CN102070683B - Method for simultaneously preparing chemical reference substances of parishin, parishin B and parishin C - Google Patents

Abstract

The invention relates to a new preparation process for simultaneously preparing parishin, parishin B and parishin C chemical reference substances. The crude extract of Gastrodia elata medicinal material is extracted, or the gastrodia elata extract is directly used as raw material, and parishin, parishin B, and parishin C with a purity of more than 98% can be obtained through resin column separation and reversed-phase high-performance liquid chromatography. Three chemical controls. The invention has simple process steps, high purity, good color and easy large-scale production.

Description

Method for preparing parishin, parishin B, parishin C chemical reference substance simultaneously

technical field

The invention relates to a new preparation process for simultaneously preparing parishin, parishin B and parishin C chemical reference substances. It mainly includes two-step high-efficiency purification of resin column separation and reversed-phase high-performance liquid chromatography, and three reference substances can be obtained at the same time by collecting fractions reasonably. 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 technique

Gastrodia elata (Gastrodia elata Bl.) is one of the 34 kinds of precious medicinal materials announced by my country, and it is mainly used for headache, dizziness, numbness of limbs, epilepsy and convulsions and other diseases. Gastrodin is the active ingredient of Gastrodia elata, and it is also the content determination component of Gastrodia elata medicinal materials in the 2005 edition of the National Pharmacopoeia of my country; while parishin, parishin B, and parishin C are citric acid substitutes of gastrodin, and their specificity and stability are better than gastrodin ( Wang Li et al., Chinese Herbal Medicine, 2006, 37(9): 1402-1405), is a potential quality control component of Gastrodia elata. The preparation of parishin reference substances is of great significance for the multi-index quality control of Gastrodia elata medicinal materials and related compounds. Yoe-ray Ku et al. successively tried to evaluate the quality of Gastrodia elata medicinal materials and related compound prescriptions with high performance liquid chromatography and capillary electrophoresis, using parishin, parishin B, and parishin C as index components (Journal of Chromatography A, 1998, 805 (1-2) : 330-336; Journal of Liquid Chromatography & Related Technologies, 1996, 19(20): 3265-3277). the

At present, parishin-like reference substances are not available in the market, and their separation and preparation methods are rarely reported. the

1) Obtain three reference substances of parishin, parishin B and parishin C at the same time. the

Jer-huei Lin etc. (Phytochemistry, 42 (2): 549-551, 1996) reported the preparation method of parishin class composition, and its preparation step has three steps: Gastrodia elata 70% ethanol extract is divided into ethanol and chloroform system through active carbon chromatography part; the above two parts were combined, suspended in water and extracted with chloroform and ethyl acetate to remove impurities; the extracted water was applied to an MCI column to obtain 3 target compounds. The preparation process of this method is relatively complicated and the preparation period is long. the

2) Obtain a single reference substance. the

Xiao-dong Yang et al. reported that 70% ethanol extract of Gastrodia elata was extracted and prepared twice in reversed-phase high-performance liquid phase to obtain parishin B 20 mg (Natural Product Research, 21(2): 180-186). The reversed-phase preparation condition was methanol- Water gradients, prepared on a smaller scale. N. Li et al. reported that the 80% ethanol extract of Gastrodia elata was extracted, sephadex LH-20 column, and reversed-phase high-performance liquid phase preparation to obtain parishin (Journal of Asian Natural Products Research, 9(4): 373-377, 2007), The reverse-phase preparation condition is ethanol-water gradient, and the scale-up preparation cost is relatively high. the

The invention adopts the gastrodia elata crude extract as a raw material, and through the two-step high-efficiency purification of resin column separation and reverse-phase high-performance liquid chromatography, reasonably collects fractions, and can obtain three reference substances in batches at the same time. the

Contents of the invention

The invention provides a new preparation method with simple process and easy large-scale production. The crude extract of Gastrodia elata can be used as raw material to simultaneously prepare three chemical reference substances of parishin, parishin B and parishin C with a purity greater than 98%. Up to 50g of reference substance can be produced per month. the

In order to achieve the above object, the technical scheme adopted in the present invention:

The crude extract of Gastrodia elata is extracted, or the Gastrodia elata extract is directly used as raw material, separated by resin column and reversed-phase high-performance liquid chromatography to obtain parishin, parishin B, and parishin C with a purity of more than 98%. A chemical reference substance;

1) Coarse separation by resin column:

Gastrodia elata medicinal material is extracted with water and/or ethanol solution to obtain crude extract, or directly purchase Gastrodia elata extract, dissolve in water or low-concentration ethanol solution, separate with resin column, first elute with water and 10% ethanol solution to remove impurities, and then use volume fraction Eluted with any ethanol solution between 20% and 30%, collected the corresponding ethanol eluate, concentrated and dried to obtain a yellow powder;

2) Purification by reversed-phase high-performance liquid chromatography:

Dissolve the yellow powder in water or a low-concentration methanol solution, filter through a microporous membrane, and separate by reversed-phase high-performance liquid chromatography. The methanol-acetic acid aqueous solution is used as the elution system, and the ultraviolet detector is monitored at 270nm. The three main components in the preparation spectrum are collected separately. Chromatographic peaks, the corresponding fractions were dried to obtain three reference substances with a purity greater than 98%, and their appearance was white powder. the

The specific process of the step 1) is: the self-made Gastrodia elata crude extract or the purchased extract are dissolved in water or an ethanol solution with a volume fraction not higher than 20%, separated by HPD 100, Diaion or AB-8 resin, and separated by 3-5 Times column volume of water, 10%, 20%, 30% ethanol solution elution;

The specific process of the step 2) is: the yellow powder is dissolved in water or a methanol solution with a volume fraction not higher than 25%, and the concentration of the sample is configured to be 50-500mg/ml; the packing material of the prepared chromatographic column is C18, and the length of the prepared column is 10- 30cm, 2-10cm in diameter; the mobile phase used is a two-phase system of methanol and aqueous solution containing acetic acid, the concentration range of acetic acid added is 0.1-1% (v/v), and the volume fraction ratio of the two-phase solvent is 25:75～ 35:65 for elution; the injection volume is 1-50ml, the flow rate is controlled at 10-250ml/min; the collection retention time is 3.5-4.5min (parishin B), 5.0-6.5min (parishin C) and 11.0-12.5 Min (parishin) fractions were dried to obtain three reference substances. the

Separate parishin, parishin B, parishin C chemical reference substance from Gastrodia elata with the present invention has following advantage and progress:

1) The present invention uses a two-step method to prepare the reference substance, and the process is simple. the

The first step uses a resin column for rough separation. Compared with the more extraction steps used in the literature, it is more specific to the target compound and can achieve the greatest degree of removal of roughness and fineness, which is beneficial to the reversed-phase preparation column in the second step. protection of. The methanol-acetic acid-water isocratic preparation method was adopted in the second step reversed-phase high-performance liquid phase preparation. Compared with the acetonitrile-phosphoric acid-water gradient method in the literature, the single injection time was 13 minutes, and there was no need between two injections. Equilibrium column greatly improves preparation flux and saves cost. the

2) The technical means adopted in the present invention are very suitable for enlarging and carrying out large-scale production. the

Raw material requirements are not high, generally commercially available crude extracts are enough, and it is easy to prepare materials in batches; the first step of rough separation uses a resin column, which can be regenerated and recycled, and the resin is cheap and easy to scale; the second step of refining uses reverse phase High-efficiency liquid phase preparation, the developed preparation method is a rapid isocratic method, which is also very suitable for large-scale production. the

3) This process achieves two-step separation to obtain three high-purity reference substances by optimizing the resin column rough separation conditions and preparing high-efficiency liquid phase refining conditions, with high yield (greater than 82%) and good color (white). the

Description of drawings

Fig. 1 is the HPLC analytical spectrum (270nm) of parishin;

Fig. 2 is the HPLC analytical spectrum (270nm) of parishin B;

Fig. 3 is the HPLC analysis spectrum (270nm) of parishin C. the

Detailed ways

Now, the present invention will be described in further detail in conjunction with the embodiments and accompanying drawings, and the embodiments are only used to illustrate the present invention, rather than to limit the present invention. the

Example 1

1. Coarse separation by resin column: 10 g of commercially available Gastrodia elata water extract is dissolved in 20% ethanol solution (the mass ratio of Gastrodia elata extract to water is 1: 4), filtered through four layers of gauze, and the clear liquid is separated by HPD-100 resin column, wherein The sample:resin ratio is 1:6 (g:ml), eluted with 3 times column volume of water, 3 times column volume of 10% ethanol solution, and 5 times column volume of 20% ethanol solution, and collected 20% ethanol solution Dematerialized, concentrated and dried to obtain 350 mg of yellow powder;

2. Reverse-phase HPLC preparative chromatographic purification: the yellow powder is dissolved in aqueous solution, the sample concentration is 50mg/ml, and filtered through a 0.45μm microporous membrane; Separation and preparation of chromatographic column packing filler is 6 μm C18 (Novapak); injection volume is 1ml, the mobile phase that adopts is methanol and the aqueous solution two-phase system that contains acetic acid, and the concentration of adding acetic acid is 0.1% (v/v), two The phase solvent was eluted with a volume fraction ratio of 25:75; the flow rate was controlled at 10ml/min, and the eluted fractions were monitored on-line by an ultraviolet detector at 270nm. Min (parishinC) and 11.5-12.5min (parishin) fractions were dried under reduced pressure at 60°C to obtain respective reference substances with a purity greater than 98% (Fig. 1-Fig. 3). Single needle injection can obtain parishin 16mg, parishin B 9mg, parishin C 5mg (60%), and reference substance parishin 112mg, parishin B 61mg, parishin C 34mg can be obtained accumulatively, the yield is about 89%, and its appearance is white powder. the

Example 2

1. Coarse separation by resin column: Gastrodia elata medicinal material 1230g, reflux extraction with 5 times the amount of 70% ethanol, 2 hours each time, 3 times. The three extracts were combined, concentrated under reduced pressure to a volume of about 3L, filtered through four layers of gauze, and the clear liquid was separated on a Diaion column. 10% ethanol solution of twice column volume and 25% ethanol solution of 4 times column volume were eluted, the 25% ethanol eluate was collected, concentrated and dried to obtain 8.61g of yellow powder;

2. Reverse-phase high-performance liquid phase preparative chromatographic purification: the yellow powder is dissolved in aqueous solution, the sample concentration is 200 mg/ml, and filtered through a 0.45 μm microporous membrane; the filtrate uses a high-performance liquid phase preparative chromatographic column with a column length of 20 cm and a diameter of 7.5 cm Separation is carried out, and the filling filler of preparation chromatographic column is 10 μm C18 (Microsorb); The injection volume is 20ml, and the mobile phase that adopts is methanol and the aqueous two-phase system that contains acetic acid, and the concentration range of adding acetic acid is 0.5% (v/v) , the two-phase solvent was eluted with a volume fraction ratio of 30:70; the flow rate was controlled at 160ml/min, and the eluted fraction was monitored on-line by an ultraviolet detector at 270nm. The fractions of -6.2min (parishin C) and 11.0-11.8min (parishin) can be dried under reduced pressure at 60 degrees to obtain each corresponding reference substance with a purity greater than 98%. Accumulatively obtained reference substances parishin 2.45g, parishin B 1.20g, parishin C 645mg, the yield is about 85%, and its appearance is white powder. the

Example 3

1. Coarse separation with resin column: Gastrodia elata medicinal material 2000g, extracted with 8 times the amount of water, extracted 3 times for 2 hours, 1.5 hours and 1 hour respectively. Combine three extracts, concentrate under reduced pressure to a volume of about 5 L, filter through four layers of gauze, and separate the supernatant on an AB-8 column. , 5 times column volume of 10% ethanol solution, 3 times column volume of 30% ethanol solution for elution, collect 30% ethanol eluate, concentrate and dry to obtain 20g of yellow powder;

2. Reversed-phase high-performance liquid chromatography preparation and purification: the yellow powder was dissolved in 25% methanol solution, the sample concentration was 500 mg/ml, and filtered through a 0.45 μm microporous membrane; the filtrate was prepared by high-performance liquid phase with a column length of 30 cm and a diameter of 10 cm The chromatographic column is separated, and the filling material of the preparation chromatographic column is 10 μmC18 (Fugi); ), the two-phase solvent is eluted with a volume fraction ratio of 35:65; the flow rate is controlled at 250ml/min, and the elution fraction is monitored on-line by an ultraviolet detector at 270nm, and the retention times are respectively collected at 3.5-4.2min (parishin B), The fractions of 5.0-6.0min (parishin C) and 11.0-12.0min (parishin) were dried under reduced pressure at 60°C to obtain the corresponding reference substances with a purity greater than 98%. The reference substances parishin 3.92g, parishin B 1.85g, parishin C 1.01g were obtained accumulatively, the yield was about 82%, and its appearance was white powder. the

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.

Images