CN101788537A - Method for measuring content of ellagic acid ingredients in euscaphis japonica medicinal materials - Google Patents
Method for measuring content of ellagic acid ingredients in euscaphis japonica medicinal materials Download PDFInfo
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Abstract
The invention provides a method for measuring content of ellagic acid ingredients in euscaphis japonica medicinal materials, which synchronously measures the contents of at least two of five ingredients of ellagic acid, 3,3'-dimethoxy ellagic acid, 3,3'-dimethoxy ellagic acid-4'-O-beta-D-xyloside, 3,3'-dimethoxy ellagic acid-4'-O-beta-D-glucoside and 3,3'-dimethoxy ellagic acid-4'-O-alpha-D-arabinoside contained in the euscaphis japonica medicinal materials by using a high efficiency liquid chromatography or an ultra-high efficiency liquid chromatography under the same chromatogram condition. In the invention, the contents of the multiple ingredients in the euscaphis japonica medicinal materials are measured under the same chromatogram condition so that the method for measuring the content of the ellagic acid ingredient in the euscaphis japonica medicinal materials has better integrity, characteristics and stability as well as high precision, favorable repeatability and high recovery rate and capability of effectively controlling the quality of the euscaphis japonica medicinal materials, thereby ensuring the effectiveness and the safety of clinical medication and laying the foundation for redevelopment and application of the euscaphis japonica medicinal materials.
Description
Technical field:
The present invention relates to the content assaying method of ellagic acid constituents in a kind of corn eyeball medicinal material, belong to the technical field of Chinese crude drug being carried out detection by quantitative.
Background technology:
The corn eyeball is the wounded in the battle or branches and leaves really of wild crow Chinese toon Euscaphis japonica (Thunb.) Dippel of Staphyleaceae plant, its former each province, the plant distribution south of the River and northeast of Yunnan, record in " Guizhou Province's Chinese crude drug, national quality of medicinal material standard " (version in 2003), " Sichuan Chinese medicinal herbal " uses its fruits and seeds.The corn eyeball is the Guizhou minority nationality medication, property suffering, sweet, flat, tool regulating qi-flowing for relieving pain, dispersing swelling and dissipating binds, the effect of dispellingging wind and arresting itching now is one of main Chinese medicinal materials in the drug standards WS-10108 of State Food and Drug Administration (ZD-0108)-2002 (trying) the blood vessels open capsule.
Blood vessels open capsule (WS-10108 (ZD-0108)-2002) has promoting blood circulation and removing blood stasis, clearing and activating the channels and collaterals function, and clinical coronary heart disease, the angina pectoris of being used for, heart blood silt has obtained one of national inventing patent (ZL02133416.1).It is less about the research of corn eyeball medicinal material that but literature survey is found, the fundamental research weakness, lack reliable qualitative-and-quantitative method and operational reference substance, only it has been carried out simple thin layer in " Guizhou Province's Chinese crude drug, national quality of medicinal material standard " (version in 2003) and differentiated.Therefore with the corn eyeball preparation blood vessels open capsule of main ingredient exploitation, its quality control standard is comparatively simple, particularly prescription main ingredient corn eyeball is not carried out the detection of projects such as assay, thereby can not guarantee the validity and the security of its clinical application well, need further research, improve its quality control standard.
Summary of the invention:
The objective of the invention is to: the content assaying method that ellagic acid constituents in a kind of corn eyeball medicinal material is provided.The present invention carries out assay simultaneously to the multiple composition in the corn eyeball medicinal material under same chromatographic condition, make that the content assaying method of ellagic acid constituents has more globality, characteristic and stability in the corn eyeball medicinal material, use for the redevelopment of the quality of effective control corn eyeball medicinal material and corn eyeball medicinal material and lay a good foundation.
Corn eyeball of the present invention is the wounded in the battle or branches and leaves really of wild crow Chinese toon Euscaphis japonica (Thunb.) Dippel of Staphyleaceae plant.
The present invention is achieved in that the content assaying method of ellagic acid constituents in the corn eyeball medicinal material is: adopt high performance liquid chromatography or Ultra Performance Liquid Chromatography method, under same chromatographic condition to ellagic acid, 3 contained in the corn eyeball medicinal material, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-at least two compositions in O-α-five compositions of D-Arabinoside carry out assay simultaneously.
Described content assaying method is with ellagic acid, 3,3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-in O-α-five kinds of reference substances of D-Arabinoside at least two kinds are for contrast, with acetonitrile: H
3PO
4Aqueous solution=6～100: 94～0 is the high performance liquid chromatography or the Ultra Performance Liquid Chromatography method of eluent gradient wash-out.
Concrete content assaying method may further comprise the steps:
(1) chromatographic condition: chromatographic column is a BEH C18 post; Acetonitrile-H
3PO
4The aqueous solution gradient elution; Column temperature is 20 ℃～50 ℃; The detection wavelength is 220～480nm;
(2) preparation of reference substance solution: take by weighing ellagic acid, 3 respectively, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-in O-α-D-Arabinoside reference substance at least two kinds, add in the rearmounted same volumetric flask of dissolve with methanol, add methyl alcohol and be diluted to scale, get reference substance solution;
(3) preparation of need testing solution: it is an amount of to get corn eyeball medicinal powder, and accurate the title decides, and it is an amount of to add methyl alcohol, extracts 2～8h, concentrates the back with the methyl alcohol dilution, gets need testing solution;
(4) assay method: measure according to the described chromatographic condition of step (1), promptly.
The chromatographic condition of abovementioned steps (1) and system suitability test are:
Instrument: Waters ACQUITY UPLC chromatograph;
Chromatographic column: ACQUITY UPLC BEH C18 post;
Moving phase: A is an acetonitrile, and B is 0.1%～1%H
3PO
4Aqueous solution is carried out gradient elution by A: B=6～100: 94～0;
Flow velocity: 0.2mL/min;
Column temperature: 40 ℃;
Detect wavelength: 235～260nm;
Sample size: 1 μ L;
Theoretical cam curve is calculated all more than 160000.
In the eluent gradient elution process, the A of eluent, B phase transformation turn to: during beginning, and A phase 6%, B phase 94% is stablized 1min; During 19min, A phase 35%, B phase 65%; During 22min, A phase 100%, B is 0 mutually.Be that according to the form below 1 carries out wash-out:
Table 1 eluent gradient wash-out table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| ??0 | ??6 | ??94 |
| ??1 | ??6 | ??94 |
| ??19 | ??35 | ??65 |
| ??22 | ??100 | ??0 |
Being prepared as of abovementioned steps (2) reference substance solution: take by weighing ellagic acid, 3 respectively, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-in O-α-D-Arabinoside reference substance at least two kinds, add dissolve with methanol, get the reference substance storing solution; Precision is measured above-mentioned reference substance storing solution respectively, puts in the same volumetric flask, adds methyl alcohol and is diluted to scale, makes the solution of concentration≤0.1mg/ml, promptly gets reference substance solution.Described five kinds of reference substances all are to adopt conventional plant method to extract separating obtained from corn eyeball medicinal material.
Being prepared as of abovementioned steps (3) need testing solution: the about 1g of corn eyeball medicinal powder decided in accurate title, place apparatus,Soxhlet's, add methanol extraction 2～8h, wash extraction apparatus with small amount of methanol, washing lotion and extract merge, concentrate and be transferred in the measuring bottle, be diluted to scale with methyl alcohol, shake up, promptly get need testing solution.
Put down in writing according to related data, the ellagic acid constituents that contains in the corn eyeball medicinal material, its parent nucleus is ellagic acid, have promoting blood circulation and removing blood stasis, blocking-up carcinogenic carcinogenic, suppress tumor cell proliferation and inducing apoptosis of tumour cell, suppress angiogenesis and effect such as anti-oxidant, and relative content is higher in medicinal material.Therefore for controlling the quality of corn eyeball medicinal material better, guarantee curative effect of medication, the applicant is with reference to Chinese Pharmacopoeia, and selected ellagic acid constituents has been set up corresponding multicomponent content assaying method as the index components of quality control.
In order to ensure content assaying method science of the present invention, reasonable, feasible, the applicant has carried out a series of experimental study and investigation to it:
One, chromatographic condition and system suitability test
1, instrument and reagent
The generation ACQUITY UPLC (U.S. water generation company comprises binary gradient pump, vacuum degassing machine, automatic sampler, column oven, diode array detector, Epower 2 workstations) of department; Agilent 1100 high performance liquid chromatograph series (U.S. Agilent Technologies comprises quaternary gradient pump, vacuum degassing machine, automatic sampler, column oven, diode array detector, Chemstation workstation); Plum Teller 1,/10 ten thousand electronic balance (AE240); Ultrapure water (Sichuan Water Technology Development Co., Ltd.).Ellagic acid, 3,3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside and 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside reference substance, all be to adopt conventional plant method from corn eyeball medicinal material, to extract to separate the preparation gained, identify through physico-chemical analysis and wave spectrum, UPLC peak area normalization method is calculated, and its purity is all greater than 98%.It is pure that acetonitrile (chromatographically pure, Hanbon Sci. ﹠ Tech. Co., Ltd.), all the other reagent are analysis.Experiment is provided by Yikang Pharmaceutical Co., Ltd., Guizhou Prov. with corn eyeball medicinal material, is accredited as the dry branch of Staphyleaceae plant corn eyeball Euscaphis japonica (Thunb.) Dippel by Guiyang Medical College associate professor Long Qingde.Sample is stored in Guizhou Province Chinese medicine ethnic drug research and development centre.
2, the selection of instrument and chromatographic column
Because Ultra Performance Liquid Chromatography (UPLC) uses the chromatographic column filler of 1.7 μ m granularities, can obtain higher post imitates, and keep constant in wideer linear velocity scope inner prop effect, thereby help improving flow rate of mobile phase, shorten analysis time, improve analysis throughput, this stationary phase has also reduced surperficial remaining silicon hydroxyl, is suitable for the analysis of traditional Chinese medicine complex system.Therefore high performance liquid chromatography (HPLC) method and Ultra Performance Liquid Chromatography (UPLC) method have been compared in test.
The corn eyeball medicinal material chromatogram result who obtains by above-mentioned two kinds of instrumental analysis and two kinds of chromatographic columns as seen, adopt the chromatographic column of UPLC and granule filler, the degree of separation of its chromatographic peak, peak shape and sensitivity etc. all are better than HPLC, and shorten analysis time nearly 1/3, so the preferred UPLC of employing measures the content of corn eyeball medicinal material.
3, the selection of moving phase
Because the compositional polarity of being measured differs bigger, the moving phase isocratic elution is difficulty comparatively, therefore gradient elution is adopted in test, the ellagic acid compounds contains phenolic hydroxyl group, with organic solvent-water is moving phase, chromatographic peak hangover is serious, so select acetonitrile-0.1%～1% phosphoric acid system for use, most chromatographic peak type symmetries in the spectrogram, degree of separation is good, analysis time is short.
On the basis of above-mentioned test the gradient condition of acetonitrile (A)-0.1%～1% phosphoric acid (B) flow phase system is optimized, moving phase is carried out gradient elution according to following table 2 described rules.
Table 2 gradient elution proportion of mobile phase changes program list
| Time (min) | Gradient 1 (A%) | Gradient 2 (A%) | Gradient 3 (A%) | Gradient 4 (A%) |
| ??0 | ??8 | ??6 | ??7 | ??6 |
| ??2 | ??8 | ??6 | ??7 | ??6 |
| ??19 | ??42 | ??41 | ??38 | ??35 |
| ??22 | ??100 | ??100 | ??100 | ??100 |
In 4 gradient, the chromatogram peak-to-peak type symmetry of gained, degree of separation are good, particularly better with 4 pairs of compound separation of being analyzed of gradient, so adopt gradient 4 to carry out wash-out.
4, measure the selection of wavelength
The ellagic acid constituents all has uv absorption, the test and Selection UV-detector.Measure through UPLC-PDA, wherein 3,3 '-the dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside has absorption maximum (to see Fig. 2-Fig. 5) at ultraviolet 245nm, ellagic acid has absorption maximum at ultraviolet 254nm, but at 245nm bigger absorption (see figure 1) is arranged also, and taking all factors into consideration selection 245nm is the assay wavelength.
5, the selection of column temperature
The influence to corn eyeball medicinal material chromatographic resolution effect has been investigated when chromatogram column temperature is 35 ℃, 40 ℃, 45 ℃ in test, and the whole looks of chromatogram differ less, and selected above-mentioned column temperature is little to the separation influence of chromatographic peak.Take all factors into consideration each local detachment degree, preferentially select 40 ℃ of column temperatures.
6, the chromatographic condition after the optimization
By instrument, moving phase, detection wavelength, column temperature isochromatic spectrum condition are optimized on the basis of investigation, obtained to be applicable to the chromatographic condition of multicomponent assay:
Instrument: Waters ACQUITY UPLC chromatograph (comprising binary gradient pump, vacuum degassing machine, automatic sampler, column oven, diode array detector, Epower 2 workstations);
Chromatographic column: ACQUITY UPLC BEH C18 post (2.1mm * 100mm, 1.7 μ m);
Moving phase: A is an acetonitrile, and B is 0.1%～1%H
3PO
4, carry out gradient elution by the regulation in the table 1;
Flow velocity: 0.2mL/min;
Column temperature: 40 ℃;
Detect wavelength: 245nm;
Sample size: 1 μ L;
Theoretical cam curve is calculated all more than 160000.
Two, the preparation of reference substance solution:
Take by weighing ellagic acid, 3 respectively, 3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid reference substance 20.23,8.84,4.78,13.30 and 6.58mg, put respectively in five 25mL measuring bottles, it is an amount of to add methyl alcohol, ultrasonicly makes dissolving, adds methyl alcohol to scale, shake up, get the reference substance storing solution.Precision is measured above-mentioned each 1mL of reference substance storing solution respectively, puts in the 10mL measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and gets reference substance solution.
Three, the preparation of need testing solution:
1, the selection of extraction solvent
Because ellagic acid compounds dissolubility in methyl alcohol is better, therefore select for use methyl alcohol as extracting solvent.
2, extracting method
Test has been compared backflow (2h), ultrasonic (1h) and Soxhlet and has been extracted (2h) three kinds of modes, three UPLC collection of illustrative plates basically identical as a result, but as can be known from five determined component content values, Soxhlet is extracted and is better than refluxing extraction and ultrasonic Extraction, so adopt with methyl alcohol is the method that solvent, Soxhlet are extracted, and sees Table 3.Simultaneously, test is also investigated Soxhlet extraction time (2h, 4h, 6h, 8h), and the result shows, extracts 6h and measured matter can be extracted fully substantially, sees Table 4.
Table 3 Different Extraction Method corn eyeball medicinal material assay result (n=2)
The different extraction time corn eyeball medicinal material assay results (n=2) of table 4
3, the need testing solution preparation method's determines
Get the about 1g of corn eyeball medicinal powder (crossing 40 orders), the accurate title, decide, and puts in the apparatus,Soxhlet's, and it is an amount of to add methyl alcohol, reflux 6h uses the small amount of methanol washing container, and washing lotion is incorporated extract into, concentrates and is transferred in the 10mL measuring bottle, be diluted to scale with methyl alcohol, shake up, promptly.
Four, the drafting of typical curve
Precision is measured reference substance solution 0.4 μ L, 0.8 μ L, 1.4 μ L, 1.6 μ L, 1.8 μ L respectively, measures by the chromatographic condition sample introduction after the above-mentioned optimization.With the sample size is horizontal ordinate, and peak area is an ordinate, the drawing standard curve.
The ellagic acid linear relationship is measured regression equation: Y=12492919.12X-61593.24, and r=0.9998, the range of linearity is: 0.032～0.146 μ g the results are shown in Table 5.
Table 5 ellagic acid linear relationship measurement result
| The sample introduction sequence number | Sample size (μ g) | Peak area |
| ??1 | ??0.032 | ??342125 |
| ??2 | ??0.064 | ??733445 |
| The sample introduction sequence number | Sample size (μ g) | Peak area |
| ??3 | ??0.113 | ??1327608 |
| ??4 | ??0.129 | ??1552441 |
| ??5 | ??0.146 | ??1733016 |
3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside linear relationship measures regression equation and is: Y=6754214.29X-11023.60, and r=0.9997, the range of linearity is: 0.014～0.063 μ g the results are shown in Table 6.
Table 63,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside linear relationship measurement result
| The sample introduction sequence number | Sample size (μ g) | Peak area |
| ??1 | ??0.014 | ??86128 |
| ??2 | ??0.028 | ??175939 |
| ??3 | ??0.049 | ??315024 |
| ??4 | ??0.056 | ??369659 |
| ??5 | ??0.063 | ??416527 |
3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside linear relationship measures regression equation and is: Y=6754214.29X-11023.60, and r=0.9998, the range of linearity is: 0.008～0.036 μ g the results are shown in Table 7.
Table 73,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside linear relationship measurement result
| The sample introduction sequence number | Sample size (μ g) | Peak area |
| ??1 | ??0.008 | ??89386 |
| ??2 | ??0.016 | ??184083 |
| ??3 | ??0.028 | ??329978 |
| ??4 | ??0.032 | ??386970 |
| ??5 | ??0.036 | ??439538 |
3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside linear relationship measures regression equation and is: Y=9068511.96X-18716.96, and r=0.9998, the range of linearity is: 0.021～0.096 μ g the results are shown in Table 8.
Table 83,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside linear relationship measurement result
| The sample introduction sequence number | Sample size (μ g) | Peak area |
| ??1 | ??0.021 | ??174461 |
| ??2 | ??0.042 | ??359480 |
| ??3 | ??0.074 | ??646305 |
| ??4 | ??0.085 | ??758685 |
| ??5 | ??0.096 | ??851271 |
3,3 '-dimethoxy ellagic acid linear relationship measures regression equation and is: Y=6953431.54X-9048.10, and r=0.9998, the range of linearity is: 0.011～0.051 μ g the results are shown in Table 9.
Table 93,3 '-dimethoxy ellagic acid linear relationship measurement result
| The sample introduction sequence number | Sample size (μ g) | Peak area |
| ??1 | ??0.011 | ??68264 |
| ??2 | ??0.022 | ??143105 |
| ??3 | ??0.039 | ??259784 |
| ??4 | ??0.045 | ??307039 |
| ??5 | ??0.051 | ??344744 |
Five, precision test
By the chromatographic condition under the content assaying method of the present invention, precision is measured corn eyeball need testing solution 1 μ L, continuous sample introduction 5 times, measure respectively ellagic acid (A), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside (B), 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside (C), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside (D), 3,3 '-dimethoxy ellagic acid (E), record peak area integration also calculates RSD, the results are shown in Table 10.
Table 10 Precision test result
By last table result as can be known, the precision of content assaying method of the present invention is good.
Six, replica test
Get 6 parts of same lot number corn eyeball medicinal material test samples, preparation method according to need testing solution under the content assaying method of the present invention prepares 6 parts of need testing solutions, measure respectively, calculate ellagic acid (A), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside (B), 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside (C), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside (D), 3,3 '-content and the RSD of dimethoxy ellagic acid (E), the results are shown in Table 11.
Table 11 replica test result
By last table result as can be known, the repeatability of content assaying method of the present invention is good.
Seven, stability test
Precision is measured the same need testing solution 1 μ L of corn eyeball, respectively at 0,1,2,4,8h measures, and the results are shown in Table 12.
Table 12 stability test result
By last table result as can be known, stable in the tested composition 8h in the need testing solution.
Eight, recovery test
Get 6 parts of the corn eyeball medicinal materials (crossing 40 mesh sieves) of measuring content, every part of about 0.5g, the accurate title, decide, (five kinds of each comfortable concentration of mixing in the reference substance solution of reference substance are respectively the mixed mixing reference substance solution 1mL that get of five kinds of reference substances of accurate respectively adding: ellagic acid reference substance 0.226mg/mL, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside reference substance 0.118mg/mL, 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside reference substance 0.121mg/mL, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside reference substance 0.142mg/mL, 3,3 '-dimethoxy ellagic acid reference substance 0.135mg/mL), room temperature volatilizes, preparation method by need testing solution under the content assaying method of the present invention prepares need testing solution, measure by the chromatographic condition under the content assaying method of the present invention, calculate, the recovery is good as a result.
The ellagic acid recovery test the results are shown in Table 13; 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside recovery test the results are shown in Table 14; 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside recovery test the results are shown in Table 15; 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside recovery test the results are shown in Table 16; 3,3 '-dimethoxy ellagic acid recovery test the results are shown in Table 17.
Table 13 ellagic acid recovery test result
Table 143,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside recovery test result
Table 153,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside recovery test result
Table 163,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside recovery test result
Table 173,3 '-dimethoxy ellagic acid recovery test result
Nine, sample size is measured
Get the corn eyeball medicinal material test sample 1g of different batches, the accurate title, decide, preparation method according to need testing solution under the content assaying method of the present invention prepares need testing solution, measure ellagic acid (A), 3 by the chromatographic condition under the content assaying method of the present invention, 3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside (B), 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside (C), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside (D), 3,3 '-content of dimethoxy ellagic acid (E), calculate.The results are shown in Table 18.
Table 18 different batches corn eyeball medicinal material assay result (n=2)
In 9 batches of samples: the average content of A is 0.4726mg/g, the average content of B is 0.3314mg/g, and the average content of C is 0.2519mg/g, and the average content of D is 0.4334mg/g, the average content of E is 0.3636mg/g, and five tested composition overall average content are 1.853mg/g.
Through comprehensive evaluation, in conjunction with each system suitability parameter situation, and through the methodology checking, indexs such as this precision of method, repeatability, the recovery are all good, improved the quality control standard of corn eyeball medicinal material, use for the redevelopment of corn eyeball medicinal material and lay a good foundation.
Compared with prior art, the present invention adopts high performance liquid chromatography or Ultra Performance Liquid Chromatography method, under same chromatographic condition, the multiple composition in the corn eyeball medicinal material is carried out assay simultaneously, make that the content assaying method of ellagic acid constituents has more globality, characteristic and stability in the corn eyeball medicinal material, and this method precision height, good reproducibility, recovery height, measurement result is accurate, can effectively control the quality of corn eyeball medicinal material, thereby guarantee the validity and the security of its clinical application, use for the redevelopment of corn eyeball medicinal material and lay a good foundation.
Description of drawings:
Fig. 1 is the ultraviolet spectrogram of ellagic acid reference substance solution.
Fig. 2 is 3,3 '-dimethoxy ellagic acid-4 '-ultraviolet spectrogram of O-β-D-glucoside reference substance solution.
Fig. 3 is 3,3 '-dimethoxy ellagic acid-4 '-ultraviolet spectrogram of O-α-D-Arabinoside reference substance solution.
Fig. 4 is 3,3 '-dimethoxy ellagic acid-4 '-ultraviolet spectrogram of O-β-D-xyloside reference substance solution.
Fig. 5 is 3,3 '-ultraviolet spectrogram of dimethoxy ellagic acid reference substance solution.
Fig. 6 is an ellagic acid (1), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside (2), 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside (3), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside (4), 3,3 '-dimethoxy ellagic acid (5) mixes the UPLC chromatogram of reference substance solution.
Fig. 7 is the UPLC chromatogram of need testing solution, chromatographic peak is respectively: ellagic acid (1), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside (2), 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside (3), 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside (4), 3,3 '-dimethoxy ellagic acid (5).
Embodiment:
Embodiments of the invention 1: the content assaying method of ellagic acid constituents is in the corn eyeball medicinal material:
Adopt the Ultra Performance Liquid Chromatography method, specifically may further comprise the steps:
(1) chromatographic condition and system suitability test:
Instrument: Waters ACQUITY UPLC chromatograph (comprising binary gradient pump, vacuum degassing machine, automatic sampler, column oven, diode array detector, Epower 2 workstations);
Chromatographic column: ACQUITY UPLC BEH C18 post (2.1mm * 100mm, 1.7 μ m);
Moving phase: A is an acetonitrile, and B is 0.1%H
3PO4 carries out gradient elution by A: B=6～100: 94～0; In the eluent gradient elution process, the A of eluent, B phase transformation turn to: during beginning, and A phase 6%, B phase 94% is stablized 1min; During 19min, A phase 35%, B phase 65%; During 22min, A phase 100%, B is 0 mutually; Be that according to the form below (table 19) carries out wash-out:
Table 19 eluent gradient wash-out table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| ??0 | ??6 | ??94 |
| ??1 | ??6 | ??94 |
| ??19 | ??35 | ??65 |
| ??22 | ??100 | ??0 |
Flow velocity: 0.2mL/min;
Column temperature: 40 ℃;
Detect wavelength: 245nm;
Sample size: 1 μ L;
Theoretical cam curve is calculated all more than 160000.
(2) preparation of reference substance solution: take by weighing ellagic acid, 3 respectively, 3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid reference substance 20.23mg, 8.84mg, 4.78mg, 13.30mg, 6.58mg, put respectively in five 25mL measuring bottles, it is an amount of to add methyl alcohol, ultrasonicly make dissolving, add methyl alcohol to scale, shake up, get the reference substance storing solution.Precision is measured above-mentioned each 1mL of reference substance storing solution respectively, puts in the 10mL measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and makes the solution of concentration≤0.1mg/ml, promptly gets reference substance solution.
(3) preparation of need testing solution: the about 1g of corn eyeball medicinal powder (crossing 40 orders) decided in accurate title, place apparatus,Soxhlet's, it is an amount of to add methyl alcohol, reflux 6h, wash extraction apparatus with small amount of methanol, washing lotion and extract merge, concentrate and be transferred in the 10mL measuring bottle, are diluted to scale with methyl alcohol, shake up, promptly get need testing solution.
(4) determination method: measure according to the described chromatographic condition of step (1), promptly.
Embodiments of the invention 2: in the corn eyeball medicinal material content assaying method of ellagic acid constituents also can for:
Adopt the Ultra Performance Liquid Chromatography method, specifically may further comprise the steps:
(1) chromatographic condition and system suitability test:
Instrument: Waters ACQUITY UPLC chromatograph;
Chromatographic column: ACQUITY UPLC BEH C18 post;
Moving phase: A is an acetonitrile, and B is 1%H
3PO4 carries out gradient elution by A: B=6～100: 94～0; In the eluent gradient elution process, the A of eluent, B phase transformation turn to: during beginning, and A phase 6%, B phase 94% is stablized 1min; During 19min, A phase 35%, B phase 65%; During 22min, A phase 100%, B is 0 mutually.
Flow velocity: 0.2mL/min;
Column temperature: 35 ℃;
Detect wavelength: 235～250nm;
Sample size: 1 μ L;
Theoretical cam curve is calculated all more than 160000.
(2) preparation of reference substance solution: take by weighing ellagic acid, 3 respectively, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside reference substance is an amount of, add dissolve with methanol, get the reference substance storing solution; Precision is measured above-mentioned reference substance storing solution respectively, puts in the same volumetric flask, adds methyl alcohol and is diluted to scale, makes the solution of concentration≤0.1mg/ml, promptly gets reference substance solution.
(3) preparation of need testing solution: the about 1g of corn eyeball medicinal powder decided in accurate title, place extraction apparatus, it is an amount of to add methyl alcohol, heating and refluxing extraction 8h, wash extraction apparatus with small amount of methanol, washing lotion and extract merge, concentrate and be transferred in the measuring bottle, are diluted to scale with methyl alcohol, shake up, promptly get need testing solution.
(4) determination method: measure according to the described chromatographic condition of step (1), promptly.
Embodiments of the invention 3: in the corn eyeball medicinal material content assaying method of ellagic acid constituents also can for:
Adopt the Ultra Performance Liquid Chromatography method, specifically may further comprise the steps:
(1) chromatographic condition and system suitability test:
Instrument: Waters ACQUITY UPLC chromatograph;
Chromatographic column: ACQUITY UPLC BEH C18 post;
Moving phase: A is an acetonitrile, and B is 0.5%H3PO4, carries out gradient elution by A: B=6～100: 94～0; In the eluent gradient elution process, the A of eluent, B phase transformation turn to: during beginning, and A phase 6%, B phase 94% is stablized 1min; During 19min, A phase 35%, B phase 65%; During 22min, A phase 100%, B is 0 mutually.
Flow velocity: 0.2mL/min;
Column temperature: 45 ℃;
Detect wavelength: 240～260nm;
Sample size: 1 μ L;
Theoretical cam curve is calculated all more than 160000.
(2) preparation of reference substance solution: take by weighing ellagic acid, 3 respectively, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside reference substance is an amount of, add dissolve with methanol, get the reference substance storing solution; Precision is measured above-mentioned reference substance storing solution respectively, puts in the same volumetric flask, adds the methyl alcohol dilution, makes the solution of concentration≤0.1mg/ml, promptly gets reference substance solution.
(3) preparation of need testing solution: the accurate title, decided the about 1g of corn eyeball medicinal powder, places extraction apparatus, adds methyl alcohol ultrasonic Extraction 2～6h, use the methanol wash extraction apparatus, washing lotion and extract merge, concentrate and be transferred in the measuring bottle, are diluted to scale with methyl alcohol, shake up, promptly get need testing solution.
(4) determination method: measure according to the described chromatographic condition of step (1), promptly.
Embodiments of the invention 4: in the corn eyeball medicinal material content assaying method of ellagic acid constituents can also for:
Adopt high performance liquid chromatography, specifically may further comprise the steps:
(1) chromatographic condition and system suitability test:
Instrument: high performance liquid chromatograph;
Chromatographic column: BEH C18 post;
Moving phase: A is an acetonitrile, and B is 0.1%～1%H3PO4, carries out gradient elution by A: B=6～100: 94～0;
Flow velocity: 0.2mL/min;
Column temperature: 35 ℃～45 ℃;
Detect wavelength: 235～260nm;
Sample size: 1 μ L;
Theoretical cam curve is calculated all more than 160000.
(2) preparation of reference substance solution: take by weighing 3 respectively, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-α-D-Arabinoside reference substance is an amount of, add in the rearmounted same volumetric flask of dissolve with methanol, add methyl alcohol and be diluted to scale, promptly get reference substance solution.
(3) preparation of need testing solution: it is an amount of to get corn eyeball medicinal powder, and accurate the title decides, and adds methanol extraction 4～8h, concentrates the back and is diluted to scale with methyl alcohol, shakes up, and promptly gets need testing solution.
(4) determination method: measure according to the described chromatographic condition of step (1), promptly.
Claims (7)
1. the content assaying method of ellagic acid constituents in the corn eyeball medicinal material, it is characterized in that: adopt high performance liquid chromatography or Ultra Performance Liquid Chromatography method, under same chromatographic condition to ellagic acid, 3 contained in the corn eyeball medicinal material, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-at least two compositions in O-α-five compositions of D-Arabinoside carry out assay simultaneously.
2. according to the content assaying method of ellagic acid constituents in the described corn eyeball of claim 1 medicinal material, it is characterized in that: described content assaying method is with ellagic acid, 3,3 '-the dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-in O-α-five kinds of reference substances of D-Arabinoside at least two kinds are contrast, with acetonitrile: H3PO4 aqueous solution=6～100: 94～0 is the high performance liquid chromatography or the Ultra Performance Liquid Chromatography method of eluent gradient wash-out.
3. according to the content assaying method of ellagic acid constituents in claim 1 or the 2 described corn eyeball medicinal materials, it is characterized in that: this method may further comprise the steps:
(1) chromatographic condition: chromatographic column is a BEH C18 post; Acetonitrile-H3PO4 aqueous solution gradient elution; Column temperature is 20 ℃～50 ℃; The detection wavelength is 220～480nm;
(2) preparation of reference substance solution: take by weighing ellagic acid, 3 respectively, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-in O-α-D-Arabinoside reference substance at least two kinds, add in the rearmounted same volumetric flask of dissolve with methanol, add methyl alcohol and be diluted to scale, get reference substance solution;
(3) preparation of need testing solution: it is an amount of to get corn eyeball medicinal powder, and accurate the title decides, and it is an amount of to add methyl alcohol, extracts 2～8h, concentrates the back with the methyl alcohol dilution, gets need testing solution;
(4) assay method: measure according to the described chromatographic condition of step (1), promptly.
4. according to the content assaying method of ellagic acid constituents in the described corn eyeball of claim 3 medicinal material, it is characterized in that: the chromatographic condition of step (1) and system suitability test are:
Instrument: Waters ACQUITY UPLC chromatograph;
Chromatographic column: ACQUITY UPLC BEH C18 post;
Moving phase: A is an acetonitrile, and B is 0.1%～1%H3PO4 aqueous solution, carries out gradient elution by A: B=6～100: 94～0;
Flow velocity: 0.2mL/min;
Column temperature: 40 ℃;
Detect wavelength: 235～260nm;
Sample size: 1 μ L;
Theoretical cam curve is calculated all more than 160000.
5. according to the content assaying method of ellagic acid constituents in the described corn eyeball of claim 4 medicinal material, it is characterized in that: in the eluent gradient elution process, the A of eluent, B phase transformation turn to: during beginning, and A phase 6%, B phase 94% is stablized 1min; During 19min, A phase 35%, B phase 65%; During 22min, A phase 100%, B is 0 mutually.
6. according to the content assaying method of ellagic acid constituents in the described corn eyeball of claim 3 medicinal material, it is characterized in that: being prepared as of step (2) reference substance solution: take by weighing ellagic acid, 3 respectively, 3 '-dimethoxy ellagic acid, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-xyloside, 3,3 '-dimethoxy ellagic acid-4 '-O-β-D-glucoside, 3,3 '-dimethoxy ellagic acid-4 '-in O-α-D-Arabinoside reference substance at least two kinds, add dissolve with methanol, get the reference substance storing solution; Precision is measured above-mentioned reference substance storing solution respectively, puts in the same volumetric flask, adds methyl alcohol and is diluted to scale, makes the solution of concentration≤0.1mg/ml, promptly gets reference substance solution.
7. according to the content assaying method of ellagic acid constituents in the described corn eyeball of claim 3 medicinal material, it is characterized in that: being prepared as of step (3) need testing solution: accurately claim to decide the about 1g of corn eyeball medicinal powder, place apparatus,Soxhlet's, add methanol extraction 2～8h, wash extraction apparatus with small amount of methanol, washing lotion and extract merge, concentrate and be transferred in the measuring bottle, are diluted to scale with methyl alcohol, shake up, promptly get need testing solution.
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| CN104730166A (en) * | 2015-03-25 | 2015-06-24 | 贵阳医学院 | Quantitative determination method for 3,3'-dimethyl ellagic acid and 3,3',4'-trimethyl ellagic acid in polygonum runcinatum |
| CN106841464A (en) * | 2017-02-06 | 2017-06-13 | 广西壮族自治区中医药研究院 | Ellagic acid and 3,3` dimethoxy ellagic acid detection method of content in a kind of China fir parasitism |
| CN107991408A (en) * | 2017-11-27 | 2018-05-04 | 江西珍视明药业有限公司 | Index components content assay method in four taste treasure's layer ice boron eye drops |
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| CN110196290A (en) * | 2019-04-02 | 2019-09-03 | 贵州医科大学 | Ellagic acid content assaying method in a kind of Rosa roxburghii Tratt |
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| CN104730166A (en) * | 2015-03-25 | 2015-06-24 | 贵阳医学院 | Quantitative determination method for 3,3'-dimethyl ellagic acid and 3,3',4'-trimethyl ellagic acid in polygonum runcinatum |
| CN104730166B (en) * | 2015-03-25 | 2016-08-17 | 贵阳医学院 | 3,3 '-dimethylellagic acid, the quantitative detecting method of 3,3 ', 4 '-trimethyl ellagic acid in a kind of Herba Polygoni Runcinati |
| CN106841464A (en) * | 2017-02-06 | 2017-06-13 | 广西壮族自治区中医药研究院 | Ellagic acid and 3,3` dimethoxy ellagic acid detection method of content in a kind of China fir parasitism |
| CN107991408A (en) * | 2017-11-27 | 2018-05-04 | 江西珍视明药业有限公司 | Index components content assay method in four taste treasure's layer ice boron eye drops |
| CN108956800A (en) * | 2018-05-18 | 2018-12-07 | 山东省食品药品检验研究院 | The measuring method of ellagic acid content in cortex moutan |
| CN108956800B (en) * | 2018-05-18 | 2021-07-23 | 山东省食品药品检验研究院 | Method for measuring content of ellagic acid in moutan bark |
| CN110196290A (en) * | 2019-04-02 | 2019-09-03 | 贵州医科大学 | Ellagic acid content assaying method in a kind of Rosa roxburghii Tratt |
| CN113533607A (en) * | 2021-01-24 | 2021-10-22 | 陕西凤丹正元生物科技有限公司 | Method for evaluating quality of peony leaf medicinal material |
| CN113533607B (en) * | 2021-01-24 | 2023-09-15 | 陕西凤丹正元生物科技有限公司 | Method for evaluating quality of peony leaf medicinal material |
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