CN103819533B - The preparation method and application of Methyl tanshinonate sodium sulfonate - Google Patents
The preparation method and application of Methyl tanshinonate sodium sulfonate Download PDFInfo
- Publication number
- CN103819533B CN103819533B CN201410093547.0A CN201410093547A CN103819533B CN 103819533 B CN103819533 B CN 103819533B CN 201410093547 A CN201410093547 A CN 201410093547A CN 103819533 B CN103819533 B CN 103819533B
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- phase
- water
- triethylamine
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses the preparation method and application of Methyl tanshinonate sodium sulfonate.This preparation method comprises the steps: to adopt high performance liquid phase reverse-phase chromatography to be separated tanshinone sulfonated liquid, and the condition of high performance liquid phase reverse-phase chromatography is as follows: mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Determined wavelength is 271nm, and retention time is 35 ~ 50min.Preparation method's preparative capacibility of the present invention is strong, preparation amount is large, the cycle is short, efficiency is high, and technological operation continuity is easy to by force carry out quality control and production.The present invention also using Methyl tanshinonate sodium sulfonate as standard substance, can carry out detection and control to the content of the Methyl tanshinonate sodium sulfonate in sodium tanshinone IIA sulfate bulk drug.
Description
Technical field
The present invention relates to pharmaceutical field, be specifically related to the preparation method and application of Methyl tanshinonate sodium sulfonate.
Background technology
The red sage root, have another name called blood ginseng, red, red, blood takes root, be the rhizome of labiate (Salvia miltiorrhza), its beginning of being used as medicine is loaded in Shennong's Herbal.The traditional Chinese medical science is thought, its bitter is slightly cold, there is dysmenorrhoea of invigorating blood circulation, effect that stasis-dispelling and pain-killing, the relieving restlessness that clears away heart-fire, cool blood disappear carbuncle, be applicable to treatment menoxenia, dysmenorrhoea, through closing, uterine bleeding, under band, coronary heart disease, atherosclerosis, hyperlipidemia, stenocardia, diseases such as beating extravasated blood, insomnia, neurasthenia, hepatitis B of falling, commonly use for gynaecology, internal medicine and external wounds card belong to the blood stasis hot person that holds concurrently.
Modern pharmacy research shows that complex chemical composition in the red sage root is mainly water-soluble salvianolic acid and fat-soluble diterpene quinones tanshinone compound.Wherein, tanshinone compound mainly comprises tanshinone Ⅰ, tanshinone IIA, tanshinone ⅡB, Isotanshinone Ⅰ, IsotanshinoneⅡ A, Cryptotanshinone, different Cryptotanshinone, methyltanshinone, hydroxyl TANSHINONES etc.
Tanshinone IIA is important effective component in red sage.Tanshinone II A sodium sulfonate (SodiumTanshinone II A Sulfonate, STS) is then the water-soluble sulfonate that tanshinone IIA obtains after sulfonation.Due to sulfonic introducing, in disease such as treatment coronary heart diseases and angina pectoris, myocardial infarction etc., Tanshinone II A sodium sulfonate is compared Tanshinone II A and is had more superiority, thus becomes main cardiovascular diseases Radix Salviae Miltiorrhizae extract Chinese medicine.
Tanshinone II A is the raw material preparing sodium tanshinone IIA sulfate, extracts from red rooted salvia medicine materical crude slice, therefore is mixed with a small amount of or that low levels other structures in pole are comparatively similar component unavoidably, as tanshinone ⅡB, Tanshinone I, Cryptotanshinone, przewaquinone A etc.To preparation high-purity tanshinone IIA sodium sulfonate, then obtain these pole low levels related component standard substance and the application of these standard substance in sodium tanshinone IIA sulfate raw material Related Component content controls, significant.
But because such compound structure is complicated, chemical complete synthesis difficulty is large, and obtain high purity reference substance becomes challenge.Studies have reported that and extract related substances from Tanshinone II A sodium sulfonate bulk drug, because its content is extremely low, HPLC purity is usually less than one of percentage, and adopt high-efficient liquid phase technique separation and Extraction, workload is large, difficulty is high.Therefore, the high purity standard substance mass-producing preparation of pole low levels component in Radix Salviae Miltiorrhizae extract, and the application in the monitoring of sodium tanshinone IIA sulfate bulk drug related substances content becomes this area insoluble technical barrier for a long time.
Methyl tanshinonate is also one of fat-soluble diterpene quinone effective constituent of the red sage root, and not yet has technology to prepare the report of Methyl tanshinonate sodium sulfonate in a large number.Prior art Patent CN200610039234.2 " application of sulfonated bodies in medicine of one group of diterpene quinone extracted from the red sage root " shows that Methyl tanshinonate sodium sulfonate can be applied to treatment coronary heart disease and (comprise stenocardia, myocardial infarction, irregular pulse, myocardial blood flow should be not enough) and antisepsis and anti-inflammation etc.Therefore, fixing quantity Methyl tanshinonate sodium sulfonate, significant to the quality-guarantee of Tanshinone II A sodium sulfonate raw material.
Summary of the invention
Technical problem to be solved by this invention is, in order to overcome Methyl tanshinonate sodium sulfonate purification difficult in prior art, efficiency is low, extracted amount is low defect, to provide the preparation method and application of Methyl tanshinonate sodium sulfonate.The preparative capacibility of preparation method of the present invention is strong, preparation amount is large, the cycle is short, purity is high, efficiency is high, and decreases the use of organic solvent, and technological operation continuity is easy to by force carry out quality control and production.
The present invention solves the problems of the technologies described above by the following technical programs.
The structure of Methyl tanshinonate sodium sulfonate of the present invention is shown below:
The invention provides the preparation method of Methyl tanshinonate sodium sulfonate, it comprises the steps: to adopt high performance liquid phase reverse-phase chromatography to be separated tanshinone sulfonated liquid;
Wherein, the condition of high performance liquid phase reverse-phase chromatography is as follows:
Mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Per-cent is the volume percent that component accounts for moving phase; Determined wavelength is 271nm, and retention time is 35 ~ 50min.
Wherein, described high performance liquid phase reverse-phase chromatography preferably comprises enriching and purifying, intermediate purification and polishing purification, and wherein, the condition optimization of described enriching and purifying is as follows:
Mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Flow velocity is 80 ~ 120mL/min(is preferably 100mL/min), determined wavelength is 271nm;
By the mixture loading 25min that tanshinone sulfonated liquid and mobile phase A are 1:4 by volume, 80% mobile phase A+20% Mobile phase B (volume fraction) balances 1min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 42 ~ 50min obtains enriching and purifying sample solution;
Adopt PrepStar SD-1 preparation system and the L & L4002 chromatographic column (internal diameter 50mm) of Varian company of the U.S., the PLRP-S anti-phase C18 polymer packing 160.0g(particle diameter 10 μm of dress Varian company, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.
Wherein, the condition optimization of described intermediate purification is as follows:
Mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Flow velocity is 80 ~ 120mL/min(is preferably 100mL/min), determined wavelength is 271nm;
By the mixture loading 10min that above-mentioned enriching and purifying sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B (volume fraction) balances 10min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 36 ~ 44min obtains intermediate purification sample solution;
Adopt PrepStar SD-1 preparation system and the L & L4002 chromatographic column (internal diameter 50mm) of Varian company of the U.S., the PLRP-S anti-phase C18 polymer packing 160.0g(particle diameter 10 μm of dress Varian company, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.
Wherein, the condition optimization of described polishing purification is as follows:
Mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Flow velocity is 20 ~ 30mL/min(is preferably 25mL/min), determined wavelength is 271nm;
By the mixture loading 10min that above-mentioned intermediate purification sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B (volume fraction) balances 10min, linear gradient elution 30min:80%A+20%B → 50%A+50%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 45 ~ 50min obtains polishing purification sample solution;
Adopt PrepStar SD-1 preparation system and the L & L4001 chromatographic column (internal diameter 25mm) of Varian company of the U.S., the anti-phase C18 silica filler 100.0g(particle diameter 10 μm of Kromasil company of dress Sweden, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.
Wherein, the red sage root that described tanshinone sulfonated liquid can be this area routine carries out the reaction solution of sulfonation reaction, obtains preferably by following step:
(1) Radix Salviae Miltiorrhizae extract is dissolved in the mixing solutions of Glacial acetic acid-diacetyl oxide, then adds the mixing solutions of the vitriol oil-Glacial acetic acid, carry out being obtained by reacting reaction solution;
(2) mixing solutions of sherwood oil, methylene dichloride and water is added the reaction solution of step (1), then add sodium chloride solution, suction filtration, obtain filter cake;
(3) with the filter cake of the mixing solutions soaking step (2) of sherwood oil-methylene dichloride, suction filtration is dry obtains crude product;
(4) with the crude product of dissolve with methanol step (3), activated carbon filtration, filtrate concentrates, and crystallisation by cooling filters, and filtrate is tanshinone sulfonated liquid.
Wherein, the mixing solutions of described Glacial acetic acid-diacetyl oxide and the volume mass of described Radix Salviae Miltiorrhizae extract are 7 ~ 9ml/g than preferably.
Wherein, the mixing solutions of described Glacial acetic acid-diacetyl oxide is preferably for Glacial acetic acid and diacetyl oxide volume ratio are 1:(2 ~ 3) mixing solutions.
Wherein, the mixing solutions of the described vitriol oil-Glacial acetic acid is preferably for the vitriol oil and Glacial acetic acid volume ratio are the mixing solutions of 1:1, and the massfraction of the described vitriol oil is preferably 98%.
Wherein, the mixing solutions of described sherwood oil, methylene dichloride and water is preferably for the volume ratio of sherwood oil, methylene dichloride and water is 1:2:(2 ~ 2.5) mixing solutions.
Wherein, the mixing solutions that the mixing solutions of described sherwood oil-methylene dichloride is preferably sherwood oil is 1:3 with methylene chloride volume ratio.
Wherein, described Radix Salviae Miltiorrhizae extract can be the Radix Salviae Miltiorrhizae extract that this area ordinary method obtains, and obtains: by red sage root extraction using alcohol preferably by following step, filters, and filtrate concentrates, dry, to obtain final product.
In the present invention, the column temperature of chromatographic column is preferably 10 ~ 30 DEG C.
In preparation method of the present invention, after described exquisite purifying terminates, also freeze-drying process can be comprised.Described freeze-drying process preferably includes following steps: concentrated by described polishing purification sample solution, lyophilize, obtains pure Methyl tanshinonate sodium sulfonate.The pressure of described concentrating under reduced pressure is preferably-0.08MPa ~-0.1MPa, the temperature of described concentrating under reduced pressure is preferably 30 ~ 40 DEG C, and described vacuum lyophilization preferably adopts the LYO-1 vacuum freeze drier of Shanghai Tofflon Science and Technology Co., Ltd. to carry out.
0.1% triethylamine in the present invention-water refers to that the volume fraction of triethylamine is the triethylamine aqueous solution of 0.1%, 0.1% triethylamine-Methanol refers to that the volume fraction of triethylamine is the triethylamine methanol solution of 0.1%, 0.1% diethylamine-water refers to that the volume fraction of diethylamine is the diethylamine aqueous solution of 0.1%, 0.1% diethylamine-methyl alcohol refers to and refers to that the volume fraction of diethylamine is the diethylamine methanol solution of 0.1%, and 0.1% ammoniacal liquor refers to that the volume fraction of ammonia is the ammonia soln of 0.1%.
Present invention also offers above-mentioned Methyl tanshinonate sodium sulfonate as standard substance in the application detecting Methyl tanshinonate sodium sulfonate content in medicine.
Wherein, described medicine is preferably sodium tanshinone IIA sulfate bulk drug.
Wherein, described application preferably comprises the steps: sodium tanshinone IIA sulfate raw material medicine solution and Methyl tanshinonate sodium sulfonate standard solution to carry out high performance liquid chromatography detection respectively, according to the peak of related substances in the retention time determination sodium tanshinone IIA sulfate bulk drug color atlas of Methyl tanshinonate sodium sulfonate standard substance color atlas, and according to the content of Methyl tanshinonate sodium sulfonate in calculated by peak area sodium tanshinone IIA sulfate bulk drug.
Wherein, the condition optimization of described high performance liquid chromatography is as follows: moving phase is methyl alcohol: 20mmol/L disodium phosphate soln=65:35(volume ratio), pH is 6.0, column temperature is 25 DEG C, determined wavelength is 271nm, and flow velocity is 1.0mL/min, sample size 20 μ L, the concentration of sodium tanshinone IIA sulfate raw material medicine solution is 0.2mg/mL, and the concentration of Methyl tanshinonate sodium sulfonate standard solution is 0.2mg/mL; Record color atlas is to 3 times of principal constituent peak retention time; Control Methyl tanshinonate sodium sulfonate single maximum contaminant peak area in sodium tanshinone IIA sulfate raw material medicine solution and must not be greater than Methyl tanshinonate sodium sulfonate standard solution peak area 3 times (single foreign matter content is no more than 2.0%), total impurities peak area sum must not be greater than Methyl tanshinonate sodium sulfonate standard solution peak area 8 times (total impurities content is no more than 5.0%).
Wherein, described Methyl tanshinonate sodium sulfonate standard solution is preferably for Methyl tanshinonate sodium sulfonate mixes formed solution with moving phase.
Wherein, described sodium tanshinone IIA sulfate raw material medicine solution is preferably for sodium tanshinone IIA sulfate bulk drug mixes formed solution with moving phase.
Without prejudice to the field on the basis of common sense, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
(1) the present invention uses and prepares high performance liquid phase reversed phase chromatography separation technology and carry out purifying Methyl tanshinonate sodium sulfonate, flow velocity height applied sample amount is large, level of automation is high, anti-phase purifying after earlier sulfonation, products obtained therefrom purity is high, preparative capacibility is strong, preparation amount is large, the cycle is short, efficiency is high and decrease the use of organic solution, and technological operation continuity is strong, is easy to carry out quality control and production.
(2) the invention also discloses a kind of detection method, using Methyl tanshinonate sodium sulfonate as standard substance, detection and control can be carried out to the content of the Methyl tanshinonate sodium sulfonate in sodium tanshinone IIA sulfate bulk drug.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Room temperature in following embodiment refers to 25 DEG C.0.1% triethylamine-water refers to that the volume fraction of triethylamine is the triethylamine aqueous solution of 0.1%, 0.1% triethylamine-Methanol refers to that the volume fraction of triethylamine is the triethylamine methanol solution of 0.1%, 0.1% diethylamine-water refers to that the volume fraction of diethylamine is the diethylamine aqueous solution of 0.1%, 0.1% diethylamine-methyl alcohol refers to and refers to that the volume fraction of diethylamine is the diethylamine methanol solution of 0.1%, and 0.1% ammoniacal liquor refers to that the volume fraction of ammonia is the ammonia soln of 0.1%.
Embodiment 1
The extraction of tanshinone compound
Take red rooted salvia (place of production: Yunnan, Ganzi, the Aba area) 3kg of pulverizing, add the ethanol 14L that massfraction is 90%, 40 DEG C of water-baths extract 2 hours.Remove medicinal material slag and earth with 350 order filter clothes and filter paper filtering respectively, filtrate is concentrated into 1.4L, 4 DEG C leave standstill 24 hours, and the filtrate filter paper filtering after leaving standstill filters the 40 DEG C of vacuum-dryings of gained solid, obtains tanshinone compound red solid powder 66.0g.
Embodiment 2
The sulfonation reaction of tanshinone compound and aftertreatment
60g tanshinone compound in Example 1, adds 140mL Glacial acetic acid and 360mL diacetyl oxide, stirs cooling suspension to 0 DEG C.Under whipped state, in 60mL Glacial acetic acid, add the 60mL98% vitriol oil, be mixed with mixed acid solution and leave standstill be cooled to room temperature.Drip mixed acid solution, maintain temperature of reaction 10 DEG C, continue insulation reaction 90min.By 300mL sherwood oil+600mL methylene dichloride+650mL purified water prepared and diluted liquid, stir and be cooled to 4 DEG C.Under whipped state, in dilution liquid, slowly add above-mentioned reaction solution, and strengthen cooling performance, guarantee system temperature is lower than 25 DEG C.In 2L purified water, add 700g sodium-chlor, fully stir preparation saturated aqueous common salt.Join in saturated aqueous common salt by the reaction solution diluted, temperature controls at 25 DEG C, and leave standstill 30min after continuing to stir 25min, vacuum filtration obtains filter cake.In the mixing solutions of 300mL sherwood oil and 900mL methylene dichloride composition, dispersion filter cake stirring at normal temperature 20min, soaks 12 hours, again vacuum filtration and dry must tanshinone sodium sulfonate crude product.Methyl alcohol 3.8L and above-mentioned tanshinone sodium sulfonate crude product is dropped in the reaction flask of 5L, stirring heating, molten clear after add the needle-use activated carbon of 16g, be heated to reflux and keep this state 50min, temperature controls at 64 ± 1 DEG C, suction filtration while hot, filtrate passes through Rotary Evaporators underpressure distillation under-0.09MPa, by feed liquid 4 DEG C of crystallisation by cooling 12 hours after steaming the methyl alcohol of about 75% volume, again feed liquid suction filtration is separated to obtain filtrate, after concentrated by rotary evaporation twice, carries out filtration clarification through 0.22 μm of organic phase millipore filtration, obtain 1L filtrate, i.e. tanshinone sulfonated liquid.
Embodiment 3
Methyl tanshinonate sodium sulfonate prepare purifying
(1) enriching and purifying: the PrepStar SD-1 preparation system and the L & L4002 chromatographic column (internal diameter 50mm) that adopt Varian company of the U.S., self-chambering the said firm PLRP-S anti-phase C18 polymer packing 160.0g (particle diameter 10 μm, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Enriching and purifying condition is: mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol, flow velocity is 100mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 25min that the tanshinone sulfonated liquid of embodiment 2 and mobile phase A are 1:4 by volume, 80% mobile phase A+20% Mobile phase B balance 1min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, enriching and purifying sample solution is obtained according to the elutriant that ultraviolet detection result collects retention time 42-48min by peak.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in tanshinone sulfonated liquid, the HPLC purity of Methyl tanshinonate sodium sulfonate is 5.70%, and retention time is 15.60min.In enriching and purifying sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 66.00%, and retention time is 15.34min.
(2) intermediate purification: the PrepStar SD-1 preparation system and the L & L4002 chromatographic column (internal diameter 50mm) that adopt Varian company of the U.S., self-chambering the said firm PLRP-S anti-phase C18 polymer packing 160.0g (particle diameter 10 μm, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Intermediate purification condition is: mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol, flow velocity is 100mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 10min that above-mentioned enriching and purifying sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B balance 10min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, intermediate purification sample solution is obtained according to the elutriant that ultraviolet detection result collects retention time 36-41min by peak.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in intermediate purification sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 81.08%, and retention time is 15.29min.
(3) polishing purification: the PrepStar SD-1 preparation system and the L & L4001 chromatographic column (internal diameter 25mm) that adopt Varian company of the U.S., self-chambering filler is the anti-phase C18 silica filler 100.0g(particle diameter 10 μm of Kromasil company of Sweden, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Exquisite purification condition is: mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol, flow velocity is 25mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 10min that above-mentioned intermediate purification sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B balance 10min, linear gradient elution 30min:80%A+20%B → 50%A+50%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 45 ~ 48min obtains polishing purification sample solution.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in polishing purification sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 99.54%, and retention time is 15.27min.
(4) freeze-drying: by above-mentioned refining sample solution 3L in Rotary Evaporators (Shensheng Science & Tech. Co., Ltd., Shanghai R1002B), under the condition of temperature 30 DEG C ~ 40 DEG C, pressure-0.08MPa ~-0.1MPa, concentrated except methyl alcohol is to 2L; Be distributed in stainless steel pallet, liquid surface height controlling is at 0.5 ~ 1.0cm, cover gauze feeding vacuum freeze drier (Shanghai Tofflon Science and Technology Co., Ltd. LYO-1) and carry out lyophilize according to pre-designed freeze-drying curve, obtain the amorphous red-brown powder 2.0g of Methyl tanshinonate sodium sulfonate.
Adopt the HPLC purity of high performance liquid chromatography testing product.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
The HPLC purity of product is 99.24%, and retention time is 15.20min.
Mass Spectrometric Identification: adopt Waters, US substance level Four bar mass spectrograph Micromass ZQESI-MS to carry out Mass Spectrometric Identification to above-mentioned refining sample, record its molecular ion peak C
20h
17o
8s
-([M-Na]
-) mass-to-charge ratio (m/z) is 417.31, meet Methyl tanshinonate sodium sulfonate mass spectrum theoretical Mr 440.40, its molecular formula is C
20h
17o
8sNa.
Nucleus magnetic resonance is identified: adopt German Bruker company Avance III400MHz nuclear magnetic resonance spectrometer to measure above-mentioned powdered product, and the carbon numbering of Methyl tanshinonate sodium sulfonate is as follows:
1h-NMR (D
2o, 400MHz, ppm) data show, two benzene hydrogenation displacement studies letter δ
h7.13 (1H, d, J=8.0Hz, H-6), and δ
h7.34 (1H, d, J=8.0Hz, H-7), and three methylene radical hydrogen chemical shifts signal δ
h2.71 (2H, m, H-1), δ
h1.62 (2H, m, H-2) and δ
h1.62 (1H, m, H-3 α), δ
h2.06 (1H, m, H-3 β), illustrate that it has naphthoquinones and furan ring structure skeleton, signal δ
h3.64 (3H, s, H-20) illustrate that this compound has the methyl of company's oxygen, hydrogen signal on C-15 disappears, and shows that this compound is sulfonated successfully, in conjunction with
13c-NMR (D
2o, 100MHz, ppm) data δ
c: 26.5 (C-1), 10.5 (C-2), 33.5 (C-3), 47.3 (C-4), 143.8 (C-5), 135.9 (C-6), 121.5 (C-7), 126.6 (C-8), 125.5 (C-9), 145.1 (C-10), 181.3 (C-11), 174.7 (C-12), 119.5 (C-13), 160.0 (C-14), 120.7 (C-15), 119.5 (C-16), 8.7 (C-17), 18.3 (C-18), 179.5 (C-19), 53.0 (C-20), determine that it is a naphthoquinones containing methyl esters and furan nucleus compounds, δ
hthe chemical shift of 2.06 (1H, m, H-3 β) is lower than tanshinone IIA, therefore knows this methyl esters by inference and is connected on C-19.To sum up, result shows that this compound is Methyl tanshinonate sodium sulfonate.
Embodiment 4
Methyl tanshinonate sodium sulfonate prepare purifying
(1) enriching and purifying: the PrepStar SD-1 preparation system and the L & L4002 chromatographic column (internal diameter 50mm) that adopt Varian company of the U.S., self-chambering the said firm PLRP-S anti-phase C18 polymer packing 160.0g (particle diameter 10 μm, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Enriching and purifying condition is: mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol, flow velocity is 100mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 25min that the tanshinone sulfonated liquid of embodiment 2 and mobile phase A are 1:4 by volume, 80% mobile phase A+20% Mobile phase B balance 1min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, enriching and purifying sample solution is obtained according to the elutriant that ultraviolet detection result collects retention time 42-49min by peak.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in tanshinone sulfonated liquid, the HPLC purity of Methyl tanshinonate sodium sulfonate is 5.70%, and retention time is 15.60min.In enriching and purifying sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 65.70%, and retention time is 15.37min.
(2) intermediate purification: the PrepStar SD-1 preparation system and the L & L4002 chromatographic column (internal diameter 50mm) that adopt Varian company of the U.S., self-chambering the said firm PLRP-S anti-phase C18 polymer packing 160.0g (particle diameter 10 μm, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Intermediate purification condition is: mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol, flow velocity is 100mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 10min that above-mentioned enriching and purifying sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B balance 10min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, intermediate purification sample solution is obtained according to the elutriant that ultraviolet detection result collects retention time 36-42min by peak.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in intermediate purification sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 80.78%, and retention time is 15.27min.
(3) polishing purification: the PrepStar SD-1 preparation system and the L & L4001 chromatographic column (internal diameter 25mm) that adopt Varian company of the U.S., self-chambering filler is the anti-phase C18 silica filler 100.0g(particle diameter 10 μm of Kromasil company of Sweden, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Exquisite purification condition is: mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol, flow velocity is 25mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 10min that above-mentioned intermediate purification sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B balance 10min, linear gradient elution 30min:80%A+20%B → 50%A+50%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 45 ~ 49min obtains polishing purification sample solution.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in polishing purification sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 98.74%, and retention time is 15.23min.
(4) freeze-drying: by above-mentioned refining sample solution 3L in Rotary Evaporators (Shensheng Science & Tech. Co., Ltd., Shanghai R1002B), under the condition of temperature 30 DEG C ~ 40 DEG C, pressure-0.08MPa ~-0.1MPa, concentrated except methyl alcohol is to 2L; Be distributed in stainless steel pallet, liquid surface height controlling is at 0.5 ~ 1.0cm, cover gauze feeding vacuum freeze drier (Shanghai Tofflon Science and Technology Co., Ltd. LYO-1) and carry out lyophilize according to pre-designed freeze-drying curve, obtain the amorphous red-brown powder 2.0g of Methyl tanshinonate sodium sulfonate.
Adopt the HPLC purity of high performance liquid chromatography testing product.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
The HPLC purity of product is 98.54%, and retention time is 15.21min.
Carry out Mass Spectrometric Identification equally and nuclear-magnetism is identified, result shows that this material is Methyl tanshinonate sodium sulfonate.
Embodiment 5
Methyl tanshinonate sodium sulfonate prepare purifying
(1) enriching and purifying: the PrepStar SD-1 preparation system and the L & L4002 chromatographic column (internal diameter 50mm) that adopt Varian company of the U.S., self-chambering the said firm PLRP-S anti-phase C18 polymer packing 160.0g (particle diameter 10 μm, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Enriching and purifying condition is: mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol, flow velocity is 100mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 25min that the tanshinone sulfonated liquid of embodiment 2 and mobile phase A are 1:4 by volume, 80% mobile phase A+20% Mobile phase B balance 1min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, enriching and purifying sample solution is obtained according to the elutriant that ultraviolet detection result collects retention time 42-50min by peak.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in tanshinone sulfonated liquid, the HPLC purity of Methyl tanshinonate sodium sulfonate is 5.70%, and retention time is 15.60min.In enriching and purifying sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 65.30%, and retention time is 15.32min.
(2) intermediate purification: the PrepStar SD-1 preparation system and the L & L4002 chromatographic column (internal diameter 50mm) that adopt Varian company of the U.S., self-chambering the said firm PLRP-S anti-phase C18 polymer packing 160.0g (particle diameter 10 μm, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Intermediate purification condition is: mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol, flow velocity is 100mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 10min that above-mentioned enriching and purifying sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B balance 10min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, intermediate purification sample solution is obtained according to the elutriant that ultraviolet detection result collects retention time 36-44min by peak.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in intermediate purification sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 80.58%, and retention time is 15.25min.
(3) polishing purification: the PrepStar SD-1 preparation system and the L & L4001 chromatographic column (internal diameter 25mm) that adopt Varian company of the U.S., self-chambering filler is the anti-phase C18 silica filler 100.0g(particle diameter 10 μm of Kromasil company of Sweden, aperture 10nm), dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm.Exquisite purification condition is: mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol, flow velocity is 25mL/min, determined wavelength is 271nm, column temperature is room temperature, by the mixture loading 10min that above-mentioned intermediate purification sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B balance 10min, linear gradient elution 30min:80%A+20%B → 50%A+50%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 45 ~ 50min obtains polishing purification sample solution.
Again by high performance liquid chromatography tracing detection HPLC purity.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
Detected result shows: in polishing purification sample solution, the HPLC purity of Methyl tanshinonate sodium sulfonate is 98.64%, and retention time is 15.22min.
(4) freeze-drying: by above-mentioned refining sample solution 3L in Rotary Evaporators (Shensheng Science & Tech. Co., Ltd., Shanghai R1002B), under the condition of temperature 30 DEG C ~ 40 DEG C, pressure-0.08MPa ~-0.1MPa, concentrated except methyl alcohol is to 2L; Be distributed in stainless steel pallet, liquid surface height controlling is at 0.5 ~ 1.0cm, cover gauze feeding vacuum freeze drier (Shanghai Tofflon Science and Technology Co., Ltd. LYO-1) and carry out lyophilize according to pre-designed freeze-drying curve, obtain the amorphous red-brown powder 2.0g of Methyl tanshinonate sodium sulfonate.
Adopt the HPLC purity of high performance liquid chromatography testing product.Testing conditions is as follows:
Instrument: Waters, US 2695-2489 high performance liquid phase system, chromatographic column: Agilent ExtendC18,4.6 × 150mm, 5 μm, moving phase: A is 0.1% triethylamine-water, B is 0.1% triethylamine-Methanol, column temperature 25 DEG C, flow velocity 1mL/min, determined wavelength 271nm, applied sample amount 20 μ L, automatic loading.Linear gradient elution is carried out according to following volume ratio: 0min:90%A+10%B → 5min:80%A+20%B → 10min:65%A+35%B → 25min:50%A+50%B → 30min:90%A+10%B with mobile phase A and Mobile phase B.
The HPLC purity of product is 98.24%, and retention time is 15.19min.
Carry out Mass Spectrometric Identification equally and nuclear-magnetism is identified, result shows that this material is Methyl tanshinonate sodium sulfonate.
Embodiment 6
Methyl tanshinonate sodium sulfonate detects the related substances in Tanshinone II A sodium sulfonate bulk drug as standard substance
The 2695-2489 high performance liquid phase system of Waters, US and chromatographic column Diamonsil C18 (4.6 × 250mm, 5 μm); Moving phase is Jia Chun ︰ 20mM disodium phosphate soln=65 ︰ 35(volume ratios), dilute phosphoric acid regulates pH to 6.0; Column temperature is 25 DEG C, and determined wavelength is 271nm, and flow velocity is 1.0mL/min; Sample size 20 μ L, gets Tanshinone II A sodium sulfonate bulk drug and Methyl tanshinonate sodium sulfonate standard substance are appropriate, adds moving phase and is mixed with respectively containing the solution of 10 μ g/mL, for Systematic pretest tests solution; The resolution of Tanshinone II A sodium sulfonate and Methyl tanshinonate sodium sulfonate and other impurity peaks should conform with the regulations, and theoretical plate number calculates should be not less than 2000 by Tanshinone II A sodium sulfonate.
Get Tanshinone II A sodium sulfonate bulk drug (lot number sees the following form) 2mg, put in 10mL measuring bottle, add moving phase solution dilution to scale, shake up, as need testing solution; Get Methyl tanshinonate sodium sulfonate 20mg, put in 100mL measuring bottle, add moving phase solution dilution to scale, shake up, as standard solution; According to chromatographic condition; get standard solution 20 μ L injecting chromatograph, regulate detection sensitivity, make principal constituent peak height be the 10-25% of full range; to get standard solution, each 20 μ L injecting chromatographs of test liquid more respectively, record color atlas is to 3 times of test liquid principal constituent peak retention time; Detect Methyl tanshinonate sodium sulfonate content: control Methyl tanshinonate sodium sulfonate single maximum contaminant peak area and must not be greater than standard solution peak area 3 times (single foreign matter content is no more than 2.0%), total impurities peak area sum must not be greater than the peak area 8 times (total impurities content is no more than 5.0%) of standard solution.The results are shown in following table.
Embodiment 7
Methyl tanshinonate sodium sulfonate detects the related substances in sodium tashinone II A sulfonate injection as standard substance
Instrument: Waters, US 2695-2489 high performance liquid phase system; Chromatographic column: DiamonsilC18 (4.6 × 250mm, 5 μm); Moving phase is Jia Chun ︰ 20mM disodium phosphate soln (65 ︰ 35), and dilute phosphoric acid regulates pH to 6.0; Column temperature is 25 DEG C, and determined wavelength is 271nm, and flow velocity is 1.0mL/min; Sample size 20 μ L, get Tanshinone II A sodium sulfonate reference substance and Methyl tanshinonate sodium sulfonate standard substance are appropriate, adding moving phase is mixed with respectively containing the solution of 10 μ g/mL, for Systematic pretest tests solution, the resolution of Tanshinone II A sodium sulfonate and Methyl tanshinonate sodium sulfonate and other impurity peaks should conform with the regulations, and theoretical plate number calculates should be not less than 2000 by Tanshinone II A sodium sulfonate.
Precision measures the sodium tashinone II A sulfonate injection (lot number see under) containing Tanshinone II A sodium sulfonate 2mg, puts in 10mL measuring bottle, adds moving phase solution dilution to scale, shake up, as need testing solution; Get Methyl tanshinonate sodium sulfonate 20mg, put in 100mL measuring bottle, add moving phase solution dilution to scale, shake up, as standard solution; According to chromatographic condition; get standard solution 20 μ L injecting chromatograph, regulate detection sensitivity, make principal constituent peak height be the 10-25% of full range; to get standard solution, each 20 μ L injecting chromatographs of need testing solution more respectively, record color atlas is to 3 times of test liquid principal constituent peak retention time; Detect Methyl tanshinonate sodium sulfonate content: control Methyl tanshinonate sodium sulfonate single maximum contaminant peak area and must not be greater than reference substance solution peak area 3 times (single foreign matter content is no more than 2.0%), total impurities peak area sum must not be greater than the peak area 8 times (total impurities content is no more than 5.0%) of reference substance solution.The results are shown in following table.
Claims (6)
1. a preparation method for Methyl tanshinonate sodium sulfonate, it comprises the steps: to adopt high performance liquid phase reverse-phase chromatography to be separated tanshinone sulfonated liquid;
Wherein, the condition of high performance liquid phase reverse-phase chromatography is as follows:
Mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Per-cent is the volume percent that component accounts for moving phase; Determined wavelength is 271nm, and retention time is 35 ~ 50min.
2. preparation method as claimed in claim 1, it is characterized in that, described high performance liquid phase reverse-phase chromatography comprises enriching and purifying, intermediate purification and polishing purification, and wherein, the condition of described enriching and purifying is as follows:
Mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Flow velocity is 80 ~ 120mL/min, and determined wavelength is 271nm;
By the mixture loading 25min that tanshinone sulfonated liquid and mobile phase A are 1:4 by volume, 80% mobile phase A+20% Mobile phase B balance 1min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 42 ~ 50min obtains enriching and purifying sample solution;
The condition of described intermediate purification is as follows:
Mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Flow velocity is 80 ~ 120mL/min, and determined wavelength is 271nm;
By the mixture loading 10min that described enriching and purifying sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B balance 10min, linear gradient elution 40min:80%A+20%B → 40%A+60%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 36 ~ 44min obtains intermediate purification sample solution;
The condition of described polishing purification is as follows:
Mobile phase A is 0.1% triethylamine-water, Mobile phase B is 0.1% triethylamine-Methanol; Or mobile phase A is 0.1% diethylamine-water, Mobile phase B is 0.1% diethylamine-methyl alcohol; Or mobile phase A is 0.1% ammoniacal liquor, Mobile phase B is methyl alcohol; Flow velocity is 20 ~ 30mL/min, and determined wavelength is 271nm;
By the mixture loading 10min that described intermediate purification sample solution and water are 1:1 by volume, 80% mobile phase A+20% Mobile phase B balance 10min, linear gradient elution 30min:80%A+20%B → 50%A+50%B is carried out according to following volume ratio with mobile phase A and Mobile phase B, again with 100% Mobile phase B regeneration 10min, collection retention time is that the elutriant of 45 ~ 50min obtains polishing purification sample solution.
3. preparation method as claimed in claim 2, it is characterized in that, the device that described enriching and purifying adopts is as follows: adopt PrepStar SD-1 preparation system and L & L4002 chromatographic column, dress PLRP-S anti-phase C18 polymer packing 160.0g, dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm; Column temperature is 10 ~ 30 DEG C;
And/or, the device that described intermediate purification adopts is as follows: adopt PrepStar SD-1 preparation system and L & L4002 chromatographic column, dress PLRP-S anti-phase C18 polymer packing 160.0g, dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm; Column temperature is 10 ~ 30 DEG C;
And/or, the device that described polishing purification adopts is as follows: adopt PrepStar SD-1 preparation system and L & L4001 chromatographic column, anti-loaded phase C18 silica filler 100.0g, dynamic axial compression system is filled to pressure 650psi, and static state is secured to post bed height 25cm; Column temperature is 10 ~ 30 DEG C.
4. preparation method as claimed in claim 2, it is characterized in that, in described enriching and purifying, described flow velocity is 100mL/min;
And/or in described intermediate purification, described flow velocity is 100mL/min;
And/or in described polishing purification, described flow velocity is 25mL/min.
5. preparation method as claimed in claim 1, is characterized in that, described tanshinone sulfonated liquid is obtained by following step:
(1) Radix Salviae Miltiorrhizae extract is dissolved in the mixing solutions of Glacial acetic acid-diacetyl oxide, then adds the mixing solutions of the vitriol oil-Glacial acetic acid, carry out being obtained by reacting reaction solution;
(2) mixing solutions of sherwood oil, methylene dichloride and water is added the reaction solution of step (1), then add sodium chloride solution, suction filtration, obtain filter cake;
(3) with the filter cake of the mixing solutions soaking step (2) of sherwood oil-methylene dichloride, suction filtration is dry obtains crude product;
(4) with the crude product of dissolve with methanol step (3), activated carbon filtration, filtrate concentrates, and crystallisation by cooling filters, and filtrate is tanshinone sulfonated liquid;
Wherein, the mixing solutions of described Glacial acetic acid-diacetyl oxide is 7 ~ 9ml/g with the volume mass ratio of described Radix Salviae Miltiorrhizae extract;
The mixing solutions of described Glacial acetic acid-diacetyl oxide is Glacial acetic acid and diacetyl oxide volume ratio is 1:(2 ~ 3) mixing solutions;
The mixing solutions of the described vitriol oil-Glacial acetic acid is the vitriol oil and Glacial acetic acid volume ratio is the mixing solutions of 1:1;
The mixing solutions of described sherwood oil, methylene dichloride and water is the volume ratio of sherwood oil, methylene dichloride and water is 1:2:(2 ~ 2.5) mixing solutions;
The mixing solutions that the mixing solutions of described sherwood oil-methylene dichloride is sherwood oil is 1:3 with methylene chloride volume ratio;
Described Radix Salviae Miltiorrhizae extract is obtained by following step: by red sage root extraction using alcohol, filters, and filtrate concentrates, dry, to obtain final product.
6. preparation method as claimed in claim 2, is characterized in that, also comprise freeze-drying process after described exquisite purifying terminates; Described freeze-drying process comprises the steps: described polishing purification sample solution to concentrate, lyophilize; Described concentrated pressure is-0.08MPa ~-0.1MPa, and described concentrated temperature is 30 ~ 40 DEG C.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410093547.0A CN103819533B (en) | 2014-03-14 | 2014-03-14 | The preparation method and application of Methyl tanshinonate sodium sulfonate |
| CN201510578612.3A CN105223288A (en) | 2014-03-14 | 2014-03-14 | The preparation method and application of methyl tanshinonate sodium sulfonate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410093547.0A CN103819533B (en) | 2014-03-14 | 2014-03-14 | The preparation method and application of Methyl tanshinonate sodium sulfonate |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510578612.3A Division CN105223288A (en) | 2014-03-14 | 2014-03-14 | The preparation method and application of methyl tanshinonate sodium sulfonate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103819533A CN103819533A (en) | 2014-05-28 |
| CN103819533B true CN103819533B (en) | 2015-10-07 |
Family
ID=50754860
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410093547.0A Active CN103819533B (en) | 2014-03-14 | 2014-03-14 | The preparation method and application of Methyl tanshinonate sodium sulfonate |
| CN201510578612.3A Pending CN105223288A (en) | 2014-03-14 | 2014-03-14 | The preparation method and application of methyl tanshinonate sodium sulfonate |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510578612.3A Pending CN105223288A (en) | 2014-03-14 | 2014-03-14 | The preparation method and application of methyl tanshinonate sodium sulfonate |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN103819533B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105301155B (en) * | 2015-07-31 | 2017-11-24 | 复旦大学附属华山医院 | A kind of method of tanshinone IIA sodium sulfonate concentration in measure human plasma |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1285907C (en) * | 2004-11-03 | 2006-11-22 | 济南百诺医药科技开发有限公司 | Tanshinone II A sodium sulfonate and method of determining tanshinone I sodium sulfonate in its preparation |
| CN100554956C (en) * | 2005-10-17 | 2009-10-28 | 秦引林 | A kind of high efficient liquid phase analysis method of sodium tanshinon |
| CN1857251A (en) * | 2006-03-31 | 2006-11-08 | 邹巧根 | Application of diterpene quinone sulfonates extracted from red sage in preparing medicine |
| CN102936275B (en) * | 2012-11-13 | 2015-04-15 | 中国人民解放军第二军医大学 | Method for separating and purifying impurities in sodium tanshinone IIA sulfonate crude drug |
-
2014
- 2014-03-14 CN CN201410093547.0A patent/CN103819533B/en active Active
- 2014-03-14 CN CN201510578612.3A patent/CN105223288A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN105223288A (en) | 2016-01-06 |
| CN103819533A (en) | 2014-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Pharmacokinetics of phenolic compounds of Danshen extract in rat blood and brain by microdialysis sampling | |
| CN104031013B (en) | A kind of utilize the isolated and purified method preparing salvianolic acid B and rosmarinic acid of high speed adverse current chromatogram | |
| CN106967036B (en) | Preparation method of EGCG | |
| CN103450145A (en) | Method for separating and preparing Brazilin and Protosappanin B from Sappanwood by using high-speed countercurrent chromatography | |
| CN103864884B (en) | The preparation method of przewaquinone A sodium sulfonate | |
| CN108276271B (en) | Method for simultaneously preparing high-purity carnosol and carnosic acid from rosemary | |
| CN110818669A (en) | Aquilaria sinensis tetrahydro 2- (2-phenethyl) chromone compound and separation method and application thereof | |
| CN103819533B (en) | The preparation method and application of Methyl tanshinonate sodium sulfonate | |
| CN101084967A (en) | Extracting and purifying technology for safflower flavonoids | |
| CN111440184B (en) | Method for preparing high-purity carnosol | |
| CN102372750A (en) | Method for simultaneously preparing albiflorin and paeoniflorin | |
| CN106957310A (en) | The high efficiency preparation method of flavonoids monomer in a kind of leaves of Hawthorn | |
| CN103819532B (en) | The preparation method and application of Tanshinone II B sodium sulfonate | |
| CN104557471B (en) | Prepare a gram grade high-purity butyl alcohol, crenulatin and the method for rhodioside from Radix Rhodiolae simultaneously | |
| CN109912582A (en) | The method of mangiferin is extracted from mango leaf | |
| CN100536868C (en) | Powder injection contg high content tanshin polyphenolic acid salts, and its preparation method | |
| CN109045087A (en) | A kind of enriching and purifying technique of Chinese sumac active component | |
| CN103822987B (en) | Application of przewatanshinquinone A sodium sulfonate | |
| CN106279091A (en) | A kind of preparation method of L-Epicatechin gallate monomer | |
| CN111454127A (en) | Extraction and purification method of honokiol | |
| CN106668234B (en) | Rose extraction and purification process for total flavonoids | |
| CN114539192B (en) | Rosin alkane type diterpenoid compound and preparation method and application thereof | |
| Pan et al. | An efficient high‐speed counter‐current chromatography method for the preparative separation of sesquiterpenoids from the rhizomes of Cyperus rotundus L. combined with evaluation of the anti‐inflammation activity in vitro and molecular docking | |
| CN116789662A (en) | Method for separating and purifying alkaloid compounds in phellodendron amurense by high-speed countercurrent chromatography | |
| CN114533719B (en) | Application of abietane diterpenoid compound in preparation of anti-inflammatory drugs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 200240 Shanghai city Minhang District Jianchuan Road No. 1317 Patentee after: Add medicine to the first biochemical pharmaceutcal corporation, Ltd in Shanghai Address before: 200240 Shanghai city Minhang District Jianchuan Road No. 1317 Patentee before: Shanghai No.1 Biochemical & Pharmaceutical Co., Ltd. |