CN102070683B - Method for simultaneously preparing chemical reference substances of parishin, parishin B and parishin C - Google Patents
Method for simultaneously preparing chemical reference substances of parishin, parishin B and parishin C Download PDFInfo
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- CN102070683B CN102070683B CN 201010566919 CN201010566919A CN102070683B CN 102070683 B CN102070683 B CN 102070683B CN 201010566919 CN201010566919 CN 201010566919 CN 201010566919 A CN201010566919 A CN 201010566919A CN 102070683 B CN102070683 B CN 102070683B
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- rhizoma gastrodiae
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Abstract
The invention relates to a new process for simultaneously preparing chemical reference substances of parishin, parishin B and parishin C. The method provided by the invention comprises the following steps: extracting rhizoma gastrodiae crude drugs to obtain a crude extract or directly using rhizoma gastrodiae extract as a raw material, and efficiently purifying the rhizoma gastrodiae extract by resin column separation and reversed-phase high-efficiency liquid phase preparative chromatography, thereby obtaining the three chemical reference substances of parishin, parishin B and parishin C, wherein the purity of each chemical reference substance is higher than 98%. The invention has the advantages of simple procedure, high purity and favorable color, and can be favor of large-scale production.
Description
Technical field
The present invention relates to a kind of new preparation process for preparing simultaneously parishin, parishin B, parishin C chemical reference substance.Mainly comprise resin column separation and two step of RP-HPLC preparative chromatography efficiently purifying, rationally collect stream part and can obtain simultaneously three kinds of reference substances.Its structural formula is as follows:
Parishin：R
1＝R
2＝R
3
Parishin?B：R
1＝R
2＝R，R
3＝H
Parishin?C：R
1＝R
3＝R，R
2＝H
Background technology
Rhizoma Gastrodiae (Gastrodia elata Bl.) is one of 34 kinds of rare medicinal herbss of China's announcement, cures mainly the diseases such as headache is dizzy, numb limbs and tense tendons, epilepsy clonus.Gastrodine is the rhizoma Gastrodiae activeconstituents, also is the assay composition of Rhizoma Gastrodiae in 2005 editions state-promulgated pharmacopoeia of China; And parishin, parishin B, parishin C are the citric acid substituent of Gastrodine, and its specificity and stability all are better than Gastrodine (Wang Li etc., herbal medicine, 2006,37 (9): 1402-1405), be the potential Quality Control composition of rhizoma Gastrodiae.Preparation parishin class reference substance is significant for many index ' s quality controls of Rhizoma Gastrodiae and relevant compound.Yoe-ray Ku etc. successively attempts with high performance liquid chromatography and capillary electrophoresis, estimate Rhizoma Gastrodiae take parishin, parishin B, parishin C as index components and reach relevant compound quality (Journal of Chromatography A, 1998,805 (1-2): 330-336; Journal ofLiquid Chromatography﹠amp; Related Technologies, 1996,19 (20): 3265-3277).
At present, parishin class reference substance has no supply on the market, and its method for separating and preparing is rare report also.
1) obtains simultaneously parishin, parishin B, three kinds of reference substances of parishin C.
(the Phytochemistry such as Jer-huei Lin, 42 (2): 549-551,1996) reported the preparation method of parishin constituents, its preparation process had three steps: rhizoma Gastrodiae 70% ethanol extraction is divided into ethanol and chloroform system position through activated carbon chromatography; More than two positions merge after the water suspendible with chloroform and ethyl acetate extraction removal of impurities; The MCI post obtains 3 target compounds on the extraction water liquid.The method preparation technology is comparatively complicated, and preparation cycle is longer.
2) obtain single reference substance.
Report rhizoma Gastrodiae 70% ethanol extractions such as Xiao-dong Yang prepare parishin B 20mg (Natural Product Research through extraction, twice RP-HPLC, 21 (2): 180-186), its anti-phase preparation condition is the methanol-water gradient, and preparative-scale is less.80% ethanol extraction of the report rhizoma Gastrodiaes such as N.Li prepares pari shin (Journal of Asian Natural Products Research through extraction, sephadex LH-20 post, RP-HPLC; 9 (4): 373-377; 2007); its anti-phase preparation condition is the alcohol-water gradient, and it is higher that preparation cost is amplified in mass-producing.
It is raw material that the present invention adopts the rhizoma Gastrodiae crude extract, by resin column separation and two step of RP-HPLC preparative chromatography efficiently purifying, rationally collects stream part, can obtain simultaneously in batches three reference substances.
Summary of the invention
The invention provides the novel preparation method of simple, the easy mass-producing of a kind of technique; can be take the rhizoma Gastrodiae crude extract as raw material; prepare simultaneously purity greater than three kinds of chemical reference substances of parishin, parishin B, parishin C of 98%, its accumulative total preparative-scale can reach monthly output 50g reference substance.
For achieving the above object, the technical solution used in the present invention:
Rhizoma Gastrodiae is through extracting to get crude extract, or directly take Rhizoma Gastrodiae extract as raw material, separate and two step of RP-HPLC preparative chromatography efficiently purifying through resin column, obtain simultaneously purity greater than three kinds of chemical reference substances of parishin, parishin B, parishin C of 98%;
1) resin column roughing out:
Rhizoma Gastrodiae water and/or ethanolic soln extract to get crude extract, or directly buy Rhizoma Gastrodiae extract, water or the dissolving of low-concentration ethanol solution, resin column separates, first remove impurity with water, 10% ethanolic soln wash-out, then with the arbitrary ethanolic soln wash-out of volume fraction between 20%-30%, collect corresponding ethanol elution thing, concentrate drying gets yellow powder;
2) the RP-HPLC preparative chromatography is refining:
Yellow powder water or the dissolving of low concentration methanol solution, filtering with microporous membrane, the RP-HPLC preparative chromatography is separated, take methyl alcohol-aqueous acetic acid as eluent system, UV-detector 270nm monitoring, collect respectively three main chromatographic peaks in the preparation collection of illustrative plates, respective streams part drying can obtain purity greater than three reference substances of 98%, and its outward appearance is white powder.
Described step 1) detailed process is: self-control rhizoma Gastrodiae crude extract or purchase extract water or volume fraction are not higher than 20% dissolve with ethanol solution, adopt HPD 100, Diaion or AB-8 resin isolation, respectively with water, 10%, 20%, the 30% ethanolic soln wash-out of 3-5 times of column volume;
Described step 2) detailed process is: yellow powder water or volume fraction are not higher than 25% dissolve with methanol solution, and the configuration sample concentration is 50～500mg/ml; The filling filler of preparative chromatography post is C18, preparation column length 10-30cm, diameter 2-10cm; The moving phase that adopts was methyl alcohol and the aqueous solution two-phase system that contains acetic acid, and the concentration range of adding acetic acid is 0.1-1% (v/v), and two-phase solvent carried out wash-out take Volume fraction as 25: 75～35: 65; Sampling volume is 1-50ml, and flow rate control is at 10-250ml/min; Collect retention time and be respectively stream part of 3.5-4.5min (parishin B), 5.0-6.5min (parishin C) and 11.0-12.5min (parishin), drying is obtained three kinds of reference substances.
With the present invention from rhizoma Gastrodiae, separate parishin, parishin B, parishin C chemical reference substance has following advantage and progress:
1) the present invention adopts two-step approach to prepare reference substance, and technique is simple.
The first step adopts the resin column roughing out, compares the more extraction step of application in the document, and target compound is had stronger specific aim, can realize farthest removing crudely and store essence, and is conducive to the protection of anti-phase preparative column in the second step.Adopt the degree preparation methods such as methyl alcohol-Acetic Acid-Water in the preparation of second step RP-HPLC, compare the acetonitrile-phosphoric acid of document-water gradient method, the single sample injection time is 13 minutes, and does not need the balance pillar between twice sample introduction, has greatly improved the preparation flux, has saved cost.
2) large-scale production is carried out in the very suitable amplification of the technique means of the present invention's employing.
Ingredient requirement is not high, and general commercially available crude extract gets final product, and is easy to get the raw materials ready in batches; Resin column is adopted in the first step roughing out, resin column can reprocessing cycle use and resin price cheap, be easy to mass-producing; Adopt the RP-HPLC preparation during second step is made with extra care, the preparation method of development is fast method such as degree of grade, also very suitable amplification scale production.
3) this technique is by optimizing resin column roughing out condition and preparation high performance liquid phase purification condition, realized that two steps separated to obtain three kinds of high purity reference substances, yield high (greater than 82%), color and luster good (white).
Description of drawings
Fig. 1 is that the HPLC of parishin analyzes collection of illustrative plates (270nm);
Fig. 2 is that the HPLC of parishin B analyzes collection of illustrative plates (270nm);
Fig. 3 is that the HPLC of parishin C analyzes collection of illustrative plates (270nm).
Embodiment
Now with accompanying drawing the present invention is described in further details in conjunction with the embodiments, embodiment only limits to illustrate the present invention, but not limitation of the invention.
Embodiment 1
1, resin column roughing out: commercially available rhizoma Gastrodiae water extract 10g is with 20% dissolve with ethanol solution (Rhizoma Gastrodiae extract is 1: 4 with the quality ratio), four layers of filtered through gauze, the HPD-100 resin column separates on the clear liquid, sample wherein: resin ratio is 1: 6 (g: ml), respectively with 20% ethanolic soln wash-out of 10% ethanolic soln of the water of 3 times of column volumes, 3 times of column volumes, 5 times of column volumes, collect 20% ethanol elution thing, concentrate drying gets yellow powder 350mg;
2, the RP-HPLC preparative chromatography is refining: yellow powder dissolves with the aqueous solution, and the configuration sample concentration is 50mg/ml, through 0.45 μ m filtering with microporous membrane; Filtrate adopts the high performance liquid preparative chromatography post of column length 10cm, diameter 2cm to separate, and the filling filler of preparative chromatography post is 6 μ m C18 (Novapak); Sampling volume was 1ml, and the moving phase of employing is methyl alcohol and the aqueous solution two-phase system that contains acetic acid, and the concentration of adding acetic acid is 0.1% (v/v), and two-phase solvent carried out wash-out take Volume fraction as 25: 75; Flow rate control is at 10ml/min, UV-detector 270nm on-line monitoring wash-out stream part, collect respectively retention time and be respectively stream part of 3.8-4.5min (parishin B), 5.8-6.5min (parishinC) and 11.5-12.5min (parishin), 60 degree drying under reduced pressure can obtain purity greater than each corresponding reference substance of 98% (Fig. 1-Fig. 3).The single needle sample introduction can obtain parishin 16mg, parishin B 9mg, parishin C 5mg (60%), accumulative total is obtained reference substance parishin 112mg, parishin B 61mg, parishin C 34mg, yield is about 89%, and its outward appearance is white powder.
Embodiment 2
1, resin column roughing out: Rhizoma Gastrodiae 1230g, 5 times of amount 70% alcohol reflux each 2 hours, extract 3 times.Merge No. three times extracting solution, be evaporated to about 3L volume, four layers of filtered through gauze, the Diaion post separates on the clear liquid, its Chinese medicinal materials: resin ratio is 1: 3 (g: ml), with 25% ethanolic soln wash-out of 10% ethanolic soln of the water of 3 times of column volumes, 3 times of column volumes, 4 times of column volumes, collect 25% ethanol elution thing respectively, concentrate drying gets yellow powder 8.61g;
2, the RP-HPLC preparative chromatography is refining: yellow powder dissolves with the aqueous solution, and the configuration sample concentration is 200mg/ml, through 0.45 μ m filtering with microporous membrane; Filtrate adopts the high performance liquid preparative chromatography post of column length 20cm, diameter 7.5cm to separate, and the filling filler of preparative chromatography post is 10 μ m C18 (Microsorb); Sampling volume was 20ml, and the moving phase of employing is methyl alcohol and the aqueous solution two-phase system that contains acetic acid, and the concentration range of adding acetic acid is 0.5% (v/v), and two-phase solvent carried out wash-out take Volume fraction as 30: 70; Flow rate control is at 160ml/min, UV-detector 270nm on-line monitoring wash-out stream part, collect respectively retention time and be respectively stream part of 3.5-4.0min (parishin B), 5.5-6.2min (parishin C) and 11.0-11.8min (parishin), 60 degree drying under reduced pressure can obtain purity greater than each corresponding reference substance of 98%.Accumulative total is obtained reference substance parishin 2.45g, parishin B 1.20g, parishin C 645mg, and yield is about 85%, and its outward appearance is white powder.
Embodiment 3
1, resin column roughing out: Rhizoma Gastrodiae 2000g, 8 times of water gagings extract, and extract 3 times, extract respectively 2 hours, 1.5 hours, 1 hour.Merge No. three times extracting solution, be evaporated to about 5L volume, four layers of filtered through gauze, the AB-8 post separates on the clear liquid, its Chinese medicinal materials: resin ratio is 1: 3 (g: ml), with 30% ethanolic soln wash-out of 10% ethanolic soln of the water of 5 times of column volumes, 5 times of column volumes, 3 times of column volumes, collect 30% ethanol elution thing respectively, concentrate drying gets yellow powder 20g;
2, the RP-HPLC preparative chromatography is refining: yellow powder 25% dissolve with methanol solution, and the configuration sample concentration is 500mg/ml, through 0.45 μ m filtering with microporous membrane; Filtrate adopts the high performance liquid preparative chromatography post of column length 30cm, diameter 10cm to separate, and the filling filler of preparative chromatography post is 10 μ mC18 (Fugi); Sampling volume was 50ml, and the moving phase of employing is methyl alcohol and the aqueous solution two-phase system that contains acetic acid, and the concentration range of adding acetic acid is 1% (v/v), and two-phase solvent carried out wash-out take Volume fraction as 35: 65; Flow rate control is at 250ml/min, UV-detector 270nm on-line monitoring wash-out stream part, collect respectively retention time and be respectively stream part of 3.5-4.2min (parishin B), 5.0-6.0min (parishinC) and 11.0-12.0min (parishin), 60 degree drying under reduced pressure can obtain purity greater than each corresponding reference substance of 98%.Accumulative total is obtained reference substance parishin 3.92g, parishin B1.85g, parishin C 1.01g, and yield is about 82%, and its outward appearance is white powder.
Claims (4)
1. the method for preparing simultaneously parishin, parishin B, parishin C chemical reference substance, it is characterized in that: Rhizoma Gastrodiae is through extracting to get crude extract, or directly take Rhizoma Gastrodiae extract as raw material, separate and two step of RP-HPLC preparative chromatography efficiently purifying through resin column, obtain simultaneously purity greater than three kinds of chemical reference substances of parishin, parishin B, parishin C of 98%; Concrete steps are as follows:
1) resin column roughing out:
Rhizoma Gastrodiae water and/or ethanolic soln extract to get crude extract, or directly buy Rhizoma Gastrodiae extract, water or the dissolving of low-concentration ethanol solution, resin column separates, first remove impurity with water, 10% ethanolic soln wash-out, then with the arbitrary ethanolic soln wash-out of volume fraction between 20%-30%, collect corresponding ethanol elution thing, concentrate drying gets yellow powder; The resin that wherein adopts in the roughing out is HPD 100, Diaion or AB-8, and described low-concentration ethanol solution refers to that volume fraction is not higher than 20% ethanolic soln,
2) the RP-HPLC preparative chromatography is refining:
Yellow powder water or the dissolving of low concentration methanol solution, filtering with microporous membrane, the RP-HPLC preparative chromatography is separated, take methyl alcohol-aqueous acetic acid as eluent system, UV-detector 270nm monitoring, collect respectively three main chromatographic peaks in the preparation collection of illustrative plates, respective streams part drying can obtain purity greater than three reference substances of 98%, and its outward appearance is white powder; Described low concentration methanol is that volume fraction is not higher than 25% methanol solution, and the configuration sample concentration is 50～500mg/ml,
The moving phase that the preparation high performance liquid phase adopts was methyl alcohol and the aqueous solution two-phase system that contains acetic acid, and the concentration range of adding acetic acid is 0.1-1% (v/v), and two-phase solvent carried out wash-out take Volume fraction as 25: 75～35: 65;
The filling filler of RP-HPLC preparative column is C18, preparation column length 10-30cm, diameter 2-10cm.
2. method according to claim 1, it is characterized in that: 1) volume of each stepwise gradient wash-out is 3-5 times of column volume in the step.
3. method according to claim 1 is characterized in that: 2) sampling volume is 1-50ml in the preparation of step RP-HPLC, and flow rate control is at 10-250ml/min.
4. method according to claim 1, it is characterized in that: 2) collect retention time in the preparation of step RP-HPLC and be respectively stream part of 3.5-4.5min (parishin B), 5.0-6.5min (parishinC) and 11.0-12.5min (parishin), drying is obtained three kinds of reference substances.
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| CN102636607B (en) * | 2012-04-05 | 2014-07-30 | 中国中医科学院中药研究所 | Quality standard of edible gastrodia elata |
| CN104744528B (en) * | 2013-12-31 | 2018-06-08 | 中国科学院大连化学物理研究所 | The method for preparing parishin E and parishin G chemical reference substances simultaneously |
| CN103896999B (en) * | 2014-04-15 | 2016-03-09 | 贵州大学 | The preparation method of the gloomy glycosides reference substance of Bali |
| CN105748500B (en) * | 2014-12-16 | 2018-08-31 | 中国科学院大连化学物理研究所 | A kind of Rhizoma Gastrodiae active ingredient composition and its application for preventing dementia |
| CN105726551A (en) * | 2016-01-28 | 2016-07-06 | 浙江大学 | Application of plant extract parishin of gastrodia elata in preparing anti-aging medicine |
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Non-Patent Citations (2)
| Title |
|---|
| 李文兰,等.《大孔吸附树脂法对天麻中天麻苷和总苷的分离纯化》.《中国医院药学杂志》.2007,第27卷(第1期),18-21. * |
| 王莉,肖红斌,梁鑫淼.《天麻化学成分研究》.《中草药》.2009,第40卷(第8期),1186-1189. * |
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