CN102048906A - Content measurement method of abrus herb capsules - Google Patents
Content measurement method of abrus herb capsules Download PDFInfo
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- CN102048906A CN102048906A CN 201110001988 CN201110001988A CN102048906A CN 102048906 A CN102048906 A CN 102048906A CN 201110001988 CN201110001988 CN 201110001988 CN 201110001988 A CN201110001988 A CN 201110001988A CN 102048906 A CN102048906 A CN 102048906A
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- methanol
- apigenin
- glucoside
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Abstract
The invention discloses a content measurement method of abrus herb capsules. In the method, a high performance liquid chromatography-ultraviolet (HPLC-UV) method is used for measuring the contents of apigenin-6,8-di-C-glucoside, apigenin-6-C-arabinose-8-C-glucoside and apigenin-6-C-glucose-8-C-arabinoside in abrus mollis hance (monarch drug in a prescription) in the abrus herb capsules under the same chromatographic condition. The method comprises the following steps: preparing a test sample solution; preparing a control sample solution; and measuring by gradient elution. The method has the advantages of strong specificity and better accuracy, precision and reproducibility, is simple and easy to operate, and has important significance for further perfecting the quality standards of the abrus herb capsules.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of content assaying method of Herba abri capsule.
Background technology
Herba abri capsule be for Guangxi Yulin pharmaceutical Co. Ltd according to folk remedy, improve the Chinese medicine famous-brand and high-quality goods form through trial-production for many years, be national Chinese medicine protection kind, once obtained the national good quality products silver medal award.In producing and selling that reached for two more than ten years and clinical use, verified have a good and clinical curative effect, on market, enjoy certain good reputation, become at present that treatment is anxious in the Chinese patent medicine, chronic hepatitis is scorching, the cholecystitis person that belongs to the syndrome of dampness-heat of liver and gallbladder and common people are daily is used for the medicine commonly used that heat-clearing and toxic substances removing, liver heat removing protect the liver.We are made up of flavour of a drug such as hair Herba Abri, Fructus Gardeniae, Herba Artemisiae Scopariaes, have the function of depressed liver-energy dispersing and function of gallbladder promoting, heat-clearing and toxic substances removing, are applicable to acute and chronic hepatitis of treatment and cholecystitis.The hair Herba Abri is our monarch drug, and the sweet cold of nature and flavor has heat-clearing and toxic substances removing, the effect of soothing liver-QI dissipating blood stasis.The crude drug source of we Herba Abri is a Guangxi special product medical material hair Herba Abri (Abrus mollis Hance), have another name called Cortex Abri, cattle day rattan etc., hair Herba Abri medicinal part is the dry herb after pulse family Abrus vegetable hair Herba Abri is removed pod, mainly is distributed in areas such as Guangdong and Guangxi Provinces, Fujian and Hainan.
Herba abri capsule is the important kind of domestic liver and gall medication, and existing national standard is ministry standard (the 18th in Chinese traditional patent formulation preparation, 151 pages), and this standard has only been recorded the thin layer chromatography of square Chinese crude drug Herba Artemisiae Scopariae and differentiated item, does not include the assay item.That version medical material Herba Abri quality standard in 2005 of Chinese Pharmacopoeia has recorded is micro-, chemical method is differentiated and project such as extractum inspection, no assay item.The present invention is on to Guangxi special product medical material hair Herba Abri chemical constituent and quality controling research basis, adopt the HPLC-UV method under same chromatographic condition, to measure the apigenin-6 of monarch drug hair Herba Abri in the preparation Herba abri capsule, 8-two-C-glucoside (apigenin-6,8-di-C-β-D glucopyranoside, Vicenin, 1), apigenin-6-C-arabinose-8-C-glucoside (apigenin 6-C-α-L-arabinopyranosyl-8-C-β-D-glucopyranoside, Isoschaftoside, 2) apigenin-6-C-glucose-8-C-galactoside (apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside, Schaffoside, 3) content, at present, the bibliographical information correlation technique is not arranged.The structural formula of chemical compound 1～3 is specific as follows
[2,3]Shown in:
1：R
1＝Glc，R
2＝Glc；2：R
1＝Ara，R
2＝Glc；3：R
1＝Glc，R
2＝Ara；
Summary of the invention
The objective of the invention is to have defective at present Herba abri capsule quality testing, provide a kind of HPLC-UV of employing method under same chromatographic condition, to measure the apigenin-6 in the monarch drug hair Herba Abri in the Herba abri capsule, the content method of 8-two-C-glucoside, apigenin-6-C-arabinose-8-C-glucoside and apigenin-6-C-glucose-8-C-galactoside, this method specificity is strong, good stability, measure accurately, help the perfect of Herba abri capsule quality standard.
The objective of the invention is to realize in the following manner:
A kind of content assaying method of Herba abri capsule is characterized in that this method may further comprise the steps:
A) preparation of need testing solution: get the content of Herba abri capsule, porphyrize is got 0.2～0.8g, add the methanol 30～70ml more than 50%, reflux 1～3 hour filters, and residue washs several times with the methanol 10～20ml more than 50%, filter, merging filtrate and washing liquid and evaporate to dryness, residue add 4～6 dissolvings of 4～6ml moisture, quantitatively are added on polyamide column, left standstill 8～12 minutes, water 5～15ml eluting discards water lotion, reuse 50～80% methanol 30～50ml eluting, collect 50～80% meoh eluates, evaporate to dryness, residue add the methanol gradation dissolving more than 50%, quantitatively move in the 10ml measuring bottle, the methanol that adds more than 50% is diluted to scale, promptly;
B) preparation of reference substance solution: take by weighing apigenin-6,8-two-C-glucoside reference substance, apigenin-6-C-arabinose-8-C-glucoside reference substance and apigenin-6-C-glucose-8-C-galactoside reference substance are an amount of, the methanol that adds more than 50% is made the mixed solution that every 1ml contains 15～35 μ g, 35～65 μ g and 15～35 μ g respectively, shake up, promptly;
C) algoscopy: adopt accurate respectively reference substance solution and the need testing solution drawn of HPLC-UV method, inject chromatograph of liquid, measure, promptly;
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; (8～10: 1) being mobile phase A, is Mobile phase B with 1～3% acetic acid, and according to the form below carries out gradient elution with methanol-isopropyl alcohol; Flow velocity: 0.8～1.2ml/min; Column temperature: 35～40 ℃; The detection wavelength is 270～274nm;
It is 50～70% methanol that the methanol of above-mentioned employing concentration more than 50% is preferably concentration.
The content assaying method of above-mentioned Herba abri capsule preferably includes following steps:
A) content of Herba abri capsule is got in the preparation of need testing solution, and porphyrize is got 0.5g, add 50% methanol 50ml, reflux 2 hours filters, residue washs with 50% methanol 15ml gradation, filters merging filtrate and washing liquid and evaporate to dryness, residue adds moisture 4 times dissolving, and successively 2.0,1.0,1.0 1.0ml quantitatively is added on polyamide column, left standstill 10 minutes, water 10ml eluting discards water lotion, reuse 60% methanol 40ml eluting, collect 60% meoh eluate, evaporate to dryness, residue add 50% methanol gradation dissolving, quantitatively move in the 10ml measuring bottle, add 50% methanol and be diluted to scale, promptly;
B) preparation of reference substance solution takes by weighing apigenin-6,8-two-C-glucoside reference substance, apigenin-6-C-arabinose-8-C-glucoside reference substance and apigenin-6-C-glucose-8-C-galactoside reference substance are an amount of, add 50% methanol and make the mixed solution that every 1ml contains 20,40 and 20 μ g respectively, shake up, promptly;
C) algoscopy: adopt accurate respectively reference substance solution and the need testing solution drawn of HPLC-UV method, inject chromatograph of liquid, measure, promptly;
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-isopropyl alcohol (9: 1) is mobile phase A, is Mobile phase B with 2% acetic acid, and described 3 stages of according to the form below are carried out gradient elution; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 272nm;
Above-mentioned polyamide column internal diameter 1.0～1.5cm, length 10～15cm, polyamide 6 0～90 order adopts wet method dress post.
The concentration of methanol of the present invention is concentration of volume percent.
Beneficial effect of the present invention compared with the prior art: this law is for the apigenin of measuring in the Herba abri capsule-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14), apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) specificity is strong, accuracy, precision, repeatability are all better, is the simple and easy to do method that is worthy to be popularized, have great importance for the quality standard that further improves Herba abri capsule.
Below be the assay research process:
Instrument and reagent: Agilent 1100 type high performance liquid chromatographs: binary geopressure gradient pump, VWD UV-detector, G1316A type column oven, Chemstation chromatographic data processing system; Chromatographic column: Zorbax SB, C
18, 5 μ m, 4.6 * 250mm, AlltechAll-Guard pre-column (C
18); Mettler Toledo XS105DU electronic balance (0.01mg); Polyamide is that column chromatography is used (60～90 orders, the biochemical factory in Taizhou, Zhejiang); Methanol is chromatographically pure; Redistilled water.Apigenin-6,8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) reference substance: the self-control of Natural Medicine Chemistry teaching and research room of China Medicine University, purity＞98%; Other used reagents and solvent are analytical pure.Herba abri capsule: Yulin Pharmaceutical Co., Ltd., Guangxi produces, lot number: 250280,250277,250243,625075.
(1) extracts determining of solvent and extracting method
This law is investigated and is extracted the influence to assay of solvent, extracting method and extraction time.Index components apigenin-6,8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) dissolubility in methanol-water are better, investigate 50% methanol, 70% methanol, each reflux, extract, 2hr of methanol, carry out assay, the content value that the result records when being the reflux, extract, solvent with 50% methanol is the highest in accordance with the law; Investigating simultaneously with 50% methanol serves as to extract solvent, and supersound extraction 30min is to assay result's influence, and the result shows 50% methanol for extracting solvent, and supersound extraction assay value is starkly lower than reflux, extract; Relatively methanol eddy extracts, and adopts 2, different extraction times such as 3hr, the result show extract 2hr after, its content value does not have significant change.Result of the test sees Table 1.
Table 1 optimization for extracting condition result of the test table
Comprehensive above result of the test, the operation of steps such as filtration when considering test simultaneously, water bath method, determine that extracting method is: get the Herba abri capsule content, porphyrize, get 0.2～0.8g, preferred 0.5g, accurate claim surely, add more than 50% (preferred 50～70%, most preferably 50%) methanol 30～70ml, 1～3 hour (preferred 2 hours) of heating in water bath backflow, filter, residue filters with (preferred 50～70%) methanol 10～20ml gradation washing more than 50% (most preferably adopting 50% methanol 15ml gradation washing), merging filtrate and washing liquid are put evaporate to dryness in the water-bath, promptly.
(2) sample-pretreating method determines
Sample is through (preferred concentration 50～70% more than 50%, most preferable concentrations 50%) after methanol eddy extracts, direct injected adopts the HPLC-UV method to measure, and the result disturbs bigger on index components chromatographic peak position, needs to increase necessary separating step to remove interference.List of references adopts C respectively
18Solid-Phase Extraction and polyamide column chromatography carry out purification, and the result shows that the employing polyamide column chromatography carries out pretreatment, and the sample size value that records is higher, can remove the interference component in the sample preferably.
In order to determine the polyamide column chromatography elution requirement, methanol extract liquid with sample, water bath method, residue is dissolved in water, and quantitatively is added on the polyamide column (60～90 orders, the internal diameter 1.0cm that have handled well, length 10cm, wet method dress post), add water 20ml respectively, 20%MeOH 20ml, 30%MeOH 20ml, 40%MeOH 20ml, 50%MeOH 20ml, 60%MeOH20ml carries out gradient elution, collect eluent (5ml/ flow point), sample introduction HPLC examines knowledge respectively, and the position that the quilt that result's demonstration is tested elutes concentrates on 30% methanol position, and it is complete that eluting 20ml gets final product eluting.
Take all factors into consideration factor such as content difference and method accuracy in the sample, so determine preferred sample pretreating method be: sample is (preferred 50～70%) methanol extract liquid and washing liquid more than 50%, evaporate to dryness, residue adds moisture time dissolving (2.0,1.0,1.0,1.0ml), quantitatively be added on polyamide column (60～90 orders of having handled well, internal diameter 1.0cm, length 10cm, wet method dress post), water 5～15ml eluting, discard water lotion, reuse 50～80% methanol 30～50ml eluting is collected 50～80% meoh eluates, evaporate to dryness, residue adds (preferred 50～70%) methanol gradation dissolving more than 50%, quantitatively move in the 10ml measuring bottle, add that (preferred 50～70%) methanol is diluted to scale more than 50%, promptly.
Best practice is: sample 50% methanol extract liquid and washing liquid, evaporate to dryness in the water-bath, residue add moisture time dissolving (2.0,1.0,1.0,1.0ml), quantitatively be added on polyamide column (60～90 orders of having handled well, internal diameter 1.0cm, length 10cm, wet method dress post), water 10ml eluting discards water lotion, reuse 60% methanol 40ml eluting, collect 60% meoh eluate, put evaporate to dryness in the water-bath, residue adds 50% methanol gradation dissolving, quantitatively move in the 10ml measuring bottle, add 50% methanol and be diluted to scale, promptly.
Below adopt optimal case of the present invention to carry out the system suitability experiment:
1) preparation of need testing solution
Get the content of Herba abri capsule, porphyrize is got about 0.5g, the accurate title, decide, and adds 50% methanol 50ml, and heating in water bath refluxed 2 hours, filter, residue washs with 50% methanol 15ml gradation, filters, merging filtrate and washing liquid are put evaporate to dryness in the water-bath, and residue adds moisture time dissolving (2.0,1.0,1.0,1.0ml), quantitatively be added on the polyamide column (60～90 orders, the internal diameter 1.0cm that have handled well, length 10cm, wet method dress post), left standstill water 10ml eluting 10 minutes, discard water lotion, reuse 60% methanol 40ml eluting is collected 60% meoh eluate, puts evaporate to dryness in the water-bath, residue adds 50% methanol gradation dissolving, quantitatively move in the 10ml measuring bottle, add 50% methanol and be diluted to scale, promptly.
2) preparation of reference substance solution
Precision takes by weighing through the apigenin of phosphorus pentoxide vacuum drying 24hr-6,8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2), and apigenin-6-C-glucose-8-C-galactoside (3) reference substance each is an amount of, add 50% methanol and make the mixing reference substance solution that every 1ml contains 33.0 μ g (1), 62.0 μ g (2), 31.8 μ g (3), promptly.
3) preparation of negative control solution
Form by prescription, get all the other outer flavour of a drug of defeathering Herba Abri, make the simulation preparation that does not contain mao Herba Abri, prepare negative control solution by the method under the need testing solution preparation by preparation technology's requirement.
4) ultraviolet detection wavelength determination
Precision takes by weighing apigenin-6, each is an amount of for 8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) reference substance, add 50% dissolve with methanol respectively and make the reference substance solution of 20 μ g/ml, in wavelength 200～500nm scope, scan respectively according to ultraviolet visible spectrophotometry (" two appendix IVA of Chinese pharmacopoeia version in 2005), the results are shown in Figure 1～4 and table 2.
The ultraviolet maximum absorption wavelength table of table 2 chemical compound 1～3
5) chromatographic condition and system suitability test
Chromatographic condition preferably adopt methanol-2% acetic acid respectively, acetonitrile-2% acetic acid, flow phase system such as acetonitrile-0.2% phosphoric acid, the chromatographic condition that the factors such as different chromatographic columns, column temperature of investigating are simultaneously determined influences such as chromatographic peak separating degree and symmetries is: Agilent Zorbax C
18(4.6 * 250mm, 5 μ m), Alltech All-guard C
18Guard column; With methanol-isopropyl alcohol (9: 1) is mobile phase A, is Mobile phase B with 2% acetic acid, carries out gradient elution by table 3, after 40min, and design high concentration organic facies chromatographic column cleaning procedure; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 272nm; Sampling volume: 20 μ l.
Table 3 gradient elution flow sheet
At above-mentioned chromatographic condition, chemical compound 1～3 all can reach baseline separation with other components, separating degree (R) all＞1.5, the chromatographic performance parameter of the index chromatographic peak of need testing solution sees Table 4.Reference substance mixed solution, Herba abri capsule need testing solution, and a HPLC collection of illustrative plates that lacks hair Herba Abri negative control solution see Fig. 4～6.By another chromatographic column (Shimadzu CLC C
186.0 the index components apigenin-6 of * theoretical tray value minimum that 150mm) records, the theoretical cam curve n=6000 of 8-two-C-glucoside (1), chemical compound 1-3 chromatographic peak all can all can reach separating degree (R＞1.5) preferably with other components.Press apigenin-6 so determine number of theoretical plate, 8-two-C-glucoside peak calculates, and should be not less than 6000.
The result shows that the retention time of each index chromatographic peak of need testing solution is consistent with reference substance, and negative control is noiseless.
Table 4 chromatographic performance parameter list
6) investigation of linear relationship
Precision takes by weighing through the apigenin of phosphorus pentoxide vacuum drying 24hr-6,8-two-C-glucoside (1) reference substance 6.10mg, apigenin-6-C-arabinose-8-C-glucoside (2) reference substance 5.80mg, apigenin-6-C-glucose-8-C-galactoside (3) reference substance 5.85mg, put in the same 25ml volumetric flask, add 50% dissolve with methanol and be diluted to scale, shake up, product mixing stock solution (chemical compound 1～3 concentration is respectively 244.0,232.0 and 234 μ g/ml) in contrast.Accurate to draw above-mentioned reference substance mixing stock solution an amount of, adds 50% methanol and dilute successively and be original content 1/2,1/4,1/8,1/16,1/32,1/64 reference substance mixed diluting solution is drawn above-mentioned each concentration and is mixed each 20 μ l of reference substance solution, by above-mentioned chromatographic condition difference sample introduction, the record chromatogram, (μ g) is abscissa with sample size, and peak area integrated value (A) is an ordinate, the drawing standard curve, data see Table 5～7, the results are shown in Figure 7～9.
Table 5 apigenin-6,8-two-C-glucoside (1) standard curve data
Table 6 apigenin-6-C-arabinose-8-C-glucoside (2) standard curve data
Table 7 apigenin-6-C-glucose-8-C-galactoside (3) standard curve data
The result shows, apigenin-6,8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) sample size is respectively 0.08～4.88,0.07～4.64, and has good linear relationship in 0.07～4.68 μ g scope.
7) minimum detectable level is measured and quantitative limit mensuration
Get above mixing reference substance solution, progressively dilute with 50% methanol, sample introduction analysis in accordance with the law, sample size when getting (S/N 〉=10) and (S/N 〉=3) signal to noise ratio is respectively minimum quantitative limit and detectability, apigenin-6, the minimum quantitative limit of 8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) is respectively 0.019,0.018,0.018 μ g, and lowest detectable limit all is about 0.01 μ g.
8) precision test
The above-mentioned mixing reference substance solution continuous sample introduction of accurate absorption 6 times, calculate the relative standard deviation (RSD) of the peak area of each index chromatographic peak, apigenin-6, the RSD value of 8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) is respectively 0.70,0.92, and 0.90%, show that this law precision is better.
9) stability test
Get the preparation Herba abri capsule, prepare need testing solution as stated above, respectively at 0,1,2,4,8,16h measures in accordance with the law, calculates the RSD (%) of each peak-to-peak area, apigenin-6 as a result, RSD (%) value of 8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) is respectively 1.92,2.17 and 0.90, and the result shows, need testing solution is basicly stable in placing 16hr, and the suggestion need testing solution is measured in placing 16hr.
10) repeatability test
Precision takes by weighing 6 parts of same lot number formulation samples, press under the assay item operation and apigenin-6 in the working sample in accordance with the law respectively, 8-two-C-glucoside (1), the content of apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3), apigenin-6 as a result, 8-two-C-glucoside (1), RSD (%) value of apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) is respectively 1.32,1.27,1.35, relative standard deviation all＜2%, the result shows that this law repeatability is better.
11) average recovery
Adopt the application of sample absorption method, get 6 parts of (1:0.133% of Herba abri capsule sample of known content; 2:0.143%; 3:0.087%), add apigenin-6 respectively, 8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3) reference substance are an amount of, measure by the method under the above-mentioned assay item in accordance with the law, calculate recovery rate, calculate recovery rate the results are shown in Table 8～10.Apigenin-6, the average recovery rate of 8-two-C-glucoside (1) is 99.10%, RSD=2.12%; The average recovery rate of apigenin-6-C-arabinose-8-C-glucoside (2) is 97.79%, RSD=2.79%; The average recovery rate of apigenin-6-C-glucose-8-C-galactoside (3) is 98.74%, RSD=4.32%.The result shows that this law has the good response rate, and accuracy is better.
Table 8 apigenin-6,8-two-C-glucoside (1) determination of recovery rates result
Table 9 apigenin-6-C-arabinose-8-C-glucoside (2) determination of recovery rates result
Table 10 apigenin-6-C-glucose-8-C-galactoside (3) determination of recovery rates result
12) sample determination
Get different lot number Herba abri capsule samples, the sample of each lot number respectively takes by weighing two parts, measure by method under the assay item in accordance with the law, apigenin in the calculation sample-6,8-two-C-glucoside (1), apigenin-6-C-arabinose-8-C-glucoside (2) and apigenin-6-C-glucose-8-C-galactoside (3).The results are shown in Table 11.
Table 11 Herba abri capsule assay result
The method of utilize setting up is measured the Herba abri capsule of different lot numbers, and from the result of assay as can be seen, the content difference of chemical compound 1-3 is bigger in the formulation samples of different lot numbers.The content limit of suggestion Herba abri capsule is: content contains apigenin-6,8-two-C-glucoside (C
27H
30O
15) must not be less than 0.03%, apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) must not be less than 0.03%, apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) must not be less than 0.02%.
Description of drawings
Fig. 1 apigenin-6, and the uv absorption spectra of 8-two-C-glucoside (1) (20.25 μ g/ml, 50%MeOH)
The uv absorption spectra of Fig. 2 apigenin-6-C-arabinose-8-C-glucoside (2) (20.21 μ g/ml, 50%MeOH)
The uv absorption spectra of Fig. 3 apigenin-6-C-glucose-8-C-galactoside (3) (21.53 μ g/ml, 50%MeOH)
Fig. 4 reference substance mixed solution HPLC chromatogram
Wherein, 1 is apigenin-6,8-two-C-glucoside
2 is apigenin-6-C-arabinose-8-C-glucoside
3 is apigenin-6-C-glucose-8-C-galactoside
Fig. 5 Herba abri capsule need testing solution HPLC chromatogram
Wherein, 1 is apigenin-6,8-two-C-glucoside
2 is apigenin-6-C-arabinose-8-C-glucoside
3 is apigenin-6-C-glucose-8-C-galactoside
Fig. 6 lacks Herba Abri negative control solution HPLC chromatogram
Fig. 7 apigenin-6,8-two-C-glucoside (1) canonical plotting
Fig. 8 apigenin-6-C-arabinose-8-C-glucoside (2) canonical plotting
Fig. 9 apigenin-6-C-glucose-8-C-galactoside (3) canonical plotting
The specific embodiment
Further set forth the present invention in the following manner:
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
A) content of Herba abri capsule is got in the preparation of need testing solution, and porphyrize is got about 0.5g, the accurate title, decide, and adds 50% methanol 50ml, and heating in water bath refluxed 2 hours, filter, residue washs several times with 50% methanol 15ml, filters, merging filtrate and washing liquid are put evaporate to dryness in the water-bath, and residue adds moisture time dissolving (2.0,1.0,1.0,1.0ml), quantitatively be added on the polyamide column (60-90 order, the internal diameter 1.0cm that have handled well, length 10cm, wet method dress post), left standstill water 10ml eluting 10 minutes, discard water lotion, reuse 60% methanol 40ml eluting is collected 60% meoh eluate, puts evaporate to dryness in the water-bath, residue adds 50% methanol gradation dissolving, quantitatively move in the 10ml measuring bottle, add 50% methanol and be diluted to scale, promptly.
B) preparation of reference substance solution takes by weighing apigenin-6,8-two-C-glucoside reference substance, apigenin-6-C-arabinose-8-C-glucoside reference substance and apigenin-6-C-glucose-8-C-galactoside reference substance are an amount of, the accurate title, decide, add 50% methanol and make the mixed solution that every 1ml contains 20,40 and 20 μ g respectively, shake up, promptly.
C) algoscopy: adopt accurate respectively reference substance solution and the need testing solution drawn of HPLC-UV method, inject chromatograph of liquid, measure, promptly;
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-isopropyl alcohol (9: 1) is mobile phase A, is Mobile phase B with 2% acetic acid, and according to the form below carries out gradient elution; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 272nm.Number of theoretical plate is pressed apigenin-6, and 8-two-C-glucoside peak calculates and is not less than 6000.
Record that content contains apigenin-6 in the Herba abri capsule, 8-two-C-glucoside (C
27H
30O
15) be no less than 0.03%, apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) be no less than 0.03%, apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) be no less than 0.02%.
Adopt this method to measure apigenin-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) and apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) lowest detectable limit all be about 0.01 μ g, 3 kinds of solution are all stable in 16 hours, apigenin-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) and apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) precision all good.
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
A) content of Herba abri capsule is got in the preparation of need testing solution, and porphyrize is got about 0.6g, the accurate title, decide, and adds 70% methanol 50ml, and heating in water bath refluxed 3 hours, filter, residue washs with 70% methanol 15ml gradation, filters, merging filtrate and washing liquid are put evaporate to dryness in the water-bath, and residue adds moisture time dissolving (2.0,1.0,1.0,1.0ml), quantitatively be added on the polyamide column (60-90 order, the internal diameter 1.0cm that have handled well, length 10cm, wet method dress post), left standstill water 15ml eluting 10 minutes, discard water lotion, reuse 70% methanol 30ml eluting is collected 70% meoh eluate, puts evaporate to dryness in the water-bath, residue adds 60% methanol gradation dissolving, quantitatively move in the 10ml measuring bottle, add 60% methanol and be diluted to scale, promptly.
B) preparation of reference substance solution takes by weighing apigenin-6,8-two-C-glucoside reference substance, apigenin-6-C-arabinose-8-C-glucoside reference substance and apigenin-6-C-glucose-8-C-galactoside reference substance are an amount of, the accurate title, decide, add 60% methanol and make the mixed solution that every 1ml contains 30,60 and 30 μ g respectively, shake up, promptly.
C) algoscopy: adopt accurate respectively reference substance solution and the need testing solution drawn of HPLC-UV method, inject chromatograph of liquid, measure, promptly;
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-isopropyl alcohol (9: 1) is mobile phase A, is Mobile phase B with 2% acetic acid, and according to the form below carries out gradient elution; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 272nm.Number of theoretical plate is pressed apigenin-6, and 8-two-C-glucoside peak calculates and is not less than 6000.
Record that content contains apigenin-6 in the Herba abri capsule, 8-two-C-glucoside (C
27H
30O
15) be no less than 0.03%, apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) be no less than 0.03%, apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) be no less than 0.02%.
Adopt this method to measure apigenin-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) and apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) lowest detectable limit all be about 0.01 μ g, 3 kinds of solution are all stable in 16 hours, apigenin-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) and apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) precision all good.
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
A) content of Herba abri capsule is got in the preparation of need testing solution, and porphyrize is got about 0.4g, the accurate title, decide, and adds 50% methanol 50ml, and heating in water bath refluxed 2 hours, filter, residue washs with 30% methanol 20ml gradation, filters, merging filtrate and washing liquid are put evaporate to dryness in the water-bath, and residue adds moisture time dissolving (2.0,1.0,1.0,1.0ml), quantitatively be added on the polyamide column (60-90 order, the internal diameter 1.0cm that have handled well, length 10cm, wet method dress post), left standstill water 8ml eluting 10 minutes, discard water lotion, reuse 50% methanol 40ml eluting is collected 50% meoh eluate, puts evaporate to dryness in the water-bath, residue adds 40% methanol gradation dissolving, quantitatively move in the 10ml measuring bottle, add 40% methanol and be diluted to scale, promptly.
B) preparation of reference substance solution takes by weighing apigenin-6,8-two-C-glucoside reference substance, apigenin-6-C-arabinose-8-C-glucoside reference substance and apigenin-6-C-glucose-8-C-galactoside reference substance are an amount of, the accurate title, decide, add 40% methanol and make the mixed solution that every 1ml contains 25,35 and 25 μ g respectively, shake up, promptly.
C) algoscopy: adopt accurate respectively reference substance solution and the need testing solution drawn of HPLC-UV method, inject chromatograph of liquid, measure, promptly;
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-isopropyl alcohol (9: 1) is mobile phase A, is Mobile phase B with 2% acetic acid, and according to the form below carries out gradient elution; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 272nm.Number of theoretical plate is pressed apigenin-6, and 8-two-C-glucoside peak calculates and is not less than 6000.
Record that content contains apigenin-6 in the Herba abri capsule, 8-two-C-glucoside (C
27H
30O
15) be no less than 0.03%, apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) be no less than 0.03%, apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) be no less than 0.02%.
Adopt this method to measure apigenin-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) and apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) lowest detectable limit all be about 0.01 μ g, 3 kinds of solution are all stable in 16 hours, apigenin-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) and apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) precision all good.
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
A) content of Herba abri capsule is got in the preparation of need testing solution, and porphyrize is got about 0.8g, the accurate title, decide, and adds methanol 70ml, and heating in water bath refluxed 1 hour, filter, residue filters with methanol 15ml gradation washing, merging filtrate and washing liquid are put evaporate to dryness in the water-bath, and residue adds moisture time dissolving (2.0,1.0,1.0,1.0ml), quantitatively be added on the polyamide column (60-90 order, the internal diameter 1.0cm that have handled well, length 10cm, wet method dress post), left standstill water 5ml eluting 12 minutes, discard water lotion, reuse 80% methanol 50ml eluting is collected 80% meoh eluate, puts evaporate to dryness in the water-bath, residue adds methanol gradation dissolving, quantitatively move in the 10ml measuring bottle, add methanol and be diluted to scale, promptly.
B) preparation of reference substance solution takes by weighing apigenin-6,8-two-C-glucoside reference substance, apigenin-6-C-arabinose-8-C-glucoside reference substance and apigenin-6-C-glucose-8-C-galactoside reference substance are an amount of, the accurate title, decide, add methanol and make the mixed solution that every 1ml contains 20,40 and 20 μ g respectively, shake up, promptly.
C) algoscopy: adopt accurate respectively reference substance solution and the need testing solution drawn of HPLC-UV method, inject chromatograph of liquid, measure, promptly;
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-isopropyl alcohol (9: 1) is mobile phase A, is Mobile phase B with 2% acetic acid, and according to the form below carries out gradient elution; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 272nm.Number of theoretical plate is pressed apigenin-6, and 8-two-C-glucoside peak calculates and is not less than 6000.
Record that content contains apigenin-6 in the Herba abri capsule, 8-two-C-glucoside (C
27H
30O
15) be no less than 0.03%, apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) be no less than 0.03%, apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) be no less than 0.02%.
Adopt this method to measure apigenin-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) and apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) lowest detectable limit all be about 0.01 μ g, 3 kinds of solution are all stable in 16 hours, apigenin-6,8-two-C-glucoside (C
27H
30O
15), apigenin-6-C-arabinose-8-C-glucoside (C
26H
28O
14) and apigenin-6-C-glucose-8-C-galactoside (C
26H
28O
14) precision all good.
List of references
[1] China Medicine University. the Chinese medicine Ci hai. Beijing: Chinese Medicine science and technology publishing house, 1996:506,1114.
[2] Liu Zhuowei, fault million unicorns, Ye Zhiwen, Zhang Xiaoqi, Wang Hao, leaf literary talent, Zhao Shouxun. the chemical constituent of hair Herba Abri aerial parts, Chinese natural drug, 2008,6 (6): 416～419.
[3] Wang Hao, Xiong Fei, Liu Zhuowei, the leaf literary talent, Zhang Luyong, still quiet, Jiang Zhenzhou, Zhao Shouxun. the application of flavone c-glycosides in preparation treatment and prevention hepatitis medicament, Chinese invention patent, CN101161668.
[4] Wang Hao. etc. Canton love-pea vine total flavone c-glycosides effective part, Preparation Method And The Use, Chinese invention patent, CN101250207.
Claims (4)
1. the content assaying method of a Herba abri capsule is characterized in that this method may further comprise the steps:
A) preparation of need testing solution: get the content of Herba abri capsule, porphyrize is got 0.2～0.8g, add the methanol 30～70ml more than 50%, reflux 1～3 hour filters, and residue washs several times with the methanol 10～20ml more than 50%, filter, merging filtrate and washing liquid and evaporate to dryness, residue add 4～6 dissolvings of 4～6ml moisture, quantitatively are added on polyamide column, left standstill 8～12 minutes, water 5～15ml eluting discards water lotion, reuse 50～80% methanol 30～50ml eluting, collect 50～80% meoh eluates, evaporate to dryness, residue add the methanol gradation dissolving more than 50%, quantitatively move in the 10ml measuring bottle, the methanol that adds more than 50% is diluted to scale, promptly;
B) preparation of reference substance solution: take by weighing apigenin-6,8-two-C-glucoside reference substance, apigenin-6-C-arabinose-8-C-glucoside reference substance and apigenin-6-C-glucose-8-C-galactoside reference substance are an amount of, the methanol that adds more than 50% is made the mixed solution that every 1ml contains 15～35 μ g, 35～65 μ g and 15～35 μ g respectively, shake up, promptly;
C) algoscopy: adopt accurate respectively reference substance solution and the need testing solution drawn of HPLC-UV method, inject chromatograph of liquid, measure, promptly;
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; (8～10: 1) being mobile phase A, is Mobile phase B with 1～3% acetic acid, and according to the form below carries out gradient elution with methanol-isopropyl alcohol; Flow velocity: 0.8～1.2ml/min; Column temperature: 35～40 ℃; The detection wavelength is 270～274nm;
2. the content assaying method of Herba abri capsule according to claim 1 is characterized in that the described concentration of this method is that all to be specially concentration be 50～70% methanol for methanol more than 50%.
3. the content assaying method of Herba abri capsule according to claim 1 is characterized in that this method may further comprise the steps:
A) preparation of need testing solution: get the content of Herba abri capsule, porphyrize is got 0.5g, add 50% methanol 50ml, reflux 2 hours filters, residue washs several times with 50% methanol 15ml, filter, merging filtrate and washing liquid and evaporate to dryness, residue add 4 dissolvings of moisture, successively 2.0,1.0,1.0,1.0ml, quantitatively be added on polyamide column, left standstill 10 minutes, water 10ml eluting, discard water lotion, reuse 60% methanol 40ml eluting is collected 60% meoh eluate, evaporate to dryness, residue adds 50% methanol gradation dissolving, quantitatively move in the 10ml measuring bottle, add 50% methanol and be diluted to scale, promptly;
B) preparation of reference substance solution takes by weighing apigenin-6,8-two-C-glucoside reference substance, apigenin-6-C-arabinose-8-C-glucoside reference substance and apigenin-6-C-glucose-8-C-galactoside reference substance are an amount of, add 50% methanol and make the mixed solution that every 1ml contains 20,40 and 20 μ g respectively, shake up, promptly;
C) algoscopy: adopt accurate respectively reference substance solution and the need testing solution drawn of HPLC-UV method, inject chromatograph of liquid, measure, promptly;
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With methanol-isopropyl alcohol (9: 1) is mobile phase A, is Mobile phase B with 2% acetic acid, and described 3 stages of according to the form below are carried out gradient elution; Flow velocity: 1.0ml/min; Column temperature: 40 ℃; The detection wavelength is 272nm;
4. according to the content assaying method of claim 1,2 or 3 described Herba abri capsules, it is characterized in that described polyamide column internal diameter 1.0～1.5cm, length 10～15cm, polyamide 6 0～90 order adopts wet method dress post.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103751359A (en) * | 2014-01-28 | 2014-04-30 | 纪磊 | Traditional Chinese medicine preparation for treating chronic hepatitis and chronic cholecystitis |
| CN105319333A (en) * | 2015-11-11 | 2016-02-10 | 广西中医药大学 | Quality detection method for Jingxuening capsule |
| CN110927291A (en) * | 2019-12-20 | 2020-03-27 | 成都普思生物科技股份有限公司 | Detection method of abrus herb extract characteristic spectrum |
| CN111024875A (en) * | 2019-12-31 | 2020-04-17 | 广东药科大学 | Construction method of abrus cantoniensis hance amide component liquid chromatography fingerprint |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250207A (en) * | 2008-04-15 | 2008-08-27 | 中国药科大学 | Canton love-pea vine total flavone c-glycosides effective part, preparation method and use thereof |
-
2011
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250207A (en) * | 2008-04-15 | 2008-08-27 | 中国药科大学 | Canton love-pea vine total flavone c-glycosides effective part, preparation method and use thereof |
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| 《中华人民共和国药典2000年版一部》 20001231 国家药典委员会 鸡骨草 151 1－4 , 1 * |
| 《中药成方制剂第18册》 19981231 卫生部 鸡骨草胶囊 151 1－4 , 1 * |
| 《安徽医药》 20091231 徐成志 鸡骨草胶囊质量标准研究 1496-1498 1－4 第13卷, 第12期 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103751359A (en) * | 2014-01-28 | 2014-04-30 | 纪磊 | Traditional Chinese medicine preparation for treating chronic hepatitis and chronic cholecystitis |
| CN105319333A (en) * | 2015-11-11 | 2016-02-10 | 广西中医药大学 | Quality detection method for Jingxuening capsule |
| CN105319333B (en) * | 2015-11-11 | 2017-04-26 | 广西中医药大学 | Quality detection method for Jingxuening capsule |
| CN110927291A (en) * | 2019-12-20 | 2020-03-27 | 成都普思生物科技股份有限公司 | Detection method of abrus herb extract characteristic spectrum |
| CN111024875A (en) * | 2019-12-31 | 2020-04-17 | 广东药科大学 | Construction method of abrus cantoniensis hance amide component liquid chromatography fingerprint |
| CN111024875B (en) * | 2019-12-31 | 2022-06-17 | 广东药科大学 | Construction method of abrus cantoniensis hance amide component liquid chromatography fingerprint |
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