CN103012148B - Method for preparing salvianolic acid A through catalytically converting salvianolic acid B - Google Patents
Method for preparing salvianolic acid A through catalytically converting salvianolic acid B Download PDFInfo
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Abstract
The invention relates to a method for preparing salvianolic acid A through catalytically converting salvianolic acid B. Water or alcohol is used for extracting a salvia miltiorrhiza extracting solution, wherein the concentration of the salvianolic acid B in the extracting solution is 1milligram/milliliter-30milligrams/milliliter or the salvianolic acid B is added with water to be diluted to 1milligram/milliliter-30milligrams/milliliter, a catalyst is added, the molar ratio of the catalyst and the salvianolic acid B is 0.1%-3.0%, alkali is added to regulate the pH to 3.0-6.5, the conversion temperature is between 100 and 140 DEG C, the conversion time is between 1 and 6 hours, cooling is carried out, acid is added to regulate the pH to 2.5-3.0, standing and centrifuging are carried out to obtain salvianolic acid A conversion liquid, the content of the salvianolic acid A is determined, wherein the catalyst is one or a plurality of ferric chloride, ruthenium trichloride, aluminium chloride, zinc chloride and palladium chloride. After the preparation method is used, the time that the salvianolic acid B is in an easily-breaking state is greatly shortened, the yield of the salvianolic acid A is obviously improved, a conversion technology is stable, and the method has strong operability and can be used for industrially producing the salvianolic acid A.
Description
Technical field
The present invention relates to a kind of method that catalyzed conversion salvianolic acid B prepares salvianolic acid A.
Background technology
Red sage formulation is the base therapy medicine of China's cardiovascular and cerebrovascular diseases, because of determined curative effect, the red sage root oneself become that China's consumption is maximum, sales volume is the highest, preparation factory is maximum, one of Chinese medicine that Clinical Dosage Form is the most complete.Red sage root active chemical mainly contains two large classes: fat-soluble tanshinone compound and water-soluble phenolic compounds.Research shows: salvianolic acid class has significant activity in anti-hepar damnification, atherosclerosis and apoptosis and Improving memory dysfunction etc.Wherein the strongest with salvianolic acid A (Salvianolic acid A) anti-oxidant activity again.
Salvianolic acid A structure is as follows:
Although as patent documentation also has the technology adopting thermal treatment Radix Salviae Miltiorrhizae extract to prepare salvianolic acid A in prior art.But the natural content of salvianolic acid A is extremely low (being about the 0.01-0.06% of red rooted salvia), and make crude drug high cost, separation and purification difficulty is excessive, seriously governs the R and D of medicine, becomes the bottleneck of its industrialization.
In addition, in prior art, experiment confirms that alkannic acid can be converted into salvianolic acid A through heated and boiled after 2 hours, infer salvianolic acid A be alkannic acid through dihydrofuran ring the reaction such as open loop, dehydration, decarboxylation and formed product [girth is new. China Medicine University's journal, 1999,30 (6): 411 ~ 416].Meanwhile, in prior art, salvianolic acid B carries out degraded by Ester hydrolysis decarboxylation and chroman ring ring-opening reaction and can change into salvianolic acid A also to have experiment to confirm, salvianolic acid B transferring structure formula is as follows,
But the conversion process of above-mentioned prior art is uncontrollable, and conversion byproducts is many, the productive rate of principal product salvianolic acid A is lower.
Moreover, although also there are some patent documentations to propose to attempt to overcome the defect existed in prior art in prior art, but be all only that simple trial heats up, change concentration of pH value or the front pressure differential self of raising conversion etc., go deep into without any a patent or prior art, study conversion reaction all sidedly and all comprise which major influence factors, more these factors are proposed as transformed the concentration of front pressure differential self without any a patent or prior art, pH value, temperature, how time etc. act synergistically each other has an impact to salvianolic acid A productive rate jointly.
On this basis, other contrivers or prior art is seldom had to propose to attempt in conversion, adding catalyzer to make reaction more abundant, having related to the associated viscera adopting catalyzer to carry out transforming in currently available technology exists only in the two parts of patent application documents submitted on April 6th, 2010 same day, these two parts of patent application documents are application number is respectively 2010101436787, the patent application that denomination of invention is " a kind of catalyzed conversion salvianolic acid B prepares the method for salvianolic acid A " and application number are 2010101436876, denomination of invention is the patent application of " a kind of method of preliminary purification salvianolic acid B conversion feedstock ".In the specification sheets of these two parts of patent applications, although disclose the technology contents that catalyzed conversion salvianolic acid B prepares salvianolic acid A, but its catalyzer is urea, the mol ratio of urea and salvianolic acid B is (0.4 ~ 0.6): 1, require to consume and add urea very high, therefore production cost is very high.Further, in above-mentioned two parts of patent application documents, concern pH value and other correlative factors work in coordination with the impact produced salvianolic acid A productive rate too.Moreover its conversion feedstock is the radix Salviae Miltiorrhizae water extract through co-chromatography preliminary purification, wherein salvianolic acid B >=50%, this all requires higher to the purity of conversion feedstock and purifying technique, and technique is also comparatively complicated, further increases production cost.More crucially, although claim in its application documents " directed transformation efficiency >=10% of the salvianolic acid A salvianolic acid B using present method to prepare, even reaches 60% ".But due to urea in fact not open loop, dehydration, decarboxylation, only have into ester effect, therefore, its transformation efficiency does not reach 60% at all, and the transformation efficiency in the specific embodiment of this application file is only up to 53%, and the transformation efficiency of multiple embodiment is only 10%, 20% and 30%.Still there is a big difference in the requirement of this and actual industrialization.
Summary of the invention
In sum, the invention provides a kind of method that catalyzed conversion salvianolic acid B prepares salvianolic acid A, the method can overcome the deficiencies in the prior art, and not only production technique is simple, and the low and salvianolic acid A transformation efficiency of cost is significantly improved.
Catalyzed conversion salvianolic acid B provided by the invention prepares the method for salvianolic acid A, wherein: the Radix Salviae Miltiorrhizae extract of water intaking extraction or ethanol-extracted, wherein in extracting solution, salvianolic acid B concentration is 1mg/ml ~ 30mg/ml or is diluted with water to 1mg/ml ~ 30mg/ml, adding with the molar percentage of salvianolic acid B is the catalyzer of 0.1% ~ 3.0%, adding alkali regulates pH to be 3.0 ~ 6.5, invert point is 100 ~ 140 DEG C, transformation time is 1 ~ 6 hour, cooling, acid adding adjusts pH to 2.5 ~ 3.0, leave standstill, centrifugal, obtain salvianolic acid A conversion fluid, measure salvianolic acid A content, wherein said catalyzer is iron(ic) chloride, ruthenium trichloride, aluminum chloride, zinc chloride, Palladous chloride (FeCl
3, RuCl
3, AlCl
3, ZnCl
2, PdCl
3) one or more.
Preferably, the Radix Salviae Miltiorrhizae extract of water intaking extraction or ethanol-extracted, wherein in extracting solution, salvianolic acid B concentration is 5mg/ml ~ 20mg/ml or is diluted with water to 5mg/ml ~ 20mg/ml, adding with the molar percentage of salvianolic acid B is the catalyzer of 0.5% ~ 2.0%, adding alkali regulates pH to be 3.5 ~ 5.5, and invert point is 110 ~ 135 DEG C, and transformation time is 2 ~ 5 hours, cooling, acid adding adjusts pH to 2.5 ~ 3.0, leaves standstill, centrifugal, obtain salvianolic acid A conversion fluid, measure salvianolic acid A content.
Preferably, the extracting method of wherein said aqueous extract is: get red rooted salvia medicine materical crude slice or particle, adds 3 ~ 15 times amount at every turn, keeps 45 ~ 95 DEG C of water temperature lixiviates to get, stir simultaneously, extract 1 ~ 3 time altogether, extract 1 ~ 4 hour at every turn with 10 ~ 50 revs/min of speed; Extracting solution is evaporated to relative density 1.10 ~ 1.25 (60 DEG C), adds ethanol and makes alcohol content 30% ~ 80%, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract, measures salvianolic acid B content.
Preferably, the extracting method of wherein said aqueous extract is: get red rooted salvia medicine materical crude slice or particle, adds 3 ~ 15 times amount at every turn and decocts extraction, extract 1 ~ 4 hour at every turn, extract 1 ~ 3 time altogether; Extracting solution is evaporated to relative density 1.10 ~ 1.25 (60 DEG C), adds ethanol and makes alcohol content 30% ~ 80%, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains conversion feedstock, measures salvianolic acid B content.
Preferably, the extracting method of wherein said alcohol extract is: get red rooted salvia medicine materical crude slice or particle, add 3 ~ 15 times amount 30% ~ 60% alcohol reflux at every turn, each extraction 1 ~ 4 hour, extracts 1 ~ 3 time, decompression recycling ethanol altogether, leave standstill, centrifugal, obtain conversion feedstock, measure salvianolic acid B content.
Preferred, wherein said red rooted salvia particle diameter of getting is about 2mm.
Preferably, wherein said catalyzer is iron(ic) chloride, ruthenium trichloride, aluminum chloride, zinc chloride or Palladous chloride.
Preferably, the wherein said alkali adjust ph alkali used that adds is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, Trisodium Citrate or sodium-acetate.
Preferably, wherein said acid adding adjusts pH acid used to be one or more of phosphoric acid, hydrochloric acid, sulfuric acid or acetic acid.
On the other hand, the present invention also provide a kind of measure salvianolic acid A and measure the method for content of danshinolic acid B be: adopt high effective liquid chromatography for measuring salvianolic acid A, salvianolic acid B, condition determination is as follows:
Take octadecylsilane chemically bonded silica as weighting agent;
Determined wavelength 286nm; Flow velocity 1.0ml/min; Column temperature 30 DEG C;
Theoretical plate number should be not less than 10000 by salvianolic acid A;
The preparation precision of reference substance solution takes in salvianolic acid A reference substance 10mg, salvianolic acid B reference substance 10mg to 100ml volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale;
The preparation of need testing solution: precision measures and is equivalent to 10mg salvianolic acid A and salvianolic acid B sample in 100ml volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale, to obtain final product;
Wash-out take methyl alcohol as mobile phase A, with 0.1 ~ 0.5% phosphoric acid for Mobile phase B, carries out gradient elution by following condition, runs 60 minutes;
When 0 ~ 10 minute, the ratio of methyl alcohol rises to 40% by 30%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 60% by 70%;
When 10 ~ 30 minutes, the ratio of methyl alcohol rises to 55% by 40%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 45% by 50%;
When 30 ~ 60 minutes, the ratio of methyl alcohol rises to 80% by 55%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 20% by 45%.
Assay method: accurate absorption reference substance solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, measures content.
The present invention is through the screening of system and optimization, first the Extraction solvent and extracting method that determine starting raw material salvianolic acid B is compared, because salvianolic acid A is better water-soluble, determine and adopt water extraction or low concentration alcohol to extract, again because salvianolic acid B thermostability is poor, determine and adopt the lixiviate of hot water temperature get and add stirring extracting method, or use low-concentration ethanol refluxing extraction, make extraction solubility lower than 100 DEG C, salvianolic acid B is kept not to be destroyed, optimum solvent consumption and extraction time is determined by orthogonal experiment, obtain the salvianolic acid B optimum extraction process adapting to suitability for industrialized production.
The present invention is compared with the prior art and shows: starting raw material red rooted salvia extracting directly can carry out the conversion that feeds intake, transform again after not needing that purifying is carried out to salvianolic acid B, namely in catalytic conversion reaction of the present invention, reaction raw materials salvianolic acid B does not need high purity, such as, do not need purity >=50% of salvianolic acid B.It is generally acknowledged that reaction raw materials is more pure better, but in catalytic conversion reaction of the present invention, the purity height of salvianolic acid B does not affect on conversion reaction effect.On the contrary, not only produce a large amount of impurity, and do not improve transformation efficiency during salvianolic acid B purity > 50%, therefore, the present invention achieves unforeseeable technique effect.
Moreover the present invention comparing by repeatedly testing, first determining and the factor of material impact is produced as transformed the concentration, pH value, temperature, time etc. of front pressure differential self to salvianolic acid A productive rate.On this basis, repeatedly test by paying a large amount of time, material and energy the concentration of temperature, pH value, time, salvianolic acid B and other correlated conditions are studied again, and how these factors act synergistically each other and jointly have an impact to salvianolic acid A productive rate.Thus determine the optimum temps, pH value, time etc. that salvianolic acid B transforms the needs control of salvianolic acid A, and salvianolic acid B initial concentration is controlled at 1mg/ml ~ 30mg/ml, thus make salvianolic acid A transformation efficiency of the present invention more obviously be better than other conversion conditions.In chemical reaction, the purity of reactant and concentration usually affect the effect of reaction.Generally there is concentration requirement to reactant, and think that concentration height specific concentration is low good.In catalytic conversion reaction of the present invention, the concentration level of salvianolic acid B does not affect conversion reaction effect.On the contrary, experiment proves that the concentration containing salvianolic acid B in the salvianolic acid B aqueous solution is not more high better, and more than 30mg/ml concentration transformation efficiency is low on the contrary, and effect is poorer.Therefore, the present invention, in cost-saving and production cycle, achieves unforeseeable technique effect, creative.When prior art does not provide the enlightenment of any technology, if those skilled in the art only infer theoretically, be impossible draw, under above-mentioned each conditional parameter, sour for pellet B is changed into the conclusion that salvianolic acid A has better changing effect.
What is more important, the present invention passes through performing creative labour, discovery is selected from iron(ic) chloride, tri-chlorination is followed closely, aluminum chloride, zinc chloride, one or more catalyzer of Palladous chloride significantly can play the effect that catalysis salvianolic acid B transforms salvianolic acid A, can the decarboxylation of catalysis salvianolic acid B and furan nucleus ring-opening reaction, substantially reduce the time that salvianolic acid B is in destructible state, and the transformation efficiency that salvianolic acid B transforms salvianolic acid A can be significantly improved, highly stable the reaching close to 60% of transformation efficiency, most cases can more than 60%, this is all impossible in any one prior art in the past.Therefore, unforeseeable technique effect is achieved.
Further, while significantly improving transformation efficiency, the catalyzer that the present invention adopts is very low relative to salvianolic acid B molar percentage, only 0.1% ~ 3.0%, this makes production cost of the present invention greatly reduce, and therefore, it is not sacrifice production cost for cost to obtain that the present invention improves transformation efficiency.On the contrary, while raising transformation efficiency, a kind of processing method being suitable for production application is provided.
Lower concentration can also mix with the salvianolic acid B of high density by the present invention, only need be made into suitable initial conversion concentration, can reach the object changing into salvianolic acid A equally.Therefore, the preparation technology of this conversion feedstock is very simple, and production cost is also very suitable for the application in actual industry while reducing.
Accompanying drawing explanation
Fig. 1. salvianolic acid B reference substance high-efficient liquid phase chromatogram;
Fig. 2. salvianolic acid B high-efficient liquid phase chromatogram in Radix Salviae Miltiorrhizae extract;
Fig. 3. salvianolic acid A reference substance high-efficient liquid phase chromatogram;
Fig. 4. salvianolic acid A high-efficient liquid phase chromatogram in salvianolic acid B catalyzed conversion liquid.
Embodiment
Namely salvianolic acid B of the present invention can adopt the method for following embodiment provided by the invention to obtain from plant as extracted the red sage root, also can commercially availablely buy, or a part is extracted the direct remix of buying of a part and is made into required extracting solution, in a word, to this not special restriction.
Embodiment 1
Get red rooted salvia medicine materical crude slice 25kg, add 8 times of water gagings at every turn and get 1.5 hours 80 DEG C of warm lixiviates, stir with 10 ~ 15 revs/min of speed simultaneously, extraction 3 times is stirred in temperature leaching altogether, filters, merging filtrate, extracting solution is evaporated to relative density 1.20 (60 DEG C), adds ethanol and makes alcohol content 70%, leaves standstill 12 hours, filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, is diluted with water to 25000ml, recording content of danshinolic acid B is 42.0mg/ml, obtain Radix Salviae Miltiorrhizae extract, refrigeration is placed, for subsequent use.
Embodiment 2
Get red rooted salvia 25kg, be ground into diameter and be about 2mm particle, add 9 times of water gagings at every turn and get 2 hours 85 DEG C of warm lixiviates, stir with 15 ~ 25 revs/min of speed simultaneously, extraction 2 times is stirred in temperature leaching altogether, filters, merging filtrate, extracting solution is evaporated to relative density 1.10 ~ 1.25 (60 DEG C), adding ethanol makes alcohol content 75%, leaves standstill 12 hours, filters, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to 25000ml, recording content of danshinolic acid B is 40.0mg/ml, obtains Radix Salviae Miltiorrhizae extract, refrigeration is placed, for subsequent use.
Embodiment 3
Get red rooted salvia medicine materical crude slice 30kg, add 10 times amount at every turn and decoct extraction, each extraction 2 hours, extracts 3 times altogether; Extracting solution is evaporated to relative density 1.10 ~ 1.20 (60 DEG C), adding ethanol makes alcohol content 70%, filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to 25000ml, recording content of danshinolic acid B is 45.0mg/ml, obtains Radix Salviae Miltiorrhizae extract, refrigeration is placed, for subsequent use.
Embodiment 4
Get red rooted salvia medicine materical crude slice 25kg, add 8 times amount 50% alcohol reflux at every turn, each extraction 2 hours, extracts 3 times altogether, decompression recycling ethanol, leave standstill, centrifugal, supernatant liquor is diluted with water to 25000ml, recording content of danshinolic acid B is 48.00mg/ml, obtains Radix Salviae Miltiorrhizae extract, and refrigeration is placed, for subsequent use.
Embodiment 5
Example 1 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 2100ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 20mg/ml, adds the ZnCl of 1.0%
25% sodium hydroxide adjusts pH to 4.5, be placed in autoclave and be incubated 4.0 hours at 120 DEG C, cooling, add 10% hydrochloric acid and adjust pH to 3.0, leave standstill, centrifugal, obtain supernatant liquor, be settled to 2000ml, recording salvianolic acid A content is 9.07mg/ml, and salvianolic acid A productive rate is 43.19% (transformation efficiency 62.77%).
Embodiment 6
Example 2 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 40000ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 1mg/ml, add the RuCl3 of 1.5%, 10% sodium bicarbonate adjusts pH to 4.6, is placed in autoclave and is incubated 3.5 hours at 125 DEG C, cooling, add 20% phosphoric acid and adjust pH to 2.8, leave standstill, centrifugal, obtain supernatant liquor, be settled to 39000ml, recording salvianolic acid A content is 0.47mg/ml, and salvianolic acid A productive rate is 42.30% (transformation efficiency 61.48%).
Embodiment 7
Example 2 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 5000ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 8.0mg/ml, adds the ZnCl of 2.5%
210% sodium carbonate adjusts pH to 4.8, be placed in autoclave and be incubated 2.5 hours at 135 DEG C, cooling, add 15% hydrochloric acid and adjust pH to 3.0, leave standstill, centrifugal, obtain supernatant liquor, be settled to 4500ml, recording salvianolic acid A content is 3.67mg/ml, and salvianolic acid A productive rate is 41.29% (transformation efficiency 60.00%).
Embodiment 8
Example 3 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 1500ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 30mg/ml, adds the FeCl of 0.8%
3pH to 4.6 adjusted by 8% potassium hydroxide, be placed in autoclave and be incubated 4.5 hours at 115 DEG C, cooling, add 15% acetic acid and adjust pH to 2.8, leave standstill, centrifugal, obtain supernatant liquor, be settled to 1500ml, recording salvianolic acid A content is 12.64mg/ml, and salvianolic acid A productive rate is 42.13% (transformation efficiency 61.23%).
Embodiment 9
Example 2 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 2500ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 16.00mg/ml, adds the AlCl of 1.5%
3with 10% Trisodium Citrate pH to 4.4, be placed in autoclave, at 130 DEG C, be incubated 3.0 hours, cooling, add 10% phosphoric acid and adjust pH to 2.7, leave standstill, centrifugal, obtain supernatant liquor, be settled to 2500ml, recording salvianolic acid A content is 6.45mg/ml, and salvianolic acid A productive rate is 40.31% (transformation efficiency 58.59%).
Embodiment 10
Example 4 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 2000ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 24.00mg/ml, adds the RuCl of 1.2%
3with 10% potassium hydroxide pH to 4.8, be placed in autoclave, at 130 DEG C, be incubated 2.0 hours, cooling, add 10 sulfuric acid and adjust pH to 3.0, leave standstill, centrifugal, obtain supernatant liquor, be settled to 2000ml, recording salvianolic acid A content is 10.02mg/ml, and salvianolic acid A productive rate is 41.75% (transformation efficiency 60.68%).
Embodiment 11
Example 4 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 4000ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 12.0mg/ml, adds the ZnCl of 0.5%
2adjust pH to 4.8 with 15% sodium carbonate, be placed in autoclave, at 125 DEG C, be incubated 2.5 hours, cooling, add 15% nitric acid and adjust pH to 3.1, leave standstill, centrifugal, obtain supernatant liquor, be settled to 4000ml, recording salvianolic acid A content is 4.94mg/ml, and salvianolic acid A productive rate is 41.17% (transformation efficiency 59.83%).
Embodiment 12
Example 2 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 8000ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 5.0mg/ml, adds the FeCl of 2.0%
3adjust pH to 4.6 with 15% Trisodium Citrate, be placed in autoclave, at 125 DEG C, be incubated 3.0 hours, cooling, add 10% hydrochloric acid and adjust pH to 3.0, leave standstill, centrifugal, obtain supernatant liquor, be settled to 8000ml, recording salvianolic acid A content is 2.08mg/ml, and salvianolic acid A productive rate is 41.60% (transformation efficiency 60.46%).
Embodiment 13
Get commercially available salvianolic acid B extract 105g (content of danshinolic acid B is 40%), add purified water and be diluted to 4200ml, obtain the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 10mg/ml, add the ZnCl of 1.0%
215% sodium-acetate pH to 5.8, be placed in autoclave and be incubated 4.5 hours at 125 DEG C, cooling, add 20% sulfuric acid and adjust pH to 3.0, leave standstill, centrifugal, obtain supernatant liquor, be settled to 4200ml, recording salvianolic acid A content is 4.15mg/ml, and salvianolic acid A productive rate is 41.50% (transformation efficiency 60.32%).
Embodiment 14
Example 1 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 2100ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 20mg/ml, adds the PdCl of 1.0%
315% sodium carbonate pH to 5.5, be placed in autoclave and be incubated 4.5 hours at 130 DEG C, cooling, add 20% hydrochloric acid and adjust pH to 3.0, leave standstill, centrifugal, obtain supernatant liquor, be settled to 4200ml, recording salvianolic acid A content is 8.15mg/ml, and salvianolic acid A productive rate is 40.75% (transformation efficiency 59.22%).
Embodiment 15
Example 3 Radix Salviae Miltiorrhizae extract 1000ml, adds purified water and is diluted to 3000ml, obtains the Radix Salviae Miltiorrhizae extract that salvianolic acid B concentration is 15mg/ml, adds the AlCl of 1.0%
315% sodium hydroxide pH to 5.5, be placed in autoclave and be incubated 4.0 hours at 135 DEG C, cooling, add 15% hydrochloric acid and adjust pH to 2.8, leave standstill, centrifugal, obtain supernatant liquor, be settled to 3000ml, recording salvianolic acid A content is 6.5lmg/ml, and salvianolic acid A productive rate is 43.40% (transformation efficiency 63.08%).
Experimental example 1: conversion feedstock preparation method
The confirmation of 1.1 Extraction solvent and extracting method
Take red rooted salvia 400g, measure content of danshinolic acid B by method under Chinese Pharmacopoeia red sage root item, be divided into 4 parts, test by following testing program:
Experiment 1: get red rooted salvia 100g, add 8 times of water gagings at every turn and get 1.5 hours 80 DEG C of warm lixiviates, warm lixiviate gets three times, united extraction liquid altogether, and measure salvianolic acid B and calculate extraction yield, result is as table 1.
Experiment 2: get red rooted salvia 100g, add 8 times of water gagings at every turn and get 1.5 hours 80 DEG C of warm lixiviates, stirs with 25 revs/min of speed simultaneously, and temperature leaching stirs extraction three times, united extraction liquid altogether, and measure salvianolic acid B and calculate extraction yield, result is as table 1.
Experiment 3: get red rooted salvia 100g, add 8 times amount soak by water 1.5 hours at every turn, decocts three times, united extraction liquid altogether, and measure salvianolic acid B and calculate extraction yield, result is as table 1.
Experiment 4: get red rooted salvia 100g, add 8 times amount 50% alcohol reflux 1.5 hours at every turn, extracts three times, united extraction liquid altogether, and measure salvianolic acid B and calculate extraction yield, result is as table 1.
Table 1. Extraction solvent and extracting method experimental result
Above-mentioned experimental result display, adopt 50% ethanol to do Extraction solvent and have impact to salvianolic acid B extraction yield, 50% extraction using alcohol is better than water extraction, but soak to add with water temperature and stir that to extract difference little, the extraction yield of two kinds of extracting modes on salvianolic acid B stirred in water extraction process and do not stir has impact, decocting extraction may be because temperature is higher, and extracting salvianolic acid B has impact.
1.2 orthogonal experiment Optimized extraction techniques
1.2.1 the optimizing research of water extraction process: according to above test-results, water extraction process is using extraction time (A), extraction time (B), quantity of solvent (C), Extracting temperature (D) four as investigation factor, each factor establishes three levels, by L9 (3
4) orthogonal table carries out orthogonal experimental design (table 2, table 3), inspection target is the extraction yield of salvianolic acid B.
Table 2 extraction factor water-glass
Table 3 extraction process orthogonal experiments table
Table 4 the results of analysis of variance table
F
0.05(2，2)＝19.00F
0.01(2，2)＝99.00
Water extraction the results of analysis of variance shows, each experimental factor is to the extraction rate of transform of salvianolic acid B there are no significant difference, therefore the water extraction condition of salvianolic acid B of the present invention for adding 3 ~ 15 times of water gagings, at 45 ~ 95 DEG C warm lixiviate get 1 ~ 3 time, stir with 10 ~ 50 revs/min of speed simultaneously, each extraction 1 ~ 4 hour or add at every turn 3 ~ 15 times amount decoct extract, each extraction 1 ~ 4 hour, extracts 1 ~ 3 time altogether.
1.2.2 the optimizing research of ethanol-extracted technique: according to above test-results, ethanol-extracted technique is using extraction time (A), extraction time (B), quantity of solvent (C), alcohol concn (D) four as investigation factor, each factor establishes three levels, by L9 (3
4) orthogonal table carries out orthogonal experimental design (table 5, table 6), inspection target is the extraction yield of salvianolic acid B.
Table 5 extraction factor water-glass
Table 6 extraction process orthogonal experiments table
Table 7 the results of analysis of variance table
F
0.05(2，2)＝19.00F
0.01(2，2)＝99.00
Alcohol reflux extracts the results of analysis of variance and shows, each experimental factor is to the extraction rate of transform of salvianolic acid B there are no significant difference, therefore the extraction conditions of this salvianolic acid B is for adding 3 ~ 15 times amount 30% ~ 60% alcohol reflux 1 ~ 3 time, extracts 1 ~ 4 hour at every turn.
1.3 salvianolic acid B raw material preparations
According to above-mentioned orthogonal experiment Optimization Technology, get red rooted salvia 10kg, add 8 times of water gagings at every turn and get 1.5 hours 80 DEG C of warm lixiviates, stir with 25 revs/min of speed simultaneously, extraction 3 times is stirred in temperature leaching altogether, filters, merging filtrate, extracting solution is evaporated to relative density 1.10 ~ 1.25 (60 DEG C), adding ethanol makes alcohol content 60%, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, vacuum-drying, salvianolic acid extract 4.15kg, recording content of danshinolic acid B is 10.25%.
Experimental example 2: salvianolic acid B transforms salvianolic acid A technics comparing
Experiment 1: the salvianolic acid B raw material getting preparation under 1.3 is about 30g, is mixed with 200ml solution, adds 10% sodium hydroxide solution adjust pH to 4.5;
Experiment 2: the salvianolic acid B raw material getting preparation under 1.3 is about 30g, is mixed with 200ml solution, adds 10% sodium hydroxide solution adjust pH to 4.5, add 1.0%ZnCl
2;
Experiment 3: get and be about 6g containing salvianolic acid B (content 51.54%) raw material, adding purified water, to be diluted to salvianolic acid B concentration be 15mg/ml solution, and add urea, making it with the mol ratio of salvianolic acid B is 0.5;
Above-mentioned each experimental group is placed in same autoclave, reacts 4.0 hours at 120 DEG C, and cooling calculates salvianolic acid A productive rate.
Table 8. salvianolic acid B transforms salvianolic acid A experimental result
Table 8 result shows, and salvianolic acid B high purity is on conversion not impact, and do not need to be purified to more than 50% and transform, salvianolic acid B is converted in salvianolic acid A process, and adjust ph, adds 1.0%ZnCl
2, greatly can improve the productive rate of salvianolic acid A.
Experimental example 3: salvianolic acid B transforms salvianolic acid A process optimization
Above-mentioned experimental study proves, salvianolic acid B purity is not affect the factor that salvianolic acid B transforms salvianolic acid A, but adds certain catalysts influence salvianolic acid B conversion salvianolic acid A, and therefore, we all add 1%ZnCl to each experimental group
2as catalyzer, other factors on affecting salvianolic acid B and be converted into salvianolic acid A: salvianolic acid B concentration (A), pH (B), temperature (C), time (D) carry out orthogonal test, each factor establishes three levels, by L9 (3
4) orthogonal table carries out test design (table 9, table 10), inspection target is the productive rate of salvianolic acid A.
Table 9. transforming agent water-glass
Table 10. conversion process orthogonal experiments table
Table 11. the results of analysis of variance table
*F
0.05(2，2)＝19.00△F
0.01(2，2)＝99.00
The results of analysis of variance shows, and is A to this orthogonal test according to the top condition that intuitive analysis optimizes
3b
2c
2d
2, factor A (salvianolic acid B concentration) has certain influence to salvianolic acid A productive rate, analyzes from K value, concentration does not affect the effect that raising salvianolic acid B is converted into salvianolic acid A higher than 30mg/ml, and the impurity formed is more, therefore, before transforming, salvianolic acid B concentration should select 1 ~ 30mg/ml; Factor B (pH), factor C (temperature) have pole significant difference to salvianolic acid A productive rate, should strictly control pH and temperature in prompting salvianolic acid B conversion process, successfully can realize transforming to salvianolic acid A of salvianolic acid B higher yields.
Experimental example 4: catalyst Z nCl
2consumption is on the impact transformed
Salvianolic acid A process optimization condition is transformed by above-mentioned salvianolic acid B, get salvianolic acid B raw material, add purified water 2001, make the solution that salvianolic acid B concentration is 15mg/ml, regulate pH to be 4.5, invert point is 120 DEG C, and transformation time is 4 hours, press and salvianolic acid B molar percent, add the catalyst Z nCl of different amount respectively
2, the results are shown in Table 12.
Table 12. catalyst Z nCl
2consumption is to transformations affect experimental result
Catalyst Z nCl
2consumption shows transformations affect experimental result, catalyst levels>=0.02% can produce 55.65% transformation efficiency, preferred catalyst consumption 0.1% one 3.0%, more preferably after 0.5% ~ 2.0%. catalyst levels > 3%, transformation efficiency no longer includes and significantly improves.
Experimental example 5: catalyzer transforms the katalysis of salvianolic acid A to salvianolic acid B
Salvianolic acid A process optimization condition is transformed by above-mentioned salvianolic acid B, get salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulate pH to be 4.5, invert point is 120 DEG C, and transformation time is 4 hours, the catalyzer adding different varieties and various dose respectively transforms, and the results are shown in Table 13.
Table 13. different catalysts transforms the impact of red A to red B
Table 13 result shows, and above 4 kinds of catalyzer all can as the catalyzer in salvianolic acid B conversion reaction, and each catalyst levels and salvianolic acid B molar percentage are 0.5% ~ 3.0%, productive rate >=40% of salvianolic acid A.Transformation efficiency is more than 60%.
Experimental example 6: salvianolic acid A, the research of salvianolic acid B analytical procedure
1. instrument and reagent
Instrument: Waters e2695 high performance liquid chromatograph, Empower2 chromatographic working station, 2998 diode-array detectors; Sartorius cp225D 100,000/electronic balance.
Chromatographic column: YMC C
18chromatographic column (250 × 4.6mm, 5 μm);
Reagent: methyl alcohol is chromatographically pure, water is ultrapure water prepared by Millipore, and other reagent are analytical pure.
Salvianolic acid B reference substance (lot number 111562-201009) all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, for assay; Salvianolic acid A reference substance is self-control, is 99.52% through purity mark content.
2. the preparation of reference substance solution and need testing solution
The preparation of 2.1 reference substance solution: precision takes salvianolic acid B respectively, salvianolic acid A reference substance is about 10mg, puts in 100ml measuring bottle, adds dissolve with methanol and be diluted to scale, shaking up, in contrast product stock solution; Precision draws above-mentioned each 1ml respectively again, puts in same 10ml measuring bottle, adds methanol dilution to scale, shake up, as mixing reference substance solution.
The preparation precision of 2.3 need testing solutions takes embodiment 1, embodiment 5 sample (being about equivalent to salvianolic acid A, each 10mg of salvianolic acid B), puts in 100ml measuring bottle, adds dissolve with methanol and be diluted to scale, shaking up, to obtain final product.
3. chromatographic condition and system suitability
Take octadecylsilane chemically bonded silica as weighting agent; Flow velocity 1.0ml/min; Determined wavelength: get mixing reference substance solution, carries out UV scanning, and result has maximum absorption at 286nm wavelength place, therefore determines that determined wavelength is 286nm; Column temperature 30 DEG C; Number of theoretical plate calculates should be not less than 10000 by salvianolic acid A peak.
During 0-10 minute, the ratio of methyl alcohol is down to 60% by the ratio that 30% rises to 40%, 0.1-0.5% phosphate aqueous solution by 70%; During 10-30 minute, the ratio of methyl alcohol is down to 45% by the ratio that 40% rises to 55%, 0.1-0.5% phosphate aqueous solution by 50%; During 30-60 minute, the ratio of methyl alcohol is down to 20% by the ratio that 55% rises to 80%, 0.1-0.5% phosphate aqueous solution by 45%.
Under these conditions, the chromatographic peak retention time of salvianolic acid A, salvianolic acid B reference substance and Radix Salviae Miltiorrhizae extract, salvianolic acid catalyzed conversion liquid is in table 14, and HPLC collection of illustrative plates is shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4.
The chromatographic peak retention time result of each reference substance of table 14.
4, the investigation of linear relationship
The above-mentioned mixing reference substance solution 0.1ml of accurate absorption, 0.2ml, 0.5ml, 1ml, 2ml, 5ml, put respectively in 10ml measuring bottle, add methanol dilution and become following concentration to be about: the series standard solution of 0.001mg/ml, 0.002mg/ml, 0.005mg/ml, 0.0lmg/ml, 0.02mg/ml, 0.05mg/ml.The each 10 μ l injection liquid chromatographies of the above-mentioned standardized solution of accurate absorption, peak area is calculated by chromatographic condition under " 3. chromatographic condition and system suitability " item, respectively with integrating peak areas value for ordinate zou, each concentrations control product sample size is X-coordinate, drawing standard curve.Result shows that mixing reference substance solution becomes good linear relationship, in table 15 in following scope.
Table 15. mixes reference substance solution linear relationship result
5. precision test
Get mixing reference substance solution, measure according to chromatographic condition under " 3. chromatographic condition and system suitability " item, repeat sample introduction 6 times.Result shows that the precision of the method is good, in table 16.
Table 16. Precision test result
6. stability test
Get need testing solution, measure according to chromatographic condition under " 3. chromatographic condition and system suitability " item, sample introduction is once at regular intervals, in result need testing solution each composition in 24 hours peak area without considerable change, show having good stability of need testing solution, in table 17.
Table 17. stability test result
7. replica test
Get this salvianolic acid A raw material, add methyl alcohol and make need testing solution 6 parts, measure according to chromatographic condition under " 3. chromatographic condition and system suitability " item.Result shows that the method repeatability is good, in table 18.
Table 18. replica test result
8. recovery test
Adopt application of sample recovery test, get the salvianolic acid A raw material 10mg of known content, accurately weighed, parallel 6 parts, a certain amount of reference substance solution is added respectively in the ratio of each component concentration-reference substance (1: 1), by 2.3 below legal system available test sample solutions, measure and calculate average recovery and the RSD of each composition.Result shows that the accuracy of the method is good, in table 19.
Table 19 recovery test result
Claims (10)
1. a catalyzed conversion salvianolic acid B prepares the method for salvianolic acid A, it is characterized in that: the Radix Salviae Miltiorrhizae extract of water intaking extraction or ethanol-extracted, wherein in extracting solution, salvianolic acid B concentration is 1mg/mL ~ 30mg/mL or is diluted with water to 1mg/mL ~ 30mg/mL, adding with the molar percentage of salvianolic acid B is the catalyzer of 0.1% ~ 3.0%, adding alkali regulates pH to be 3.0 ~ 6.5, invert point is 100 ~ 140 DEG C, transformation time is 1 ~ 6 hour, cooling, acid adding adjusts pH to 2.5 ~ 3.0, leave standstill, centrifugal, obtain salvianolic acid A conversion fluid, measure salvianolic acid A content, wherein said catalyzer is iron(ic) chloride, ruthenium trichloride, aluminum chloride, zinc chloride, one or more of Palladous chloride.
2. a kind of catalyzed conversion salvianolic acid B according to claim 1 prepares the method for salvianolic acid A, it is characterized in that: the Radix Salviae Miltiorrhizae extract of water intaking extraction or ethanol-extracted, wherein in extracting solution, salvianolic acid B concentration is 5mg/mL ~ 20mg/mL or is diluted with water to 5mg/mL ~ 20mg/mL, adding with the molar percentage of salvianolic acid B is the catalyzer of 0.5% ~ 2.0%, adding alkali regulates pH to be 3.5 ~ 4.5, invert point is 110 ~ 135 DEG C, transformation time is 2 ~ 5 hours, cooling, acid adding adjusts pH to 2.5 ~ 3.0, leave standstill, centrifugal, obtain salvianolic acid A conversion fluid, measure salvianolic acid A content.
3. a kind of catalyzed conversion salvianolic acid B described in claim 1 or 2 prepares the method for salvianolic acid A, the extracting method of wherein said aqueous extract is: get red rooted salvia medicine materical crude slice or particle, add 3 ~ 15 times of water gagings keeps 45 ~ 95 DEG C of water temperature lixiviates to get at every turn, stir with 10 ~ 50 revs/min of speed simultaneously, extract 1 ~ 3 time altogether, extract 1 ~ 4 hour at every turn; Extracting solution is evaporated to relative density and is determined as 1.10 ~ 1.25 at 60 DEG C, adds ethanol and makes alcohol content 30% ~ 80%, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract, measures salvianolic acid B content.
4. a kind of catalyzed conversion salvianolic acid B described in claim 1 or 2 prepares the method for salvianolic acid A, the extracting method of wherein said aqueous extract is: get red rooted salvia medicine materical crude slice or particle, add 3 ~ 15 times amount water boiling and extraction at every turn, extract 1 ~ 4 hour at every turn, extract 1 ~ 3 time altogether; Extracting solution is evaporated to relative density and is determined as 1.10 ~ 1.25 at 60 DEG C, adds ethanol and makes alcohol content 30% ~ 80%, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains conversion feedstock, measures salvianolic acid B content.
5. a kind of catalyzed conversion salvianolic acid B described in claim 1 or 2 prepares the method for salvianolic acid A, the extracting method of wherein said alcohol extract is: get red rooted salvia medicine materical crude slice or particle, add 3 ~ 15 times amount 30% ~ 60% alcohol reflux at every turn, each extraction 1 ~ 4 hour, extracts 1 ~ 3 time, decompression recycling ethanol altogether, leave standstill, centrifugal, obtain conversion feedstock, measure salvianolic acid B content.
6. a kind of catalyzed conversion salvianolic acid B according to claim 5 prepares the method for salvianolic acid A, and wherein said gets red rooted salvia particle diameter 2mm.
7. a kind of catalyzed conversion salvianolic acid B described in claim 1 or 2 prepares the method for salvianolic acid A, and wherein said catalyzer is iron(ic) chloride, ruthenium trichloride, aluminum chloride, zinc chloride or Palladous chloride.
8. a kind of catalyzed conversion salvianolic acid B described in claim 1 or 2 prepares the method for salvianolic acid A, wherein said add alkali adjust ph alkali used be sodium hydroxide, potassium hydroxide, Trisodium Citrate one or more.
9. a kind of catalyzed conversion salvianolic acid B described described in claim 1 prepares the method for salvianolic acid A, and wherein said acid adding adjusts pH acid used to be one or more of phosphoric acid, hydrochloric acid, sulfuric acid or acetic acid.
10. a kind of catalyzed conversion salvianolic acid B according to claim 2 prepares the method for salvianolic acid A, and the method for wherein said mensuration salvianolic acid A and mensuration content of danshinolic acid B is: adopt high effective liquid chromatography for measuring salvianolic acid A, salvianolic acid B, condition determination is as follows:
Take octadecylsilane chemically bonded silica as weighting agent;
Determined wavelength 286nm; Flow velocity 1.0mL/min; Column temperature 30 DEG C;
Theoretical plate number should be not less than 10000 by salvianolic acid A;
The preparation precision of reference substance solution takes in salvianolic acid A reference substance 10mg, salvianolic acid B reference substance 10mg to 100mL volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale;
The preparation of need testing solution: precision measures and is equivalent to 10mg salvianolic acid A and 10mg salvianolic acid B sample in 100mL volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale, to obtain final product;
Wash-out take methyl alcohol as mobile phase A, with 0.1 ~ 0.5% phosphoric acid for Mobile phase B, carries out gradient elution by following condition, runs 60 minutes;
When 0 ~ 10 minute, the ratio of methyl alcohol rises to 40% by 30%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 60% by 70%;
When 10 ~ 30 minutes, the ratio of methyl alcohol rises to 55% by 40%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 45% by 50%;
When 30 ~ 60 minutes, the ratio of methyl alcohol rises to 80% by 55%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 20% by 45%;
Assay method: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures content.
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