CN102507825A - Detecting method for effective components of traditional Chinese medicine Hugu capsules for preventing and treating osteoporosis - Google Patents
Detecting method for effective components of traditional Chinese medicine Hugu capsules for preventing and treating osteoporosis Download PDFInfo
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Abstract
The invention relates to a detecting method for traditional Chinese medicine components, in particular to a detecting method for effective components of traditional Chinese medicine Hugu capsules for preventing and treating osteoporosis, which is used for detecting effective components of Hugu capsules by means of multi-wavelength high-performance liquid chromatography. The detecting method includes the specific steps: a, preparing sample solution to be detected; b, preparing mixed reference sample solution; c, respectively detecting the sample solution to be detected and the mixed reference sample solution by means of high performance liquid chromatography; and d, comparing a high performance liquid chromatogram of the sample solution to be detected with that of the mixed reference sample solution. The detecting method can be used for simultaneously detecting chlorogenic acid, 2, 3, 5, 4'-tetrahydrostibene-2-O-beta-D-glucoside, ferulic acid, naringin and icariin and the content of the five effective components in the Hugu capsules. When the effective components in the Hugu capsules are detected by the method, high performance liquid chromatograms of standard substances of the five components are omitted, time and reagents can be reduced, and the detecting method is simple, convenient, high in precision and fine in reproducibility.
Description
Technical field
The present invention relates to the detection method of traditional Chinese medicine ingredients, be specifically related to a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating.
Background technology
Chinese medicine protects capsule and is processed by Chinese crude drugs such as the fleece-flower root, icariin, cultivated land, tortoise plastron, Morinda officinalis, the bark of eucommia, teasel root, the rhizome of davallia, Radix Angelicae Sinensis, Chinese yams, has the merit of tonifying kidney and benefiting sperm.Protect capsule be used to treat lumbar vertebrae pain that middle-aged and old syndrome of deficiency of kidney essence occurs, soft unable, can not be prudent, the lower limb impotence weak, walk with difficulty, or talagia, complexion dull, send out take off, sexual hypoesthesia, dizziness and tinnitus, pink tongue tongue thin white, red tongue with thin fur is white; And above-mentioned symptom appears in sufferers of osteoporosis face usually.Protecting capsule is compound Chinese medicinal preparation, and the primary standard that detects about effective constituent only detects single components such as the fleece-flower root, icariin, cultivated land respectively.And protect in preparation technology's the process of capsule in understanding, we find to protect the existing quality standard of capsule, and to detect effective constituent not comprehensive, and complex operation, can not satisfy the requirement of quality monitoring.
Summary of the invention
The objective of the invention is to deficiency to prior art; A kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating is provided; Detection method of protecting the effective constituent of capsule provided by the present invention can be carried out detection by quantitative to the plurality of Chinese composition that protects capsule simultaneously; The detection composition is complete, and is simple to operate, can satisfy the requirement of quality monitoring.
To achieve these goals, the present invention adopts following technical scheme:
A kind ofly prevent and treat the detection method that osteoporosis Chinese medicine protects the effective constituent of capsule; The said Chinese prescription that protects capsule is the fleece-flower root, barrenwort, prepared rhizome of rehmannia, tortoise plastron, Morinda officinalis, the bark of eucommia, teasel root, the rhizome of davallia, Radix Angelicae Sinensis and Chinese yam; It is characterized in that: said detection method of protecting the effective constituent of capsule is to utilize the high performance liquid chromatography detection of multi-wavelength to protect the various effective constituents in the capsule, and concrete step is:
A, prepare sample solution to be checked: get and protect the capsule content, be mixed with the sample solution to be checked of drug 4mg/ml～20mg/ml with solvent;
B, preparation mix reference substance solution: get 2; 3; 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin mix the back and are mixed with the reference substance solution that each constituent concentration is 1 μ g/ml～200 μ g/ml with solvent;
C, then with sample solution to be checked with mix reference substance solution and carry out high performance liquid chromatography by following method respectively and detect:
With the octadecylsilane chemically bonded silica is chromatographic column filler; By sample size is 1 μ L～20 μ L sample introductions; Column temperature is 10 ℃～40 ℃; Use by the moving phase of F phase, G phase and H phase composition and carry out gradient elution with the flow velocity of 0.3ml/min～1.5ml/min and by the proportioning of table 1; Adopt simultaneously and detect the ultraviolet light multi-wavelength detection that wavelength is 267nm～273nm, 280～286nm, 317nm～323nm and 322nm～328nm, obtain sample solution to be checked and the high-efficient liquid phase chromatogram that mixes reference substance solution;
F phase, G phase and H proportioning table mutually in table 1 moving phase
| Time/minute | F/% | G/% | H/% |
| 0 | 5～20 | 90～60 | 5～20 |
| 2～7 | 5～20 | 90～60 | 5～20 |
| 5～14 | 7～30 | 86～40 | 7～30 |
| 10～23 | 11～35 | 78～30 | 11～35 |
| 15～35 | 15～40 | 70～20 | 15～40 |
| 20～55 | 20～45 | 60～10 | 20～45 |
Wherein, the number percent in the table 1 is percent by volume;
Said gradient elution step is: 0 minute, the percent by volume of F phase was 5%～20% in the moving phase, and the percent by volume of G phase is 90%～60% in the moving phase, and the percent by volume of H phase is 5%～20% in the moving phase; 2 minutes～7 minutes, F was 5%～20% mutually, and G is 90%～60% mutually, and H is 5%～20% mutually; 5 minutes～14 minutes, F was 7%～30% mutually, and G is 86%～40% mutually, and H is 7%～30% mutually; 10 minutes～23 minutes, F was 11%～35% mutually, and G is 78%～30% mutually, and H is 11%～35% mutually; 15 minutes～35 minutes, F was 15%～40% mutually, and G is 70%～20% mutually, and H is 15%～40% mutually; 20 minutes～55 minutes, F was 20%～45% mutually, and G is 60%～10% mutually, and H is 20%～45% mutually; Wherein, above number percent is percent by volume;
D, relatively with the high-efficient liquid phase chromatogram of sample solution to be checked and the high-efficient liquid phase chromatogram that mixes reference substance solution; With the identical peak of retention time of corresponding composition in the high-efficient liquid phase chromatogram that mixes reference substance solution promptly is the characteristic peak with this composition same substance, and calculates content according to peak area by external standard method.
Wherein, each peak is followed successively by chlorogenic acid, 2,3,5 in the high-efficient liquid phase chromatogram of said mixing reference substance solution, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin;
Said preparation sample solution to be checked with mix the used solvent of reference substance solution and can be: 10%～95% ethanol, 10%～100% methyl alcohol or the solvent identical with moving phase, said preparation sample solution to be checked and the dissolution mechanism that mixes reference substance solution are that Chinese medicine such as ultrasonic, refluxing extraction commonly usedly extracts, the purifying mode.
The F of said moving phase is methyl alcohol mutually; The H of said moving phase is acetonitrile mutually; The G of said moving phase is 0.1%～2% acetic acid mutually.
The preparation method of said G phase is: add entry in the analytically pure glacial acetic acid and be mixed with 0.1%～2% acetic acid.
Sample solution to be checked of the present invention can adopt following method preparation: precision takes by weighing and protects capsule content medicine in right amount; Handle with dissolution with solvents and employing traditional Chinese medicine extraction purification process commonly used then; Use solvent to be settled to concentration at last for containing content medicine 4mg/ml～20mg/ml; Filter, get final product.
Preferably, said moving phase F phase during gradient elution, G are as shown in table 2 with H proportioning mutually mutually.
F phase, G phase and H proportioning table mutually in table 2 moving phase
| Time/minute | F/% | G/% | H/% |
| 0 | 7～15 | 86～70 | 7～15 |
| 3～6 | 7～15 | 86～70 | 7～15 |
| 6～12 | 9～20 | 82～60 | 9～20 |
| 12～20 | 14～25 | 72～50 | 14～25 |
| 20～30 | 20～30 | 60～40 | 20～30 |
| 30～45 | 25～35 | 50～30 | 25～35 |
Wherein, the number percent in the table 2 is percent by volume;
Said gradient elution step is: 0 minute, the percent by volume of F phase was 7%～15% in the moving phase, and the percent by volume of G phase is 86%～70% in the moving phase, and the percent by volume of H phase is 7%～15% in the moving phase; 3 minutes～6 minutes, F was 7%～15% mutually, and G is 86%～70% mutually, and H is 7%～15% mutually; 6 minutes～12 minutes, F was 9%～20% mutually, and G is 82%～60% mutually, and H is 9%～20% mutually; 12 minutes～20 minutes, F was 14%～25% mutually, and G is 72%～50% mutually, and H is 14%～25% mutually; 20 minutes～30 minutes, F was 20%～30% mutually, and G is 60%～40% mutually, and H is 20%～30% mutually; 30 minutes～45 minutes, F was 25%～35% mutually, and G is 50%～30% mutually, and H is 25%～35% mutually; Wherein, above number percent is percent by volume.
Preferred, said moving phase F phase during gradient elution, G are as shown in table 3 with H proportioning mutually mutually.
F phase, G phase and H proportioning table mutually in table 3 moving phase
| Time/minute | F/% | G/% | H/% |
| 0 | 10 | 80 | 10 |
| 5 | 10 | 80 | 10 |
| 8 | 13 | 74 | 13 |
| 15 | 17 | 66 | 17 |
| 25 | 25 | 50 | 25 |
| 35 | 30 | 40 | 30 |
Wherein, the number percent in the table 3 is percent by volume;
Said gradient elution step is: 0 minute, the percent by volume of F phase was 10% in the moving phase, and the percent by volume of G phase is 80% in the moving phase, and the percent by volume of H phase is 10% in the moving phase; 5 minutes, F was 10% mutually, and G is 80% mutually, and H is 10% mutually; 8 minutes, F was 13% mutually, and G is 74% mutually, and H is 13% mutually; 15 minutes, F was 17% mutually, and G is 66% mutually, and H is 17% mutually; 25 minutes, F was 25% mutually, and G is 50% mutually, and H is 25% mutually; 35 minutes, F was 30% mutually, and G is 40% mutually, and H is 30% mutually; Wherein, above number percent is percent by volume.
Preferably, the detection wavelength of said ultraviolet light multi-wavelength detection is 270nm, 283nm, 320nm and 325nm.
Preferably, the flow velocity of said moving phase is 0.8 ml/min.
Preferably, said column temperature is 30 ℃.
The ownership at each peak in the high-efficient liquid phase chromatogram of mixing reference substance solution according to the invention is confirmed through following method: prepare 2 respectively; 3; 5; The standard solution of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin; The high-efficient liquid phase chromatogram that under same chromatographic condition, prepares each independent standard solution and said mixing reference substance solution then respectively, the ownership of confirming to mix each peak in the reference substance solution chromatogram according to the retention time at the peak of corresponding composition in each independent standard solution chromatogram.Through revision test repeatedly; Finding to mix in the reference substance solution in the said moving phase has five kinds of compositions that the fixing peak that goes out is arranged, and these five kinds of compositions are chlorogenic acid, 2,3; 5; Therefore 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin, can confirm to mix the ownership at five peaks in the reference substance solution high-efficient liquid phase chromatogram.
The present invention compared with prior art, beneficial effect is:
1) owing to has the ownership at five peaks to be respectively chlorogenic acid, 2 in the mixing reference substance solution high-efficient liquid phase chromatogram; 3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin; Therefore; When the effective constituent of capsule is protected in detection, do not need to prepare respectively again the high-efficient liquid phase chromatogram of each standard items of above five kinds of compositions, thereby saved time and reagent.
2) detection method of the present invention adopts the method for high-efficient liquid phase chromatogram to carry out qualitative and quantitative detection to protecting capsule; Characteristic according to the effective constituent of protecting capsule; Moving phase is selected the acetic acid of methyl alcohol, acetonitrile and 0.1%～2%, and follow procedure continuously changes in the moving phase each ratio between mutually and carry out gradient elution, reaches the purpose that multiple composition in the capsule is protected in accurate separation; Become component selections to detect wavelength to each wash-out simultaneously, thereby further improve the degree of accuracy that detects.
3) adopt detection method of the present invention; Can be simultaneously to 2 in the effective constituent of protecting capsule; 3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, these five kinds of compositions of aurantiin carry out detection by quantitative; Can investigate more all sidedly and protect main effective constituent composition and ratio situation of change in the capsule, reduce analytical procedure and reduced composition variation and error at measurment that the sample pre-treatments link possibly cause.
4) detection method of the present invention have easy, stable, precision is high, favorable reproducibility, the advantage that is easy to grasp.
Description of drawings
To be that the present invention is a kind of prevent and treat the high-efficient liquid phase chromatogram of mixing reference substance solution of embodiment 1 of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule to Fig. 1.
To be that the present invention is a kind of prevent and treat the high-efficient liquid phase chromatogram of sample solution to be checked of embodiment 1 of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule to Fig. 2.
Embodiment
Below in conjunction with embodiment the present invention is further described.
embodiment 1.
(1) instrument and reagent.
Instrument: Agilent1200 high performance liquid chromatograph (online degasser, quaternary pump, automatic sampler, column oven, PDAD); The Kromasil100-5C18 chromatographic column; Sartorius 100,000/balance; KQ5200DB type numerical control supersonic washer; Milli-pore-Q ultrapure water preparing instrument.
Reference substance: 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin.
Sample to be checked: protect capsule.
Other reagent: acetonitrile (chromatographically pure), methyl alcohol, glacial acetic acid (analyzing pure).
(2) detection method.
The preparation of a, mixing reference substance solution: precision takes by weighing 2,3, and 5; 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin are an amount of, the dissolve with methanol with 100%, constant volume; Shake up; Membrane filtration, making chlorogenic acid content is 80 μ g/ml, 2,3; 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside content is that 150 μ g/ml, ferulaic acid content are that 100 μ g/ml, naringin content are that 80 μ g/ml, icariin content are the mixing reference substance solution of 90 μ g/ml.
The preparation of b, sample solution to be checked: get the content that protects capsule and cross sieve No. four, get then about 0.2g carry out precision claim fixed after, place the volumetric flask of 25ml, accurate 80% methyl alcohol that adds is to scale mark; In heating-up temperature is to carry out under 57 ℃ ultrasonic it being dissolved fully, makes the sample solution that concentration is 8mg/ml, shakes up filtering with microporous membrane.
C, chromatographic column are filler with the octadecylsilane chemically bonded silica; Get and mix control sample solution 20 μ L sample introductions; Column temperature is 30 ℃, and flow velocity 0.8ml/min carries out gradient elution by table 4; And adopt and detect the ultraviolet light multi-wavelength detection that wavelength is 270nm, 283nm, 320nm and 325nm, obtain mixing the high-efficient liquid phase chromatogram of reference substance solution.
F phase, G phase and H proportioning table mutually in table 4 moving phase
| Time/minute | F/% | G/% | H/% |
| 0 | 10 | 80 | 10 |
| 5 | 10 | 80 | 10 |
| 8 | 13 | 74 | 13 |
| 15 | 17 | 66 | 17 |
| 25 | 25 | 50 | 25 |
| 35 | 30 | 40 | 30 |
Wherein, the G of moving phase is 1% acetic acid mutually, and its preparation method is: analytically pure glacial acetic acid is added water be mixed with 1% acetum; The F of moving phase is methyl alcohol mutually, and the H of moving phase is acetonitrile mutually.
Obtain the high-efficient liquid phase chromatogram of sample solution to be checked according to the method described above, wherein, the sample size of sample solution to be checked is 20 μ L, flow velocity 0.8ml/min.
(3) result relatively.
See Fig. 1, the ownership at A, B, C, D, each peak of E is respectively chlorogenic acid, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin in the high-efficient liquid phase chromatogram of mixing reference substance solution.
See Fig. 1 and Fig. 2; The retention time and the peak area at the high-efficient liquid phase chromatogram of sample solution more to be checked and each peak of high-efficient liquid phase chromatogram that mixes reference substance solution; Can know that chlorogenic acid, 2 is represented at A, B, C, D, each peak of E respectively in the high-efficient liquid phase chromatogram of sample solution to be checked; 3,5, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin; Chlorogenic acid contents is 0.3mg/mg in this sample solution to be checked, 2,3; 5, the content of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside is 1.6mg/mg, and content of ferulic acid is 0.3mg/mg; The content of aurantiin is 0.4mg/mg, and content Determination of Icariin is 2.6mg/mg.
embodiment 2.
(1) instrument and reagent.
Instrument: Agilent1200 high performance liquid chromatograph (online degasser, quaternary pump, automatic sampler, column oven, PDAD); The Kromasil100-5C18 chromatographic column; Sartorius 100,000/balance; KQ5200DB type numerical control supersonic washer; Milli-pore-Q ultrapure water preparing instrument.
Reference substance: 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin.
Sample to be checked: protect capsule.
Other reagent: acetonitrile (chromatographically pure), methyl alcohol, glacial acetic acid (analyzing pure).
(2) detection method.
The preparation of a, mixing reference substance solution: precision takes by weighing 2,3, and 5; 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin are an amount of, the dissolve with ethanol with 80%, constant volume; Shake up; Membrane filtration, making chlorogenic acid content is 75 μ g/ml, 2,3; 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside content is that 200 μ g/ml, ferulaic acid content are that 90 μ g/ml, naringin content are that 95 μ g/ml, icariin content are the mixing reference substance solution of 150 μ g/ml.
The preparation of b, sample solution to be checked: get the content that protects capsule and cross sieve No. four, get then about 0.2g carry out precision claim fixed after, place the volumetric flask of 25ml, accurate 80% methyl alcohol that adds is to scale mark; In heating-up temperature is to carry out under 57 ℃ ultrasonic it being dissolved fully, makes the sample solution that concentration is 15mg/ml, shakes up filtering with microporous membrane.
C, chromatographic column are filler with the octadecylsilane chemically bonded silica; Get and mix control sample solution 20 μ L sample introductions; Column temperature is 40 ℃, and flow velocity 1.5ml/min carries out gradient elution by table 5; And adopt and detect the ultraviolet light multi-wavelength detection that wavelength is 267nm, 280nm, 323nm and 328nm, obtain mixing the high-efficient liquid phase chromatogram of reference substance solution.
F phase, G phase and H proportioning table mutually in table 5 moving phase
| Time/minute | F/% | G/% | H/% |
| 0 | 8 | 75 | 15 |
| 6 | 8 | 75 | 12 |
| 10 | 20 | 80 | 15 |
| 20 | 23 | 60 | 20 |
| 30 | 28 | 48 | 23 |
| 45 | 35 | 35 | 32 |
Wherein, the G of moving phase is 2% acetic acid mutually, and its preparation method is: analytically pure glacial acetic acid is added water be mixed with 2% acetum; The F of moving phase is methyl alcohol mutually, and the H of moving phase is acetonitrile mutually.
Obtain the high-efficient liquid phase chromatogram of sample solution to be checked according to the method described above, wherein, the sample size of sample solution to be checked is 15 μ L, flow velocity 1.5ml/min.
(3) result relatively.
See Fig. 1, the ownership at A, B, C, D, each peak of E is respectively chlorogenic acid, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin in the high-efficient liquid phase chromatogram of mixing reference substance solution.
See Fig. 1 and Fig. 2; The retention time and the peak area at the high-efficient liquid phase chromatogram of sample solution more to be checked and each peak of high-efficient liquid phase chromatogram that mixes reference substance solution; Can know that chlorogenic acid, 2 is represented at A, B, C, D, each peak of E respectively in the high-efficient liquid phase chromatogram of sample solution to be checked; 3,5, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin; Chlorogenic acid contents is 0.4mg/mg in this sample solution to be checked, 2,3; 5, the content of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside is 1.8mg/mg, and content of ferulic acid is 0.5mg/mg; The content of aurantiin is 0.6mg/mg, and content Determination of Icariin is 2.9mg/mg.
Embodiment 3.
(1) instrument and reagent.
Instrument: Agilent1200 high performance liquid chromatograph (online degasser, quaternary pump, automatic sampler, column oven, PDAD); The Kromasil100-5C18 chromatographic column; Sartorius 100,000/balance; KQ5200DB type numerical control supersonic washer; Milli-pore-Q ultrapure water preparing instrument.
Reference substance: 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin.
Sample to be checked: protect capsule.
Other reagent: acetonitrile (chromatographically pure), methyl alcohol, glacial acetic acid (analyzing pure).
(2) detection method.
The preparation of a, mixing reference substance solution: precision takes by weighing 2,3, and 5; 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin are an amount of, the dissolve with ethanol with 95%, constant volume; Shake up; Membrane filtration, making chlorogenic acid content is 100 μ g/ml, 2,3; 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside content is that 160 μ g/ml, ferulaic acid content are that 80 μ g/ml, naringin content are that 1055 μ g/ml, icariin content are the mixing reference substance solution of 120 μ g/ml.
The preparation of b, sample solution to be checked: get the content that protects capsule and cross sieve No. four, get then about 0.3g carry out precision claim fixed after, place the volumetric flask of 25ml, accurate 80% methyl alcohol that adds is to scale mark; In heating-up temperature is to carry out under 57 ℃ ultrasonic it being dissolved fully, makes the sample solution that concentration is 20mg/ml, shakes up filtering with microporous membrane.
C, chromatographic column are filler with the octadecylsilane chemically bonded silica; Get and mix control sample solution 20 μ L sample introductions; Column temperature is 20 ℃, and flow velocity 0.5ml/min carries out gradient elution by table 6; And adopt and detect the ultraviolet light multi-wavelength detection that wavelength is 273nm, 286nm, 317nm and 322nm, obtain mixing the high-efficient liquid phase chromatogram of reference substance solution.
F phase, G phase and H proportioning table mutually in table 6 moving phase
| Time/minute | F/% | G/% | H/% |
| 0 | 15 | 72 | 13 |
| 4 | 15 | 72 | 12 |
| 6 | 20 | 75 | 20 |
| 12 | 25 | 65 | 20 |
| 20 | 28 | 48 | 23 |
| 30 | 32 | 45 | 35 |
Wherein, the G of moving phase is 0.1% acetic acid mutually, and its preparation method is: analytically pure glacial acetic acid is added water be mixed with 0.1% acetum; The F of moving phase is methyl alcohol mutually, and the H of moving phase is acetonitrile mutually.
Obtain the high-efficient liquid phase chromatogram of sample solution to be checked according to the method described above, wherein, the sample size of sample solution to be checked is 10 μ L, flow velocity 0.5ml/min.
(3) result relatively.
See Fig. 1, the ownership at A, B, C, D, each peak of E is respectively chlorogenic acid, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin in the high-efficient liquid phase chromatogram of mixing reference substance solution.
See Fig. 1 and Fig. 2; The retention time and the peak area at the high-efficient liquid phase chromatogram of sample solution more to be checked and each peak of high-efficient liquid phase chromatogram that mixes reference substance solution; Can know that chlorogenic acid, 2 is represented at A, B, C, D, each peak of E respectively in the high-efficient liquid phase chromatogram of sample solution to be checked; 3,5, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin; Chlorogenic acid contents is 0.4mg/mg in this sample solution to be checked, 2,3; 5, the content of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside is 2.0mg/mg, and content of ferulic acid is 0.6mg/mg; The content of aurantiin is 0.8mg/mg, and content Determination of Icariin is 3.1mg/mg.
Should be noted that at last; Above embodiment is only in order to explain technical scheme of the present invention; But not to the restriction of protection domain of the present invention, although with reference to preferred embodiment the present invention has been done explanation at length, those of ordinary skill in the art is to be understood that; Can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the essence and the scope of technical scheme of the present invention.
Claims (9)
1. prevent and treat the detection method that osteoporosis Chinese medicine protects the effective constituent of capsule for one kind; The said Chinese prescription that protects capsule is the fleece-flower root, barrenwort, prepared rhizome of rehmannia, tortoise plastron, Morinda officinalis, the bark of eucommia, teasel root, the rhizome of davallia, Radix Angelicae Sinensis and Chinese yam; It is characterized in that: said detection method of protecting the effective constituent of capsule is to utilize the high performance liquid chromatography detection of multi-wavelength to protect the various effective constituents in the capsule, and concrete step is:
A, prepare sample solution to be checked: get and protect the capsule content, be mixed with the sample solution to be checked of drug 4mg/ml～20mg/ml with solvent;
B, preparation mix reference substance solution: get 2; 3; 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, icariin, chlorogenic acid, aurantiin mix the back and are mixed with the reference substance solution that each constituent concentration is 1 μ g/ml～200 μ g/ml with solvent;
C, then with sample solution to be checked with mix reference substance solution and carry out high performance liquid chromatography by following method respectively and detect:
With the octadecylsilane chemically bonded silica is chromatographic column filler; By sample size is 1 μ L～20 μ L sample introductions; Column temperature is 10 ℃～40 ℃; Use by F phase, G mutually and the moving phase of H phase composition carry out gradient elution with the flow velocity of 0.3ml/min～1.5ml/min, adopting simultaneously and detecting wavelength is that the ultraviolet light multi-wavelength of 267nm～273nm, 280～286nm, 317nm～323nm and 322nm～328nm detects, and obtains sample solution to be checked and the high-efficient liquid phase chromatogram that mixes reference substance solution;
D, relatively with the high-efficient liquid phase chromatogram of sample solution to be checked and the high-efficient liquid phase chromatogram that mixes reference substance solution; With the identical peak of retention time of corresponding composition in the high-efficient liquid phase chromatogram that mixes reference substance solution promptly is the characteristic peak with this composition same substance, and calculates content according to peak area by external standard method;
Wherein, said gradient elution step is: 0 minute, the percent by volume of F phase was 5%～20% in the moving phase, and the percent by volume of G phase is 90%～60% in the moving phase, and the percent by volume of H phase is 5%～20% in the moving phase; 2 minutes～7 minutes, F was 5%～20% mutually, and G is 90%～60% mutually, and H is 5%～20% mutually; 5 minutes～14 minutes, F was 7%～30% mutually, and G is 86%～40% mutually, and H is 7%～30% mutually; 10 minutes～23 minutes, F was 11%～35% mutually, and G is 78%～30% mutually, and H is 11%～35% mutually; 15 minutes～35 minutes, F was 15%～40% mutually, and G is 70%～20% mutually, and H is 15%～40% mutually; 20 minutes～55 minutes, F was 20%～45% mutually, and G is 60%～10% mutually, and H is 20%～45% mutually; Wherein, above number percent is percent by volume;
Wherein, each peak is followed successively by chlorogenic acid, 2,3,5 in the high-efficient liquid phase chromatogram of said mixing reference substance solution, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-glucoside, forulic acid, aurantiin, icariin.
2. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 1 is characterized in that: said preparation sample solution to be checked can be with the used solvent of mixing reference substance solution: 10%～95% ethanol, 10%～100% methyl alcohol or the solvent identical with moving phase.
3. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 1, it is characterized in that: the F of said moving phase is methyl alcohol mutually; The H of said moving phase is acetonitrile mutually; The G of said moving phase is 0.1%～2% acetic acid mutually.
4. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 3, it is characterized in that: the preparation method of said G phase is: add entry in the analytically pure glacial acetic acid and be mixed with 0.1%～2% acetic acid.
5. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 1; It is characterized in that: said gradient elution step is: 0 minute; The percent by volume of F phase is 7%～15% in the moving phase; The percent by volume of G phase is 86%～70% in the moving phase, and the percent by volume of H phase is 7%～15% in the moving phase; 3 minutes～6 minutes, F was 7%～15% mutually, and G is 86%～70% mutually, and H is 7%～15% mutually; 6 minutes～12 minutes, F was 9%～20% mutually, and G is 82%～60% mutually, and H is 9%～20% mutually; 12 minutes～20 minutes, F was 14%～25% mutually, and G is 72%～50% mutually, and H is 14%～25% mutually; 20 minutes～30 minutes, F was 20%～30% mutually, and G is 60%～40% mutually, and H is 20%～30% mutually; 30 minutes～45 minutes, F was 25%～35% mutually, and G is 50%～30% mutually, and H is 25%～35% mutually; Wherein, above number percent is percent by volume.
6. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 5; It is characterized in that: said gradient elution step is: 0 minute; The percent by volume of F phase is 10% in the moving phase; The percent by volume of G phase is 80% in the moving phase, and the percent by volume of H phase is 10% in the moving phase; 5 minutes, F was 10% mutually, and G is 80% mutually, and H is 10% mutually; 8 minutes, F was 13% mutually, and G is 74% mutually, and H is 13% mutually; 15 minutes, F was 17% mutually, and G is 66% mutually, and H is 17% mutually; 25 minutes, F was 25% mutually, and G is 50% mutually, and H is 25% mutually; 35 minutes, F was 30% mutually, and G is 40% mutually, and H is 30% mutually; Wherein, above number percent is percent by volume.
7. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 1 is characterized in that: the detection wavelength that said ultraviolet light multi-wavelength detects is 270nm, 283nm, 320nm, 325nm.
8. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 1, it is characterized in that: the flow velocity of said moving phase is 0.8 ml/min.
9. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 1, it is characterized in that: said chromatographic column column temperature is 30 ℃.
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