CN102475739A - Radix Salviae Miltiorrhizae water extract and preparation method thereof - Google Patents
Radix Salviae Miltiorrhizae water extract and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a Radix Salviae Miltiorrhizae water extract and a preparation method of the Radix Salviae Miltiorrhizae water extract. The preparation method of the Radix Salviae Miltiorrhizae water extract is convenient for operation and suitable for industrialized large-scale production; and the obtained water extract has a high extraction rate of salvianolic acid B, effectively reduces the content of salvianolic acid E which is difficult to be removed, and can be used for conveniently preparing high-purity salvianolic acid B.
Description
Technical field
The present invention relates to a kind of Radix Salviae Miltiorrhizae water extract and preparation method thereof.
Background technology
Salviamiltiorrhizabung derives from the dry root and rhizome of Labiatae salvia Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bunge).Have stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, the effect of the relieving restlessness that clears away heart-fire, be mainly used in the treatment of cardiovascular and cerebrovascular disease.Research shows that active component wherein has pharmacologically actives such as antithrombotic, anti-platelet aggregation, antioxidation, antitumor.
The active component of Radix Salviae Miltiorrhizae can be divided into water solublity and fat-soluble two parts, and wherein water-soluble portion is to be the phenolic acid compound of representative with the salvianolic acid B; Fat-soluble active ingredient is to be the diterpene phenanthrenequione compounds of representative with the tanshinone.Research shows that salvianolic acid B is content the strongest the highest, the active chemical compound in the red sage root water soluble ingredient, is that the drug development of effective ingredient also receives publicity day by day with the salvianolic acid B.
Though the separation purifying technique of red sage root water soluble ingredient is started late, development rapidly.Main method has decoction and alcohol sedimentation technique, high-speed countercurrent chromatography, CO
2Supercritical extraction etc.These methods can only be brought up to higher level (purity about 95%, yield about 85%) with salvianolic acid B purity and yield, are difficult to prepare pure article (purity＞99%).
The inventor discloses in CN101638401A through biotransformation method and column chromatography the purity of salvianolic acid B has been brought up to more than 99%, is the present the highest achievement in research of isolation and purification method moderate purity about salvianolic acid B.Remove the low content component salvianolic acid E in the red sage root water soluble ingredient but this method is still difficult, it is the progress of the drug research of effective ingredient that the existence of this impurity has hindered with the salvianolic acid B.
Summary of the invention
Technical problem to be solved by this invention be overcome in the prior art in the Radix Salviae Miltiorrhizae water extract content of salvianolic acid B not high, contain the defective that more difficulty is removed the component salvianolic acid E, a kind of Radix Salviae Miltiorrhizae water extract and preparation method thereof is provided.The extract yield of salvianolic acid B is high in this water extract, and effectively reduces the difficult content that removes the component salvianolic acid E, utilizes this Radix Salviae Miltiorrhizae water extract can prepare highly purified salvianolic acid B easily.
Therefore, the present invention relates to a kind of Radix Salviae Miltiorrhizae water extract, wherein, the relative amount of salvianolic acid E is below 0.4%, as 0.1%～0.4%; Preferable is below 0.3%, as 0.2%～0.3%.Wherein, the content of salvianolic acid B is generally 67～90%; Preferable is 72～84%.
Wherein, the content of salvianolic acid E in the relative amount of described salvianolic acid E (%)=Radix Salviae Miltiorrhizae water extract/(in the Radix Salviae Miltiorrhizae water extract in the content of salvianolic acid B+Radix Salviae Miltiorrhizae water extract the content of salvianolic acid E) * 100%.
The invention further relates to the method for preparing of above-mentioned Radix Salviae Miltiorrhizae water extract, the method includes the steps of: Radix Salviae Miltiorrhizae is extracted in pH is 3.5～4.5 aqueous acid, get final product.
Wherein, described acid is preferable is in hydrochloric acid, oxalic acid, acetic acid and the citric acid one or more, and better is hydrochloric acid.What the pH of described aqueous acid was preferable is 4.0.The consumption of described aqueous acid is preferable is 7～9 times of Radix Salviae Miltiorrhizae volume, and better is 8 times.
In the method for the present invention, other conditions can adopt the typical conditions of existing Radix Salviae Miltiorrhizae water extract method for preparing, and optimum condition is following:
What wherein, described Radix Salviae Miltiorrhizae was preferable is red rooted salvia.What described Radix Salviae Miltiorrhizae was preferable pulverizes the back use for process, and what the particle diameter after the pulverizing was preferable is 30～200 orders; Better is 50 orders.
Wherein, what the temperature of described extraction was preferable is 70～80 ℃, and better is 75 ℃.
Wherein, what the time of described extraction was preferable is 50～70 minutes, and better is 60 minutes.
Among the present invention, best, the method for preparing of described Radix Salviae Miltiorrhizae water extract comprises the following step: add the water of 7～9 times of volumes in the red rooted salvia, transfer pH to 3.5～4.5 with acid, under 70～80 ℃, extracted 50～70 minutes, get final product.
Wherein, the consumption of described water is preferable is 8 times of red rooted salvia volume.What described pH was preferable is 4.0.What described acid was preferable is hydrochloric acid.That the concentration of described acid is preferable is 1mol/L.What the temperature of described extraction was preferable is 75 ℃.What the time of described extraction was preferable is 60 minutes.
The invention further relates to the Radix Salviae Miltiorrhizae water extract that makes by said method.
The invention still further relates to a kind of method for preparing of salvianolic acid B, the above-mentioned water extract that makes is purified through post processing, get final product.
Wherein, the method for described post processing can be the conventional post-processing approach that carries out to the Radix Salviae Miltiorrhizae water extract in method that existing bibliographical information crosses or this area.Preferable, can be referring to CN101638401A.What described post-processing approach was preferable comprises the steps:
(1) the Radix Salviae Miltiorrhizae water extract is carried out biotransformation;
(2) step (1) gained material is separated with reverse resin column, get final product.
Wherein, the record that can walk in page 2 last column referring to CN101638401A description page 1 the reciprocal the 4th of the scope of application of each technical characterictic in step (1) and the step (2) and preferable range.
In the preferred embodiments of the present invention, the biotransformation described in the step (1) preferable for to transform with the Fusarium graminearum resting cell; What the temperature of described biotransformation was preferable is 20～35 ℃; What the time of described biotransformation was preferable is 24～90 hours; Described in the step (2) use the isolating eluent of reversed-phase resin post preferable be water or methanol aqueous solution.
On the basis that meets this area general knowledge, but above-mentioned each preferred feature combination in any among the present invention promptly gets each preferred embodiments of the present invention.
Raw material described in the present invention or reagent except that specifying, all commercially available getting.
Positive progressive effect of the present invention is: Radix Salviae Miltiorrhizae water extract preparation of the present invention is convenient; Be suitable for large-scale industrialization production; The extract yield of salvianolic acid B can reach more than 92% in this water extract; And effectively reduce the difficult content that removes the component salvianolic acid E, thereby advantage is provided for preparing highly purified salvianolic acid B.
Description of drawings
Fig. 1 is the HPLC spectrogram of embodiment 1 gained water extract.
The specific embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.
Used raw material or reagent is except that specifying among the embodiment, all commercially available getting.
Red rooted salvia: available from Nanyang, Henan Chinese Medicinal Materials Markets, it is subsequent use to pulverize 50 mesh sieves.
Salvianolic acid B reference substance: self-control, purity >=99%.
The HPLC testing conditions:
Chromatographic column: Agilent C18 reversed phase chromatographic column (5 μ m, 250 * 4.6mm);
Column temperature: 40 ℃;
Mobile phase: aqueous formic acid (mass percent 0.5%): acetonitrile=4: 1 (volume ratio);
Flow velocity: 1ml/min; Sample size: 10 μ l;
Detect wavelength: 288nm.
Under above-mentioned testing conditions, the retention time of salvianolic acid B and salvianolic acid E is respectively 31.5min, 33.8min.
The quality percentage composition of salvianolic acid B records about content of danshinolic acid B method for measuring in the red rooted salvia according to the Pharmacopoeia of the People's Republic of China (2005 editions) division of traditional Chinese drugs in the medical material.That is:
Chromatographic condition and system availability test are filler with the octadecylsilane chemically bonded silica; With methanol-acetonitrile-formic acid-water (30: 10: 1: 59) be mobile phase; The detection wavelength is 286nm.Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000.
It is an amount of that the preparation precision of reference substance solution takes by weighing the salvianolic acid B reference substance, adds 75% methanol and process the solution that every 1ml contains 0.14mg, promptly gets.
The about 0.2g of these article powder (crossing sieve No. three) is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask, the accurate 75% methanol 50ml that adds; Claim to decide weight, reflux 1 hour is taken out, and puts cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up with 75% methanol; Filter, get subsequent filtrate, promptly get.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and promptly get.
Measure through said method, the percentage composition of salvianolic acid B is 3.6% in the used medical material of this test.
Among the present invention, the computational methods of the extract yield of salvianolic acid B are following:
Wherein,
The used quality of medicinal material of percentage composition * test of salvianolic acid B in the content of salvianolic acid B (mg)=medical material in the medical material
Among the embodiment; The content of salvianolic acid E in the relative amount of salvianolic acid E (%)=water extract in the water extract/(in the water extract in the content+water extract of salvianolic acid B the content of salvianolic acid E) * 100%; Accordingly, can obtain through the calculated by peak area of the two in the HPLC testing result of water extract.
Finally obtain the yield of salvianolic acid B=(quality of the high-purity danshinolic acid B that the extraction process flow process finally obtains/when extraction is initial in the red rooted salvia quality of salvianolic acid B) * 100%
Embodiment 1
Take by weighing the 10g Danshen Root, add 70ml water, transfer pH to 3.5 with the hydrochloric acid of 1mol/L, in 75 ℃ of water-baths, extract 60min, filter, the HPLC purity of salvianolic acid B and salvianolic acid E is respectively 79.463% and 0.202% (result sees Fig. 1) in the mensuration water extract; The extract yield that calculates salvianolic acid B in the water extract is 92.1%, and the relative amount of salvianolic acid E is 0.253%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.234g, purity is 99.7%, yield is 65%.
Embodiment 2
Take by weighing the 10g Danshen Root; Add 80ml water, transfer pH to 3.5, in 75 ℃ of water-baths, extract 60min with the hydrochloric acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 81.191% and 0.164% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 92.3%, and the relative amount of salvianolic acid E is 0.202%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.241g, purity is 99.5%, yield is 67%.
Embodiment 3
Take by weighing the 10g Danshen Root; Add 90ml water, transfer pH to 3.5, in 75 ℃ of water-baths, extract 60min with the oxalic acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 75.261% and 0.182% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 92.6%, and the relative amount of salvianolic acid E is 0.241%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.245g, purity is 99.4%, yield is 68%.
Embodiment 4
Take by weighing the 10g Danshen Root; Add 80ml water, transfer pH to 4.0, in 75 ℃ of water-baths, extract 60min with the hydrochloric acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 80.798% and 0.172% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 93.3%, and the relative amount of salvianolic acid E is 0.213%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.252g, purity is 99.8%, yield is 70%.
Embodiment 5
Take by weighing the 10g Danshen Root; Add 80ml water, transfer pH to 4.5, in 75 ℃ of water-baths, extract 60min with the acetic acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 72.903% and 0.201% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 93.5%, and the relative amount of salvianolic acid E is 0.274%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.227g, purity is 99.8%, yield is 63%.
Embodiment 6
Take by weighing the 10g Danshen Root; Add 80ml water, transfer pH to 4.0, in 70 ℃ of water-baths, extract 65min with the hydrochloric acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 72.376% and 0.147% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 92.5%, and the relative amount of salvianolic acid E is 0.203%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.256g, purity is 99.9%, yield is 71%.
Embodiment 7
Take by weighing the 10g Danshen Root; Add 80ml water, transfer pH to 4.0, in 80 ℃ of water-baths, extract 65min with the hydrochloric acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 75.407% and 0.220% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 93.7%, and the relative amount of salvianolic acid E is 0.291%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.248g, purity is 99.6%, yield is 69%.
Embodiment 8
Take by weighing the 10g Danshen Root; Add 70ml water, transfer pH to 4.0, in 75 ℃ of water-baths, extract 60min with the citric acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 83.228% and 0.194% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 93.1%, and the relative amount of salvianolic acid E is 0.232%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.241g, purity is 99.5%, yield is 67%.
Embodiment 9
Take by weighing the 10g Danshen Root; Add 70ml water, transfer pH to 4.0, in 75 ℃ of water-baths, extract 65min with the hydrochloric acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 74.807% and 0.176% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 93.5%, and the relative amount of salvianolic acid E is 0.235%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.263g, purity is 99.8%, yield is 73%.
Embodiment 10
Take by weighing the 10g Danshen Root; Add 70ml water, transfer pH to 4.0, in 75 ℃ of water-baths, extract 70min with the hydrochloric acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 80.102% and 0.207% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 93.7%, and the relative amount of salvianolic acid E is 0.258%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.266g, purity is 99.7%, yield is 74%.
Comparative Examples 1 (comparing) with the method among the CN101638401A
Take by weighing the 10g Danshen Root; Add 40ml water, in 90 ℃ of water-baths, extract twice, filter; Merge filtrating twice; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 66.808% and 0.510% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 89.7%, and the relative amount of salvianolic acid E is 0.758%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.187g, purity is 97.4%, yield is 52%.
Can be found out that by Comparative Examples 1 different extracting modes all has considerable influence to the extract yield of salvianolic acid B in the water extract, the relative amount of salvianolic acid E, the relative amount of salvianolic acid E is with the purity and the yield of appreciable impact final products salvianolic acid B.
Comparative Examples 2
Take by weighing the 10g Danshen Root; Add 80ml water, transfer pH to 1.0, in 75 ℃ of water-baths, extract 65min with the hydrochloric acid of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 63.224% and 0.225% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 73.5%, and the relative amount of salvianolic acid E is 0.354%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.191g, purity is 87.4%, yield is 53%.
Comparative Examples 3
Take by weighing the 10g Danshen Root; Add 70ml water, transfer pH to 10.0, in 75 ℃ of water-baths, extract 65min with the sodium hydrate aqueous solution of 1mol/L; Filter; The HPLC purity of salvianolic acid B and salvianolic acid E is respectively 63.272% and 2.260% in the mensuration water extract, and the extract yield that calculates salvianolic acid B in the water extract is 69.4%, and the relative amount of salvianolic acid E is 3.452%.
Above-mentioned water extract is transferred pH to 2.0 with the hydrochloric acid of 1mol/L,, merge organic facies with ethyl acetate extraction twice; Concentrating under reduced pressure is removed ethyl acetate, and adding 200ml pH is 7.0 phosphate buffer dissolving in the residue, adds 20g Fusarium graminearum wet thallus; 28 ℃ of biotransformations 36 hours, remove rosmarinic acid, reuse resin XAD-2 absorption; Elder generation is with the water elution remove impurity of 5 times of column volumes; The methanol aqueous solution eluting resin column of reuse volumetric concentration 45% is removed less than the impurity of salvianolic acid B to retention time fully, and the methanol aqueous solution eluting resin column of reuse volumetric concentration 55% is complete to the salvianolic acid B eluting, collects HPLC purity greater than 99% eluent; Finally obtain salvianolic acid B 0.162g, purity is 80.8%, yield is 45%.
Can find out the extract yield of salvianolic acid B and the relative amount of salvianolic acid E in the change meeting appreciable impact water extract of extracting solution pH by Comparative Examples 2,3.The pH of extracting solution crosses when hanging down, and sour environment is unfavorable for the stripping of water soluble ingredient; When the pH of extracting solution was too high, salvianolic acid B was unstable under strong alkaline condition, is prone to be destroyed.Therefore the too high or too low acquisition that all is unfavorable for high yield high-purity danshinolic acid B of extracting solution pH.
Claims (16)
1. a Radix Salviae Miltiorrhizae water extract is characterized in that, wherein the relative amount of salvianolic acid E is below 0.4%.
2. Radix Salviae Miltiorrhizae water extract as claimed in claim 1 is characterized in that, wherein the relative amount of salvianolic acid E is 0.1～0.4%.
3. Radix Salviae Miltiorrhizae water extract as claimed in claim 2 is characterized in that, wherein the relative amount of salvianolic acid E is 0.2～0.3%.
4. like each described Radix Salviae Miltiorrhizae water extract of claim 1～3, it is characterized in that wherein the content of salvianolic acid B is 67～90%.
5. Radix Salviae Miltiorrhizae water extract as claimed in claim 4 is characterized in that, wherein the content of salvianolic acid B is 72～84%.
6. like the method for preparing of each described Radix Salviae Miltiorrhizae water extract of claim 1～5, it is characterized in that comprising following steps: Radix Salviae Miltiorrhizae is extracted, get final product in pH is 3.5～4.5 aqueous acid.
7. the method for preparing of Radix Salviae Miltiorrhizae water extract as claimed in claim 6 is characterized in that, described Radix Salviae Miltiorrhizae is for using through pulverizing the back, and the particle diameter after the pulverizing is 30～200 orders.
8. the method for preparing of Radix Salviae Miltiorrhizae water extract as claimed in claim 6 is characterized in that, described acid is one or more in hydrochloric acid, oxalic acid, acetic acid and the citric acid.
9. the method for preparing of Radix Salviae Miltiorrhizae water extract as claimed in claim 6 is characterized in that, the pH of described aqueous acid is 4.0.
10. the method for preparing of Radix Salviae Miltiorrhizae water extract as claimed in claim 6 is characterized in that, the consumption of described aqueous acid is 7～9 times of Radix Salviae Miltiorrhizae volume.
11. the method for preparing of Radix Salviae Miltiorrhizae water extract as claimed in claim 6 is characterized in that, the temperature of described extraction is 70～80 ℃.
12. the method for preparing of Radix Salviae Miltiorrhizae water extract as claimed in claim 6 is characterized in that, the time of described extraction is 50～70 minutes.
13. the method for preparing of Radix Salviae Miltiorrhizae water extract as claimed in claim 6 is characterized in that, described method for preparing comprises the following step: the water that adds 7～9 times of volumes in the red rooted salvia; Transfer pH to 3.5～4.5 with acid; Under 70～80 ℃, extracted 50～70 minutes, get final product.
14. the method for preparing of a salvianolic acid B is characterized in that, will purify through post processing like each described Radix Salviae Miltiorrhizae water extract of claim 1～5, gets final product.
15. the method for preparing of salvianolic acid B as claimed in claim 14 is characterized in that, described post-processing approach comprises the steps:
(1) the Radix Salviae Miltiorrhizae water extract is carried out biotransformation;
(2) step (1) gained material is separated with the reversed-phase resin post, get final product.
16. method for preparing as claimed in claim 15; It is characterized in that; Biotransformation described in the step (1) is for to transform with the Fusarium graminearum resting cell, and the temperature of described biotransformation is 20～35 ℃, and the time of described biotransformation is 24～90 hours; The isolating eluent of reversed-phase resin post that uses described in the step (2) is water or methanol aqueous solution.
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