CN108558976A - The preparation method of low polarity rare ginsenoside Δ PPD and Δ PPT - Google Patents
The preparation method of low polarity rare ginsenoside Δ PPD and Δ PPT Download PDFInfo
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Abstract
The present invention provides the preparation methods for including the quasi- monomeric compound Δ PPD of rare ginsenoside (including Δ (20 21) PPD and isomer Δ (20 22) PPD) and the quasi- monomeric compound Δ PPT of rare ginsenoside (including 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
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 technology
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 of the official International Cancer Research Center (IARC) of subordinate negative by World Health Organization
The latest edition of duty《World's cancer report》Swift and violent proliferation situation will be presented in prediction, global cases of cancer, by 2012 14,000,000
People, fast 19,000,000 people for increasing to 2025,24,000,000 people were up to 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 death , Fen Do to account for the 21.9% and 26.8% of global total amount.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 patterns 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
Therapy, the invasion of Chang Yinwei cancer cells spreads to adjacent tissue or far-end transfer and effect is limited；The therapy of radiotherapy, then
It is limited to injure caused by other internal normal structures；The therapy of drug, it is pernicious for late period dispersivity and metastatic
Tumour is most basic therapy.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 pattern 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 suppressing or eliminating tumour；Although biological therapy without too big toxic side effect,
Since technology requires tight, complex process, price is high, and numerous cancer patients and family members are difficult to bear, influence it and controlled in cancer
It popularizes in treatment field.
Since there are the research and development of above-mentioned various limitations, natural antitumor drug to 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 pattern 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
Active ingredient 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 includes mainly：It is 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 antitumor 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, it is referred to as low polarity rare ginsenoside, includes mainly that prototype ginsenoside is logical
Cometabolism derivative (C-k, Rg3, Rh2, Rh1, aPPD, aPPT etc.) and the side chain desugar simultaneously crossed glycosidic bond degradation and generated
It is dehydrated conversion derivative (Rg5, Rk1, Rh3, Rk2, Δ (20-21) PPD, the 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 preparation method be: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, what which was provided prepares Δ (20-22) PPT and the side PPD Δ (20-22)
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 isomers, has Δ (20-
22) bioactivity similar PPT；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
Prepared by protopanaxatriol group raw material or the production of raw material monomer Ginsenoside F1, or need to utilize expensive protopanoxadiol
Prepared by group raw material or the production of raw material monomer ginseng saponin C-K, be that industrial mass manufacturing cost is high, cannot 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
Family enterprise can simultaneously on a large scale manufacture high-content (>Rare metabolism ginsenoside 30-90%) and rare conversion ginseng soap
Glycosides, including the higher Δ of active anticancer (20-21) PPT, Δ (20-22) PPT, Δ (20-21) PPD and Δ (20-22)
PPD.Therefore, the preparation method of the low polarity rare ginsenoside of research above two, to finding the stronger ingredient of activity and development
Anti-cancer agent will have great importance.
Invention content
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
Caulking gum 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
Water dissolution is used after the normal propyl alcohol layer decompressing and extracting of reaction solution, then uses the low polar substances of n-hexane extraction, subsequently uses acetic acid second
Ester extracts, and 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 simultaneously concentrate；
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：Silica gel the first ethyl acetate of addition and n-hexane solvent is taken to stir evenly dress column, first acetic acid
The volume ratio of ethyl acetate n-hexane is 1 in ethyl ester and n-hexane solvent:2, the concentrate and silicon that the merging filtrate is obtained
Glue mixes sample, and will mix the silica gel after sample it is finely ground after fill column, first rinsing 3 with first ethyl acetate and hexane solution retains
Volume, the 2nd and the 3rd retention volume collect quasi- monomeric compound Δ PPD, then with the second ethyl acetate and hexane solution 1:1 punching 3
A retention volume rinses altogether 6 retention volumes, ethyl acetate n-hexane in second ethyl acetate and n-hexane solvent
Volume ratio is 1:1, the 5th and the 6th retention volume 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 are 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 is 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 give gen-seng haulms powder extracts；
In a preferred example, it is described by gen-seng haulms break into powder use 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 give Ginseng stem leaf powder ethanol extract, further includes：By second
Alcohol recycling is reused, and impregnates 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 on a large scale be divided 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 first stone.
Description of the drawings
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 implementation mode
The cracking reaction of glycosidic bond occurs during steaming for the main component ginsenoside in Araliaceae extract
Although the rare saponin component new with Rh1, Rk3, Rh4, Rg3, Rg5, Rk1 etc. can be generated after the dehydration of side chain,
The stronger rare saponins of activity are formed to the glycosidic bond of further break off remnant：Rh2、Rk2、Rh3、aPPT、aPPD、Δ
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 ingredients,
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 extensive 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 prototype Araliaceae heating cracking, extraction is directly used, 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
Caulking gum 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
Water dissolution is used after the normal propyl alcohol layer decompressing and extracting of reaction solution, then uses the low polar substances of n-hexane extraction, subsequently uses acetic acid second
Ester extracts, and 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 simultaneously concentrate；
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：Silica gel the first ethyl acetate of addition and n-hexane solvent is taken to stir evenly dress column, first ethyl acetate and n-hexane
The volume ratio of ethyl acetate n-hexane is 1 in solvent:2, the concentrate and silica gel mixed sample that the merging filtrate is obtained, and will mix
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 with the second ethyl acetate and hexane solution 1:13 retention volumes of punching, rinse altogether 6 guarantors
Stay volume, the volume ratio of ethyl acetate n-hexane is 1 in second ethyl acetate and n-hexane solvent:1, the 5th and the 6th retains
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 are 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 give gen-seng haulms powder extracts；
In a preferred example, it is described by gen-seng haulms break into powder use 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 give Ginseng stem leaf powder ethanol extract, further includes：By second
Alcohol recycling is reused, and impregnates 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 condition 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 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 give 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.
Caulking gum 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 alcohols, stirring after ten minutes, are added 60 grams of metallic sodiums, when reaction is released to no hydrogen, 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, water dissolution is used after normal propyl alcohol layer decompressing and extracting, 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 flow：
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
Rare ginsenoside crude product extracts
Purpose：Active principle is extracted using dissolving 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 simultaneously concentrates.
Silica gel post separation Δ PPD, Δ PPT
Purpose：Using silica gel post separation Δ PPD, Δ PPT and remove most of pigment
Method：Take 90g silica gel (200-300 mesh) that 300ml (ethyl acetate is added：N-hexane 1:2) solution stirs evenly dress column, silicon
The long 20cm wide 4cm of rubber column gel column specification.Concentrate and 15g silica gel mixed samples, and will mix the silica gel after sample it is finely ground after fill column.Retention volume is big
It is generally 200ml, first uses ethyl acetate：N-hexane 1:23 retention volumes of punching, then use ethyl acetate：N-hexane 1:1 reservation of punching 3
Volume totally 6 retention volumes.2nd, 3 retention volumes collect Δ PPD, the 5th, 6 retention volumes collect Δ PPT.
It presses in Δ PPD and quickly prepares
Purpose：It prepares purification Δ (20-22) PPD and removes most of pigment
Chromatographic column：It is pressed in C18 40g 40-60um instruments Agela and quickly prepares Detection wavelength：203 flow velocity 20ml/min
Method：95% acetonitrile water is isocratic, appearance time 15-25min, sample size 300mg.Remainder is Δ (20-21)
PPD
It is prepared by Δ PPD, Δ PPT high-voltage high-speed
Purpose：It purifies Δ PPD and Δ PPT and removes depigmentaton
Chromatographic column：Waters Symmetry C18 7um 19*250 instrument Aglient Detection wavelengths：203nm flow velocitys:
10ml/min
Δ PPT preparation methods：70% acetonitrile is isocratic, appearance time 20-25min, sample size 100mg.
Δ PPD preparation methods：90% acetonitrile is isocratic, and appearance time 15-20min, sample size 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 solutions are concentrated into the muddy total 100- of addition 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 solutions 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
Extraction and preparation：
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 possible)；It is then placed in
8h is dried in vacuo at 80 DEG C in baking oven and obtains dry boiling Ginseng stem leaf powder, impregnated two days with 95% ethyl alcohol of 5kg, filtering,
Filtrate is concentrated to give 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) raw material pre-treatment：It is crushed after the impurity such as silt are removed in the washing of American Ginseng sun-dried ginseng, crosses 40 mesh sieve；
(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
It is 1.5MPa, Steam explosion treatment 60 minutes to steam explosion pressure inside the tank；
(3) biological complex enzyme is handled：Raw material after steam explosion is adjusted into water content as 40% with water, 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, 5h is hydrolyzed 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) it detects：Sampling ginseng powder raw material carries out batch examining, and detects (including the monomer ginsenoside of ginsenoside in sample
Rg3, Rh2), panaxan, ginseng protein content；
(6) it packs：Through examining qualified ginseng powder to be packed by every barrel of 25kg, seal 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 saponins are added, stirs lower heating
It being hydrolyzed 1 hour, temperature is controlled at 190 DEG C, and 50 times of water dilution is added after being cooled to room temperature, then is extracted with ethyl acetate,
Recycle ethyl acetate, obtain product 7g, through TLC prove wherein to contain Ginsenoside Rg3, Rh2, Rh1, Rg2 and protopanoxadiol and
Protopanaxatriol.
Comparative example 3
Radix Notoginseng 18kg (specifications：Countless heads are purchased from Yunnan) it is ground into powdered (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, three times with petroleum ether extraction, water intaking is mutually extracted four times with n-butanol,
N-butanol layer concentrates, and there are arasaponin n-butanol extract 1.78kg.
It takes total saponin extracts 100g to be dissolved in 1300ml n-butanols, heats, stirring, sodium ethoxide is added, and (chemistry is pure, pure
Degree：80%) 132.6g (1.56mol, concentration：1.2mol/L), lead to oxygen, 90 DEG C are reacted 65 hours, and reaction terminates.Reaction solution is cooled to
Room temperature, the washing being saturated with n-butanol use water dissolution, ethyl acetate extraction, ethyl acetate washed with water to wash after n-butanol layer concentration,
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) measures
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 that compound (A3) measures 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 positions C-21 or C-22 on the positions 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 (5)
1. a kind of preparation method of rare ginsenoside comprising：In the pyrolysis Araliaceae containing low polarity rare ginsenoside
Normal propyl alcohol, metallic sodium, benzoyl peroxide, and logical oxygen are sequentially added in plant stem-leaf extract, and reaction solution is heated instead
Should be made, wherein the pyrolysis Araliaceae containing low polarity rare ginsenoside preferably be selected from wild ginseng, red ginseng, it is black ginseng,
American Ginseng and Radix Notoginseng；
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 caulking gum pipe, condenser pipe, the rubber tube other end to connect funnel, the funnel immerses the beaker equipped with atoleine,
Optional, normal propyl alcohol is added and stirs 10 minutes later,
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
Water dissolution is used after propyl alcohol layer decompressing and extracting, is then used the low polar substances of n-hexane extraction, is subsequently extracted with ethyl acetate, is 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 filters after shaking up standing, after the ethyl acetate ultrasound that filter residue adds is obtained by filtration, is added
N-hexane shake up standing after filter, merging filtrate simultaneously concentrates；
Optional, the addition ethyl acetate solution ultrasound 30min.
3. preparation method according to claim 1, 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 in first ethyl acetate and n-hexane solvent:2, the merging is filtered
The concentrate and silica gel mixed sample that liquid obtains, and will mix the silica gel after sample it is finely ground after fill column, first with first ethyl acetate and just
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
Ethyl diacetate and hexane solution 1:1 punching 3 retention volumes, altogether rinse 6 retention volumes, second ethyl acetate and
The volume ratio of ethyl acetate n-hexane is 1 in n-hexane solvent:1, the 5th and the 6th retention volume collects Δ (20-21) PPT and Δ
(20-22)PPT；
Optional, silica gel is 200-300 mesh,
Optional, the long 20cm wide 4cm of silicagel column specification after column are filled,
Optional, the chromatographic column retention volume after the dress column is 200ml.
4. preparation method according to claim 1, which is characterized in that 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 give the cauline leaf powder extracts；
It is optional, it is described the cauline leaf is broken into powder to use pulverizer,
Optional, the boiling in steam autoclave is 120 DEG C, boiling 20h under 0.15MPa pressure,
Optional, by cauline leaf powder vacuum drying described in boiling to be placed in oven and dried 8h at 80 DEG C,
Optional, described the step of filtrate is concentrated to give the cauline leaf powder ethanol extract, further includes：Ethyl alcohol is recycled and is repeated
It uses, impregnates filter residue 5 times, merging finally obtains stem-leaf extract described in boiling.
5. the rare ginsenoside prepared by preparation method according to any one of claim 1 to 4.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1508145A (en) * | 2002-12-13 | 2004-06-30 | 中国科学院大连化学物理研究所 | Anti-tumour ginseng saponin aglycone derivatives |
| WO2004054595A1 (en) * | 2002-12-13 | 2004-07-01 | Dalian Institute Of Chemical Physics | A method for preparing low polar ginsenoside and aglucon thereof by catalytic pyrolysis |
| CN1569882A (en) * | 2004-04-29 | 2005-01-26 | 上海中药创新研究中心 | Process for preparing protopanoxadiol and protopanaxatriol |
| CN1958595A (en) * | 2005-11-01 | 2007-05-09 | 中山以诺生物科技有限公司 | Method for preparing protopanoxadiol and protopanaxatriol by using synergistic oxidation and alkaline bydrolysis of oxide and hyperoxide |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1508145A (en) * | 2002-12-13 | 2004-06-30 | 中国科学院大连化学物理研究所 | Anti-tumour ginseng saponin aglycone derivatives |
| WO2004054595A1 (en) * | 2002-12-13 | 2004-07-01 | Dalian Institute Of Chemical Physics | A method for preparing low polar ginsenoside and aglucon thereof by catalytic pyrolysis |
| CN1569882A (en) * | 2004-04-29 | 2005-01-26 | 上海中药创新研究中心 | Process for preparing protopanoxadiol and protopanaxatriol |
| CN1958595A (en) * | 2005-11-01 | 2007-05-09 | 中山以诺生物科技有限公司 | Method for preparing protopanoxadiol and protopanaxatriol by using synergistic oxidation and alkaline bydrolysis of oxide and hyperoxide |
Non-Patent Citations (1)
| Title |
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| KANG, KI SUNG等: "Protective effect of ginseng sapogenins against 2,2"-azobis (1-aminopropane) dihydrochloride (AAPH)-induced LLC-PK1 cell damage", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 》 * |
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