CN102675408B - A kind of preparation method of Tanshinone II A - Google Patents
A kind of preparation method of Tanshinone II A Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of Tanshinone II A, it comprises the following step: fat soluble ingredient of red sage root macropore reverse phase absorption resin is carried out column chromatography for separation; Wherein, the parameter of described macropore reverse phase absorption resin is as follows: particle diameter is 50 ~ 100 μm, and aperture is
Description
Technical field
The present invention is specifically related to a kind of preparation method of Tanshinone II A.
Background technology
The red sage root is one of the most frequently used medicine in China's traditional medicine, derives from the dry root and rhizome of the Chun Xing section salvia red sage root (SalviamiltiorrhizaBunge).The red sage root is recorded in Shennong's Herbal the earliest, is listed in top grade medicine, is mainly used in the treatment of cardiovascular and cerebrovascular diseases.Within 2005, " pharmacopeia " (division of traditional Chinese drugs) is recorded, and the red sage root has effect of stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, the relieving restlessness that clears away heart-fire.
The activeconstituents of the red sage root is divided into fat-soluble and water-soluble two portions.Salvia-soluble part take salvianolic acid B as the phenolic acid compound of representative, and the fat-soluble active ingredient of the red sage root is TANSHINONES, it is terpene phenanthrenequione compounds, its chemical composition mainly contains Tanshinone I, Tanshinone II A, Cryptotanshinone etc., they are all the tetracyclic compounds of diterpene quinones, in red rooted salvia and its preparation, Tanshinone II A, the content of Cryptotanshinone is the highest.Regulation in " pharmacopeia ", tanshinone in salvia miltiorrhiza bunge IIA content is no less than 0.2%, and content of danshinolic acid B is no less than 3%.
Red sage root medicine commercially available at present can be divided into two large classes according to administering mode: (1) red sage root oral pharmaceutical: mainly contain Radix Salviae Miltiorrhizae Tabellae, FUFANG DANSHEN PIAN and FUFANG DANSHEN DIWAN several; (2) injection of danshen medicine: mainly contain Radix Salviae Miltiorrhizae Injection, sodium tanshinone IIA sulfate injection liquid, poly phenolic acid of Radix Salviae Miltiorrhizae saline injection, wherein, both effective constituents are clear and definite, and quality control is strict, are the Radix Salviae Miltiorrhizae Injectiones that quality on market is higher.
China's red sage root output is huge, cheap, but red sage root fat-soluble active ingredient content is lower, and its drug price is higher.Improve its main active ingredient Tanshinone II A content, for the further exploitation of salviamiltiorrhizabung, have great importance.
" separation and purification of Tanshinone II A " (Meng Zhaoquan that Meng Zhao congruence people delivers, Chen Hongnan, Qian Jie etc. the separation and purification [J] of Tanshinone II A. Chinese experimental pharmacology of traditional Chinese medical formulae magazine .2010.16 (2): 6-7) middle employing macroporous adsorbent resin HPD-100 purification, after purifying, purity reaches 42%, and yield is 80%.
Summary of the invention
Technical problem to be solved by this invention is in the extraction and separation method of existing Tanshinone II A, and yield and purity are all very low, and cannot realize industrialized defect, and provides a kind of preparation method of Tanshinone II A.Preparation method of the present invention not only product yield is high, and can reach very high product purity.Method of the present invention can be carried out amplification and be produced, and has certain using value.
The present inventor, through large quantity research, finds to adopt a kind of macropore reverse phase absorption resin of special selection to be separated fat soluble ingredient of red sage root, can obtain the Tanshinone II A of very high yield.The present inventor also optimizes absorb-elute condition further, make the yield of Tanshinone II A be greater than 85%, and purity is greater than 70%, and makes the purity of Tanshinone II A reach more than 98% by twice crystallization, and yield is greater than 60%.
Therefore, the present invention relates to a kind of preparation method of Tanshinone II A, it comprises the following step: fat soluble ingredient of red sage root macropore reverse phase absorption resin is carried out column chromatography for separation; Wherein, the parameter of described macropore reverse phase absorption resin is as follows: particle diameter is 50 ~ 100 μm, and aperture is
medium is polystyrene-divinylbenzene.Described aperture is preferably
what described macropore reverse phase absorption resin was better is the macropore reverse phase absorption fat that Shenzhen Na Wei scientific & technical corporation produces, and model is NMPS100, and its particle diameter is: 50-100 μm, and aperture is
resin matrix is polystyrene-divinylbenzene.
Described macropore reverse phase absorption resin before use, preferably first carries out pre-treatment, and pretreated method can be this resinoid method of pre-treatment of this area routine.Preferred method is as follows: rinse resin with aqueous acid, then washes neutrality with deionized water.Wherein, the acid in described aqueous acid can be mineral acid, example hydrochloric acid, and its volumetric molar concentration is preferably 0.5 ~ 2.5N, preferred 1N.Aqueous acid is preferably the aqueous acetone solution of acid, and the volumetric concentration of acetone is preferably 60 ~ 90%, preferably 70%.The consumption of aqueous acid is preferably 2 ~ 5 column volumes, preferably 2 ~ 3 column volumes.Better pre-treatment step is as follows: 70% aqueous acetone solution containing 1N hydrochloric acid is rinsed resin, and consumption is 3BV, then washes neutrality with deionized water.
Wherein, described fat soluble ingredient of red sage root can be obtained by the method for the extraction fat soluble ingredient of red sage root of this area routine, preferably comprises the following step: tanshinol extract being mixed in pH is in the water of 5 ~ 8, filters, obtains filter residue.Wherein, described pH value is preferably 6 ~ 7.Before filtration, preferably also carry out the step mixed of vibrating.
Wherein, described tanshinol extract can be obtained by the method preparing tanshinol extract of this area routine, preferably comprises the following step: soaked by the red rooted salvia aqueous ethanolic solution after pulverizing, then centrifugal, gets supernatant liquor, concentrated.Wherein, the particle diameter of the red rooted salvia after described pulverizing is preferably 20 ~ 50 orders, and better is 20 orders.Described aqueous ethanolic solution with pulverize after the volume mass of red rooted salvia be 5 ~ 10L/Kg than preferably, that better is 6 ~ 8L/Kg.The volumetric concentration of described aqueous ethanolic solution is preferably 70% ~ 100%, preferably more than 90%.The temperature of described immersion is preferably 60 ~ 80 DEG C.The time of described immersion is preferably 0.5 ~ 2 hour, and better is 1 hour.Described immersion and centrifugal number of times are preferably 1 ~ 3 time, and better is 2 times.Described is concentrated preferably dry for being concentrated into, by Rotary Evaporators evaporate to dryness at the temperature of 35 ~ 50 DEG C (preferably 40 DEG C).
The preparation method of the best of described tanshinol extract is as follows: the red sage root is done pulverizing medicinal materials and cross 20 mesh sieves, with the aqueous solution of 70 ~ 100% ethanol centrifuging and taking supernatant liquor after soak extraction 1h at 60 ~ 80 DEG C, the aqueous solution of wherein said ethanol is 6 ~ 8L/Kg with the volume mass ratio of the dry medicinal material of the red sage root, this operation repetition twice, merge twice supernatant liquor, steam to dry at 40 DEG C of backspins, obtain tanshinol extract.
In the present invention, described carry out with macropore reverse phase absorption resin method and the condition of carrying out column chromatography for separation with macropore reverse phase absorption resin that the method for column chromatography for separation and condition all can be this area routine, the present inventor is through large quantity research, find out following particularly preferred step and condition: with the aqueous ethanolic solution of volumetric concentration 60% ~ 95% for eluent, fat soluble ingredient of red sage root macropore reverse phase absorption resin is carried out column chromatography for separation.The kind of macropore reverse phase absorption resin ditto described in.
Preferably, in described column chromatography for separation, the elutriant that the purity of collecting Tanshinone II A is greater than 70%.
Wherein, the step of described column chromatography for separation is preferably as follows: after fat soluble ingredient of red sage root upper prop, first carry out wash-out removal of impurities with the aqueous ethanolic solution of volumetric concentration 60% ~ 70%, wash-out is carried out again with the aqueous ethanolic solution of volumetric concentration 90% ~ 95%, the elutriant that the purity of collecting Tanshinone II A is greater than 70%.
Wherein, described volumetric concentration is the consumption of the aqueous ethanolic solution of 60% ~ 70% is preferably 2-4 column volume, preferably 3 column volumes, and the consumption of the aqueous ethanolic solution of volumetric concentration 90% ~ 95% is preferably 2 ~ 4 column volumes, preferably 3 column volumes.
In the present invention, the detection method of the purity of Tanshinone II A can be the detection method of this area routine, as by methods such as HPLC or GC, with Tanshinone II A sterling for contrast detects.
In the present invention, before fat soluble ingredient of red sage root macropore reverse phase absorption resin is carried out column chromatography for separation, preferably, first fat soluble ingredient of red sage root is dissolved in the aqueous ethanolic solution of volumetric concentration 60% ~ 70%, obtain the ethanolic soln of fat soluble ingredient of red sage root, then upper prop carries out column chromatography for separation.
In the present invention, the quality (or volume) of described macropore reverse phase absorption resin can be selected by this area Conventional wisdom, and preferably, the red rooted salvia after aforesaid pulverizing and the volume ratio of resin are 50 ~ 200g/L, and that better is 100 ~ 150g/L.The specification of resin column can be the conventional specification in this area, preferably for aspect ratio is 5: 1 ~ 3: 1, and preferably 4: 1.
In the present invention, after macropore reverse phase absorption resin column chromatographic separation terminates, preferably also comprise the following step: crystallisation step column chromatography for separation gained material being carried out this area routine, obtains Tanshinone II A crystal.
Wherein, described crystallisation step preferably comprises the following step:
(1) elutriant of column chromatography for separation gained is concentrated, obtain enriched material, with acetone solution, in gained acetone soln, drip distilled water to solution occurs muddy, gained turbid solution is left standstill 24 ~ 72 hours under 2-8 DEG C of (preferably 4 DEG C) condition (best 48 hours), obtain Tanshinone II A coarse crystal;
(2) by the distillation washing of step (1) gained coarse crystal, distilled water is added again with after acetone solution, occur muddy to solution, gained turbid solution is left standstill 24 ~ 72 hours under 2-8 DEG C of (preferably 4 DEG C) condition (best 48 hours), obtain Cryptotanshinone IIA crystal.
In step (1), described simmer down to routine operation, can be concentrated into dry.The volume of acetone is preferably for dissolve to enriched material, and better, its volume is 2 ~ 4 times (preferably 2 times) of described enriched material volume.According to this area general knowledge, after standing crystallization, obtain Tanshinone II A coarse crystal by suction filtration.
In step (2), the number of times of described distillation washing can be 1 ~ 3 time, and the consumption of each distilled water wash and the volume mass of coarse crystal are 10 ~ 20L/kg than preferably.The volume of acetone described in step (2) preferably for dissolving to coarse crystal, 2 ~ 4 times (preferably 2 times) of what its volume was better is described coarse crystal volume.
In the present invention, after above-mentioned crystallisation step, the purity of the crystal obtained is greater than 98%, and yield is greater than 60% (HPLC detection).
In the present invention, best, the preparation method of described Tanshinone II A comprises the following step:
1, get a certain amount of red sage root to do pulverizing medicinal materials and cross 20 mesh sieves, with the aqueous solution of volumetric concentration 70-100% ethanol centrifuging and taking supernatant liquor after soak extraction 1h at 60-80 DEG C, described aqueous ethanolic solution with pulverize and sieve after the volume mass of red rooted salvia than for 6-8L/Kg, this operation repetition twice, merge twice supernatant liquor, steam to dry at 40 DEG C of backspins, obtain tanshinol extract.
2, by the water dissolution tanshinol extract of pH7-8, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root
3, with the dissolve with ethanol fat soluble ingredient of red sage root of volumetric concentration 60-70%, upper macropore reverse phase absorption resin NMPS-100 adsorbs, with the 60-70% ethanol removal of impurities of 3 times of column volumes, with the ethanol elution resin of 90-95%, Fraction collection, the sample HPLC of Fraction collection is analyzed, purity is reached the sample mix of more than 70%.
4, mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drip distilled water to solution occurs muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtains Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, then adds a small amount of distilled water with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtain Tanshinone II A crystal, HPLC detects its purity and is greater than 98%.
In the present invention, above-mentioned each preferred feature can under the prerequisite without prejudice to this area general knowledge arbitrary combination, obtain each preferred embodiments of the present invention.
Except specified otherwise, the reagent that the present invention is used and raw material are all commercially.
Positive progressive effect of the present invention is:
(1) in preparation method of the present invention, the Tanshinone II A obtained not only yield is higher, and can reach very high purity.
(2) method of the present invention can carry out amplification production, has certain using value.
Embodiment
Further illustrate the present invention by embodiment below, but the present invention is not limited.
In following each embodiment:
The red sage root is originated: the dry medicinal material of the red sage root (place of production: Henan)
HPLC condition is as follows:
Methyl alcohol: water=75: 25
Sample size 10 μ l column temperature 35 DEG C of flow velocity 0.8ml/min
Stand-by time 20min determined wavelength UV270nm
AgilentEclipsepiusC18 post 5 μm of 4.6 × 250nm
BV in embodiment represents column volume, is this area conventional unit.
In following embodiment, the Tanshinone II A content in raw material red rooted salvia all measures by this area common detection methods, and the concentration of aqueous acetone solution and aqueous ethanolic solution is volumetric concentration.
Embodiment 1
(Tanshinone II A content was 0.53% (weight percent) through pulverizing the red rooted salvia powder of 20 mesh sieves to get 100g, add 600mL centrifugal after soak extraction 1h at 60 DEG C containing the aqueous solution of 70% ethanol, get supernatant liquor, this operation repetition twice, merge twice supernatant liquor, steam removing ethanol at 40 DEG C of backspins, obtain tanshinol extract concentrated solution.
By the water dissolution tanshinol extract of pH7, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root.With the dissolve with ethanol fat soluble ingredient of red sage root of 70%.
Get pretreated macropore reverse phase absorption resin NMPS-1001L (resin pre-treatment step: adopt 70% aqueous acetone solution containing 1N hydrochloric acid to rinse resin, consumption is about 3BV, neutrality is washed again with deionized water) be loaded in Φ 80 × 350mm chromatography column, by NMPS-100 on fat soluble ingredient of red sage root ethanolic soln.First with the aqueous solution removal of impurities of 5BV containing 60% ethanol after upper prop, then wash down concentrated for Tanshinone II A containing 90% ethanol with 4BV, Fraction collection elutriant, and detected by HPLC, Tanshinone II A purity is greater than the elutriant mixing of 70%.The purity of mixed solution is 75.6%, and the yield of this step is 86.5%.
Mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drips distilled water to solution occur muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtain Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, a small amount of distilled water is added again with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtains Tanshinone II A crystal, and HPLC detects its purity and is greater than 98.2%, crystalline quality is 323.8mg, yield 61.1%.
Embodiment 2
(Tanshinone II A content was 0.53% (weight percent) through pulverizing the red rooted salvia powder of 20 mesh sieves to get 100g, add 700mL centrifugal after soak extraction 1h at 70 DEG C containing the aqueous solution of 80% ethanol, get supernatant liquor, this operation repetition twice, merge twice supernatant liquor, steam removing ethanol at 40 DEG C of backspins, obtain tanshinol extract concentrated solution.
By the water dissolution tanshinol extract of pH7, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root.With the dissolve with ethanol fat soluble ingredient of red sage root of 60%.
Get pretreated macropore reverse phase absorption resin NMPS-1001L (resin pre-treatment step: adopt 70% aqueous acetone solution containing 1N hydrochloric acid to rinse resin, consumption is about 3BV, neutrality is washed again with deionized water) be loaded in Φ 80 × 350mm chromatography column, by NMPS-100 on fat soluble ingredient of red sage root ethanolic soln.First with the aqueous solution removal of impurities of 5BV containing 60% ethanol after upper prop, then wash down concentrated for Tanshinone II A containing 90% ethanol with 4BV, Fraction collection elutriant, and detected by HPLC, Tanshinone II A purity is greater than the elutriant mixing of 70%.The purity of mixed solution is 75.2%, and the yield of this step is 86.1%.
Mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drips distilled water to solution occur muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtain Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, a small amount of distilled water is added again with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtains Tanshinone II A crystal, and HPLC detects its purity and is greater than 98.1%, crystalline quality is 320.1mg, yield 60.4%.
Embodiment 3
(Tanshinone II A content was 0.53% (weight percent) through pulverizing the red rooted salvia powder of 20 mesh sieves to get 100g, add 700mL centrifugal after soak extraction 1h at 80 DEG C containing the aqueous solution of 90% ethanol, get supernatant liquor, this operation repetition twice, merge twice supernatant liquor, steam removing ethanol at 40 DEG C of backspins, obtain tanshinol extract concentrated solution.
By the water dissolution tanshinol extract of pH7, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root.With the dissolve with ethanol fat soluble ingredient of red sage root of 70%.
Get pretreated macropore reverse phase absorption resin NMPS-1001L (resin pre-treatment step: adopt 70% aqueous acetone solution containing 1N hydrochloric acid to rinse resin, consumption is about 3BV, neutrality is washed again with deionized water) be loaded in Φ 80 × 350mm chromatography column, by NMPS-100 on fat soluble ingredient of red sage root ethanolic soln.First with the aqueous solution removal of impurities of 5BV containing 60% ethanol after upper prop, then wash down concentrated for Tanshinone II A containing 95% ethanol with 4BV, Fraction collection elutriant, and detected by HPLC, Tanshinone II A purity is greater than the elutriant mixing of 70%.The purity of mixed solution is 76.4%, and the yield of this step is 86.9%.
Mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drips distilled water to solution occur muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtain Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, a small amount of distilled water is added again with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtains Tanshinone II A crystal, and HPLC detects its purity and is greater than 98.5%, crystalline quality is 330.2mg, yield 62.3%.
Embodiment 4
(Tanshinone II A content was 0.53% (weight percent) through pulverizing the red rooted salvia powder of 20 mesh sieves to get 100g, add 800mL centrifugal after soak extraction 1h at 70 DEG C containing the aqueous solution of 100% ethanol, get supernatant liquor, this operation repetition twice, merge twice supernatant liquor, steam removing ethanol at 40 DEG C of backspins, obtain tanshinol extract concentrated solution.
By the water dissolution tanshinol extract of pH7, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root.With the dissolve with ethanol fat soluble ingredient of red sage root of 70%.
Get pretreated macropore reverse phase absorption resin NMPS-1001L (resin pre-treatment step: adopt 70% aqueous acetone solution containing 1N hydrochloric acid to rinse resin, consumption is about 3BV, neutrality is washed again with deionized water) be loaded in Φ 80 × 350mm chromatography column, by NMPS-100 on fat soluble ingredient of red sage root ethanolic soln.First with the aqueous solution removal of impurities of 5BV containing 60% ethanol after upper prop, then wash down concentrated for Tanshinone II A containing 95% ethanol with 4BV, Fraction collection elutriant, and detected by HPLC, Tanshinone II A purity is greater than the elutriant mixing of 70%.The purity of mixed solution is 77.3%, and the yield of this step is 87.3%.
Mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drips distilled water to solution occur muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtain Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, a small amount of distilled water is added again with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtains Tanshinone II A crystal, and HPLC detects its purity and is greater than 98.9%, crystalline quality is 333.9mg, yield 63.0%.
Embodiment 5
(Tanshinone II A content was 0.53% (weight percent) through pulverizing the red rooted salvia powder of 20 mesh sieves to get 100g, add 800mL centrifugal after soak extraction 1h at 70 DEG C containing the aqueous solution of 100% ethanol, get supernatant liquor, this operation repetition twice, merge twice supernatant liquor, steam removing ethanol at 40 DEG C of backspins, obtain tanshinol extract concentrated solution.
By the water dissolution tanshinol extract of pH8, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root.With the dissolve with ethanol fat soluble ingredient of red sage root of 70%.
Get pretreated macropore reverse phase absorption resin NMPS-1001L (resin pre-treatment step: adopt 70% aqueous acetone solution containing 1N hydrochloric acid to rinse resin, consumption is about 3BV, neutrality is washed again with deionized water) be loaded in Φ 80 × 350mm chromatography column, by NMPS-100 on fat soluble ingredient of red sage root ethanolic soln.First with the aqueous solution removal of impurities of 5BV containing 70% ethanol after upper prop, then wash down concentrated for Tanshinone II A containing 90% ethanol with 4BV, Fraction collection elutriant, and detected by HPLC, Tanshinone II A purity is greater than the elutriant mixing of 70%.The purity of mixed solution is 78.8%, and the yield of this step is 88.2%.
Mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drips distilled water to solution occur muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtain Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, a small amount of distilled water is added again with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtains Tanshinone II A crystal, and HPLC detects its purity and is greater than 99.3%, crystalline quality is 345.1mg, yield 65.1%.
Embodiment 6
(Tanshinone II A content was 0.53% (weight percent) through pulverizing the red rooted salvia powder of 20 mesh sieves to get 100g, add 600mL centrifugal after soak extraction 1h at 80 DEG C containing the aqueous solution of 80% ethanol, get supernatant liquor, this operation repetition twice, merge twice supernatant liquor, steam removing ethanol at 40 DEG C of backspins, obtain tanshinol extract concentrated solution.
By the water dissolution tanshinol extract of pH8, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root.With the dissolve with ethanol fat soluble ingredient of red sage root of 70%.
Get pretreated macropore reverse phase absorption resin NMPS-1001L (resin pre-treatment step: adopt 70% aqueous acetone solution containing 1N hydrochloric acid to rinse resin, consumption is about 3BV, neutrality is washed again with deionized water) be loaded in Φ 80 × 350mm chromatography column, by NMPS-100 on fat soluble ingredient of red sage root ethanolic soln.First with the aqueous solution removal of impurities of 5BV containing 60% ethanol after upper prop, then wash down concentrated for Tanshinone II A containing 90% ethanol with 4BV, Fraction collection elutriant, and detected by HPLC, Tanshinone II A purity is greater than the elutriant mixing of 70%.The purity of mixed solution is 76.9%, and the yield of this step is 87.0%.
Mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drips distilled water to solution occur muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtain Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, a small amount of distilled water is added again with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtains Tanshinone II A crystal, and HPLC detects its purity and is greater than 98.6%, crystalline quality is 279.7mg, yield 61%.
Embodiment 7
(Tanshinone II A content was 0.53% (weight percent) through pulverizing the red rooted salvia powder of 20 mesh sieves to get 100g, add 700mL centrifugal after soak extraction 1h at 80 DEG C containing the aqueous solution of 70% ethanol, get supernatant liquor, this operation repetition twice, merge twice supernatant liquor, steam removing ethanol at 40 DEG C of backspins, obtain tanshinol extract concentrated solution.
By the water dissolution tanshinol extract of pH7, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root.With the dissolve with ethanol fat soluble ingredient of red sage root of 60%.
Get pretreated macropore reverse phase absorption resin NMPS-1001L (resin pre-treatment step: adopt 70% aqueous acetone solution containing 1N hydrochloric acid to rinse resin, consumption is about 3BV, neutrality is washed again with deionized water) be loaded in Φ 80 × 350mm chromatography column, by NMPS-100 on fat soluble ingredient of red sage root ethanolic soln.First with the aqueous solution removal of impurities of 5BV containing 70% ethanol after upper prop, then wash down concentrated for Tanshinone II A containing 95% ethanol with 4BV, Fraction collection elutriant, and detected by HPLC, Tanshinone II A purity is greater than the elutriant mixing of 70%.The purity of mixed solution is 75.9%, and the yield of this step is 86.9%.
Mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drips distilled water to solution occur muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtain Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, a small amount of distilled water is added again with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtains Tanshinone II A crystal, and HPLC detects its purity and is greater than 98.4%, crystalline quality is 330.2mg, yield 62.8%.
Embodiment 8
(Tanshinone II A content was 0.53% (weight percent) through pulverizing the red rooted salvia powder of 20 mesh sieves to get 100g, add 800mL centrifugal after soak extraction 1h at 70 DEG C containing the aqueous solution of 90% ethanol, get supernatant liquor, this operation repetition twice, merge twice supernatant liquor, steam removing ethanol at 40 DEG C of backspins, obtain tanshinol extract concentrated solution.
By the water dissolution tanshinol extract of pH8, filter after vibration mixing, filter residue is fat soluble ingredient of red sage root.With the dissolve with ethanol fat soluble ingredient of red sage root of 60%.
Get pretreated macropore reverse phase absorption resin NMPS-1001L (resin pre-treatment step: adopt 70% aqueous acetone solution containing 1N hydrochloric acid to rinse resin, consumption is about 3BV, neutrality is washed again with deionized water) be loaded in Φ 80 × 350mm chromatography column, by NMPS-100 on fat soluble ingredient of red sage root ethanolic soln.First with the aqueous solution removal of impurities of 5BV containing 70% ethanol after upper prop, then wash down concentrated for Tanshinone II A containing 95% ethanol with 4BV, Fraction collection elutriant, and detected by HPLC, Tanshinone II A purity is greater than the elutriant mixing of 70%.The purity of mixed solution is 78.2%, and the yield of this step is 87.6%.
Mixed solution is concentrated into dry, with 2 times of sample volume acetone solutions, in acetone soln, slowly drips distilled water to solution occur muddy, turbid solution is positioned over the crystallization of 4 DEG C of refrigerator hold over night, obtain Tanshinone II A coarse crystal, suction filtration, distillation washing coarse crystal once, a small amount of distilled water is added again with after 2 times of sample volume acetone solutions, hold over night crystallization in 4 DEG C of refrigerators, obtains Tanshinone II A crystal, and HPLC detects its purity and is greater than 99.0%, crystalline quality is 340.3mg, yield 64.2%.
Claims (9)
1. a preparation method for Tanshinone II A, it comprises the following step: fat soluble ingredient of red sage root macropore reverse phase absorption resin is carried out column chromatography for separation; Wherein, described macropore reverse phase absorption resin is the macropore reverse phase absorption fat that Shenzhen Na Wei scientific & technical corporation produces, and model is NMPS100, and its particle diameter is: 50-100 μm, and aperture is
resin matrix is polystyrene-divinylbenzene; The step of described column chromatography for separation is as follows: after fat soluble ingredient of red sage root upper prop, first carry out wash-out removal of impurities with the aqueous ethanolic solution of volumetric concentration 60% ~ 70%, wash-out is carried out again with the aqueous ethanolic solution of volumetric concentration 90% ~ 95%, the elutriant that the purity of collecting Tanshinone II A is greater than 70%; Described fat soluble ingredient of red sage root is obtained by following method: tanshinol extract being mixed in pH is in the water of 5 ~ 8, filters, obtains filter residue; Described tanshinol extract is obtained by following method: soaked by the red rooted salvia aqueous ethanolic solution after pulverizing, then centrifugal, gets supernatant liquor, concentrated.
2. preparation method as claimed in claim 1, is characterized in that: before use, first carry out pre-treatment, pretreated method is as follows: rinse resin with aqueous acid, then washes neutrality with deionized water for described macropore reverse phase absorption resin.
3. preparation method as claimed in claim 1, is characterized in that: described pH value is 6 ~ 7.
4. preparation method as claimed in claim 1, it is characterized in that: wherein, the particle diameter of the red rooted salvia after described pulverizing is 20 ~ 50 orders; Described aqueous ethanolic solution with pulverize after the volume mass ratio of red rooted salvia be 5 ~ 10L/Kg; The volumetric concentration of described aqueous ethanolic solution is 70% ~ 100%; The temperature of described immersion is 60 ~ 80 DEG C; The time of described immersion is 0.5 ~ 2 hour; Described immersion and centrifugal number of times are 1 ~ 3 time.
5. preparation method as claimed in claim 1, it is characterized in that: described volumetric concentration is the consumption of the aqueous ethanolic solution of 60% ~ 70% is 2-4 column volume, the consumption of the aqueous ethanolic solution of volumetric concentration 90% ~ 95% is 2 ~ 4 column volumes.
6. preparation method as claimed in claim 1, it is characterized in that: before fat soluble ingredient of red sage root macropore reverse phase absorption resin is carried out column chromatography for separation, first fat soluble ingredient of red sage root is dissolved in the aqueous ethanolic solution of volumetric concentration 60% ~ 70%, obtain the ethanolic soln of fat soluble ingredient of red sage root, then upper prop carries out column chromatography for separation.
7. preparation method as claimed in claim 1, is characterized in that: after macropore reverse phase absorption resin column chromatographic separation terminates, also comprise the following step: crystallisation step column chromatography for separation gained material being carried out this area routine, obtains Tanshinone II A crystal; Wherein, described crystallisation step comprises the following step:
(1) elutriant of column chromatography for separation gained is concentrated, obtain enriched material, with acetone solution, in gained acetone soln, drip distilled water to solution occur muddy, gained turbid solution is left standstill more than 20 hours under 2-8 DEG C of condition, obtains Tanshinone II A coarse crystal;
(2) by the distillation washing of step (1) gained coarse crystal, then add distilled water with after acetone solution, occur to solution muddy, gained turbid solution is left standstill more than 20 hours under 2-8 DEG C of condition, obtains Tanshinone II A crystal.
8. preparation method as claimed in claim 7, is characterized in that: in step (1) or (2), the described temperature left standstill by gained turbid solution is 4 DEG C.
9. preparation method as claimed in claim 7, it is characterized in that: in step (1), the volume of acetone is 2 ~ 4 times of described enriched material volume;
And/or in step (2), the number of times of described distillation washing is 1 ~ 3 time, and the consumption of each distilled water wash is 10 ~ 20L/kg with the volume mass ratio of coarse crystal;
And/or in step (2), the volume of described acetone is 2 ~ 4 times of described coarse crystal volume.
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