CN101169355A - Total gastrodine and bound gastrodine detection method - Google Patents
Total gastrodine and bound gastrodine detection method Download PDFInfo
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Abstract
The invention relates to a detecting method for total gastrodin and combined gastrodin. The method comprises the steps as follows: the materials of the detected object which at least comprises the ingredients of combined gastrodin is hydrolyzed under an alkali condition, and then the contents of the dissociative gastrodin ingredients are detected to obtain the contents of master gastrodin in the materials of the detected object, which comprises dissociative gastrodin and combined gastrodin. The contents of the combined gastrodin in the detected object are obtained after deducting the dissociative gastrodin which is obtained through the prior detecting method in the detected object from the master gastrodin, thereby providing a reliable method for objective and accurate detection and quality control on the contents of gastrodin component of raw material or preparation in medicine preparation and/or medicine use, and further being beneficial for ensuring the product quality, increasing the medicine efficiency, and decreasing the adverse influence due to inaccurate contents of active ingredients.
Description
Technical field
The present invention relates to a kind of detection method to the Gastrodin composition, particularly to the total gastrodin content in the tested product that contain free state and combined state Gastrodin simultaneously, and/or in conjunction with the detection method of gastrodin content.
Background technology
Rhizoma Gastrodiae is a kind of conventional Chinese medicine that records in the Chinese Pharmacopoeia, and the dry tuber for orchid Gastrodia elata Bl. has the effect that suppressing hyperactive liver for calming endogenous wind ends convulsion, and it is dizzy to can be used for having a headache, extremity numbness, child convulsion, epilepsy clonus, lockjaw etc.With the rhizoma Gastrodiae is the medicine of effective constituent, and the gastrodia tuber preparation (as whole day fiber crops capsule) and the compound tall gastrodia tuber preparation (as gastrodia tuber refreshment capsule, gastrodia tuber and fleece-flower root sheet, Tianma Wan, big Ligusticum wallichii ball etc.) of folk prescription arranged.So far, Chinese scholars has been done number of research projects to aspects such as the chemical constitution of rhizoma Gastrodiae, pharmacological actions, and has progressively set up the quality control standard of rhizoma Gastrodiae medicinal material.
There are some researches show that Gastrodin is the active component of rhizoma Gastrodiae, so this composition is as the quality control index composition of rhizoma Gastrodiae medicinal material and preparation thereof.As, 2005 editions " content of Gastrodin is as the method for quality control in HPLC method mensuration rhizoma Gastrodiae medicinal material that Chinese pharmacopoeia is recorded and the whole day fiber crops capsule; Zhang Leis etc. are to " compound gastrodia elata particle " (" Anhui medicine " 2006; 10 (12): 919-920), (" Chinese medicine company " 2005:14 (19): 55-56), Pan Zhenyu etc. are to " medicine containing gastrodia tuber for curing wind syndrome of head sheet " (" traditional Chinese medicine Leader " 2005:11 (5): 61-63) wait that gastrodin content also adopts the HPLC method to carry out method for measuring respectively in the preparation to " exceedingly high oral liquid " for Li Honggang etc.
Existing research is found, chemical constitution is except that Gastrodin (promptly free Gastrodin) in the rhizoma Gastrodiae medicinal material, also contain the various ester constituents (Wang Li: " rhizoma Gastrodiae chemical substance basis and method of quality control research " that another kind of Gastrodin and citric acid (citric acid) condensation forms, " Postgraduate School, Chinese Academy of Sciences's doctorate paper " p10), as the gloomy glycosides of Bali, the gloomy glycosides B of Bali, the gloomy glycosides C of Bali etc.Gastrodin can be described as with the various ester constituents of citric acid condensation and combines Gastrodin, Gastrodin and be total Gastrodin that it contains in conjunction with the Gastrodin sum.
The gloomy glycosides of Bali: R
1=R
2=R
3=R
The gloomy glycosides B:R of Bali
1=R
2=R, R
3=H
The gloomy glycosides C:R of Bali
1=R
3=R, R
2=H)
Find through the inventor's further investigation, in the rhizoma Gastrodiae medicinal material, in conjunction with the content of Gastrodin far above " method of Chinese pharmacopoeia directly detects the content of the Gastrodin (Gastrodin promptly dissociates) that obtains by 2005 editions.Deep research also finds can have same or analogous drug activity in conjunction with Gastrodin and Gastrodin.For example; Gastrodin (free state) and the gloomy glycosides of Bali (combined state) show to the protective effect test findings of in-vitro simulated cerebral ischemia rat brain capillary endothelium (BMEC); each test dose (12.5 of the gloomy glycosides of Gastrodin and Bali; 25,50,100; 200mg/L) can obviously strengthen the BMEC vigor; improve the survival rate of impaired endothelial cell (EC), impel EC NO secretion to increase, show that the gloomy glycosides of Gastrodin and Bali all has the certain protection effect to the rat BMEC ischemic injuries of in vitro culture.
Because present content detection and method of quality control only can be realized the free Gastrodin in rhizoma Gastrodiae medicinal material and the preparation thereof is detected, the inherent quality that is difficult to objective reality ground reflection rhizoma Gastrodiae medicinal material and preparation thereof, and as the total amount of bringing into play whole active components of drug action in vivo, in drug manufacture and clinical use to the control of actual mass and to the grasp of result of treatment and bad reaction and to medicinal material rationally, the aspects such as utilization of saving, all have obvious defects.
Summary of the invention
Based on above-mentioned situation, the present invention will provide a kind of can be to total Gastrodin in rhizoma Gastrodiae medicinal material and the preparation thereof and/or the method that detects in conjunction with gastrodin content.
The detection method of the said total Gastrodin of the present invention is to contain tested product raw material in conjunction with the Gastrodin composition at least after the alkali condition hydrolysis, to detect the content of its free state Gastrodin composition again.The wherein said tested product raw material that contains at least in conjunction with the Gastrodin composition is meant in the tested product raw material also can be to contain simultaneously in conjunction with Gastrodin and Gastrodin (promptly free Gastrodin) two constituents for only containing in conjunction with the Gastrodin composition.
To after basic hydrolysis, contain the detection of the tested product of free state Gastrodin composition by aforesaid way of the present invention, can adopt at present existing use and/or the various detection methods of report are carried out, for example can adopt as the high performance liquid chromatography of 2005 editions Chinese Pharmacopoeia appendix VI D content and detect this free state Gastrodin composition.
Since in conjunction with Gastrodin be a class by in the Gastrodin structure-ester type compound that OH is become with citric acid, therefore be easy to its hydrolysis the Gastrodin composition that obtains dissociating with the alkali condition of gentleness by present usual manner.For example,, said hydrolysis reaction is carried out smoothly and finish to the heating condition that is no more than 80 ℃ at normal temperature, wherein preferred hydrolysis temperature is 25 ℃-60 ℃.Said basic hydrolysis is generally carried out in greater than alkaline solutions such as 12 aqueous solution, dilute alcohol solutions at pH, can more help the complete of hydrolysis reaction, and under the more weak condition of alkalescence, carry out, then may cause the incomplete of hydrolysis reaction, for example can be preferably the hydrolysis environment of alkali concn 〉=0.25mol/L.Used alkaline components generally can be selected as alkali metal hydroxide commonly used such as NaOH, potassium hydroxide.
Said method of the present invention according to the different situations of tested product raw material, can adopt following different modes or approach to carry out respectively in the specific implementation.
A kind of mode be can to this tested product raw material elder generation's water or can with the miscible organic solvent of water at least a this is contained the tested product raw material that combines the Gastrodin composition at least and extracts after, after again extract being carried out basic hydrolysis, with resulting hydrolysate as tested product and detect wherein total Gastrodin component content.This kind mode is specially adapted to the sampling amount of tested product raw material when big, obtain its extract earlier after, can reduce to be hydrolyzed the volume of material greatly, help subsequent operation.
Another kind of mode is after earlier this tested product raw material being carried out said basic hydrolysis, again water or can with the miscible organic solvent of water at least a hydrolysate is extracted, with resulting extract as tested product and detect wherein total Gastrodin component content.The sampling amount that this kind mode generally is applicable to tested product raw material more than hour situation.
Saidly in above-mentioned two kinds of processing modes can be the polar organic solvent that comprises ethanol, methyl alcohol, acetonitrile, acetone, and serve as preferred all to adopt the mixed solvent of forming by water and said polar organic solvent with the miscible organic solvent of water.
The result of contrast test shows that there is not the otherness of statistical significance in above-mentioned two kinds of different processing modes to the influence of testing result.
Detection method with total Gastrodin in rhizoma Gastrodiae medicinal material and the preparation thereof is an example, and a kind of canonical process of the above-mentioned detection method of the present invention can be undertaken by following mode:
(1) preparation of need testing solution: it is an amount of to get rhizoma Gastrodiae medicinal material and preparation thereof, and accurate the title decides, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds claims to decide weight, heating and refluxing extraction 3 hours, put the cold weight that claims to decide again, supply the weight that subtracts mistake with Diluted Alcohol, filter, get subsequent filtrate 10ml, evaporate to dryness, residue adds the dissolving of 5%NaOH solution, is transferred in the 10ml measuring bottle, and adds 5%NaOH solution to scale, shake up, accurate this solution 1ml that draws puts in the 5ml tool plug test tube, 40 ℃ of water-bath hydrolysis 2 hours, take out, with volume ratio is that 3: 97 acetonitrile-water mixed solution is transferred in the 10ml measuring bottle, transfers to neutrality or faintly acid (for example pH5～7) with hydrochloric acid, and the acetonitrile-water mixed solution that adds volume ratio and be 3: 97 is diluted to scale, shake up, filter, get subsequent filtrate, promptly get need testing solution;
(2) preparation of reference substance solution: press the method preparation of regulation under a rhizoma Gastrodiae assay of Pharmacopoeia of the People's Republic of China version in 2005 item, in contrast product solution;
(3) assay method: the method for pressing regulation under a rhizoma Gastrodiae assay of Pharmacopoeia of the People's Republic of China version in 2005 item is measured.
In the above-mentioned hydrolytic process of the present invention, owing to all be hydrolyzed into Gastrodin in conjunction with Gastrodin in the tested product raw material into free state, so testing result is the content of the total Gastrodin that exists with various states in the tested product raw material.And, can only directly detect the content of the Gastrodin that in tested product raw material, exists with free state by having the method for using and/or reporting (comprising Chinese Pharmacopoeia) at present.Therefore, obtain the deduction gastrodin content that direct detection obtains to this tested product raw material that does not carry out above-mentioned basic hydrolysis in total Gastrodin component content by said method of the present invention, its difference is the content in conjunction with Gastrodin in these tested product.
Be appreciated that thus, above-mentioned detection method of the present invention can be objective, truly, obtain the content of total Gastrodin composition of existing with different conditions in rhizoma Gastrodiae medicinal material or its various forms pharmaceutical preparation exactly, thereby for being implemented in pharmacy and/or the medication to all control of active component contents in the inherent quality of medicinal raw material or the preparation, grasp, provide objective, accurately and the scientific and reliable detection method, guaranteeing product quality, improve curative effect of medication, minimizing is because of the inaccurate adverse effect that may cause of active component content, and to medicinal material rationally, save to utilize and improve aspect such as its utilization factor, all have actively and realistic meanings.
Embodiment below in conjunction with embodiment is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.Do not breaking away under the above-mentioned technological thought situation of the present invention, various replacements or change according to ordinary skill knowledge and customary means are made all should comprise within the scope of the invention.
Embodiment
Total Gastrodin in the embodiment 1 rhizoma Gastrodiae medicinal material and detect in conjunction with gastrodin content
The preparation of need testing solution: tested rhizoma Gastrodiae medicinal material is in 80 ℃ of drying under reduced pressure, pulverize, get the about 0.8g of powder (crossing sieve No. four), the accurate title, decide, put in the tool plug conical flask, accurate adding Diluted Alcohol (by " the compound method preparation of 2005 editions one appendix p98 Diluted Alcohol of Chinese pharmacopoeia) 50ml, claim to decide weight, heating and refluxing extraction 3 hours is put cold title again and is decided weight, supplies the weight that subtracts mistake with Diluted Alcohol, filter, get subsequent filtrate 10ml evaporate to dryness, residue adds the dissolving of 3: 97 acetonitrile-water mixed solution of volume ratio (following all herewith), is transferred in the 10ml measuring bottle, and be diluted to scale with acetonitrile-water (3: 97) mixed solution, shake up, filter, get the need testing solution of subsequent filtrate as (dissociating) Gastrodin.
Get above-mentioned subsequent filtrate 10ml more in addition, evaporate to dryness, residue adds the dissolving of 5% (w) NaOH solution, be transferred in the 10ml measuring bottle, and with the 5%NaOH solution dilution to scale, shake up, accurate this solution 1ml that draws puts in the 5ml tool plug test tube, 40 ℃ of hydrolysis 2 hours, take out, be transferred in the 10ml measuring bottle, transfer pH to neutrality or faintly acid with hydrochloric acid with acetonitrile-water (3: 97) mixed solution, add acetonitrile-water (3: 97) mixed solution and be diluted to scale, shake up, filter, get the need testing solution of subsequent filtrate as total Gastrodin.
The preparation of reference substance solution: precision takes by weighing at 1 hour Gastrodin reference substance of 80 ℃ of drying under reduced pressure an amount of, adds moving phase and makes the solution that every 1ml contains 50ug.
Assay method: the high performance liquid chromatography according to 2005 editions Chinese Pharmacopoeia appendix VI D regulations is measured.
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; With acetonitrile-0.05% phosphoric acid solution (3: 97) is moving phase; The detection wavelength is 220nm.Number of theoretical plate calculates by the Gastrodin peak should be not less than 5000.
Respectively accurate reference substance solution 10 μ l and need testing solution 5～10 μ l of drawing inject liquid chromatograph, measure, and calculate, and promptly get the Gastrodin that dissociates, total Gastrodin and in conjunction with the content of Gastrodin.
Testing result: this routine tested medicinal material sample (calculating with dry product) contains total Gastrodin 2.85%, and Gastrodin (dissociating) 0.64% is 2.21% (w%) in conjunction with Gastrodin.
Embodiment 2: the total gastrodin content in the rhizoma Gastrodiae medicinal material detects
The preparation of need testing solution: tested rhizoma Gastrodiae medicinal material is pulverized in 80 ℃ of drying under reduced pressure, gets the about 0.2g of powder (crossing sieve No. four), the accurate title, decide, and puts in the tool plug conical flask, adds 5%KOH Diluted Alcohol 50ml, after room temperature was placed and was hydrolyzed in 4 hours, hydrochloric acid was regulated pH to neutral, heating and refluxing extraction 3 hours, put cold, be transferred in the 100ml measuring bottle, thin up shakes up to scale, filter, get subsequent filtrate as need testing solution.
The preparation of reference substance solution, chromatographic condition and system suitability test are all with embodiment 1.
Determination method: according to the high effective liquid chromatography for measuring of 2005 editions Chinese Pharmacopoeia appendix VI D regulations.
Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject liquid chromatograph, measure, and calculate.
Testing result: this routine tested medicinal material is pressed dry product and is calculated, and contains total Gastrodin 2.87%.
Embodiment 3: free Gastrodin in the rhizoma Gastrodiae medicinal material, total Gastrodin and in conjunction with the detection of gastrodin content
The preparation of need testing solution: other gets a collection of rhizoma Gastrodiae medicinal material in 80 ℃ of drying under reduced pressure, pulverize, get the about 0.8g of powder (crossing sieve No. four), accurate title is fixed, put in the tool plug conical flask, the accurate 50% methyl alcohol 50ml that adds claims to decide weight, heating and refluxing extraction 3 hours, put the cold weight that claims to decide again, supply the weight that subtracts mistake with 50% methyl alcohol, filter, get the need testing solution of subsequent filtrate as (dissociating) Gastrodin.Get subsequent filtrate 10ml more in addition, evaporate to dryness, residue add the dissolving of 2%NaOH solution, be transferred in the 10ml measuring bottle, and with the 2%NaOH solution dilution to scale, shake up, accurate this solution 2ml that draws puts in the 5ml tool plug test tube, 40 ℃ of hydrolysis 2 hours, take out, be transferred in the 10ml measuring bottle, transfer PH to neutrality or faintly acid with hydrochloric acid with methyl alcohol, add methyl alcohol and be diluted to scale, shake up, filter, get the need testing solution of subsequent filtrate as total Gastrodin.
The preparation of reference substance solution: precision takes by weighing at 1 hour Gastrodin reference substance of 80 ℃ of drying under reduced pressure an amount of, adds methyl alcohol and makes the solution that every 1ml contains 50ug.
Determination method: the high performance liquid chromatography according to 2005 editions Chinese Pharmacopoeia appendix VI D regulations is measured.
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filling agent; (0.1mol/L potassium dihydrogen phosphate and 0.1mol/L sodium dihydrogen phosphate mixed in equal amounts)-water (10: 3: 87) is moving phase with methyl alcohol-phosphate solution; The detection wavelength is 270nm.Number of theoretical plate calculates by the Gastrodin peak should be not less than 2000.
Respectively accurate reference substance solution and each 5 μ l of need testing solution of drawing inject liquid chromatograph, measure, and calculate, and promptly get the Gastrodin that dissociates, total Gastrodin and in conjunction with the content of Gastrodin.
Testing result: these routine tested product are pressed dry product and are calculated, and contain Gastrodin 0.29%, and total Gastrodin 1.64% is in conjunction with Gastrodin 1.35%.
Embodiment 4: free Gastrodin in the Tianma Wan, total Gastrodin and in conjunction with the content detection of Gastrodin
The preparation of need testing solution: tested Tianma Wan is pulverized, get the about 10g of powder (crossing sieve No. four), the accurate title, decide, put in the tool plug conical flask, the accurate 70% ethanol 100ml that adds claims to decide weight, heating and refluxing extraction 4 hours, put cold title again and decide weight, supply the weight that subtracts mistake, filter with 70% ethanol, get subsequent filtrate 20ml, evaporate to dryness, residue add the dissolving of acetonitrile-water (3: 97) mixed solution, are transferred in the 10ml measuring bottle, and be diluted to scale with acetonitrile-water (3: 97) mixed solution, shake up, filter, get the need testing solution of subsequent filtrate as (dissociating) Gastrodin.
Get filtrate 20ml evaporate to dryness more in addition, residue adds the dissolving of 1%NaOH solution, is transferred in the 10ml measuring bottle, and with the 1%NaOH solution dilution to scale, shake up accurate this solution 1ml that draws, put in the 5ml tool plug test tube, 60 ℃ of hydrolysis 2 hours are taken out, be transferred in the 10ml measuring bottle with acetonitrile-water (3: 97) mixed solution, transfer PH to neutrality or faintly acid with hydrochloric acid, add acetonitrile-water (3: 97) mixed solution and be diluted to scale, shake up, filter, get the need testing solution of filtrate as total Gastrodin.
Determination method: according to the high effective liquid chromatography for measuring of 2005 editions Chinese Pharmacopoeia appendix VI D regulations.
Preparation/the chromatographic condition of reference substance solution and system suitability test are all with embodiment 1.
Respectively accurate each 5～10 μ l of reference substance solution and need testing solution that draw inject liquid chromatograph, measure, and calculate, and promptly get the Gastrodin that dissociates, total Gastrodin and in conjunction with the content of Gastrodin.
Testing result: the every 1g of these routine tested product contains Gastrodin 0.22mg, and total Gastrodin 0.91mg is in conjunction with Gastrodin 0.69mg.
Embodiment 5: the content detection of total Gastrodin in the gastrodia tuber refreshment capsule
The preparation of need testing solution: get the detected about 0.6g of gastrodia tuber refreshment capsule 's content, the accurate title, decide, and puts in the tool plug conical flask, the 30% ethanolic solution 50ml that adds 10%NaOH, room temperature is placed 2 hours (hydrolysis), transfers pH to neutral with hydrochloric acid, heating and refluxing extraction 3 hours, put cold, be transferred in the 100ml measuring bottle, thin up shakes up to scale, filter, get subsequent filtrate as need testing solution.
Determination method: according to the high effective liquid chromatography for measuring of 2005 editions Chinese Pharmacopoeia appendix VI D regulations.
The preparation of chromatographic condition and system suitability test, reference substance solution is with embodiment 1.
Accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing inject liquid chromatograph, measure, and calculate, promptly.
Testing result: the every capsules of these routine tested product contains total Gastrodin 3.10mg.
Embodiment 6: the content detection of total Gastrodin in the compound gastrodia elata particle
The preparation of need testing solution: get the detected about 2.5g of compound gastrodia elata particle, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 100ml that adds, claim to decide weight, heating and refluxing extraction 2 hours is put cold title again and is decided weight, supplies the weight that subtracts mistake with methyl alcohol, filter, get subsequent filtrate 20ml evaporate to dryness, residue adds the dissolving of 5%NaOH solution, is transferred in the 10ml measuring bottle, and with the 5%NaOH solution dilution to scale, shake up, accurate this solution 1ml that draws puts in the 5ml tool plug test tube, 40 ℃ of hydrolysis 2 hours, take out, be transferred in the 10ml measuring bottle, transfer pH to neutrality or faintly acid with hydrochloric acid with acetonitrile-water (3: 97) mixed solution, add acetonitrile-water (3: 97) mixed solution and be diluted to scale, shake up, filter, get the need testing solution of subsequent filtrate as total Gastrodin.
Determination method: according to the high effective liquid chromatography for measuring of 2005 editions Chinese Pharmacopoeia appendix VI D regulations.
The preparation of chromatographic condition and system suitability test, reference substance solution is with embodiment 1.
Accurate respectively reference substance solution and each 5～10 μ l of need testing solution of drawing inject liquid chromatograph, measure, and calculate, promptly.
Testing result: the every 1g of these routine tested product contains total Gastrodin 1.74mg.
Embodiment 7: the content detection of total Gastrodin in the exceedingly high oral liquid
The preparation of need testing solution: get detected exceedingly high oral liquid 5ml, evaporate to dryness, residue adds the dissolving of 8%NaOH solution, be transferred in the 10ml measuring bottle, and with the 8%NaOH solution dilution to scale, shake up, accurate this solution 1ml that draws puts in the 5ml tool plug test tube, room temperature is placed (hydrolysis) 3 hours, take out, be transferred in the 10ml measuring bottle, transfer pH to neutrality or faintly acid with hydrochloric acid with acetonitrile-water (3: 97) mixed solution, add acetonitrile-water (3: 97) mixed solution and be diluted to scale, shake up, filter, get the need testing solution of subsequent filtrate as total Gastrodin.
Determination method: according to the high effective liquid chromatography for measuring of 2005 editions Chinese Pharmacopoeia appendix VI D regulations.
The preparation of chromatographic condition and system suitability test, reference substance solution is with embodiment 1.
Accurate respectively reference substance solution and each 5～10 μ l of need testing solution of drawing inject liquid chromatograph, measure, and calculate, promptly.
Testing result: the every 1ml of these routine tested product contains total Gastrodin 0.81mg.
Embodiment 8: extract choice of Solvent and influence
Get the about 0.8g of rhizoma Gastrodiae medicinal powder, totally 9 parts, the accurate title, decide, put in the tool plug conical flask, precision adds entry respectively, 30% ethanol, Diluted Alcohol, 70% ethanol, 30% methyl alcohol, 50% methyl alcohol, 70% methyl alcohol, 30% acetone, each 50ml of 30% acetonitrile claims to decide weight, heating and refluxing extraction 3 hours, put the cold weight that claims to decide again, supply the weight that subtracts mistake with the corresponding solvent that extracts respectively, filter, get subsequent filtrate 10ml, evaporate to dryness, residue adds the dissolving of acetonitrile-water (3: 97) mixed solution, is transferred in the 10ml measuring bottle, and is diluted to scale with acetonitrile-water (3: 97) mixed solution, shake up, as the need testing solution of free Gastrodin.
Get above-mentioned filtrate 10ml more respectively in addition, evaporate to dryness, residue add the dissolving of 5%NaOH solution, be transferred in the 10ml measuring bottle, and add 5%NaOH solution, shake up to scale, accurate this solution 1ml that draws puts in the 5ml tool plug test tube, 40 ℃ of water-bath hydrolysis 2 hours, take out, be transferred in the 10ml measuring bottle with acetonitrile-water (3: 97) mixed solution, transfer pH to neutrality or faintly acid, add acetonitrile-water (3: 97) mixed solution and be diluted to scale with hydrochloric acid, shake up, as the need testing solution of total Gastrodin.
Respectively according to " a rhizoma Gastrodiae assay of Chinese pharmacopoeia version in 2005 the item method of regulation is down measured, and calculates, and promptly gets the content of dissociate Gastrodin and total Gastrodin, and the result is as shown in table 1.
Table 1 extracts choice of Solvent and influence
| Extract solvent | Water | 30% ethanol | Diluted Alcohol | 70% ethanol | 30% methyl alcohol | 50% methyl alcohol | 70% methyl alcohol | 30% acetone | 30% acetonitrile |
| Free Gastrodin % | 0.71 | 0.65 | 0.63 | 0.64 | 0.64 | 0.63 | 0.62 | 0.58 | 0.60 |
| Total Gastrodin % | 2.95 | 2.90 | 2.84 | 2.88 | 2.85 | 2.86 | 2.81 | 2.65 | 2.73 |
| In conjunction with Gastrodin % | 2.24 | 2.25 | 2.21 | 2.24 | 2.21 | 2.23 | 2.19 | 2.07 | 2.13 |
Illustrate: (1) is in conjunction with Gastrodin=total Gastrodin-Gastrodin dissociates:
(2) Diluted Alcohol is by " the compound method preparation of 98 pages of Diluted Alcohols of 2005 editions one appendix of Chinese pharmacopoeia.
Table 1 is the result show, water or moisture ethanol, methyl alcohol, acetone, acetonitrile all can propose free Gastrodin and preferably in conjunction with Gastrodin.
Embodiment 9: the selection of extracting method and influence
Get the about 0.8g of rhizoma Gastrodiae medicinal powder, totally 7 parts, the accurate title, decide, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds, claim to decide weight, wherein, 3 duplicate samples difference sonicated 20,40,60 minutes, 4 duplicate samples were distinguished heating and refluxing extraction 1 in addition, 2,3,4 hours, above-mentioned sample is put and is claimed to decide weight again after cold, supply the weight that subtracts mistake with Diluted Alcohol, filter, get subsequent filtrate 10ml, evaporate to dryness, residue adds the dissolving of acetonitrile-water (3: 97) mixed solution, is transferred in the 10ml measuring bottle, and is diluted to scale with acetonitrile-water (3: 97) mixed solution, shake up, as the need testing solution of free Gastrodin.
Get filtrate 10ml more respectively in addition, evaporate to dryness, residue add the dissolving of 5%NaOH solution, be transferred in the 10ml measuring bottle, and add 5%NaOH solution, shake up to scale, accurate this solution 1ml that draws puts in the 5ml tool plug test tube, 40 ℃ of water-bath hydrolysis 2 hours, take out, be transferred in the 10ml measuring bottle with acetonitrile-water (3: 97) mixed solution, transfer pH to neutrality or faintly acid, add acetonitrile-water (3: 97) mixed solution and be diluted to scale with hydrochloric acid, shake up, as the need testing solution of total Gastrodin.According to " a rhizoma Gastrodiae assay of Chinese pharmacopoeia version in 2005 the item method of regulation is down measured, and calculates, and promptly gets the content of dissociate Gastrodin and total Gastrodin, and the result is as shown in table 2.
The selection of table 2 extracting method and influence
| Extracting method | The ultrasonic Extraction time (minute) | Reflux extracting time (hour) | |||||
| 20 | 40 | 60 | 1 | 2 | 3 | 4 | |
| Free Gastrodin % | 0.58 | 0.61 | 0.62 | 0.60 | 0.62 | 0.64 | 0.65 |
| Total Gastrodin % | 2.60 | 2.71 | 2.75 | 2.67 | 2.78 | 2.86 | 2.89 |
| In conjunction with Gastrodin % | 2.02 | 2.10 | 2.13 | 2.07 | 2.16 | 2.22 | 2.25 |
Illustrate: with table 1
By table 2 as seen, ultrasonic or refluxing extraction all can propose free Gastrodin preferably and in conjunction with Gastrodin, be good with refluxing extraction 2-4 hour.
Embodiment 10: the selection of hydrolysis temperature and influence
Get the about 0.8g of rhizoma Gastrodiae medicinal powder, the accurate title, decide, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds, claim to decide weight, heating and refluxing extraction 3 hours is put the cold weight that claims to decide again, supply the weight that subtracts mistake with Diluted Alcohol, filter, get filtrate 10ml, evaporate to dryness, residue adds the dissolving of 5%NaOH solution, be transferred in the 10ml measuring bottle, and add 5%NaOH solution, shake up to scale, accurate this solution 1ml that draws, totally 6 parts, put respectively in the 5ml tool plug test tube, in refrigerator cold-storage (4 ℃), 25 ℃, 40 ℃, 60 ℃, 80 ℃, 100 ℃ of placements (hydrolysis) 2 hours, take out, be transferred in the 10ml measuring bottle with acetonitrile-water (3: 97) mixed solution, transfer pH to neutrality or faintly acid, add acetonitrile-water (3: 97) mixed solution and be diluted to scale with hydrochloric acid, shake up, as the need testing solution of total Gastrodin.By " a rhizoma Gastrodiae assay of Chinese pharmacopoeia version in 2005 the item method of regulation is down measured, and calculates, and promptly gets the content of total Gastrodin, and the result is as shown in table 3.
The selection of table 3 hydrolysis temperature
| Hydrolysis temperature | 4℃ | 25℃ | 40℃ | 60℃ | 80℃ | 100℃ |
| Total gastrodin content (%) | 2.82 | 2.84 | 2.84 | 2.81 | 2.73 | 2.25 |
By table 3 result as seen, the preference temperature of basic hydrolysis is 4 ℃-80 ℃, all can will be hydrolyzed into free Gastrodin in conjunction with Gastrodin preferably, and optimum hydrolysis temperature wherein is 25 ℃-60 ℃.
Embodiment 11: the alkali concn of hydrolysising condition is selected and influence
Get the about 1.6g of rhizoma Gastrodiae medicinal powder, the accurate title, decide, and puts in the tool plug conical flask, the accurate Diluted Alcohol 100ml that adds, claim to decide weight, heating and refluxing extraction 3 hours is put the cold weight that claims to decide again, supply the weight that subtracts mistake with Diluted Alcohol, filter, get filtrate 10ml, totally 8 parts, the difference evaporate to dryness, residue adds 0.5% respectively, 1%, 2%, 4%, 6%, 8%, 10%, the dissolving of 20%NaOH solution is transferred in the 10ml measuring bottle, and adds corresponding N aOH solution to scale, shake up, accurate this solution 1ml that draws puts in the 5ml tool plug test tube, 40 ℃ of water-bath hydrolysis 2 hours, take out, be transferred in the 10ml measuring bottle with acetonitrile-water (3: 97) mixed solution, transfer PH to neutrality or faintly acid, add acetonitrile-water (3: 97) mixed solution and be diluted to scale with hydrochloric acid, shake up, as the need testing solution of total Gastrodin.According to " a rhizoma Gastrodiae assay of Chinese pharmacopoeia version in 2005 the item method of regulation is down measured, and calculates, and promptly gets the content of total Gastrodin, and the result is as shown in table 4.
The alkali concn of table 4 hydrolysising condition is selected and influence
| Alkali concn (%) | 0.5％ | 1％ | 2％ | 4％ | 6％ | 8％ | 10％ | 20％ |
| Total gastrodin content (%) | 2.63％ | 2.72 | 2.84 | 2.84 | 2.86 | 2.87 | 2.86 | 2.89 |
By table 4 result as seen, alkali concn be 〉=1% (or 〉=0.25mol/L), pH value of solution can both will be hydrolyzed into free Gastrodin well all greater than under 12 the condition in conjunction with Gastrodin.
Embodiment 12: the selection of hydrolysis time and influence
Get the about 0.8g of rhizoma Gastrodiae medicinal powder, the accurate title, decide, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds, claim to decide weight, heating and refluxing extraction 3 hours is put the cold weight that claims to decide again, supply the weight that subtracts mistake with Diluted Alcohol, filter, get filtrate 10ml, evaporate to dryness, residue adds the dissolving of 1%NaOH solution, be transferred in the 10ml measuring bottle, and add 1%NaOH solution, shake up to scale, the accurate place near the steps solution 1ml that draws, totally 5 parts, put respectively in the 5ml tool plug test tube, in 40 ℃ of placement (hydrolysis) 0.5,1,2,4,8 hours, take out, be transferred in the 10ml measuring bottle with acetonitrile-water (3: 97) mixed solution, transfer pH to neutral or acid, add acetonitrile-water (3: 97) mixed solution and be diluted to scale with hydrochloric acid, shake up, as the need testing solution of total Gastrodin.
Other gets filtrate 10ml, and 2 parts, evaporate to dryness, residue add 5%NaOH and the dissolving of 10%NaOH solution respectively, are equipped with need testing solution with legal system.According to " a rhizoma Gastrodiae assay of Chinese pharmacopoeia version in 2005 the item method of regulation is down measured, and calculates, and promptly gets the content of total Gastrodin, and the result is as shown in table 5.
The selection of table 5 hydrolysis time and influence
| Hydrolysis time (h) | 1％NaOH | 5％NaOH | 10％NaOH | ||||||||||||
| 0.5 | 1 | 2 | 4 | 8 | 0.5 | 1 | 2 | 4 | 8 | 0.5 | 1 | 2 | 4 | 8 | |
| Total gastrodin content (%) | 2.79 | 2.82 | 2.84 | 2.85 | 2.84 | 2.85 | 2.87 | 2.84 | 2.83 | 2.85 | 2.86 | 2.84 | 2.82 | 2.81 | 2.79 |
By table 5 result as seen, the suitable time of basic hydrolysis is 0.5-8 hour.
Further research is also found, hydrolysis temperature, alkali concn and hydrolysis time three to tested product raw material in the inventive method can interrelated and cooperations.For example, when adopting the basic hydrolysis of low concentration, can make in conjunction with the Gastrodin hydrolysis complete by prolonging hydrolysis time and/or improving hydrolysis temperature.As, adopt the 0.50%NaOH room temperature to place the hydrolysis effect of spend the night (about 20 hours), can be suitable with 2 hours effect of room temperature hydrolysis in 5%NaOH solution.
Detection method of the present invention according to traditional Chinese medicine quality standard method of analysis verification guide principle, is investigated linear relationship, stability, precision, reappearance, average recovery etc., and the result all meets the requirements.
In addition, with in contrast, after the method for employing the foregoing description 1 is hydrolyzed, detect its gastrodin content in conjunction with the pure product of the gloomy glycosides of the Bali of Gastrodin.Testing result shows, the actual Gastrodin amount that records is 98.6% of a Theoretical Calculation amount, show that it is thoroughly with completely to the hydrolysis in conjunction with Gastrodin that the present invention adopts above-mentioned basic hydrolysis mode, total Gastrodin detection method of setting up on this basis is feasible, and the accuracy of testing result has enough confidence levels.
Claims (10)
1. the detection method of total Gastrodin is characterized in that detecting the content of free state Gastrodin composition wherein again with containing tested product raw material in conjunction with the Gastrodin composition at least after the alkali condition hydrolysis.
2. the detection method of total Gastrodin as claimed in claim 1, the temperature that it is characterized in that said basic hydrolysis is that normal temperature is to the heating condition that is no more than 80 ℃.
3. the detection method of total Gastrodin as claimed in claim 2, the temperature that it is characterized in that said basic hydrolysis is 25 ℃-60 ℃.
4. the detection method of total Gastrodin as claimed in claim 1 is characterized in that said basic hydrolysis is for carrying out in greater than 12 alkaline solution in the pH value.
5. as the detection method of the described total Gastrodin of one of claim 1 to 4, it is characterized in that the said tested product raw material that contains at least in conjunction with the Gastrodin composition comprises rhizoma Gastrodiae medicinal material or its extract, and the preparation that contains rhizoma Gastrodiae medicinal material or its extract, this tested product raw material elder generation's water or can with the miscible organic solvent of water at least a this is contained the tested product raw material that combines the Gastrodin composition at least and extracts after, after again extract being carried out basic hydrolysis, with resulting hydrolysate as tested product and detect wherein total Gastrodin component content, said can with the miscible organic solvent of water for comprising ethanol, methyl alcohol, acetonitrile, the polar organic solvent of acetone.
6. the detection method of total Gastrodin as claimed in claim 5 is characterized in that the said mixed solvent that the extraction employing of tested product raw material is made up of water and said polar organic solvent.
7. as the detection method of the described total Gastrodin of one of claim 1 to 4, it is characterized in that the said tested product raw material that contains at least in conjunction with the Gastrodin composition comprises rhizoma Gastrodiae medicinal material or its extract, and the preparation that contains rhizoma Gastrodiae medicinal material or its extract, after earlier this tested product raw material being carried out said basic hydrolysis, again water or can with the miscible organic solvent of water at least a hydrolysate is extracted, with resulting extract as tested product and detect wherein total Gastrodin component content, said can with the miscible organic solvent of water for comprising ethanol, methyl alcohol, acetonitrile, the polar organic solvent of acetone.
8. the detection method of total Gastrodin as claimed in claim 7 is characterized in that the said mixed solvent that the extraction employing of tested product is made up of water and said polar organic solvent.
9. the detection method of total Gastrodin in rhizoma Gastrodiae medicinal material and the preparation thereof is characterized in that being undertaken by following mode:
(1) preparation of need testing solution: it is an amount of to get rhizoma Gastrodiae medicinal material and preparation thereof, and accurate the title decides, and puts in the tool plug conical flask, the accurate Diluted Alcohol 50ml that adds claims to decide weight, heating and refluxing extraction 3 hours, put the cold weight that claims to decide again, supply the weight that subtracts mistake with Diluted Alcohol, filter, get subsequent filtrate 10ml, evaporate to dryness, residue adds the dissolving of 5%NaOH solution, is transferred in the 10ml measuring bottle, and adds 5%NaOH solution to scale, shake up, accurate this solution 1ml that draws puts in the 5ml tool plug test tube, 40 ℃ of water-bath hydrolysis 2 hours, take out, with volume ratio is that 3: 97 acetonitrile-water mixed solution is transferred in the 10ml measuring bottle, transfers pH to neutrality or faintly acid with hydrochloric acid, and the acetonitrile-water mixed solution that adds volume ratio and be 3: 97 is diluted to scale, shake up, filter, get subsequent filtrate, promptly get need testing solution;
(2) preparation of reference substance solution: press the method preparation of regulation under a rhizoma Gastrodiae assay of Pharmacopoeia of the People's Republic of China version in 2005 item, in contrast product solution;
(3) assay method: the method for pressing regulation under a rhizoma Gastrodiae assay of Pharmacopoeia of the People's Republic of China version in 2005 item is measured.
10. in conjunction with the detection method of Gastrodin, it is characterized in that by the described method of claim 1 by after containing the tested product that contain total Gastrodin composition of tested product raw material in conjunction with the Gastrodin composition at least and detecting through obtaining after the alkali condition hydrolysis, deduction directly detects the gastrodin content that obtains to this tested product raw material that does not carry out said basic hydrolysis from the total Gastrodin component content that obtains, and is the content in conjunction with Gastrodin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115554256A (en) * | 2022-09-23 | 2023-01-03 | 乐泰药业有限公司 | Preparation method and quality control method of gastrodin tablets |
| CN116459318A (en) * | 2023-05-29 | 2023-07-21 | 昆明理工大学 | Preparation method of gastrodia elata hydrolysate |
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| KR100540033B1 (en) * | 2002-01-18 | 2005-12-29 | 주식회사 팬제노믹스 | Crude Drug Compositions for treating or preventing arthritic diseases and the process for preparing them |
| CN1515904A (en) * | 2003-01-10 | 2004-07-28 | 贵州宏宇药业有限公司 | Method for determining gastrodine content |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115554256A (en) * | 2022-09-23 | 2023-01-03 | 乐泰药业有限公司 | Preparation method and quality control method of gastrodin tablets |
| CN115554256B (en) * | 2022-09-23 | 2024-04-02 | 乐泰药业有限公司 | Preparation method and quality control method of gastrodin tablet |
| CN116459318A (en) * | 2023-05-29 | 2023-07-21 | 昆明理工大学 | Preparation method of gastrodia elata hydrolysate |
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