CN108558976B - The preparation method of low polarity rare ginsenoside Δ PPD and Δ PPT - Google Patents

Abstract

The present invention provides the preparation methods for including the quasi- monomeric compound Δ PPD of rare ginsenoside (comprising Δ (20-21) PPD and isomer Δ (20-22) PPD) and the quasi- monomeric compound Δ PPT of rare ginsenoside (comprising Δ (20-21) PPT and isomer Δ (20-22) PPT), compared to the existing method for preparing rare ginsenoside, this preparation method is simple, cost is relatively low, simultaneously on this basis, the present invention also provides prepare rare ginsenoside monomeric compound Δ (20-21) PPD, Δ (20-22) PPD and rare ginsenoside monomeric compound Δ (20-21) PPT, the method of Δ (20-22) PPT；The method by simple column chromatography for separation technology, can low cost, high efficiency and on a large scale separation prepare the quasi- monomeric compound Δ PPD of rare ginsenoside and its monomer component and the quasi- monomeric compound Δ PPT of rare ginsenoside and its monomer component.

Description

The preparation method of low polarity rare ginsenoside Δ PPD and Δ PPT

Technical field

The present invention relates to field of medicaments, and in particular to the preparation method of a kind of low polarity rare ginsenoside.

Background technique

Cancer is to torture the second-biggest-in-the-world disease of human life, and the death rate is only second to cardiovascular and cerebrovascular disease, is that the mankind are dead One of main factor died.Organize cancer mechanism, the official International Cancer Research Center (IARC) of subordinate negative by World Health Organization The latest edition " report of world's cancer " of duty predicts that swift and violent proliferation situation will be presented in global cases of cancer, by 2012 14,000,000 People, fast 19,000,000 people for increasing to 2025, were up to 24,000,000 people by 2035 year by year.Report also shows that the whole world was new in 2012 Increasing cases of cancer has nearly half to appear in Asia, and wherein most ranks first in China, the newly-increased cases of cancer height of China.2012 Newly-increased 3,070,000 cancer patients of China simultaneously cause about 2,200,000 people dead, account for the 21.9% and 26.8% of global total amount respectively.WHO Data be slightly below China statistics.2012 annual datas of national tumour Register publication show that China is annual newly-increased Cases of cancer about 3,500,000, it is therefore dead that there are about 2,500,000 people.

There are mainly three types of modes for the common method for the treatment of of cancer now: operation, radiotherapy and drug therapy, and which is selected and is controlled Treatment method then depends on position, grade malignancy, development degree and the patient body state of tumour.In Three models, operation Treatment method, the invasion of Chang Yinwei cancer cell spreads to adjacent tissue or far-end transfer and effect is limited；The treatment method of radiotherapy, then It is limited to the injury caused by other internal normal tissues；The treatment method of drug, it is pernicious for advanced stage dispersivity and metastatic Tumour is most basic treatment method.In past decades, though the chemotherapy for being conceived to direct killing tumour cell has significantly Development and progress becomes the backbone of tumor pharmacother, but this treatment mode is poor to the slow solid tumor effect of proliferation, drug Selectivity is small, toxicity is more and serious defect becomes the important limiting factor in clinical treatment.After operation, radiation and chemotherapy The 4th kind of mode later is the biological therapy of tumour, mainly passes through the effect of tumor host defense mechanism or biological agent The biologically of body itself is adjusted, to suppress or eliminate tumour；Although biological therapy without too big toxic side effect, Since technical requirements are tight, complex process, price is high, and numerous cancer patients and family members are difficult to bear, influence it and control in cancer It popularizes in treatment field.

Since there are above-mentioned various limitations, the research and development of natural antitumor drug achieve more and more concerns.It is natural anti- Cancer drug either in inhibition or killing tumor cell, adjustment body's immunity, improvement symptom and feature and mitigates chemicotherapy In toxic side effect, or in the conditioning after being ill of tumour, all have important function.As a result, natural plants new treatment will become after The 5th kind of mode after operation, radiotherapy, chemotherapy and biotherapy.

Araliaceae (araliaceae) Panax (Pana ×) plant, such as ginseng (P.ginseng), American Ginseng (P.quenquefolinus), Radix Notoginseng (P.notoginseng), panax japonicus (P.uaponicus), cucurbitaceae genus gynostemma Gynostemma pentaphylla (Gynostemma oentaphyllum Thrunb Mak) etc. is the rare traditional medicinal plant of China, main Effective component is dammarane type four-ring triterpenoid ginseng saponin series compound.Have now been found that the prototype ginseng in Araliaceae Saponin(e has more than 60 kinds, can be divided into two major classes: 1) glycol group ginsenoside (Ra, Rb1, Rb2, Rb3, Rc, Rd etc.)；2) triol group people Join saponin(e (Re, Rf, Rg1, Rg2, Rh1 etc.), is all made of glucoside member and sugar, is generally dissolved easily in water, curative effect specifically includes that immune Regulatory function, improving micro_circulation effect adjust digestive function, enhancing memory and learning ability, anti-aging, tranquilizing the mind etc., but not Show apparent anti-tumor activity.

Low polarity rare ginsenoside content in former panax species is little, exists only in wild ginseng or red ginseng, black In ginseng and the ginseng and Radix Notoginseng product of processings such as ripe Radix Notoginseng, it is also only ten thousand/it is several, and it is insoluble in water, usually only it is dissolved in ethyl alcohol Or in the low polar organic solvents such as ethyl acetate, referred to as low polarity rare ginsenoside mainly includes that prototype ginsenoside is logical Cometabolism derivative (C-k, Rg3, Rh2, Rh1, aPPD, aPPT etc.) and the side chain desugar simultaneously crossing glycosidic bond degradation and generating It is dehydrated conversion derivative (Rg5, Rk1, Rh3, Rk2, Δ (20-21) PPD, Δ (20-22) with more double bond structures formed PPD, Rh4, Rk3, F4, Rg6, Δ (20-21) PPT, Δ (20-22) PPT etc.).Low polarity rare ginsenoside is in addition to general Except the logical original bioactivity of prototype ginsenoside, also show that prototype saponin(e do not have is antitumor, antiviral etc. completely new Pharmaceutical activity, have high medical value and application prospect.

Hasegawa show husband (Japan Kokai 8-291194) report the preparation method comprises the following steps: sour water solution is natural or 3 hydroxyls Free ginsenoside, it is mono- that hydrolysate can get Δ (20-22) PPT and Δ (20-22) PPD after routine and chromatographic isolation Product.The primary product of sour water solution ginsenoside is the panaxatriol and panoxadiol of side chain cyclisation, Δ (20-22) PPT and Δ (20-22) PPD is only micro by-product, thus, Δ (20-22) PPT and the side PPD Δ (20-22) are prepared provided by the patent Legal low to conversion ratio, product yield is low, isolates and purifies difficulty height, is not suitable with industrial production.·

Chinese patent CN1249076C, CN1249077C and CN1269835C report new compound: 1,3 β, 6 α, 12 β-three hydroxyls -20 (21), and 24 (25)-diene-dammarane [dammar-3 β, 6 α, 12 β-trihydro × yl-20 (21), 24 (25)-diene, abbreviation Δ (20-21) PPT.Δ (20-21) PPT is Δ (20-22) PPT configurational isomer, has Δ (20- 22) PPT similar bioactivity；And 2: 3 β, 12 β-dihydroxy -20 (21), 24 (25)-diene-Da Ma burn [dammar-3 β, 12 β-dihydroxyl-20 (21), 24 (25)-diene, abbreviation Δ (20-21) PPD].Δ (20-21) PPD is Δ (20-22) PPD Configurational isomer, with the similar bioactivity of Δ (20-22) PPD, above-mentioned these types compound is needed using expensive Protopanaxatriol group raw material or the production preparation of raw material monomer Ginsenoside F1, or need to utilize expensive protopanoxadiol Group raw material or the production preparation of raw material monomer ginseng saponin C-K, are that industrial mass preparation cost is high, not can effectively solve Δ The contradiction of the supply shortage of (20-22) PPT and Δ (20-22) PPD.Until up to now, still without one on domestic and international market Enterprise, family can manufacture the rare metabolism ginsenoside and rare conversion ginseng soap of high-content (> 30-90%) on a large scale simultaneously Glycosides, including the higher Δ of anticancer activity (20-21) PPT, Δ (20-22) PPT, Δ (20-21) PPD and Δ (20-22) PPD.Therefore, the preparation method for studying above two low polarity rare ginsenoside, to the stronger ingredient of discovery activity and development Anti-cancer agent will have great importance.

Summary of the invention

The technical problem to be solved in the present invention is to provide include the quasi- monomeric compound Δ PPD of rare ginsenoside and standard The preparation method of monomeric compound Δ PPT, on this basis, the present invention also each provide the quasi- monomer of rare ginsenoside monomer The method for preparing purified of compound Δ PPD and quasi- monomeric compound Δ PPT.

First aspect present invention provides the preparation method of above-mentioned rare ginsenoside comprising: dilute containing low polarity Normal propyl alcohol, metallic sodium, benzoyl peroxide are sequentially added in the pyrolysis Araliaceae stem-leaf extract for having ginsenoside, and is led to Oxygen, and reaction solution heating reaction is made, wherein the pyrolysis Araliaceae containing low polarity rare ginsenoside is excellent Selected from wild ginseng, red ginseng, black ginseng, American Ginseng and Radix Notoginseng；

In a preferred example, the heating reaction carries out in four-hole bottle, and the four-hole bottle is passed through equipped with thermometer, oxygen Seal rubber pipe, condenser pipe are connected on pipe, top, the rubber tube other end connects funnel, and the funnel, which immerses, is equipped with liquid stone The beaker of wax,

In a preferred example, normal propyl alcohol is added to stir 10 minutes later,

In a preferred example, metallic sodium is added, reaction to no hydrogen is released,

In a preferred example, the heating reaction is reacted 24 hours for 86 DEG C,

In a preferred example, reaction solution after reaction, is cooled to room temperature, then washed reaction liquid by the heating, will It is dissolved after the normal propyl alcohol layer decompressing and extracting of reaction solution with water, then uses the low polar substances of n-hexane extraction, subsequently use acetic acid second Ester extraction, is evaporated ethyl acetate portion and obtains rare ginsenoside.

In a preferred example, ethyl acetate solution ultrasound is added in the rare ginsenoside obtained, added just Hexane filters after shaking up standing, after the ethyl acetate ultrasound that filter residue adds is obtained by filtration, after the n-hexane of addition shakes up standing Filtering, merging filtrate are simultaneously concentrated；

In a preferred example, the addition ethyl acetate solution ultrasound 30min.

The second aspect of the present invention additionally provides the quasi- monomeric compound Δ PPD of rare ginsenoside and quasi- monomeric compound Δ The purification process of PPT comprising: take silica gel the first ethyl acetate of addition and n-hexane solvent to stir evenly dress column, first acetic acid The volume ratio of ethyl acetate n-hexane is 1:2, the concentrate and silicon that the merging filtrate is obtained in ethyl ester and n-hexane solvent Glue mixes sample, and will mix the silica gel after sample it is finely ground after fill column, first rinse 3 with first ethyl acetate and hexane solution and retain Volume, the 2nd and the 3rd retention volume collects quasi- monomeric compound Δ PPD, then rushes 3 with the second ethyl acetate and hexane solution 1:1 A retention volume rinses altogether 6 retention volumes, ethyl acetate n-hexane in second ethyl acetate and n-hexane solvent Volume ratio is that the retention volume of 1:1, the 5th and the 6th collects quasi- monomeric compound Δ PPT；

In a preferred example, silica gel is 200-300 mesh,

In a preferred example, the long 20cm wide 4cm of silicagel column specification after column is filled,

In a preferred example, the chromatographic column retention volume after the dress column is 200ml.

In a preference of first aspect present invention and second aspect, the heat containing low polarity rare ginsenoside Solution Araliaceae stem-leaf extract is made with the following method: after gen-seng haulms are broken into powder, Ginseng stem leaf powder being placed on steaming Then boiling Ginseng stem leaf powder is dried in vacuo to obtain dry boiling Ginseng stem leaf powder, with 95% second by boiling in vapour pressure cooker Alcohol impregnates dry boiling Ginseng stem leaf powder, then filters, filtrate is concentrated to get gen-seng haulms powder extracts；

It is in a preferred example, described that gen-seng haulms are broken into powder using pulverizer,

In a preferred example, the boiling in steam autoclave be 120 DEG C, boiling 20h under 0.15MPa pressure,

In a preferred example, by the vacuum drying of boiling Ginseng stem leaf powder to be placed in oven and dried 8h at 80 DEG C,

In a preferred example, described the step of filtrate is concentrated to get Ginseng stem leaf powder ethanol extract further include: by second Alcohol recycling is reused, and is impregnated filter residue 5 times, and merging finally obtains boiling extract of Radix Ginseng stem and leaf.

Terminology used in the present invention have it is defined below, unless otherwise described:

The term as used herein " rare ginsenoside " means to mainly contain one or more rare ginsenoside singulations Close object Δ (20-21) PPD, Δ (20-22) PPD and Δ (20-21) PPT, Δ (20-22) PPT or the quasi- monomer of rare ginsenoside Compound Δ PPD and Δ PPT.

The term as used herein " the quasi- monomeric compound Δ PPD of rare ginsenoside " means to have the following structure the rare of formula Ginsenoside monomer Δ (20-21) PPD:

And have the following structure the compound of isomer rare ginsenoside Δ (20-22) PPD of formula a kind of:

The term as used herein " the quasi- monomeric compound Δ PPT of rare ginsenoside " means to have the following structure the rare of formula Ginsenoside monomer Δ (20-21) PPT:

And have the following structure the compound of isomer rare ginsenoside Δ (20-22) PPT of formula a kind of:

The present invention provides include the quasi- monomeric compound Δ PPD of rare ginsenoside and the quasi- singulation of rare ginsenoside The preparation method for closing the composition of object Δ PPT, compared to the existing method for preparing rare ginsenoside, this preparation method is simple, Cost is relatively low, while on this basis, and the present invention also provides prepare rare ginsenoside monomer Δ PPD and rare ginsenoside The method of monomer Δ PPT, the method low cost, high efficiency and can be divided on a large scale by simple column chromatography for separation technology From the quasi- monomeric compound Δ PPD of rare ginsenoside and the quasi- monomeric compound Δ PPT of rare ginsenoside is prepared, further to open Hair related drugs lay the foundation.

Detailed description of the invention

Fig. 1 is the schematic arrangement of rare ginsenoside monomer Δ (20-21) PPD.

Fig. 2 is the schematic arrangement of rare ginsenoside monomer Δ (20-22) PPD.

Fig. 3 is the schematic arrangement of rare ginsenoside monomer Δ (20-21) PPT.

Fig. 4 is the schematic arrangement of rare ginsenoside monomer Δ (20-22) PPT.

Specific embodiment

The cracking reaction of glycosidic bond occurs during steaming for the main component ginsenoside in Araliaceae extract Although the rare saponin component new with can produce Rh1, Rk3, Rh4, Rg3, Rg5, Rk1 etc. after the dehydration of side chain, The stronger rare saponins of activity: Rh2, Rk2, Rh3, aPPT, aPPD, Δ are formed to the glycosidic bond of further break off remnant The ingredients such as PPT, Δ PPD are relatively difficult.It, can be further although being reacted under conditions of strong acid or weak acid (high temperature) after steaming The glycosidic bond of break off remnant, but many side reactions can also occur simultaneously, such as: the ring-closure reaction of side chain, pendant double bonds Addition reaction etc., can only isolated a small amount of rare saponin(e Rh2, Rk2, Rh3, aPPT, aPPD, Δ PPT, Δ PPD ingredient, Preparation can not be mass produced and realize really application.It, can if carrying out dual oxide reaction after steaming under alkaline condition To avoid the above side chain side reaction, the large scale preparation of rare saponin component is realized.Meanwhile it is not pre- in the present invention It first carries out complex process and expensive protoplast joins two/triol group raw material or prepared by the separation of monomeric compound raw material, But dual oxide alkaline hydrolysis again after directlying adopt prototype Araliaceae heating cracking, extracting, it is finally separating purifying, saves technique Cost is conducive to industrialized production.

First aspect present invention provides the synthesis preparation method of above-mentioned rare ginsenoside composition comprising: containing Normal propyl alcohol, metallic sodium, peroxidating are sequentially added in the pyrolysis Araliaceae stem-leaf extract for having low polarity rare ginsenoside Benzoyl, and logical oxygen, and reaction solution heating reaction is made, wherein the pyrolysis containing low polarity rare ginsenoside Araliaceae preferably is selected from wild ginseng, red ginseng, black ginseng, American Ginseng and Radix Notoginseng；

In a preferred example, the heating reaction carries out in four-hole bottle, and the four-hole bottle is passed through equipped with thermometer, oxygen Seal rubber pipe, condenser pipe are connected on pipe, top, the rubber tube other end connects funnel, and the funnel, which immerses, is equipped with liquid stone The beaker of wax,

In a preferred example, normal propyl alcohol is added to stir 10 minutes later,

In a preferred example, metallic sodium is added, reaction to no hydrogen is released,

In a preferred example, the heating reaction is reacted 24 hours for 86 DEG C,

In a preferred example, reaction solution after reaction, is cooled to room temperature, then washed reaction liquid by the heating, will It is dissolved after the normal propyl alcohol layer decompressing and extracting of reaction solution with water, then uses the low polar substances of n-hexane extraction, subsequently use acetic acid second Ester extraction, is evaporated ethyl acetate portion and obtains rare ginsenoside.

In a preferred example, ethyl acetate solution ultrasound is added in the rare ginsenoside obtained, added just Hexane filters after shaking up standing, after the ethyl acetate ultrasound that filter residue adds is obtained by filtration, after the n-hexane of addition shakes up standing Filtering, merging filtrate are simultaneously concentrated；

In a preferred example, the addition ethyl acetate solution ultrasound 30min.

The second aspect of the present invention additionally provides the purifying side of rare ginsenoside quasi- monomeric compound Δ PPD and Δ PPT Method comprising: take silica gel the first ethyl acetate of addition and n-hexane solvent to stir evenly dress column, first ethyl acetate and n-hexane The volume ratio of ethyl acetate n-hexane is 1:2, the concentrate and silica gel mixed sample that the merging filtrate is obtained, and mixing in solvent Column is filled after silica gel after sample is finely ground, first with first ethyl acetate and hexane solution 3 retention volumes of flushing, the 2nd and the 3rd Retention volume collects Δ PPD, then rushes 3 retention volumes with the second ethyl acetate and hexane solution 1:1, rinses 6 guarantors altogether Volume is stayed, the volume ratio of ethyl acetate n-hexane is the reservation of 1:1, the 5th and the 6th in second ethyl acetate and n-hexane solvent Volume collection Δ PPT；

In a preferred example, silica gel is 200-300 mesh,

In a preferred example, the long 20cm wide 4cm of silicagel column specification after column is filled,

In a preferred example, the chromatographic column retention volume after the dress column is 200ml.

In a preference of first aspect present invention and second aspect, the extract of Radix Ginseng stem and leaf is made with the following method : after gen-seng haulms are broken into powder, Ginseng stem leaf powder is placed on boiling in steam autoclave, then by boiling Ginseng stem leaf powder Vacuum drying obtains dry boiling Ginseng stem leaf powder, and dry boiling Ginseng stem leaf powder is impregnated with 95% ethyl alcohol, is then filtered, Filtrate is concentrated to get gen-seng haulms powder extracts；

It is in a preferred example, described that gen-seng haulms are broken into powder using pulverizer,

In a preferred example, the boiling in steam autoclave be 120 DEG C, boiling 20h under 0.15MPa pressure,

In a preferred example, by the vacuum drying of boiling Ginseng stem leaf powder to be placed in oven and dried 8h at 80 DEG C,

In a preferred example, described the step of filtrate is concentrated to get Ginseng stem leaf powder ethanol extract further include: by second Alcohol recycling is reused, and is impregnated filter residue 5 times, and merging finally obtains boiling extract of Radix Ginseng stem and leaf.

Unless specifically indicated, term used herein has the general sense in fields of the present invention.

Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation Property, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according in the art Technology or conditions described in document are carried out according to product description.Reagents or instruments used without specified manufacturer, Being can be with conventional products that are commercially available.

Embodiment 1 prepares low polarity rare ginsenoside composition and low polarity rare ginsenoside monomer Δ from ginseng The method of (20-21) PPT, Δ (20-22) PPT, Δ (20-21) PPD and Δ (20-22) PPD

Present embodiments provide a kind of low polarity rare ginsenoside monomer Δ (20-21) PPT for the formula of having the following structure Preparation method:

And have the following structure the preparation method of low polarity rare ginsenoside monomer Δ (20-22) PPT of formula a kind of:

Meanwhile the present embodiment additionally provides low polarity rare ginsenoside composition and has the following structure a kind of low of formula The preparation method of polarity rare ginsenoside monomer Δ (20-21) PPD:

And have the following structure the preparation method of low polarity rare ginsenoside monomer Δ (20-22) PPD of formula a kind of:

(1) it extracts and prepares:

The gen-seng haulms (1.0kg) for originating from Jilin bought from Chinese medicine wholesale market, are placed after breaking into powder with pulverizer In medical steam autoclave, the boiling 20h under 120 DEG C, 0.15MPa pressure, (Ginseng stem leaf powder will avoid mixed with water as far as possible It closes)；It is then placed in baking oven the vacuum drying 8h at 80 DEG C and obtains dry boiling Ginseng stem leaf powder, soaked with 95% ethyl alcohol of 5kg Bubble two days, filtering, filtrate are concentrated to get boiling Ginseng stem leaf powder ethanol extract.Ethyl alcohol recycling, which is reused, impregnates filter residue 5 times, Merging finally obtains boiling extract of Radix Ginseng stem and leaf 650g.

Seal rubber pipe is connected on equipped with thermometer, oxygen access tube, top, and (the rubber tube other end connects a funnel, leaching Enter in the beaker equipped with atoleine), in the four-hole bottle of the 2L of condenser pipe, be added 200 grams of boiling extract of Radix Ginseng stem and leaf and 1500ml normal propyl alcohol, stirring after ten minutes, are added 60 grams of metallic sodiums, when reaction to no hydrogen is released, benzoyl peroxide are added 10 grams, start logical oxygen, and reaction solution is heated to 86 DEG C and is reacted 24 hours.After reaction, reaction solution is cooled to room temperature, water It washes three times, is dissolved after normal propyl alcohol layer decompressing and extracting with water, first use the low polar substances of n-hexane extraction, then extracted with ethyl acetate It takes, is evaporated ethyl acetate portion and obtains rare ginsenoside crude mixture 70g, crude product is refined using following silicon column chromatographic technique Rare ginsenoside monomer.

(2) separating technology process:

In diagram, low polarity rare ginsenoside monomer Δ PPT₁Refer to Δ (20-21) PPT, Δ PPT₂Refer to Δ (20-22) PPT, Δ PPD₁Refer to Δ (20-21) PPD, Δ PPD₂Refer to Δ (20-22) PPD.

(3) operating process and process conditions

The extraction of rare ginsenoside crude product

Purpose: active principle is extracted using dissolution sex differernce removal pigment.

Method: it takes rare ginsenoside crude product 20g that the ethyl acetate solution ultrasound 30min of 200ml is added, adds The n-hexane of 200ml filters after shaking up standing, and 100ml is being added just after adding the ethyl acetate ultrasound 30min of 100ml in filter residue Hexane filters after shaking up standing, and merging filtrate is simultaneously concentrated.

Silica gel post separation Δ PPD, Δ PPT

Purpose: using silica gel post separation Δ PPD, Δ PPT and most of pigment is removed

Method: it takes 90g silica gel (200-300 mesh) that 300ml (ethyl acetate: n-hexane 1:2) solution is added and stirs evenly dress column, silicon The long 20cm wide 4cm of rubber column gel column specification.Concentrate and 15g silica gel mixed sample, and will mix the silica gel after sample it is finely ground after fill column.Retention volume is big It is generally 200ml, first use ethyl acetate: n-hexane 1:2 rushes 3 retention volumes, then with ethyl acetate: n-hexane 1:1 rushes 3 reservations Volume totally 6 retention volumes.2nd, 3 retention volumes collect Δ PPD, the 5th, 6 retention volumes collection Δ PPT.

Quickly preparation is pressed in Δ PPD

Purpose: preparation purification Δ (20-22) PPD simultaneously removes most of pigment

Chromatographic column: quickly preparation Detection wavelength: 203 flow velocity 20ml/min is pressed in C18 40g 40-60um instrument Agela

Method: 95% acetonitrile water is isocratic, appearance time 15-25min, sample volume 300mg.Remainder is Δ (20-21) PPD

Δ PPD, the preparation of Δ PPT high-voltage high-speed

Purpose: purification Δ PPD and Δ PPT simultaneously removes depigmentaton

Chromatographic column: Waters Symmetry C18 7um 19*250 instrument Aglient Detection wavelength: 203nm flow velocity: 10ml/min

Δ PPT preparation method: 70% acetonitrile is isocratic, appearance time 20-25min, sample volume 100mg.

Δ PPD preparation method: 90% acetonitrile is isocratic, and appearance time 15-20min, sample volume 100mg goes the purity of depigmentation Higher Δ PPD, Δ PPT method for crystallising

Purpose: it is purified using method for crystallising

Δ PPD method for crystallising: 70% or more Δ PPD2 preparation solution is concentrated into the muddy addition total 100- of methanol by several times of liquid 200ml is threaded to volume 20ml and is put in 4 degree of refrigerators standings.

Δ PPT method for crystallising: a small amount of methanol 15ml is added to being completely dissolved in 70% or more Δ PPT2 preparation solution after being spin-dried for Naturally volatilization crystallization.

Embodiment 2 prepares low polarity rare ginsenoside composition and low polarity rare ginsenoside monomer Δ from Radix Notoginseng The preparation method of (20-21) PPT, Δ (20-2122) PPT, Δ (20-21) PPD and Δ (20-22) PPD

It extracts and prepares:

The notoginseng haulm (1.0kg) bought from Chinese medicine wholesale market, is placed on medical steam after breaking into powder with pulverizer In pressure cooker, the boiling 20h under 120 DEG C, 0.15MPa pressure, (notoginseng haulm powder will avoid mixing with water as far as possible)；It is then placed in 8h is dried in vacuo in baking oven at 80 DEG C and obtains dry boiling Ginseng stem leaf powder, is impregnated two days with 95% ethyl alcohol of 5kg, filtering, Filtrate is concentrated to get boiling notoginseng haulm powder ethanol extract.Ethyl alcohol recycling, which is reused, impregnates filter residue 5 times, and merging finally obtains Boiling Panax Notoginseng Folium Saponins 550g.

Separating-purifying and method for crystallising are the same as embodiment 1.

Comparative example 1: in heated or slightly sour environment, hypoglycemic hydrolysis occurs for the general ginsenoside part in ginseng or Radix Notoginseng can Generate the secondary saponin(e of ginseng

(1) it raw material pre-treatment: is crushed after the impurity such as silt are removed in the washing of American Ginseng sun-dried ginseng, crosses 40 meshes；

(2) steam explosion is handled: ginseng powder is soaked with 10% acetic acid, is made its water content 35%, is placed in steam-explosion jar, is passed through steam To steam explosion pressure inside the tank be 1.5MPa, Steam explosion treatment 60 minutes；

(3) biological complex enzyme is handled: raw material after steam explosion being adjusted water content with water as 40%, is 0.5% by material ratio It (includes cellulase 25%, alpha-amylase 25%, zytase 10%, laccase 5%, albumen that biological complex enzyme preparation, which is added, in amount Enzyme 20%, pectase 10%, mannase 5%), it is uniformly mixed, hydrolyzes 5h under the conditions of pH5.5,30 DEG C；

(4) dry sterilization: obtained reaction system will be handled through (3), be dried under reduced pressure, microwave sterilization；

(5) detect: sampling ginseng powder raw material carries out batch examining, ginsenoside in test sample (including monomer ginsenoside Rg3, Rh2), panaxan, ginseng protein content；

(6) it packs: being examined qualified ginseng powder by every barrel of 25kg packaging, sealed up for safekeeping.

But these needs react at higher temperature and stronger acid condition, and product is easy to happen side chain cyclisation at this time And the secondary saponin(e of ginseng for forming panoxadiol and panaxatriol type, while also occurring that other side reactions, it is difficult on a large scale Prepare the secondary saponin(e of the stronger ginseng of pharmacological activity (Rk1/Rg5, Rk3/Rh4 and Rk2/Rh3).

On the other hand, preparing the secondary saponin(e of ginseng (Rg3, Rh1, Rh2) can also be carried out by Base hydrolysis method, in alkalinity Under the conditions of hydrolyze, Rh1, aPPT can be obtained in triol group ginsenoside；Rg3, Rh2, aPPD can be obtained in glycol group ginsenoside.

Comparative example 2

10gNaOH is dissolved in the ethylene glycol solution of 100ml, 10g panax quinquefolium saponin is added, stirs lower heating It being hydrolyzed 1 hour, temperature is controlled at 190 DEG C, 50 times of water dilution is added after being cooled to room temperature, then be extracted with ethyl acetate, Recycle ethyl acetate, obtain product 7g, through TLC prove wherein containing Ginsenoside Rg3, Rh2, Rh1, Rg2 and protopanoxadiol and Protopanaxatriol.

Comparative example 3

Radix Notoginseng 18kg (specification: countless heads are purchased from Yunnan) is pulverized into powder (100-200 mesh), with 95% ethyl alcohol of 30kg It impregnates two days, filtering, filtrate is concentrated to give Radix Notoginseng ethanol extract, and ethyl alcohol recycling, which is reused, impregnates filter residue six times, final accumulative Radix Notoginseng ethanol extract 3.37kg is obtained, is dissolved in water, is extracted three times with petroleum ether, water intaking is mutually extracted four times with n-butanol, N-butanol layer concentration, there are arasaponin n-butanol extract 1.78kg.

It takes total saponin extracts 100g to be dissolved in 1300ml n-butanol, heats, stirring, sodium ethoxide is added, and (chemistry is pure, pure Degree: 80%) 132.6g (1.56mol, concentration: 1.2mol/L) leads to oxygen, and 90 DEG C are reacted 65 hours, and reaction terminates.Reaction solution is cooled to Room temperature, the washing being saturated with n-butanol are dissolved after n-butanol layer concentration with water, and ethyl acetate extracts, and ethyl acetate washed with water is washed, It is dry.After concentration, is purified through silica gel column chromatography [1~5% methanol/chloroform solution gradient elution], obtains protopanoxadiol (A2) 6g, It is 97.93% that HPLC, which measures purity,；It is 99.95% that protopanaxatriol (A3) 11g, HPLC, which measure purity,.Compound (A3) measurement Physicochemical data be coincident with literature value: Chen Yingjie et al, Journal of Shenyang College of Pharmacy, 1987,4 (4), 282-289.

The physicochemical data of compound (A3) measurement is as follows:

1H NMR (300MHz, CDCl3): δ 5.18 (d, 1H), 4.14 (m, 1H), 3.6 (m, 1H), 3.2 (s, 1H), 2.15- 1.61 (m, 20H), 1.58-1.17 (m, 12H), 1.05 (s, 5H), 1.01 (s, 3H), 0.95 (d, 8H).

13C NMR (300MHz, CDCl3): 132.1,125.2,78.8,74.7,71.0,68.9,61.4,53.7,51.7, 49.8,47.7,47.2,41.2,39.6,39.4,39.1,34.8,31.4,31.3,31.2,27.3,27.2,26.7,26.0, 22.6,18.0,17.5,17.4,17.1,15.8.

It is above-mentioned to be shared using Base hydrolysis method the disadvantage is that target product low yield, reaction temperature is high, especially basic hydrolysis people C-20 spatial configurations do not change when joining saponin(e, and-the H on-OH and the position C-21 or C-22 on the position C-20 will not be dehydrated And double bond is formed between C-20 and C-21 or C-22, therefore, it is impossible to directly prepare the secondary saponin(e of novel ginseng by basic hydrolysis (Rk1/Rg5, Rk3/Rh4, Rk2/Rh3 etc.).

Claims (3)

1. a kind of preparation method of rare ginsenoside comprising: the pyrolysis slender acanthopanax containing low polarity rare ginsenoside Section's plant stem-leaf extract is made with the following method: after the pyrolysis Araliaceae cauline leaf is broken into powder, by the cauline leaf powder It is placed on boiling in steam autoclave, cauline leaf powder described in boiling then is dried in vacuo to obtain cauline leaf powder described in dry boiling, Cauline leaf powder described in dry boiling is impregnated with 95% ethyl alcohol, then filters, filtrate is concentrated to get the cauline leaf powder extracts；? Normal propyl alcohol, metallic sodium, peroxide are sequentially added in pyrolysis Araliaceae stem-leaf extract containing low polarity rare ginsenoside Change benzoyl, and logical oxygen, and reaction solution heating reaction is made, wherein the heat containing low polarity rare ginsenoside It solves Araliaceae and is selected from ginseng, American Ginseng and Radix Notoginseng；The rare ginsenoside is to contain one or more rare ginseng soaps Glycosides monomeric compound Δ (20-21) PPD, Δ (20-22) PPD and Δ (20-21) PPT, Δ (20-22) PPT or rare ginseng soap Glycosides quasi- monomeric compound Δ PPD and Δ PPT；

It is optional, it is described that the cauline leaf is broken into powder using pulverizer,

Optional, the boiling in steam autoclave is 120 DEG C, boiling 20h under 0.15MPa pressure,

Optional, by the vacuum drying of cauline leaf powder described in boiling to be placed in oven and dried 8h at 80 DEG C,

Optional, described the step of filtrate is concentrated to get the cauline leaf powder ethanol extract further include: ethyl alcohol is recycled and is repeated It using, impregnates filter residue 5 times, merging finally obtains stem-leaf extract described in boiling,

Optional, the heating reaction carries out in four-hole bottle, and the four-hole bottle on thermometer, oxygen access tube, top equipped with connecting There are seal rubber pipe, condenser pipe, the rubber tube other end connects funnel, and the funnel immerses the beaker that atoleine is housed,

Optional, it is added after normal propyl alcohol stirring 10 minutes,

Optional, metallic sodium is added, reaction to no hydrogen is released,

Optional, the heating reaction is reacted 24 hours for 86 DEG C,

Optional, reaction solution after reaction, is cooled to room temperature, then washed reaction liquid, just by reaction solution by the heating It is dissolved after propyl alcohol layer decompressing and extracting with water, then uses the low polar substances of n-hexane extraction, be subsequently extracted with ethyl acetate, be evaporated Ethyl acetate portion obtains rare ginsenoside.

2. preparation method according to claim 1, which is characterized in that ethyl acetate is added in the rare ginsenoside obtained Solution ultrasound, the n-hexane added filter after shaking up standing, after the ethyl acetate ultrasound that filter residue adds is obtained by filtration, are added N-hexane shake up standing after filter, merging filtrate is simultaneously concentrated；

Optional, the addition ethyl acetate solution ultrasound 30min.

3. preparation method according to claim 2, which is characterized in that silica gel the first ethyl acetate of addition and n-hexane solvent is taken to stir Even dress column, the volume ratio of ethyl acetate n-hexane is 1:2 in first ethyl acetate and n-hexane solvent, and merging filtrate is obtained The concentrate and silica gel mixed sample arrived, and will mix the silica gel after sample it is finely ground after fill column, first with first ethyl acetate and n-hexane Solution rinses 3 retention volumes, and the 2nd and the 3rd retention volume collects Δ (20-21) PPD and Δ (20-22) PPD, then with the second second Acetoacetic ester and hexane solution 1:1 rush 3 retention volumes, rinse 6 retention volumes altogether, second ethyl acetate and just oneself The volume ratio of ethyl acetate n-hexane is that the retention volume of 1:1, the 5th and the 6th collects Δ (20-21) PPT and Δ (20- in alkane solvents 22)PPT；

Optional, silica gel is 200-300 mesh,

Optional, the long 20cm wide 4cm of silicagel column specification after filling column,

Optional, the chromatographic column retention volume after the dress column is 200ml.