CN103575850A - Detection method for Gymnadenia conopsea medicinal material - Google Patents
Detection method for Gymnadenia conopsea medicinal material Download PDFInfo
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Abstract
The invention relates to a detection method for a Gymnadenia conopsea medicinal material. The Gymnadenia conopsea medicinal material is detected by one or more of thin layer chromatographic identification, polysaccharide content detection, feature spectrum peak and gastrodin content detection methods; the thin layer chromatographic identification method has high specificity and high sensitivity; the internal quality of the Gymnadenia conopsea medicinal material can be further measured by performing content detection on polysaccharide and gastrodin; feature spectrum with pertinence and the detection method thereof are selected through tests, so that the Gymnadenia conopsea can be detected accurately.
Description
Technical field
The present invention relates to a kind of detection method of medicinal material, particularly a kind of detection method of hand ginseng medicinal material, belongs to medical technical field.
Background technology
Hand ginseng, hide name " prosperous drawing ", piece root for the orchid family hand ginseng platymiscium hand ginseng Gymnadenia conopsea (L.) R.Br., has good Tianjing Busuiye medicinal, effects such as increasing intelligence of building up health, promote the production of body fluid to quench thirst, calm the nerves, be hide, medicinal herbs most in use in anaesthetic kidney tonifying prescription.
At present, less for the detection method research of hand ginseng, weak foundation.Document < < HPLC measures the content > > (CHINA JOURNAL OF CHINESE MATERIA MEDICA of 4 kinds of effective constituents in prosperous the drawing of Tibetan medicine conic gymnadenia tuber, 14 phases in 2009, abbreviation " document 1 ") a kind of 4 kinds of intelligence development constituents dactylorhin B that simultaneously measure have been introduced, loroglossin, dactylorhin A, the method of militarine, adopt C18 reverse-phase chromatographic column, with methanol-water gradient elution (0min15% → 10min20% → 20min45% → 35min70%), flow velocity 0.7mlmin-1, at 222nm wavelength place, detect 4 kinds of effective constituents content.The range of linearity is dactylorhin B0.2-4.0 μ g, l-oroglossin0.01-5.0 μ g, dactylorhin A0.08-4.5 μ g, militarine0.08-4.5 μ g.Document < < HPLC measures Gastrodin > > (Chinese patent drug in the Tibetan medicine conic gymnadenia tuber of Qinghai-Tibet different regions, 5 phases in 2011, abbreviation " document 2 ") introduced the assay method of Gastrodin in a kind of hand ginseng, adopt Hypersil ODS-2 (4.6mm * 250mm, 5 μ m) chromatographic column, mobile phase is methanol-water (containing 0.04% phosphoric acid)=8: 92, volumetric flow rate 1mL/min, detect wavelength 222nm, 30 ℃ of column temperatures.Result Gastrodin sample size is good linear relationship with peak area within the scope of 1.400～8.400 μ g, r=0.9999, and average recovery rate is that 99.14%, RSD is 0.69% (n=9).
The above-mentioned detection method for hand ginseng medicinal material is perfect not to the utmost.For the method for document 1, the standard items of 4 kinds of compositions are difficult to obtain, and complicated operation is loaded down with trivial details, and experimental result is unstable, and repeatability is poor; For the method for document 2, depend merely on a kind of method that detects component content, for the detection of the Chinese crude drug of complicated component, representative poor.
Summary of the invention
For overcoming the deficiencies in the prior art, the invention provides a kind of detection method of hand ginseng medicinal material.
Technical scheme of the present invention is as follows:
Hand is joined a detection method for medicinal material, comprises one or more in following method:
The tlc identification method of A, hand ginseng medicinal material:
Get hand ginseng medicinal material fine powder 0.2-1g, add water-saturated n-butanol 10-30ml, ultrasonic processing 20-40min, filter, filtrate evaporate to dryness, residue adds water 2ml to be made to dissolve, by D101 macroporous resin column, first, with 20ml washing, discard filtrate, then with 60%-80% ethanol 25ml wash-out, collect filtrate, evaporate to dryness, residue adds 70%-80% methyl alcohol 1ml and dissolves, as need testing solution; Separately get hand ginseng control medicinal material, with legal system for control medicinal material solution; Draw respectively need testing solution, each 10 μ l of control medicinal material solution, point is in silica G thin layer version, ethyl acetate-methanol-water mixture that the volume ratio of take is 8.5-9:0.8-1.2:0.2-0.3 is developping agent expansion, take out, dry, spray, with 10% phosphomolybdic acid ethanol solution, is heated to clear spot at 105 ℃; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
The content determination of B, total polysaccharide:
1) preparation of contrast solution: get anhydrous dextrose reference substance, add the solution that water is mixed with 0.1mg/ml;
2) preparation of typical curve: precision measures above-mentioned contrast solution 0ml, 0.1ml, 0.3ml, 0.5ml, 0.8ml, 1ml, be placed in respectively the tool plug test tube of 10ml, respectively add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool, to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, at 582nm place, measure respectively absorbance, and draw absorbance-concentration standard curve;
3) need testing solution preparation and determination method: get hand ginseng medicinal material fine powder 0.5-5g, accurately weighed, put in round-bottomed flask, add ethanol 100ml refluxing extraction 1 hour, after water bath heat preservation 2 hours, filter, discard filtrate, residue and filter paper are placed in to flask, adding distil water 100ml, heating and refluxing extraction 2 hours, filter while hot, residue and flask wash with water 3 times, each 20ml, merging filtrate and washing lotion, let cool, be transferred in 250ml measuring bottle, add water to scale, shake up, precision measures the tool plug dry test-tube that 1ml is placed in 10ml, add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, in 582nm place, measure absorbance, and according to the content of polysaccharide in typical curve calculating hand ginseng,
The content detection method of C, characteristic spectrum peak and Gastrodin:
1) chromatographic condition and system flexibility: take octadecylsilane chemically bonded silica as filling agent, detect wavelength 222nm, 40 ℃ of column temperatures, number of theoretical plate calculates and should be not less than 2000 by Gastrodin peak, take acetonitrile as mobile phase A, the 0.05mol/L potassium dihydrogen phosphate that every 100ml of take adds 0.1ml phosphoric acid is Mobile phase B, adopt mobile phase A, Mobile phase B to carry out gradient elution simultaneously, 0min → 10min → 45min → 70min → 90min, according to the above-mentioned time period, mobile phase A: 3% → 3% → 17% → 17% → 25%; Mobile phase B: 97% → 97% → 83% → 83% → 75%;
2) preparation of reference substance solution: get Gastrodin reference substance, add methyl alcohol and dissolve and dilute, make the reference substance solution that every 1ml methyl alcohol contains 0.1mg Gastrodin;
3) preparation of need testing solution: get hand ginseng medicinal material fine powder 1-5g, accurately weighed, precision adds water-saturated n-butanol solution 25ml, weighed weight, ultrasonic extraction 1h, let cool, more weighed weight, with water-saturated n-butanol, supply the weight of less loss, shake up, filter, after filtrate evaporate to dryness, add methyl alcohol 5ml and dissolve, obtain;
4) determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In gained test sample characteristic spectrum, present 5 characteristic peaks, wherein 1 peak is corresponding with Gastrodin peak retention time, for S peak, calculate the ratio at other 4 characteristic peak relative retention times and S peak, 4 ratios are: 1.83,5.14,5.65,6.12, difference is in ± 10%, simultaneously according to calculated by peak area gastrodin content.
The present invention is preferred, and the tlc identification method of hand ginseng medicinal material is:
Get hand ginseng medicinal material fine powder 0.5g, add water-saturated n-butanol 10ml, ultrasonic processing 30min, filter, filtrate evaporate to dryness, residue adds water 2ml to be made to dissolve, by internal diameter, be 1.5cm, post height is the D101 macroporous resin column of 12cm, first with 20ml, washes, discard filtrate, with 70% ethanol 25ml wash-out, collect filtrate, evaporate to dryness again, residue adds 70% methyl alcohol 1ml and dissolves, as need testing solution; Separately get hand ginseng control medicinal material, with legal system for control medicinal material solution; Draw respectively need testing solution, each 10 μ l of control medicinal material solution, put in silica G thin layer version, ethyl acetate-methanol-water mixture that the volume ratio of take is 9:1:0.2 is developping agent expansion, take out, dry, spray, with 10% phosphomolybdic acid ethanol solution, is heated to clear spot at 105 ℃; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
The present invention is preferred, and in the preparation of content determination step 3) need testing solution and determination method of total polysaccharide, the sampling amount of hand ginseng medicinal material fine powder is 2g.
The present invention is preferred, and in the preparation of the content detection method step 3) need testing solution of characteristic spectrum peak and Gastrodin, the sampling amount of hand ginseng medicinal material fine powder is 2g.
The pass of weight portion of the present invention and parts by volume is g/ml or kg/L.
Beneficial effect of the present invention
The invention provides a kind of detection method of hand ginseng medicinal material, by hand, join the discriminating of medicinal material, and/or hand is joined total determination of polysaccharide, and/or the detection of the characteristic spectrum of hand ginseng, can measure the content of Gastrodin, doublely can utilize 5 characteristic spectrum peak fingerprints to differentiate hands ginseng, opponent's ginseng detects targetedly.Thin-layer identification method specificity is strong, highly sensitive; Polysaccharide and Gastrodin are carried out to the interior quality that content detection can further be weighed hand ginseng medicinal material, and the present invention has also selected by experiment and has had more characteristic spectrum and detection method thereof targetedly, detection hand ginseng that can be clearer and more definite.Result shows that method is simple and feasible, has good accuracy and precision, can effectively ensure the quality of products.
Accompanying drawing explanation
Fig. 1 is the chromatogram of object of reference Gastrodin;
Fig. 2 is the chromatogram of hand ginseng medicinal material, and wherein peak 1 is S peak, and peak 1-5 is characteristic peak;
Wherein, horizontal ordinate is the time, unit: minute (Minutes); Ordinate is voltage, unit: volt (Volts).
Specific implementation method
Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.
Experimental example 1, tlc identification method
(Qinghai gold is scolded Tibetan medicine medicine company incorporated company and is provided to get hand ginseng control medicinal material, lot number is 111021) powder 0.5g, hand ginseng medicinal material sample (scold Tibetan medicine medicine company incorporated company and provide by Qinghai gold, lot number is 120801) powder 0.5g, compound hand ginseng ball (scold Tibetan medicine medicine company incorporated company and provide by Qinghai gold, lot number is 20111215) powder 3g, operation repetitive is as follows: add water-saturated n-butanol 10ml, ultrasonic processing 30min, filter, filtrate evaporate to dryness, residue adds water 2ml to be made to dissolve, by D101 macroporous resin column (internal diameter 1.5cm, the high 12cm of post), 20ml washing, discard filtrate, with 70% ethanol 25ml wash-out, collect filtrate, evaporate to dryness, residue adds 70% methyl alcohol 1ml and dissolves, make respectively control medicinal material solution, need testing solution 1 and need testing solution 2, draw respectively each 10 μ l of above-mentioned three kinds of solution, put on silica gel g thin-layer plate, ethyl acetate-methanol-water mixture that the volume ratio of take is 9:1:0.2, as developping agent launches, takes out, and dries, and spray, with 10% phosphomolybdic acid ethanol solution, is heated to clear spot at 105 ℃.
In the collection of illustrative plates of hand ginseng and compound hand ginseng ball, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
The assay method of experimental example 2, total polysaccharide
1. instrument, reagent and test sample
Instrument: electronic balance: Shimadzu AUW220D type; Shimadzu UV-2450 type uv-spectrophotometric instrument.
Reference substance: anhydrous dextrose reference substance.(Chinese pharmaceutical biological product is identified institute; Lot number: 110833-200503)
Sample: hand ginseng medicinal material (Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), lot number is respectively: 120801,120802,120803.
2. total determination of polysaccharide
The preparation of contrast solution: get anhydrous dextrose reference substance 10mg, put in the volumetric flask of 100ml, be dissolved in water and be diluted to scale, shake up, obtain.
The preparation of typical curve: precision measures above-mentioned contrast solution 0ml, 0.1ml, 0.3ml, 0.5ml, 0.8ml, 1ml, be placed in respectively the tool plug test tube of 10ml, respectively add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool, to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, at 582nm place, measure respectively absorbance, and draw absorbance-concentration standard curve;
Need testing solution preparation and determination method: get hand ginseng fine powder 0.2g, accurately weighed, put in round-bottomed flask, add ethanol 100ml refluxing extraction 1 hour, after water bath heat preservation 2 hours, filter, discard filtrate, residue and filter paper are placed in to flask, adding distil water 100ml, heating and refluxing extraction 2 hours, filter while hot, residue and flask wash with water 3 times, each 20ml, merging filtrate and washing lotion, let cool, be transferred in 250ml measuring bottle, add water to scale, shake up, precision measures the tool plug dry test-tube that 1ml is placed in 10ml, add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, in 582nm place, measure absorbance, and according to the content of polysaccharide in typical curve calculating hand ginseng.
3. reappearance test
Get 6 parts, 120801 lot number hand ginseng medicinal material sample, by method under total determination of polysaccharide item, measure, calculate total polyoses content and relative standard deviation thereof.In Table 1, result shows that the method repeatability is good.
The total determination of polysaccharide reproducible test results of table 1
4. recovery test
Precision takes 6 parts, the same a collection of hand ginseng medicinal material sample of known total polyoses content, and (lot number: 120801), precision adds anhydrous dextrose reference substance respectively, by method mensuration under total determination of polysaccharide item, calculate recovery rate and relative standard deviation thereof.Result shows: this assay method measurement result is accurate.The results are shown in Table 2.
Table 2 recovery experimental result
5. three batches of total determination of polysaccharide of medicinal material
Get three batches of Tibetan medicine material hand ginsengs, by method under total determination of polysaccharide item, measure, calculate total polyoses content in hand ginseng.The results are shown in Table 3.
The total determination of polysaccharide result of table 3
Experimental example 3, hand ginseng characteristic peak are determined and gastrodin content assay method
1. instrument, reagent and test sample
Instrument: high performance liquid chromatograph: Agilent 1260 types; Electronic balance: Shimadzu AUW220D type.
Reference substance: (Chinese pharmaceutical biological product is identified institute, lot number: 110807-200205 to Gastrodin reference substance.）
Sample: hand ginseng medicinal material (Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), lot number is respectively: 120801,120802,120803,1200804,120805,120806.
2. liquid phase chromatogram condition
Take octadecylsilane chemically bonded silica as filling agent, detect wavelength 222nm, 40 ℃ of column temperatures, number of theoretical plate calculates and should be not less than 2000 by Gastrodin peak, take acetonitrile as mobile phase A, the 0.05mol/L potassium dihydrogen phosphate that every 100ml of take adds 0.1ml phosphoric acid is Mobile phase B, adopts mobile phase A, Mobile phase B to carry out gradient elution simultaneously
3. the preparation of object of reference solution
Get Gastrodin reference substance, add methyl alcohol and make every 1ml containing the solution of Gastrodin 0.1mg, obtain.
4. the preparation of need testing solution
Get hand ginseng medicinal material sample powder 2.0g, accurately weighed, precision adds water-saturated n-butanol solution 25ml, weighed weight, ultrasonic extraction 1h, lets cool, more weighed weight, supplies the weight of less loss with water-saturated n-butanol, shake up, filter, after filtrate evaporate to dryness, add methyl alcohol 5ml and dissolve, obtain.
5. hand ginseng medicinal material standard feature peak collection of illustrative plates is selected
Get 6 batches of Tibetan medicine hand ginseng medicinal materials, be prepared into respectively need testing solution, under above-mentioned liquid phase chromatogram condition, precision is drawn object of reference solution and each 10 μ l of need testing solution respectively, and injection liquid chromatography, records chromatogram, sees Fig. 1 and Fig. 2.Collection of illustrative plates according to 6 batch samples, the characteristic fingerprint pattern peak of selected hand ginseng medicinal material: should present 5 characteristic peaks in chromatogram, wherein 1 peak should be consistent with the retention time at object of reference Gastrodin peak, is designated as S peak, and calculate the ratio at all the other 4 characteristic peak relative retention times and S peak.
Result shows, the selected relative retention time at 5 common characteristic peaks and the RSD of relative peak area are all less than 5%, meet fingerprint map analyzing requirement.According to standard finger-print, the ratio at 5 characteristic peak relative retention times and S peak is: 1.00(peak 1), 1.83(peak 2), 5.14(peak 3), 5.65(peak 4), 6.12(peak 5), the results are shown in Table 4 and Fig. 2.
Table 4 common characteristic peak relative retention time table
6. hand is joined the content of Gastrodin in medicinal material
According to peak area, by external standard method, calculate, in above-mentioned 6 batches of hands ginseng medicinal material, the average content of Gastrodin is 0.321%.
Following examples all can realize the effect of above-mentioned experimental example, wherein
Hand ginseng control medicinal material: Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided, and lot number is 111021.
Hand ginseng medicinal material, Qinghai gold is scolded Tibetan medicine medicine company incorporated company and is provided, and lot number is 120801.
Embodiment 1
A, thin-layer chromatography are differentiated
Get hand ginseng control medicinal material powder 0.5g, hand ginseng sample powder 0.5g, operation repetitive is as follows: add water-saturated n-butanol 10ml, ultrasonic processing 30min, filter, filtrate evaporate to dryness, residue adds water 2ml to be made to dissolve, by D101 macroporous resin column (internal diameter 1.5cm, the high 12cm of post), 20ml washing, discard filtrate, with 70% ethanol 25ml wash-out, collect filtrate, evaporate to dryness, residue adds 70% methyl alcohol 1ml and dissolves, and makes respectively control medicinal material solution, need testing solution; Draw respectively each 10 μ l of above-mentioned 2 kinds of solution, put in silica G thin layer version, ethyl acetate-methanol-water mixture that the volume ratio of take is 9:1:0.2, as developping agent launches, takes out, and dries, and spray, with 10% phosphomolybdic acid ethanol solution, is heated to clear spot at 105 ℃.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
The assay of B, total polysaccharide
1) instrument, reagent and test sample
Instrument: electronic balance: Shimadzu AUW220D type; Shimadzu UV ?2450 type uv-spectrophotometric instrument.
Reference substance: anhydrous dextrose reference substance.(Chinese pharmaceutical biological product is identified institute; Lot number: 110833-200503)
2) preparation of contrast solution: get anhydrous dextrose reference substance 10mg, put in the volumetric flask of 100ml, be dissolved in water and be diluted to scale, shake up, obtain.
3) preparation of typical curve: precision measures above-mentioned contrast solution 0ml, 0.1ml, 0.3ml, 0.5ml, 0.8ml, 1ml, be placed in respectively the tool plug test tube of 10ml, respectively add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool, to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, at 582nm place, measure respectively absorbance, and draw absorbance-concentration standard curve;
4) determination method: get hand ginseng medicinal material sample fine powder 2g, accurately weighed, put in round-bottomed flask, add ethanol 100ml refluxing extraction 1 hour, after water bath heat preservation 2 hours, filter, discard filtrate, residue and filter paper are placed in to flask, adding distil water 100ml, heating and refluxing extraction 2 hours, filter while hot, residue and flask wash with water 3 times, each 20ml, merging filtrate and washing lotion, let cool, be transferred in 250ml measuring bottle, add water to scale, shake up, precision measures the tool plug dry test-tube that 1ml is placed in 10ml, add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, in 582nm place, measure absorbance, and according to the content of polysaccharide in typical curve calculating hand ginseng, result is 34.55%.
C, characteristic spectrum and gastrodin content detection method:
1) chromatographic condition and system flexibility: take octadecylsilane chemically bonded silica as filling agent, detect wavelength 222nm, 40 ℃ of column temperatures, number of theoretical plate calculates and should be not less than 2000 by Gastrodin peak, take acetonitrile as mobile phase A, the 0.05mol/L potassium dihydrogen phosphate that every 100ml of take adds 0.1ml phosphoric acid is Mobile phase B, adopt mobile phase A, Mobile phase B to carry out gradient elution simultaneously, 0min → 10min → 45min → 70min → 90min, according to the above-mentioned time period, mobile phase A: 3% → 3% → 17% → 17% → 25%; Mobile phase B: 97% → 97% → 83% → 83% → 75%;
2) preparation of reference substance solution: get Gastrodin reference substance, add methyl alcohol and dissolve and dilute, make the reference substance solution that every 1ml methyl alcohol contains 0.1mg Gastrodin;
3) preparation of need testing solution: get hand ginseng medicinal material sample powder 2g, accurately weighed, precision adds water-saturated n-butanol solution 25ml, weighed weight, ultrasonic extraction 1h, let cool, more weighed weight, with water-saturated n-butanol, supply the weight of less loss, shake up, filter, after filtrate evaporate to dryness, add methyl alcohol 5ml and dissolve, obtain;
4) determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In gained test sample characteristic spectrum, present 5 characteristic peaks, wherein 1 peak is corresponding with Gastrodin peak retention time, for S peak, calculate the ratio at other 4 characteristic peak relative retention times and S peak, 4 ratios are: 1.82,5.15,5.65,6.13, difference is in ± 10%, and simultaneously according to calculated by peak area gastrodin content, result is 0.323%.
Embodiment 2:
A, thin-layer chromatography are differentiated
Get hand ginseng control medicinal material powder 1g, hand ginseng sample powder 1g, operation repetitive is as follows: add water-saturated n-butanol 30ml, ultrasonic processing 40min, filter, filtrate evaporate to dryness, residue adds water 2ml to be made to dissolve, by D101 macroporous resin column (internal diameter 1.5cm, the high 12cm of post), 20ml washing, discard filtrate, with 60% ethanol 25ml wash-out, collect filtrate, evaporate to dryness, residue adds 70% methyl alcohol 1ml and dissolves, and makes respectively control medicinal material solution, need testing solution; Draw respectively each 10 μ l of above-mentioned 2 kinds of solution, put in silica G thin layer version, ethyl acetate-methanol-water mixture that the volume ratio of take is 8.5:1.2:0.3 is developping agent expansion, take out, dry, spray, with 10% phosphomolybdic acid ethanol solution, is heated to clear spot at 105 ℃.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
The assay of B, total polysaccharide
1) instrument, reagent and test sample
Instrument: electronic balance: Shimadzu AUW220D type; Shimadzu UV ?2450 type uv-spectrophotometric instrument.
Reference substance: anhydrous dextrose reference substance.(Chinese pharmaceutical biological product is identified institute; Lot number: 110833-200503)
2) preparation of contrast solution: get anhydrous dextrose reference substance 10mg, put in the volumetric flask of 100ml, be dissolved in water and be diluted to scale, shake up, obtain.
3) preparation of typical curve: precision measures above-mentioned contrast solution 0ml, 0.1ml, 0.3ml, 0.5ml, 0.8ml, 1ml, be placed in respectively the tool plug test tube of 10ml, respectively add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool, to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, at 582nm place, measure respectively absorbance, and draw absorbance-concentration standard curve;
4) determination method: get hand ginseng medicinal material sample fine powder 5g, accurately weighed, put in round-bottomed flask, add ethanol 100ml refluxing extraction 1 hour, after water bath heat preservation 2 hours, filter, discard filtrate, residue and filter paper are placed in to flask, adding distil water 100ml, heating and refluxing extraction 2 hours, filter while hot, residue and flask wash with water 3 times, each 20ml, merging filtrate and washing lotion, let cool, be transferred in 250ml measuring bottle, add water to scale, shake up, precision measures the tool plug dry test-tube that 1ml is placed in 10ml, add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, in 582nm place, measure absorbance, and according to the content of polysaccharide in typical curve calculating hand ginseng, result is 34.64%.
C, characteristic spectrum and gastrodin content detection method:
1) chromatographic condition and system flexibility: take octadecylsilane chemically bonded silica as filling agent, detect wavelength 222nm, 40 ℃ of column temperatures, number of theoretical plate calculates and should be not less than 2000 by Gastrodin peak, take acetonitrile as mobile phase A, the 0.05mol/L potassium dihydrogen phosphate that every 100ml of take adds 0.1ml phosphoric acid is Mobile phase B, adopt mobile phase A, Mobile phase B to carry out gradient elution simultaneously, 0min → 10min → 45min → 70min → 90min, according to the above-mentioned time period, mobile phase A: 3% → 3% → 17% → 17% → 25%; Mobile phase B: 97% → 97% → 83% → 83% → 75%;
2) preparation of reference substance solution: get Gastrodin reference substance, add methyl alcohol and dissolve and dilute, make the reference substance solution that every 1ml methyl alcohol contains 0.1mg Gastrodin;
3) preparation of need testing solution: get hand ginseng medicinal material sample powder 5g, accurately weighed, precision adds water-saturated n-butanol solution 25ml, weighed weight, ultrasonic extraction 1h, let cool, more weighed weight, with water-saturated n-butanol, supply the weight of less loss, shake up, filter, after filtrate evaporate to dryness, add methyl alcohol 5ml and dissolve, obtain;
4) determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In gained test sample characteristic spectrum, present 5 characteristic peaks, wherein 1 peak is corresponding with Gastrodin peak retention time, for S peak, calculate the ratio at other 4 characteristic peak relative retention times and S peak, 4 ratios are: 1.84,5.13,5.66,6.13, difference is in ± 10%, and simultaneously according to calculated by peak area gastrodin content, result is 0.320%.
Embodiment 3
A, thin-layer chromatography are differentiated
Get hand ginseng control medicinal material powder 0.2g, hand ginseng sample powder 0.2g, operation repetitive is as follows: add water-saturated n-butanol 10ml, ultrasonic processing 20min, filter, filtrate evaporate to dryness, residue adds water 2ml to be made to dissolve, by D101 macroporous resin column, 20ml washing, discards filtrate, with 80% ethanol 25ml wash-out, collect filtrate, evaporate to dryness, residue adds 80% methyl alcohol 1ml and dissolves, and makes respectively control medicinal material solution, need testing solution; Draw respectively each 10 μ l of above-mentioned 2 kinds of solution, put in silica G thin layer version, ethyl acetate-methanol-water mixture that the volume ratio of take is 9:0.8:0.2, as developping agent launches, takes out, and dries, and spray, with 10% phosphomolybdic acid ethanol solution, is heated to clear spot at 105 ℃.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
The assay of B, total polysaccharide
1) instrument, reagent and test sample
Instrument: electronic balance: Shimadzu AUW220D type; Shimadzu UV ?2450 type uv-spectrophotometric instrument.
Reference substance: anhydrous dextrose reference substance.(Chinese pharmaceutical biological product is identified institute; Lot number: 110833-200503)
2) preparation of contrast solution: get anhydrous dextrose reference substance 10mg, put in the volumetric flask of 100ml, be dissolved in water and be diluted to scale, shake up, obtain.
3) preparation of typical curve: precision measures above-mentioned contrast solution 0ml, 0.1ml, 0.3ml, 0.5ml, 0.8ml, 1ml, be placed in respectively the tool plug test tube of 10ml, respectively add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool, to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, at 582nm place, measure respectively absorbance, and draw absorbance-concentration standard curve;
4) determination method: get hand ginseng medicinal material sample fine powder 0.5g, accurately weighed, put in round-bottomed flask, add ethanol 100ml refluxing extraction 1 hour, after water bath heat preservation 2 hours, filter, discard filtrate, residue and filter paper are placed in to flask, adding distil water 100ml, heating and refluxing extraction 2 hours, filter while hot, residue and flask wash with water 3 times, each 20ml, merging filtrate and washing lotion, let cool, be transferred in 250ml measuring bottle, add water to scale, shake up, precision measures the tool plug dry test-tube that 1ml is placed in 10ml, add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, in 582nm place, measure absorbance, and according to the content of polysaccharide in typical curve calculating hand ginseng, result is 34.71%.
C, characteristic spectrum and gastrodin content detection method:
1) chromatographic condition and system flexibility: take octadecylsilane chemically bonded silica as filling agent, detect wavelength 222nm, 40 ℃ of column temperatures, number of theoretical plate calculates and should be not less than 2000 by Gastrodin peak, take acetonitrile as mobile phase A, the 0.05mol/L potassium dihydrogen phosphate that every 100ml of take adds 0.1ml phosphoric acid is Mobile phase B, adopt mobile phase A, Mobile phase B to carry out gradient elution simultaneously, 0min → 10min → 45min → 70min → 90min, according to the above-mentioned time period, mobile phase A: 3% → 3% → 17% → 17% → 25%; Mobile phase B: 97% → 97% → 83% → 83% → 75%;
2) preparation of reference substance solution: get Gastrodin reference substance, add methyl alcohol and dissolve and dilute, make the reference substance solution that every 1ml methyl alcohol contains 0.1mg Gastrodin;
3) preparation of need testing solution: get hand ginseng medicinal material sample powder 1g, accurately weighed, precision adds water-saturated n-butanol solution 25ml, weighed weight, ultrasonic extraction 1h, let cool, more weighed weight, with water-saturated n-butanol, supply the weight of less loss, shake up, filter, after filtrate evaporate to dryness, add methyl alcohol 5ml and dissolve, obtain;
4) determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In gained test sample characteristic spectrum, present 5 characteristic peaks, wherein 1 peak is corresponding with Gastrodin peak retention time, for S peak, calculate the ratio at other 4 characteristic peak relative retention times and S peak, 4 ratios are: 1.81,5.15,5.64,6.12, difference is in ± 10%, and simultaneously according to calculated by peak area gastrodin content, result is 0.325%.
Claims (4)
1. a detection method for hand ginseng medicinal material, is characterised in that, comprises one or more in following method:
The tlc identification method of A, hand ginseng medicinal material:
Get hand ginseng medicinal material fine powder 0.2-1g, add water-saturated n-butanol 10-30ml, ultrasonic processing 20-40min, filter, filtrate evaporate to dryness, residue adds water 2ml to be made to dissolve, by D101 macroporous resin column, first, with 20ml washing, discard filtrate, then with 60%-80% ethanol 25ml wash-out, collect filtrate, evaporate to dryness, residue adds 70%-80% methyl alcohol 1ml and dissolves, as need testing solution; Separately get hand ginseng control medicinal material, with legal system for control medicinal material solution; Draw respectively need testing solution, each 10 μ l of control medicinal material solution, point is in silica G thin layer version, ethyl acetate-methanol-water mixture that the volume ratio of take is 8.5-9:0.8-1.2:0.2-0.3 is developping agent expansion, take out, dry, spray, with 10% phosphomolybdic acid ethanol solution, is heated to clear spot at 105 ℃; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
The content determination of B, total polysaccharide:
1) preparation of contrast solution: get anhydrous dextrose reference substance, add the solution that water is mixed with 0.1mg/ml;
2) preparation of typical curve: precision measures above-mentioned contrast solution 0ml, 0.1ml, 0.3ml, 0.5ml, 0.8ml, 1ml, be placed in respectively the tool plug test tube of 10ml, respectively add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool, to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, at 582nm place, measure respectively absorbance, and draw absorbance-concentration standard curve;
3) need testing solution preparation and determination method: get hand ginseng medicinal material fine powder 0.5-5g, accurately weighed, put in round-bottomed flask, add ethanol 100ml refluxing extraction 1 hour, after water bath heat preservation 2 hours, filter, discard filtrate, residue and filter paper are placed in to flask, adding distil water 100ml, heating and refluxing extraction 2 hours, filter while hot, residue and flask wash with water 3 times, each 20ml, merging filtrate and washing lotion, let cool, be transferred in 250ml measuring bottle, add water to scale, shake up, precision measures the tool plug dry test-tube that 1ml is placed in 10ml, add water to 2.0ml, shake up, in ice bath, slowly drip w/v 0.2% By Anthrone Sulphuric acid solution to scale, mix, after letting cool to water bath heat preservation 10 minutes, take out, use immediately ice bath cooling 10 minutes, in 582nm place, measure absorbance, and according to the content of polysaccharide in typical curve calculating hand ginseng,
The content detection method of C, characteristic spectrum peak and Gastrodin:
1) chromatographic condition and system flexibility: take octadecylsilane chemically bonded silica as filling agent, detect wavelength 222nm, 40 ℃ of column temperatures, number of theoretical plate calculates and should be not less than 2000 by Gastrodin peak, take acetonitrile as mobile phase A, the 0.05mol/L potassium dihydrogen phosphate that every 100ml of take adds 0.1ml phosphoric acid is Mobile phase B, adopt mobile phase A, Mobile phase B to carry out gradient elution simultaneously, 0min → 10min → 45min → 70min → 90min, according to the above-mentioned time period, mobile phase A: 3% → 3% → 17% → 17% → 25%; Mobile phase B: 97% → 97% → 83% → 83% → 75%;
2) preparation of reference substance solution: get Gastrodin reference substance, add methyl alcohol and dissolve and dilute, make the reference substance solution that every 1ml methyl alcohol contains 0.1mg Gastrodin;
3) preparation of need testing solution: get hand ginseng medicinal material fine powder 1-5g, accurately weighed, precision adds water-saturated n-butanol solution 25ml, weighed weight, ultrasonic extraction 1h, let cool, more weighed weight, with water-saturated n-butanol, supply the weight of less loss, shake up, filter, after filtrate evaporate to dryness, add methyl alcohol 5ml and dissolve, obtain;
4) determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;
In gained test sample characteristic spectrum, present 5 characteristic peaks, wherein 1 peak is corresponding with Gastrodin peak retention time, for S peak, calculate the ratio at other 4 characteristic peak relative retention times and S peak, 4 ratios are: 1.83,5.14,5.65,6.12, difference is in ± 10%, simultaneously according to calculated by peak area gastrodin content.
2. the detection method of hand ginseng medicinal material according to claim 1, is characterised in that, tlc identification method is:
Get hand ginseng medicinal material fine powder 0.5g, add water-saturated n-butanol 10ml, ultrasonic processing 30min, filter, filtrate evaporate to dryness, residue adds water 2ml to be made to dissolve, by internal diameter, be 1.5cm, post height is the D101 macroporous resin column of 12cm, first with 20ml, washes, discard filtrate, with 70% ethanol 25ml wash-out, collect filtrate, evaporate to dryness again, residue adds 70% methyl alcohol 1ml and dissolves, as need testing solution; Separately get hand ginseng control medicinal material, with legal system for control medicinal material solution; Draw respectively need testing solution, each 10 μ l of control medicinal material solution, put in silica G thin layer version, ethyl acetate-methanol-water mixture that the volume ratio of take is 9:1:0.2 is developping agent expansion, take out, dry, spray, with 10% phosphomolybdic acid ethanol solution, is heated to clear spot at 105 ℃; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
3. the detection method of hand ginseng medicinal material according to claim 1, is characterised in that, in the preparation of content determination step 3) need testing solution and determination method of total polysaccharide, the sampling amount of hand ginseng medicinal material fine powder is 2g.
4. the detection method of hand ginseng medicinal material according to claim 1, is characterised in that, in the preparation of the content detection method step 3) need testing solution of characteristic spectrum peak and Gastrodin, the sampling amount of hand ginseng medicinal material fine powder is 2g.
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