴定其结构分别为(+)Dihydromyricetin(60.5mg),通过HPLC检测分析化合物的纯度≥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(+)Dihydromyricetin(1)活性单体化合物的方法,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 dihydromyricetin from vine tea tissue, by being separated from vine tea tissue using silica gel column chromatography and half preparation efficient liquid phase, purifying dihydromyricetin, it is analyzed using LC-HR-ESI MS, 1D and 2D NMR spectra data, it identifies that its structure is respectively (+) Dihydromyricetin (60.5mg), purity >=98% of compound 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 (+) Dihydromyricetin (1) active monomer compound, make full use of vine tea old leaf, inferior leaf, avoid the vine tea wasting of resources, great economic benefit is created, may advantageously facilitate vine tea industry development.)

1. a kind of method for isolating and purifying dihydromyricetin 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 and identifying dihydromyricetin from vine tea tissue described in accordance with the claim 1, special Sign is that general flavone is extracted medicinal extract by step b and 100-200 mesh purification on normal-phase silica gel mixed in equal amounts mixes sample, and 5 times of quality 300-400 mesh are just Phase silica gel fills column, number A column, with solvent petroleum ether, ethyl acetate, methanol elution, elution program 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, 100% petroleum ether of A-1 elution fraction；A-2 elution fraction 80% Petroleum ether, 20% ethyl acetate；50% petroleum ether of A-3 elution fraction, 50% 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 is washed De- 50% ethyl acetate of component, 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；90% chloroform of B-6 elution fraction, 10% 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, 10% acetone of C-1 elution fraction, 90% water；C-2 elution fraction 20% acetone, 80% water；30% acetone of C-3 elution fraction, 70% water；40% acetone of C-4 elution fraction, 60% water；C-5 elution 50% acetone of component, 50% water；60% acetone of C-6 elution fraction, 40% water；70% acetone of C-7 elution fraction, 30% water；C-8 80% acetone of elution fraction, 20% water.

3. a kind of method for isolating and purifying and identifying dihydromyricetin from vine tea tissue described in accordance with the claim 1, special Sign be step C by described by the component C-3 containing targeted activity compound, it is pure with Shimadzu efficient liquid phase C18 reverse phase post separation 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 72%, B phase 28%；Time:23min, A phase 72%, B phase 28%；Time: 28min, A phase 0%, B phase 100%；Time:27min, A phase 0%, B phase 100%；Time:27.1min, A phase 72%, B phase 28%；Time:30min, stop；Detection wavelength 270nm, 28%B equality affords compound dihydromyricetin: Rt= 12.5min。

4. a kind of identification method of dihydromyricetin, it is characterised in that the MS of dihydromyricetin reactive compound structure,¹H NMR Spectrum,¹³C H NMR spectroscopy, DEPT135NMR spectrum identification: [M+H] of compound dihydromyricetin⁺321.1 being accredited as dihydromyricetin (+) Dihydromyricetin monomeric 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 dihydromyricetin list from vine tea tissue The method of body compound, present invention relates particularly to a kind of methods for isolating and purifying and identifying dihydromyricetin 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.Separate and identify in vine tea some column rich contents, such as dihydromyricetin of the flavonoids with important physiological function, Myricetin and texifolin isoreactivity ingredient.The present inventor studies dihydromyricetin in discovery vine tea young tender stem tip (the Lichuan place of production) Content be 186.31 ± 10.43mg/g, dihydromyricetin be accumulated in vine tea young tender stem tip tissue be up to 18%.Study table Bright dihydromyricetin is lived with anti-oxidant, anti-neuroinflamation, neuroprotection, anti-senile dementia disease, antibacterial, a variety of physiology such as anticancer Property.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 sufficiently It utilizes, causes the waste of vine tea resource.

Summary of the invention

It is an object of the invention to efficiently, economically isolate and purify and identify dihydro from vine tea old leaf, inferior leaf The method of myricetin (+) Dihydromyricetin monomeric compound makes full use of vine tea old leaf, inferior leaf, avoids resource unrestrained Take, to create economic benefit.

A kind of method that dihydromyricetin 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 for isolating and purifying and identifying dihydromyricetin from vine tea tissue of the present invention, it is characterised in that step b General flavone is extracted into medicinal extract 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, 100% petroleum ether of A-1 elution fraction；80% petroleum ether of A-2 elution fraction, 20% ethyl acetate；50% petroleum ether of A-3 elution fraction, 50% ethyl acetate；20% petroleum ether of A-4 elution fraction, 80% second Acetoacetic ester；100% ethyl acetate of A-5 elution fraction；80% ethyl acetate of A-6 elution fraction, 20% methanol；A-7 elution fraction 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；90% chloroform of B-6 elution fraction, 10% 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, 10% acetone of C-1 elution fraction, 90% water；C-2 elution 20% acetone of component, 80% water；30% acetone of C-3 elution fraction, 70% water；40% acetone of C-4 elution fraction, 60% water；C-5 50% acetone of elution fraction, 50% water；60% acetone of C-6 elution fraction, 40% water；70% acetone of C-7 elution fraction, 30% Water；80% acetone of C-8 elution fraction, 20% water；

A kind of method isolating and purifying and identify dihydromyricetin from vine tea tissue of the present invention, it is characterised in that Step C by described by the component C-3 containing targeted activity compound, 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 are washed De- program is pressed: Time:0.01min, A phase 72%, B phase 28%；Time:23min, A phase 72%, B phase 28%；Time:28min, A phase 0%, B phase 100%；Time:27min, A phase 0%, B phase 100%；Time:27.1min, A phase 72%, B phase 28%； Time:30min, stop；Detection wavelength 270nm, 28%B equality affords compound dihydromyricetin: Rt= 12.5min。

A kind of identification method of dihydromyricetin of the present invention, it is characterised in that dihydromyricetin reactive compound structure MS、¹H H NMR spectroscopy,¹³C H NMR spectroscopy, the identification of DEPT135 H NMR spectroscopy: [M+H] of compound dihydromyricetin⁺321.1, it is accredited as Dihydromyricetin (+) Dihydromyricetin monomeric compound.

The 1H (400MHz) and 13C (101MHz) NMR data of dihydromyricetin compound 1

Note:^aWith CD₃OD is test solvent；

One kind of the present invention isolates and purifies and identifies dihydromyricetin (+) from vine tea old leaf, inferior leaf The method of Dihydromyricetin, 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-ESIMS, 1D and 2D NMR spectra data, identify that its structure is (+)Dihydromyricetin.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 (+) Dihydromyricetin (1) monomeric compound, makes full use of vine tea old leaf, inferior leaf, avoids the wasting of resources, To create economic benefit.

2) present invention solves efficiently, economically to isolate and purify and identify (+) from vine tea old leaf, inferior leaf The method of Dihydromyricetin (1) monomeric compound, makes full use of vine tea old leaf, inferior leaf, avoids the wasting of resources, creates Great economic benefit may advantageously facilitate 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 dihydromyricetin Element is analyzed using LC-HR-ESI MS, 1D and 2D NMR spectra data, identifies that its structure is respectively (+) Dihydromyricetin (60.5mg) tests and analyzes purity >=98% of compound by HPLC.The present invention solves efficient, warp Ji ground separates from vine tea old leaf, inferior leaf, purifies and identifies (+) Dihydromyricetin (1) active monomer compound Method makes full use of vine tea old leaf, inferior leaf, avoids 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 (+) Dihydromyricetin monomeric compound；

Attached drawing 2 is (+) Dihydromyricetin 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.