CN102507825B - 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 made 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 for the treatment of lumbar vertebrae pain that middle-aged and old syndrome of deficiency of kidney essence occur, soft unable, can not be prudent, the lower limb impotence weak, walk with difficulty, or talagia, complexion dull, send out de-, 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, about the primary standard of effective substance, only detects respectively the single components such as the fleece-flower root, icariin, cultivated land.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 meet the requirement of quality monitoring.
Summary of the invention
The object of the invention is to for the deficiencies in the 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 quantitatively detect the plurality of Chinese composition that protects capsule simultaneously, the detection composition is complete, simple to operate, can meet 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 described 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: described 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 capsule, and concrete step is:
A, prepare sample solution to be checked: get and protect the capsule content, with solvent, be mixed with the sample solution to be checked containing medicine 4mg/ml～20mg/ml;
B, preparation mix reference substance solution: get 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, icariin, chlorogenic acid, aurantiin are mixed with solvent the reference substance solution that each constituent concentration is 1 μ g/ml～200 μ g/ml after mixing;
C, then by sample solution to be checked with mix reference substance solution and carry out by the following method respectively the high performance liquid chromatography detection:
Take octadecylsilane chemically bonded silica as chromatographic column filler, by sample size, be 1 μ L～20 μ L sample introductions, column temperature is 10 ℃～40 ℃, use mobile phase by F phase, G phase and H phase composition with the flow velocity of 0.3ml/min～1.5ml/min and carry out gradient elution by the proportioning of table 1, adopt and detect the ultraviolet light multi-wavelength detection that wavelength is 267nm～273nm, 280～286nm, 317nm～323nm and 322nm～328nm simultaneously, obtain sample solution to be checked and mix the high-efficient liquid phase chromatogram of reference substance solution;
The proportioning table of F phase, G phase and H phase in table 1 mobile 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 table 1 is percent by volume;
Described gradient elution step is: 0 minute, in mobile phase, the percent by volume of F phase was 5%～20%, and in mobile phase, the percent by volume of G phase is 90%～60%, and in mobile phase, the percent by volume of H phase is 5%～20%; 2 minutes～7 minutes, F was that 5%～20%, G is that 90%～60%, H is 5%～20% mutually mutually mutually; 5 minutes～14 minutes, F was that 7%～30%, G is that 86%～40%, H is 7%～30% mutually mutually mutually; 10 minutes～23 minutes, F was that 11%～35%, G is that 78%～30%, H is 11%～35% mutually mutually mutually; 15 minutes～35 minutes, F was that 15%～40%, G is that 70%～20%, H is 15%～40% mutually mutually mutually; 20 minutes～55 minutes, F was that 20%～45%, G is that 60%～10%, H is 20%～45% mutually mutually mutually; Wherein, above number percent is percent by volume;
D, by the high-efficient liquid phase chromatogram of sample solution to be checked and the high-efficient liquid phase chromatogram that mixes reference substance solution relatively, the peak identical with the retention time of corresponding composition in the high-efficient liquid phase chromatogram that mixes reference substance solution be the characteristic peak with this composition same substance, and calculate content according to peak area by external standard method.
Wherein, in the high-efficient liquid phase chromatogram of described mixing reference substance solution, each peak is followed successively by chlorogenic acid, 2,3,5, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin;
Described preparation sample solution to be checked with mix reference substance solution solvent used and can be: 10%～95% ethanol, 10%～100% methyl alcohol or the solvent identical with mobile phase, the Chinese medicines such as that described preparation sample solution to be checked and the dissolution mechanism that mixes reference substance solution are is ultrasonic, refluxing extraction are commonly used to be extracted, the purifying mode.
The F of described mobile phase is methyl alcohol mutually; The H of described mobile phase is acetonitrile mutually; The G of described mobile phase is 0.1%～2% acetic acid mutually.
The preparation method of described G phase is: in analytically pure glacial acetic acid, add water to be mixed with 0.1%～2% acetic acid.
Sample solution to be checked of the present invention can adopt following method preparation: precision takes and protects in right amount capsule content medicine, then by dissolution with solvents and adopt traditional Chinese medicine extraction purification process commonly used to be processed, finally with solvent, be settled to concentration for containing content medicine 4mg/ml～20mg/ml, filter, get final product.
Preferably, the proportioning of described mobile phase F phase, G phase and H phase is as shown in table 2 during gradient elution.
The proportioning table of F phase, G phase and H phase in table 2 mobile 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 table 2 is percent by volume;
Described gradient elution step is: 0 minute, in mobile phase, the percent by volume of F phase was 7%～15%, and in mobile phase, the percent by volume of G phase is 86%～70%, and in mobile phase, the percent by volume of H phase is 7%～15%; 3 minutes～6 minutes, F was that 7%～15%, G is that 86%～70%, H is 7%～15% mutually mutually mutually; 6 minutes～12 minutes, F was that 9%～20%, G is that 82%～60%, H is 9%～20% mutually mutually mutually; 12 minutes～20 minutes, F was that 14%～25%, G is that 72%～50%, H is 14%～25% mutually mutually mutually; 20 minutes～30 minutes, F was that 20%～30%, G is that 60%～40%, H is 20%～30% mutually mutually mutually; 30 minutes～45 minutes, F was that 25%～35%, G is that 50%～30%, H is 25%～35% mutually mutually mutually; Wherein, above number percent is percent by volume.
Preferred, during gradient elution, the proportioning of described mobile phase F phase, G phase and H phase is as shown in table 3.
The proportioning table of F phase, G phase and H phase in table 3 mobile 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 table 3 is percent by volume;
Described gradient elution step is: 0 minute, in mobile phase, the percent by volume of F phase was 10%, and in mobile phase, the percent by volume of G phase is 80%, and in mobile phase, the percent by volume of H phase is 10%; 5 minutes, F was that 10%, G is that 80%, H is 10% mutually mutually mutually; 8 minutes, F was that 13%, G is that 74%, H is 13% mutually mutually mutually; 15 minutes, F was that 17%, G is that 66%, H is 17% mutually mutually mutually; 25 minutes, F was that 25%, G is that 50%, H is 25% mutually mutually mutually; 35 minutes, F was that 30%, G is that 40%, H is 30% mutually mutually mutually; Wherein, above number percent is percent by volume.
Preferably, the detection wavelength of described ultraviolet light multi-wavelength detection is 270nm, 283nm, 320nm and 325nm.
Preferably, the flow velocity of described mobile phase is 0.8 ml/min.
Preferably, described column temperature is 30 ℃.
The ownership at each peak in the high-efficient liquid phase chromatogram of mixing reference substance solution of the present invention is determined by the following method: prepare respectively 2,3,5, the standard solution of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, icariin, chlorogenic acid, aurantiin, then prepare respectively the high-efficient liquid phase chromatogram of each independent standard solution and described mixing reference substance solution under same chromatographic condition, according to the retention time at the peak of corresponding composition in each independent standard solution chromatogram, determine the ownership of mixing each peak in the reference substance solution chromatogram.Through revision test repeatedly, find to mix in reference substance solution and have five kinds of compositions that the fixing peak that goes out is arranged in described mobile phase, these five kinds of compositions are chlorogenic acid, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin, therefore, can determine the ownership of mixing five peaks in the reference substance solution high-efficient liquid phase chromatogram.
Compared with prior art, beneficial effect is in the present invention:
1) owing to mixing in the reference substance solution high-efficient liquid phase chromatogram, there is the ownership at five peaks to be respectively chlorogenic acid, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, 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 the detection of quantitative and qualitative analysis to protecting capsule, characteristic according to the effective constituent of protecting capsule, mobile phase is selected the acetic acid of methyl alcohol, acetonitrile and 0.1%～2%, and follow procedure continuously changes in mobile phase each ratio between mutually and carries out gradient elution, reach accurate separation and protect the purpose of Multiple components in capsule, to each wash-out, become component selections to detect wavelength simultaneously, thereby further improve the degree of accuracy detected.
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-Glucose glycosides, forulic acid, icariin, chlorogenic acid, these five kinds of compositions of aurantiin quantitatively detect, can investigate more all sidedly and protect main effective constituent composition and ratio situation of change in capsule, reduce analytical procedure and reduced composition variation and error at measurment that the sample pre-treatments link may cause.
4) detection method of the present invention have advantages of easy, stable, precision is high, favorable reproducibility, be easy to grasp.
The accompanying drawing explanation
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 illustrated.
embodiment 1.
(1) instrument and reagent.
Instrument: Agilent1200 high performance liquid chromatograph (online degasser, quaternary pump, automatic sampler, column oven, diode array detector); The Kromasil100-5C18 chromatographic column; Sartorius 100,000/balance; KQ5200DB type numerical control ultrasonic cleaner; Milli-pore-Q ultrapure water preparing instrument.
Reference substance: 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, 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 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, icariin, chlorogenic acid, aurantiin are appropriate, methyl alcohol with 100% dissolves, constant volume, shake up, membrane filtration, making chlorogenic acid content is 80 μ g/ml, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides content is that 150 μ g/ml, ferulaic acid content are that 100 μ g/ml, naringin content are the mixing reference substance solution that 80 μ g/ml, Icariin content are 90 μ g/ml.
The preparation of b, sample solution to be checked: get the content that protects capsule and cross sieve No. four, then get about 0.2g carry out accurately weighed after, be placed in the volumetric flask of 25ml, precision adds 80% methyl alcohol to scale mark; Be to carry out under 57 ℃ ultrasonic it being dissolved fully in heating-up temperature, make the sample solution that concentration is 8mg/ml, shake up filtering with microporous membrane.
C, chromatographic column be take octadecylsilane chemically bonded silica as filler, get and mix control sample solution 20 μ L sample introductions, column temperature is 30 ℃, flow velocity 0.8ml/min, carry out gradient elution by table 4, and adopt the ultraviolet light multi-wavelength detection that the detection wavelength is 270nm, 283nm, 320nm and 325nm, obtain mixing the high-efficient liquid phase chromatogram of reference substance solution.
The proportioning table of F phase, G phase and H phase in table 4 mobile 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 mobile phase is 1% acetic acid mutually, and its preparation method is: analytically pure glacial acetic acid is added to water and be mixed with 1% acetum; The F of mobile phase is methyl alcohol mutually, and the H of mobile phase is acetonitrile mutually.
Obtain according to the method described above the high-efficient liquid phase chromatogram of sample solution to be checked, 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, in the high-efficient liquid phase chromatogram of mixing reference substance solution, the ownership at A, B, C, D, each peak of E is respectively chlorogenic acid, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin.
See Fig. 1 and Fig. 2, 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, in the high-efficient liquid phase chromatogram of known sample solution to be checked, A, B, C, D, each peak of E represent respectively chlorogenic acid, 2,3,5, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin; The content of this sample solution Content of Chlorogenic Acid to be checked is 0.3mg/mg, 2,3,5, the content of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides is 1.6mg/mg, and the content of forulic acid is 0.3mg/mg, the content of aurantiin is 0.4mg/mg, and the content 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, diode array detector); The Kromasil100-5C18 chromatographic column; Sartorius 100,000/balance; KQ5200DB type numerical control ultrasonic cleaner; Milli-pore-Q ultrapure water preparing instrument.
Reference substance: 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, 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 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, icariin, chlorogenic acid, aurantiin are appropriate, ethanol with 80% dissolves, constant volume, shake up, membrane filtration, making chlorogenic acid content is 75 μ g/ml, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides content is that 200 μ g/ml, ferulaic acid content are that 90 μ g/ml, naringin content are the mixing reference substance solution that 95 μ g/ml, Icariin content are 150 μ g/ml.
The preparation of b, sample solution to be checked: get the content that protects capsule and cross sieve No. four, then get about 0.2g carry out accurately weighed after, be placed in the volumetric flask of 25ml, precision adds 80% methyl alcohol to scale mark; Be to carry out under 57 ℃ ultrasonic it being dissolved fully in heating-up temperature, make the sample solution that concentration is 15mg/ml, shake up filtering with microporous membrane.
C, chromatographic column be take octadecylsilane chemically bonded silica as filler, get and mix control sample solution 20 μ L sample introductions, column temperature is 40 ℃, flow velocity 1.5ml/min, carry out gradient elution by table 5, and adopt the ultraviolet light multi-wavelength detection that the detection wavelength is 267nm, 280nm, 323nm and 328nm, obtain mixing the high-efficient liquid phase chromatogram of reference substance solution.
The proportioning table of F phase, G phase and H phase in table 5 mobile 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 mobile phase is 2% acetic acid mutually, and its preparation method is: analytically pure glacial acetic acid is added to water and be mixed with 2% acetum; The F of mobile phase is methyl alcohol mutually, and the H of mobile phase is acetonitrile mutually.
Obtain according to the method described above the high-efficient liquid phase chromatogram of sample solution to be checked, 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, in the high-efficient liquid phase chromatogram of mixing reference substance solution, the ownership at A, B, C, D, each peak of E is respectively chlorogenic acid, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin.
See Fig. 1 and Fig. 2, 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, in the high-efficient liquid phase chromatogram of known sample solution to be checked, A, B, C, D, each peak of E represent respectively chlorogenic acid, 2,3,5, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin; The content of this sample solution Content of Chlorogenic Acid to be checked is 0.4mg/mg, 2,3,5, the content of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides is 1.8mg/mg, and the content of forulic acid is 0.5mg/mg, the content of aurantiin is 0.6mg/mg, and the content 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, diode array detector); The Kromasil100-5C18 chromatographic column; Sartorius 100,000/balance; KQ5200DB type numerical control ultrasonic cleaner; Milli-pore-Q ultrapure water preparing instrument.
Reference substance: 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, 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 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, icariin, chlorogenic acid, aurantiin are appropriate, ethanol with 95% dissolves, constant volume, shake up, membrane filtration, making chlorogenic acid content is 100 μ g/ml, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides content is that 160 μ g/ml, ferulaic acid content are that 80 μ g/ml, naringin content are the mixing reference substance solution that 1055 μ g/ml, Icariin content are 120 μ g/ml.
The preparation of b, sample solution to be checked: get the content that protects capsule and cross sieve No. four, then get about 0.3g carry out accurately weighed after, be placed in the volumetric flask of 25ml, precision adds 80% methyl alcohol to scale mark; Be to carry out under 57 ℃ ultrasonic it being dissolved fully in heating-up temperature, make the sample solution that concentration is 20mg/ml, shake up filtering with microporous membrane.
C, chromatographic column be take octadecylsilane chemically bonded silica as filler, get and mix control sample solution 20 μ L sample introductions, column temperature is 20 ℃, flow velocity 0.5ml/min, carry out gradient elution by table 6, and adopt the ultraviolet light multi-wavelength detection that the detection wavelength is 273nm, 286nm, 317nm and 322nm, obtain mixing the high-efficient liquid phase chromatogram of reference substance solution.
The proportioning table of F phase, G phase and H phase in table 6 mobile 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 mobile phase is 0.1% acetic acid mutually, and its preparation method is: analytically pure glacial acetic acid is added to water and be mixed with 0.1% acetum; The F of mobile phase is methyl alcohol mutually, and the H of mobile phase is acetonitrile mutually.
Obtain according to the method described above the high-efficient liquid phase chromatogram of sample solution to be checked, 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, in the high-efficient liquid phase chromatogram of mixing reference substance solution, the ownership at A, B, C, D, each peak of E is respectively chlorogenic acid, 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin.
See Fig. 1 and Fig. 2, 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, in the high-efficient liquid phase chromatogram of known sample solution to be checked, A, B, C, D, each peak of E represent respectively chlorogenic acid, 2,3,5, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin; The content of this sample solution Content of Chlorogenic Acid to be checked is 0.4mg/mg, 2,3,5, the content of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides is 2.0mg/mg, and the content of forulic acid is 0.6mg/mg, the content of aurantiin is 0.8mg/mg, and the content of icariin is 3.1mg/mg.
Finally should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention; but not limiting the scope of the invention; although with reference to preferred embodiment, the present invention has been done to explain; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.
Claims (8)
1. prevent and treat the detection method that osteoporosis Chinese medicine protects the effective constituent of capsule for one kind, the described 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: described 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 capsule, and concrete step is:
A, prepare sample solution to be checked: get and protect the capsule content, with solvent, be mixed with the sample solution to be checked containing medicine 4mg/ml～20mg/ml;
B, preparation mix reference substance solution: get 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, icariin, chlorogenic acid, aurantiin are mixed with solvent the reference substance solution that each constituent concentration is 1 μ g/ml～200 μ g/ml after mixing;
C, then by sample solution to be checked with mix reference substance solution and carry out by the following method respectively the high performance liquid chromatography detection:
Take octadecylsilane chemically bonded silica as chromatographic column filler, by sample size, be 1 μ L～20 μ L sample introductions, column temperature is 10 ℃～40 ℃, use by the mobile 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, adopt and detect the ultraviolet light multi-wavelength detection that wavelength is 267nm～273nm, 280～286nm, 317nm～323nm and 325nm～328nm simultaneously, obtain sample solution to be checked and mix the high-efficient liquid phase chromatogram of reference substance solution;
D, by the high-efficient liquid phase chromatogram of sample solution to be checked and the high-efficient liquid phase chromatogram that mixes reference substance solution relatively, the peak identical with the retention time of corresponding composition in the high-efficient liquid phase chromatogram that mixes reference substance solution be the characteristic peak with this composition same substance, and calculate content according to peak area by external standard method;
Wherein, described gradient elution step is: 0 minute, in mobile phase, the percent by volume of F phase was 5%～20%, and in mobile phase, the percent by volume of G phase is 90%～60%, and in mobile phase, the percent by volume of H phase is 5%～20%; 2 minutes～7 minutes, F was that 5%～20%, G is that 90%～60%, H is 5%～20% mutually mutually mutually; 5 minutes～14 minutes, F was that 7%～30%, G is that 86%～40%, H is 7%～30% mutually mutually mutually; 10 minutes～23 minutes, F was that 11%～35%, G is that 78%～30%, H is 11%～35% mutually mutually mutually; 15 minutes～35 minutes, F was that 15%～40%, G is that 70%～20%, H is 15%～40% mutually mutually mutually; 20 minutes～55 minutes, F was that 20%～45%, G is that 60%～10%, H is 20%～45% mutually mutually mutually; Wherein, above number percent is percent by volume;
Wherein, in the high-efficient liquid phase chromatogram of described mixing reference substance solution, each peak is followed successively by chlorogenic acid, 2,3,5, the characteristic peak of 4 '-tetrahydroxystilbene-2-O-β-D-Glucose glycosides, forulic acid, aurantiin, icariin;
The F of described mobile phase is methyl alcohol mutually; The H of described mobile phase is acetonitrile mutually; The G of described mobile phase is 0.1%～2% acetic acid mutually.
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: described preparation sample solution to be checked with mix reference substance solution solvent used and be: 10%～95% ethanol, 10%～100% methyl alcohol or the solvent identical with mobile 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 preparation method of described G phase is: in analytically pure glacial acetic acid, add water to be mixed with 0.1%～2% acetic acid.
4. 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: described gradient elution step is: 0 minute, in mobile phase, the percent by volume of F phase is 7%～15%, in mobile phase, the percent by volume of G phase is 86%～70%, and in mobile phase, the percent by volume of H phase is 7%～15%; 3 minutes～6 minutes, F was that 7%～15%, G is that 86%～70%, H is 7%～15% mutually mutually mutually; 6 minutes～12 minutes, F was that 9%～20%, G is that 82%～60%, H is 9%～20% mutually mutually mutually; 12 minutes～20 minutes, F was that 14%～25%, G is that 72%～50%, H is 14%～25% mutually mutually mutually; 20 minutes～30 minutes, F was that 20%～30%, G is that 60%～40%, H is 20%～30% mutually mutually mutually; 30 minutes～45 minutes, F was that 25%～35%, G is that 50%～30%, H is 25%～35% mutually mutually mutually; Wherein, above number percent is percent by volume.
5. a kind of detection method that osteoporosis Chinese medicine protects the effective constituent of capsule of preventing and treating according to claim 4, it is characterized in that: described gradient elution step is: 0 minute, in mobile phase, the percent by volume of F phase is 10%, in mobile phase, the percent by volume of G phase is 80%, and in mobile phase, the percent by volume of H phase is 10%; 5 minutes, F was that 10%, G is that 80%, H is 10% mutually mutually mutually; 8 minutes, F was that 13%, G is that 74%, H is 13% mutually mutually mutually; 15 minutes, F was that 17%, G is that 66%, H is 17% mutually mutually mutually; 25 minutes, F was that 25%, G is that 50%, H is 25% mutually mutually mutually; 35 minutes, F was that 30%, G is that 40%, H is 30% mutually mutually 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 1, it is characterized in that: the detection wavelength of described ultraviolet light multi-wavelength detection is 270nm, 283nm, 320nm, 325nm.
7. 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 described mobile phase is 0.8 ml/min.
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: described chromatographic column column temperature is 30 ℃.
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| CN105056190A (en) * | 2015-08-14 | 2015-11-18 | 李再宝 | Traditional Chinese medicine composition for promoting fracture healing and detection method of effective components |
| CN108272943B (en) * | 2018-03-08 | 2021-03-30 | 广东安诺药业股份有限公司 | A pharmaceutical composition for treating osteoporotic fracture, and its preparation method |
| CN114509507B (en) * | 2020-11-16 | 2024-03-12 | 上海新亚药业邗江有限公司 | Quantitative method for simultaneously measuring multiple indexes in polygonatum sibiricum Zanyu capsules |
| CN114720600A (en) * | 2022-04-07 | 2022-07-08 | 广东安诺药业股份有限公司 | Method for detecting stilbene glucoside component in polygonum multiflorum prepared by bone-protecting capsule |
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