CN109765320A - A kind of content assaying method for spray agent for treating tendon and bone injury - Google Patents
A kind of content assaying method for spray agent for treating tendon and bone injury Download PDFInfo
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Abstract
The present invention relates to a kind of content assaying methods for spray agent for treating tendon and bone injury, more particularly to effective component Paeoniflorin in spray agent for treating tendon and bone injury, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the content assaying method of 7 kinds of ingredients such as precious leaves of pulse plants glycosides I, preparation including test solution, the preparation of reference substance solution, the measurement of HPLC method, using content assaying method provided by the invention, plurality of active ingredients can be measured simultaneously, detection line is steady, separating degree is good, accuracy in detection is high, stability is good, shorten the assay time, improve detection efficiency, for the quality for controlling spray agent for treating tendon and bone injury, provide a kind of more comprehensive quantitative detecting method, stability to investigate spray agent for treating tendon and bone injury simultaneously, provide the method for the more comprehensive stability quantitative expedition of a kind of pair of effective component.
Description
Technical field
The present invention relates to field of medicine invention, and in particular to a kind of content assaying method for spray agent for treating tendon and bone injury.
Background technique
Spray agent for treating tendon and bone injury be by runcinate knotweed herb, radix paeoniae rubra, Herba Epimedii, pheretima, wild aconite root, menthol, it is extracted made of in
Medicine compound preparation has activating microcirculation and removing stasis medicinal, the effect of swelling and pain relieving, is used for soft tissue injury, in spray agent for treating tendon and bone injury it is effective at
Dividing is: Paeoniflorin, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I etc., but muscles and bones wound is spraying
There is no a kind of detection methods of science for the crucial effective component of these in agent, achieve more comprehensive quality testing.
Quantitative target in the existing quality standard of spray agent for treating tendon and bone injury is the Paeoniflorin in radix paeoniae rubra, and radix paeoniae rubra is in prescription
Dosage is few, for runcinate knotweed herb, in quality standard, is also only identified in a manner of thin-layer chromatography, this standard is too simple
It is single, it is difficult to which that effective control is realized to the quality of product.
Existing literature Xu Chaofei, the quality research of seedling medicine " spray agent for treating tendon and bone injury " and quality standard promote [D], Kweiyang medicine
Institute, 2015, disclose it is a kind of with HPLC while measuring 4 kinds of ingredients 3 in spray agent for treating tendon and bone injury, 3'- dimethylellagic acid, 3,3',
The method of 4'- trimethyl ellagic acid, Paeoniflorin, icariin, this method determine 4 kinds of ingredients in a kind of Chinese materia medica preparation simultaneously
Content, but influence muscles and bones hurt quality ingredient it is very much, only measuring wherein 4 kinds of ingredients can not be in effective testing product
Effective component, methyl ellagic acid therein just account for 2 kinds, and experiment shows that effective component is more in Herba Epimedii, only measures icariin
Obviously the effective component in Herba Epimedii cannot be effectively detected, so in the measurement spray agent for treating tendon and bone injury that method can not be scientific
Active constituent content, and if operated multiple for the effective component in spray agent for treating tendon and bone injury repeatedly with different reference substance measurements
It is miscellaneous, it is inefficient.
Therefore, for the content of effective component in more preferable more accurately measurement spray agent for treating tendon and bone injury, promote and control muscles and bones
The quality for hurting spray, the present invention provides it is a kind of it is easy to operate, specificity is strong, separating degree is good, stability is high, efficiently and can be same
When in spray agent for treating tendon and bone injury 7 kinds of effective components carry out assay method.
Summary of the invention
For existing problem, originally the purpose for invention is to provide one kind and is used for while measuring effective in spray agent for treating tendon and bone injury
Ingredient Paeoniflorin, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the content assaying method of precious leaves of pulse plants glycosides I and general
This method is studied applied to spray agent for treating tendon and bone injury study on the stability, and this method is easy to operate, specificity is strong, separating degree is good, stablizes
Property it is high, efficiently, and 7 kinds of ingredients can be measured simultaneously, effective detection is realized to the quality of product.
A kind of content assaying method for spray agent for treating tendon and bone injury of the present invention, specifically: use high-efficient liquid phase color
Spectrometry carries out assay to 7 kinds of effective components in spray agent for treating tendon and bone injury simultaneously, and 7 kinds of effective components of the assay are specific
Are as follows: Paeoniflorin, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I, the content assaying method packet
Include following steps: the preparation of test solution, the preparation of reference substance solution, the measurement of HPLC method.
The specific steps of sample solution preparation method of the present invention are as follows: stringing bone fracture 2~10ml of spray, precision are inhaled
It taking, sets in 100~200ml evaporating dish, be evaporated, 2~10ml water is added to dissolve, lysate is transferred in 25~50ml test tube, add 5~
20ml water-saturated n-butanol solution in test tube, extraction, then plus 5~20ml water-saturated n-butanol solution extraction, take upper layer to clarify
Liquid is set in 100~200ml evaporating dish, is evaporated, dissolved with methanol, is settled in 2~10ml volumetric flask, with filtering with microporous membrane,
Take subsequent filtrate as test solution.
Preferably, test solution of the present invention the preparation method comprises the following steps: take 6~8ml of spray agent for treating tendon and bone injury, precision is inhaled
It takes, sets in 100ml evaporating dish, be evaporated, 5ml water is added to dissolve, lysate is transferred in 25ml test tube, adds 10ml water-saturated n-butanol
Solution in test tube, extraction, then plus 10ml water-saturated n-butanol solution extraction, take supernatant liquid, set in 100ml evaporating dish,
It is evaporated, is dissolved with methanol, is settled in 10ml volumetric flask, with filtering with microporous membrane, take subsequent filtrate as test solution.
The specific steps of reference substance solution preparation method of the present invention are as follows: take icariin reference substance, methanol is added to dissolve
It is 0.15~0.32mg/ml to concentration, it is spare；Epimedin A reference substance is taken, adding methanol to be dissolved to concentration is 1~2mg/ml, standby
With；Epimedin B reference substance is taken, adding methanol to be dissolved to concentration is 0.8~2mg/ml, spare；Epimedin C reference substance is taken, methanol is added
Being dissolved to concentration is 0.01~0.1mg/ml, spare；Take precious leaves of pulse plants glycosides I reference substance, add methanol be dissolved to concentration be 0.02~
0.03mg/ml, it is spare；Ellagic acid reference substance is taken, adding methanol to be dissolved to concentration is 0.8~2mg/ml, spare；Paeoniflorin is taken to compare
Product, adding methanol to be dissolved to concentration is 0.8~2mg/ml, spare；By reference substance solution Paeoniflorin, icariin, the tan flower of preparation
Acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I, take respectively 1~2ml, 1~1.5ml, 1~1.5ml, 100~150ul,
50~100ul, 250~300ul, 100~150ul are made into the mixed reference substance solution of 7 kinds of ingredients.
Preferably, the specific steps of reference substance solution preparation method of the present invention are as follows: take icariin reference substance, add first
It is 0.236mg/ml that alcohol, which is dissolved to concentration, spare；Epimedin A reference substance is taken, adding methanol to be dissolved to concentration is 1.5mg/ml, standby
With；Epimedin B reference substance is taken, adding methanol to be dissolved to concentration is 1.2mg/ml, spare；Epimedin C reference substance is taken, methanol is added to dissolve
It is 0.05mg/ml to concentration, it is spare；Precious leaves of pulse plants glycosides I reference substance is taken, adding methanol to be dissolved to concentration is 0.026mg/ml, spare；Take tan
Sour reference substance is spent, adding methanol to be dissolved to concentration is 1.2mg/ml, spare；Paeoniflorin reference substance is taken, adds methanol to be dissolved to concentration to be
1.43mg/ml spare；
By the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the precious leaves of pulse plants
Glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be settled to 5ml respectively and is made into the mixed of 7 kinds of ingredients
Close reference substance solution.
The chromatographic condition of HPLC method measurement of the present invention are as follows: Agilent ZORBA*S8~C18 column；Inner diameter size is
4.6*150mm,5um；Mobile phase B is the potassium dihydrogen phosphate of 0.05mol/L, and mobile phase C is methanol, and mobile phase D is acetonitrile；
0.85~1ml/min of flow velocity, Detection wavelength 0~23min:230nm, 23~54min:254nm, 54~137min:270nm, column
35 DEG C of temperature carries out gradient elution.
Gradient elution method of the present invention are as follows:
| Time | Mobile phase B | Mobile phase C | Mobile phase D | Flow velocity |
| 0~16min | 92%~90% | —— | 8%~10% | 1ml/min |
| 16~20min | 90% | —— | 10% | 1ml/min |
| 20~22min | 90%~88% | —— | 10%~12% | 1ml/min |
| 22~33min | 88%~83% | —— | 12%~17% | 1ml/min |
| 33min~50min | 83%~79% | —— | 17%~21% | 1ml/min |
| 50~51min | 79%~77.5% | —— | 21%~22.5% | 1~0.85ml/min |
| 51~80min | 77.5% | —— | 22.5% | 0.85ml/min |
| 80~80.01min | 77.5%~67% | 0~11% | 22.5%~22% | 0.85~1ml/min |
| 80.01~97min | 67%~65% | 11%~0% | 22%~35% | 1ml/min |
| 97~107min | 65% | —— | 35% | 1ml/min |
| 107~117min | 65%~50% | —— | 35%~50% | 1ml/min |
| 117~137min | 50%~35% | —— | 50~65% | 1ml/min |
Preferably, gradient elution method of the present invention are as follows:
| Time | Mobile phase | Mobile phase C | Mobile phase D | Flow velocity |
| 0-14min | 92%-90% | —— | 8%-10% | 1ml/min |
| 14-17.5min | 90% | —— | 10% | 1ml/min |
| 17.5-22min | 90%-88% | —— | 10%-12% | 1ml/min |
| 22-33min | 88%-83% | —— | 12%-17% | 1ml/min |
| 33-50min | 83%-79% | —— | 17%-21% | 1ml/min |
| 50-51min | 79%-77.5% | —— | 21%-22.5% | 1-0.85ml/min |
| 51-80min | 77.5% | —— | 22.5% | 0.85ml/min |
| 80-80.01min | 77.5%-67% | 0-11% | 22.5%-22% | 0.85-1ml/min |
| 80.01-97min | 67%-65% | 11%-0% | 22%-35% | 1ml/min |
| 97-107min | 65% | —— | 35% | 1ml/min |
| 107-117min | 65%-50% | —— | 35%-50% | 1ml/min |
| 117-137min | 50%-35% | —— | 50-65% | 1ml/min |
HPLC method measuring method of the present invention are as follows: the accurate absorption test solution and control solution each 5 of difference~
10uL, inject high performance liquid chromatograph, measurement, calculate to get.
Through practical measurement, the content of Paeoniflorin is 2000~2500ug/ml in spray agent for treating tendon and bone injury of the present invention；Tan flower
The content of acid is 400~500ug/ml；The content of Epimedin A is 20~30ug/ml；The content of Epimedin B is 30~40ug/
ml；The content of epimedin C is 20~30ug/ml；The content of icariin is 100~150ug/ml；The content of precious leaves of pulse plants glycosides I is 10
~15ug/ml.
Through practical measurement, it is preferred that the content of Paeoniflorin is 2028ug/ml in spray agent for treating tendon and bone injury of the present invention；Tan
The content for spending acid is 476ug/ml；The content of Epimedin A is 25ug/ml；The content of Epimedin B is 38ug/ml；Epimedin C
Content is 23ug/ml；The content of icariin is 148ug/ml；The content of precious leaves of pulse plants glycosides I is 11ug/ml.
The present invention will simultaneously hurt the method that 7 kinds of effective components carry out assay in spray agent for treating tendon and bone injury applied to muscles and bones
The stability study of spray is investigated.
Compared with the prior art, the present invention has the following advantages:
1, [D] Guiyang Medical College is promoted with the quality research of Xu Chaofei seedling medicine " spray agent for treating tendon and bone injury " and quality standard,
Technology disclosed in 2015 is compared, and documents determine a kind of content of 4 kinds of ingredients in Chinese materia medica preparation simultaneously, and the present invention is same
When measure the content of 7 kinds of effective component, overcome while in the prior art cannot be effectively 7 kinds of effective component in detection product
Defect.
2, the present invention establishes for the first time while measuring the content assaying method of 7 kinds of effective component in spray agent for treating tendon and bone injury, real
Measurement while having showed 7 kinds of ingredients effectively has detected 7 kinds in spray agent for treating tendon and bone injury in three main medicinal materials and most represents
Property effective component Paeoniflorin, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I content, be muscle
The foundation in the liquid-phase fingerprint library of bone fracture spray provides a kind of measurement scheme rapidly and efficiently.
3, a kind of quantitative expedition method of stability for having more comprehensive effective component is provided.
4, detection method of content of the invention, detection baseline is steady, separating degree is high, accuracy in detection is high, stability is good.
Detailed description of the invention:
The HPLC chromatogram of mixed reference substance solution in Fig. 1 embodiment 1, wherein the corresponding compound in 1~7 peak is respectively
1, Paeoniflorin；2, ellagic acid；3, Epimedin A；4, Epimedin B；5, epimedin C；6, icariin；7, precious leaves of pulse plants glycosides I.
The HPLC chromatogram of test solution in Fig. 2 embodiment 1, wherein the corresponding compound in 1~7 peak is 1, Chinese herbaceous peony respectively
Medicine glycosides；2, ellagic acid；3, Epimedin A；4, Epimedin B；5, epimedin C；6, icariin；7, precious leaves of pulse plants glycosides I.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Instrument involved in following embodiment includes: that 1100 high performance liquid chromatograph of Agilent (includes: online de-
Mechanism of qi, quaternary pump, autosampler, DAD detector, the U.S.), Agilent chromatographic work station (the limited public affairs of U.S.'s Agilent Technologies
Department)；CH~250 model supersonic wave cleaning machine (innovates moral ultrasonic electronic research institute in Beijing)；The electronic balance of XS~105 (Switzerland Mei Te
Le~support benefit company)；Liquid-transfering gun (range is 20~200 μ L and 100~1000 μ L, German eppendorf company).Reagent packet
Include: reference substance (is purchased from Sichuan Wei Keqi Biotechnology Co., Ltd): Paeoniflorin (lot number: wkq18032104)；Ellagic acid (batch
Number: wkq18020502)；Epimedin A (lot number: wkq17040509)；Epimedin B (lot number: wkq17032809)；Epimedin C
(lot number: wkq17040701)；Precious leaves of pulse plants glycosides I (lot number: wkq16081303)；Icariin (is purchased from: Chinese pharmaceutical biological product inspection
Determine institute) (lot number: 110737~200414).
Embodiment 1
In accordance with the following methods detect spray agent for treating tendon and bone injury in effective component Paeoniflorin, icariin, ellagic acid, Epimedin A,
The content of Epimedin B, epimedin C, precious leaves of pulse plants glycosides I.
Chromatographic condition: Agilent ZORBA*S8~C18 column；Inner diameter size is 4.6*150mm, 5um, and Mobile phase B is
The potassium dihydrogen phosphate of 0.05mol/L, mobile phase C be methanol, mobile phase D be acetonitrile, 0~23min:230nm of Detection wavelength,
23~54min:254nm, 54~137min:270nm, 35 DEG C of column temperature, gradient elution method are as follows:
| Time | Mobile phase B | Mobile phase C | Mobile phase D | Flow velocity |
| 0~14min | 92%~90% | —— | 8%~10% | 1ml/min |
| 14~17.5min | 90% | —— | 10% | 1ml/min |
| 17.5~22min | 90%~88% | —— | 10%~12% | 1ml/min |
| 22~33min | 88%~83% | —— | 12%~17% | 1ml/min |
| 33~50min | 83%~79% | —— | 17%~21% | 1ml/min |
| 50~51min | 79%~77.5% | —— | 21%~22.5% | 1~0.85ml/min |
| 51~80min | 77.5% | —— | 22.5% | 0.85ml/min |
| 80~80.01min | 77.5%~67% | 0~11% | 22.5%~22% | 0.85~1ml/min |
| 80.01~97min | 67%~65% | 11%~0% | 22%~35% | 1ml/min |
| 97~107min | 65% | —— | 35% | 1ml/min |
| 107~117min | 65%~50% | —— | 35%~50% | 1ml/min |
| 117~137min | 50%~35% | —— | 50~65% | 1ml/min |
The preparation of test solution: taking spray agent for treating tendon and bone injury 5ml, and precision is drawn, sets in 100ml evaporating dish, be evaporated, add
The dissolution of 5ml water, lysate are transferred in 25ml test tube, add 10ml water-saturated n-butanol solution in test tube, are extracted, then plus 10ml
The extraction of water-saturated n-butanol solution, takes supernatant liquid, sets in 100ml evaporating dish, be evaporated, dissolved with methanol, be settled to 10ml
In volumetric flask, with filtering with microporous membrane, take subsequent filtrate as test solution.
The preparation of reference substance solution: icariin reference substance, Epimedin A reference substance, Epimedin B reference substance, epimedin C are taken
Reference substance, treasured leaves of pulse plants glycosides I reference substance, ellagic acid reference substance and Paeoniflorin reference substance, add methanol to dissolve, concentration, which is respectively prepared, is
Epimedin A reference substance solution that icariin reference substance solution, the concentration of 0.236mg/ml is 1.5mg/ml, concentration 1.2mg/
The treasured that epimedin C reference substance solution that Epimedin B reference substance solution, the concentration of ml is 0.05mg/ml, concentration are 0.026mg/ml
The Paeoniflorin control that ellagic acid reference substance solution that leaves of pulse plants glycosides I reference substance solution, concentration are 1.2mg/ml, concentration are 1.43mg/ml
Product solution, it is spare, by the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C,
Precious leaves of pulse plants glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be settled to 5ml respectively and is made into 7 kinds of ingredients
Mixed reference substance solution.
High effective liquid chromatography for measuring: accurate respectively to draw each 10uL of test solution and control solution, injection is efficient
Liquid chromatograph, measurement, calculate to get.
The HPLC chromatogram of mixed reference substance solution is shown in Fig. 1.
The HPLC chromatogram of test solution is shown in Fig. 2.
Study on the stability: above-mentioned content assaying method is applied to study on the stability.
Embodiment 2
In accordance with the following methods detect spray agent for treating tendon and bone injury in effective component Paeoniflorin, icariin, ellagic acid, Epimedin A,
The content of Epimedin B, epimedin C, precious leaves of pulse plants glycosides I.
Chromatographic condition: Agilent ZORBA*S8~C18 column；Inner diameter size is 4.6*150mm, 5um, and Mobile phase B is
The potassium dihydrogen phosphate of 0.05mol/L, mobile phase C be methanol, mobile phase D be acetonitrile, 0~23min:230nm of Detection wavelength,
23~54min:254nm, 54~137min:270nm, 35 DEG C of column temperature, gradient elution method are as follows:
The preparation of test solution: taking spray agent for treating tendon and bone injury 10ml, and precision is drawn, sets in 100ml evaporating dish, be evaporated, add
5ml aqueous dissolution, lysate are transferred in 25ml test tube, add 10ml water-saturated n-butanol solution in test tube, extraction, then plus
The extraction of 10ml water-saturated n-butanol solution, takes supernatant liquid, sets in 100ml evaporating dish, be evaporated, dissolved with methanol, is settled to
In 5ml volumetric flask, with filtering with microporous membrane, take subsequent filtrate as test solution.
The preparation of reference substance solution: icariin reference substance, Epimedin A reference substance, Epimedin B reference substance, epimedin C are taken
Reference substance, treasured leaves of pulse plants glycosides I reference substance, ellagic acid reference substance and Paeoniflorin reference substance, add methanol to dissolve, concentration, which is respectively prepared, is
Epimedin A reference substance solution that icariin reference substance solution, the concentration of 0.236mg/ml is 1.5mg/ml, concentration 1.2mg/
The treasured that epimedin C reference substance solution that Epimedin B reference substance solution, the concentration of ml is 0.05mg/ml, concentration are 0.026mg/ml
The Paeoniflorin control that ellagic acid reference substance solution that leaves of pulse plants glycosides I reference substance solution, concentration are 1.2mg/ml, concentration are 1.43mg/ml
Product solution, it is spare, by the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C,
Precious leaves of pulse plants glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be settled to 5ml respectively and is made into 7 kinds of ingredients
Mixed reference substance solution.
High effective liquid chromatography for measuring: accurate respectively to draw each 10uL of test solution and control solution, injection is efficient
Liquid chromatograph, measurement, calculate to get.
Study on the stability: above-mentioned content assaying method is applied to study on the stability.
Embodiment 3
In accordance with the following methods detect spray agent for treating tendon and bone injury in effective component Paeoniflorin, icariin, ellagic acid, Epimedin A,
The content of Epimedin B, epimedin C, precious leaves of pulse plants glycosides I.
Chromatographic condition: Agilent ZORBA*S8~C18 column；Inner diameter size is 4.6*150mm, 5um, and Mobile phase B is
The potassium dihydrogen phosphate of 0.05mol/L, mobile phase C be methanol, mobile phase D be acetonitrile, 0~23min:230nm of Detection wavelength,
23~54min:254nm, 54~137min:270nm, 35 DEG C of column temperature, gradient elution method is as follows:
| Time | Mobile phase B | Mobile phase C | Mobile phase D | Flow velocity |
| 0~14min | 92%~90% | —— | 8%~10% | 1ml/min |
| 14~17.5min | 90% | —— | 10% | 1ml/min |
| 17.5~22min | 90%~88% | —— | 10%~12% | 1ml/min |
| 22~33min | 88%~83% | —— | 12%~17% | 1ml/min |
| 33~50min | 83%~79% | —— | 17%~21% | 1ml/min |
| 50~51min | 79%~77.5% | —— | 21%~22.5% | 1~0.85ml/min |
| 51~80min | 77.5% | —— | 22.5% | 0.85ml/min |
| 80~80.01min | 77.5%~67% | 0~11% | 22.5%~22% | 0.85~1ml/min |
| 80.01~97min | 67%~65% | 11%~0% | 22%~35% | 1ml/min |
| 97~107min | 65% | —— | 35% | 1ml/min |
| 107~117min | 65%~50% | —— | 35%~50% | 1ml/min |
| 117~137min | 50%~35% | —— | 50~65% | 1ml/min |
The preparation of test solution: taking spray agent for treating tendon and bone injury 8ml, and precision is drawn, sets in 100ml evaporating dish, be evaporated, add
5ml aqueous dissolution, lysate are transferred in 25ml test tube, add 10ml water-saturated n-butanol solution in test tube, extraction, then plus
The extraction of 10ml water-saturated n-butanol solution, takes supernatant liquid, sets in 100ml evaporating dish, be evaporated, dissolved with methanol, is settled to
In 5ml volumetric flask, with filtering with microporous membrane, take subsequent filtrate as test solution.
The preparation of reference substance solution: icariin reference substance, Epimedin A reference substance, Epimedin B reference substance, epimedin C are taken
Reference substance, treasured leaves of pulse plants glycosides I reference substance, ellagic acid reference substance and Paeoniflorin reference substance, add methanol to dissolve, concentration, which is respectively prepared, is
Epimedin A reference substance solution that icariin reference substance solution, the concentration of 0.236mg/ml is 1.5mg/ml, concentration 1.2mg/
The treasured that epimedin C reference substance solution that Epimedin B reference substance solution, the concentration of ml is 0.05mg/ml, concentration are 0.026mg/ml
The Paeoniflorin control that ellagic acid reference substance solution that leaves of pulse plants glycosides I reference substance solution, concentration are 1.2mg/ml, concentration are 1.43mg/ml
Product solution, it is spare.
By the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the precious leaves of pulse plants
Glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be settled to 5ml respectively and is made into the mixed of 7 kinds of ingredients
Close reference substance solution.
High effective liquid chromatography for measuring: accurate respectively to draw each 10uL of test solution and control solution, injection is efficient
Liquid chromatograph, measurement, calculate to get.
Study on the stability: above-mentioned content assaying method is applied to study on the stability.
Embodiment 4
In accordance with the following methods detect spray agent for treating tendon and bone injury in effective component Paeoniflorin, icariin, ellagic acid, Epimedin A,
The content of Epimedin B, epimedin C, precious leaves of pulse plants glycosides I.
Chromatographic condition: Agilent ZORBA*S8~C18 column；Inner diameter size is 4.6*150mm, 5um, and Mobile phase B is
The potassium dihydrogen phosphate of 0.05mol/L, mobile phase C be methanol, mobile phase D be acetonitrile, 0~23min:230nm of Detection wavelength,
23~54min:254nm, 54~137min:270nm, 35 DEG C of column temperature.Gradient elution method is as follows:
| Time | Mobile phase B | Mobile phase C | Mobile phase D | Flow velocity |
| 0~14min | 92%~90% | —— | 8%~10% | 1ml/min |
| 14~17.5min | 90% | —— | 10% | 1ml/min |
| 17.5~22min | 90%~88% | —— | 10%~12% | 1ml/min |
| 22~33min | 88%~83% | —— | 12%~17% | 1ml/min |
| 33~50min | 83%~79% | —— | 17%~21% | 1ml/min |
| 50~51min | 79%~77.5% | —— | 21%~22.5% | 1~0.85ml/min |
| 51~80min | 77.5% | —— | 22.5% | 0.85ml/min |
| 80~80.01min | 77.5%~67% | 0~11% | 22.5%~22% | 0.85~1ml/min |
| 80.01~97min | 67%~65% | 11%~0% | 22%~35% | 1ml/min |
| 97~107min | 65% | —— | 35% | 1ml/min |
| 107~117min | 65%~50% | —— | 35%~50% | 1ml/min |
| 117~137min | 50%~35% | —— | 50~65% | 1ml/min |
The preparation of test solution: taking spray agent for treating tendon and bone injury 5ml, and precision is drawn, sets in 100ml evaporating dish, be evaporated, add
The dissolution of 5ml water, lysate are transferred in 25ml test tube, add 10ml ethyl acetate solution in test tube, are extracted, then plus 10ml acetic acid
The extraction of ethyl ester solution, takes supernatant liquid, sets in 100ml evaporating dish, be evaporated, dissolved with methanol, be settled in 10ml volumetric flask,
With filtering with microporous membrane, take subsequent filtrate as test solution.
The preparation of reference substance solution: icariin reference substance, Epimedin A reference substance, Epimedin B reference substance, epimedin C are taken
Reference substance, treasured leaves of pulse plants glycosides I reference substance, ellagic acid reference substance and Paeoniflorin reference substance, add methanol to dissolve, concentration, which is respectively prepared, is
Epimedin A reference substance solution that icariin reference substance solution, the concentration of 0.236mg/ml is 1.5mg/ml, concentration 1.2mg/
The treasured that epimedin C reference substance solution that Epimedin B reference substance solution, the concentration of ml is 0.05mg/ml, concentration are 0.026mg/ml
The Paeoniflorin control that ellagic acid reference substance solution that leaves of pulse plants glycosides I reference substance solution, concentration are 1.2mg/ml, concentration are 1.43mg/ml
Product solution, it is spare.
By the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the precious leaves of pulse plants
Glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be settled to 5ml respectively and is made into the mixed of 7 kinds of ingredients
Close reference substance solution.
High effective liquid chromatography for measuring: it is accurate respectively to draw each 5uL of test solution and control solution, inject efficient liquid
Chromatography, measurement, calculate to get.
Study on the stability: above-mentioned content assaying method is applied to study on the stability.
Embodiment 5
In accordance with the following methods detect spray agent for treating tendon and bone injury in effective component Paeoniflorin, icariin, ellagic acid, Epimedin A,
The content of Epimedin B, epimedin C, precious leaves of pulse plants glycosides I.
Chromatographic condition: Agilent ZORBA*S8~C18 column；Inner diameter size is 4.6*150mm, 5um, and Mobile phase B is
The potassium dihydrogen phosphate of 0.05mol/L, mobile phase C be methanol, mobile phase D be acetonitrile, 0~23min:230nm of Detection wavelength,
23~54min:254nm, 54~137min:270nm, 35 DEG C of column temperature, gradient elution method is as follows:
| Time | Mobile phase B | Mobile phase C | Mobile phase D | Flow velocity |
| 0~16min | 92%~90% | —— | 8%~10% | 1ml/min |
| 16~20min | 90% | —— | 10% | 1ml/min |
| 20~22min | 90%~88% | —— | 10%~12% | 1ml/min |
| 22~33min | 88%~83% | —— | 12%~17% | 1ml/min |
| 33min~50min | 83%~79% | —— | 17%~21% | 1ml/min |
| 50~51min | 79%~77.5% | —— | 21%~22.5% | 1~0.85ml/min |
| 51~80min | 77.5% | —— | 22.5% | 0.85ml/min |
| 80~80.01min | 77.5%~67% | 0~11% | 22.5%~22% | 0.85~1ml/min |
| 80.01~97min | 67%~65% | 11%~0% | 22%~35% | 1ml/min |
| 97~107min | 65% | —— | 35% | 1ml/min |
| 107~117min | 65%~50% | —— | 35%~50% | 1ml/min |
| 117~137min | 50%~35% | —— | 50~65% | 1ml/min |
The preparation of test solution: taking spray agent for treating tendon and bone injury 5ml, and precision is drawn, sets in 100ml evaporating dish, be evaporated, add
The dissolution of 5ml water, lysate are transferred in 25ml test tube, add 10ml water-saturated n-butanol solution in test tube, are extracted, then plus 10ml
The extraction of water-saturated n-butanol solution, takes supernatant liquid, sets in 100ml evaporating dish, be evaporated, dissolved with methanol, be settled to 10ml
In volumetric flask, with filtering with microporous membrane, take subsequent filtrate as test solution.
The preparation of reference substance solution: icariin reference substance, Epimedin A reference substance, Epimedin B reference substance, epimedin C are taken
Reference substance, treasured leaves of pulse plants glycosides I reference substance, ellagic acid reference substance and Paeoniflorin reference substance, add methanol to dissolve, concentration, which is respectively prepared, is
Epimedin A reference substance solution that icariin reference substance solution, the concentration of 0.236mg/ml is 1.5mg/ml, concentration 1.2mg/
The treasured that epimedin C reference substance solution that Epimedin B reference substance solution, the concentration of ml is 0.05mg/ml, concentration are 0.026mg/ml
The Paeoniflorin control that ellagic acid reference substance solution that leaves of pulse plants glycosides I reference substance solution, concentration are 1.2mg/ml, concentration are 1.43mg/ml
Product solution, it is spare.
By the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the precious leaves of pulse plants
Glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be settled to 5ml respectively and is made into the mixed of 7 kinds of ingredients
Close reference substance solution.
High effective liquid chromatography for measuring: accurate respectively to draw each 10uL of test solution and control solution, injection is efficient
Liquid chromatograph, measurement, calculate to get.
Above embodiments testing result shows that, using the content of the method for the present invention detection bone fracture spray, baseline is steady, sample
Each target component Paeoniflorin, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I are separated in product,
Illustrate to determine its 7 kinds of effective components while content assaying method of the present invention for spray agent for treating tendon and bone injury can be effectively, and point
, accuracy in detection height high from degree, stability are good.
Study on the stability: above-mentioned content assaying method is applied to study on the stability.
Detection method of content screening test:
In order to further verify effectiveness of the invention, inventor has done a series of test and has been most suitable for this hair to filter out
Bright reagent, parameter, method etc., specific as follows:
Test example 1 prepares test solution using sodium hydrate aqueous solution, tests each ingredient separation situation.
In accordance with the following methods detect spray agent for treating tendon and bone injury in effective component Paeoniflorin, icariin, ellagic acid, Epimedin A,
The content of Epimedin B, epimedin C, precious leaves of pulse plants glycosides I:
Chromatographic condition: Agilent ZORBA*S8~C18 column；Inner diameter size is 4.6*150mm, 5um, and Mobile phase B is
The potassium dihydrogen phosphate of 0.05mol/L, mobile phase C be methanol, mobile phase D be acetonitrile, 0~23min:230nm of Detection wavelength,
23~54min:254nm, 54~137min:270nm, 35 DEG C of column temperature, gradient elution method are as follows:
| Time | Mobile phase B | Mobile phase C | Mobile phase D | Flow velocity |
| 0~14min | 92%~90% | —— | 8%~10% | 1ml/min |
| 14~17.5min | 90% | —— | 10% | 1ml/min |
| 17.5~22min | 90%~88% | —— | 10%~12% | 1ml/min |
| 22~33min | 88%~83% | —— | 12%~17% | 1ml/min |
| 33~50min | 83%~79% | —— | 17%~21% | 1ml/min |
| 50~51min | 79%~77.5% | —— | 21%~22.5% | 1~0.85ml/min |
| 51~80min | 77.5% | —— | 22.5% | 0.85ml/min |
| 80~80.01min | 77.5%~67% | 0~11% | 22.5%~22% | 0.85~1ml/min |
| 80.01~97min | 67%~65% | 11%~0% | 22%~35% | 1ml/min |
| 97~107min | 65% | —— | 35% | 1ml/min |
| 107~117min | 65%~50% | —— | 35%~50% | 1ml/min |
| 117~137min | 50%~35% | —— | 50~65% | 1ml/min |
The preparation of test solution: taking spray agent for treating tendon and bone injury 5ml, and precision is drawn, sets in 100ml evaporating dish, be evaporated, add
The dissolution of 5ml sodium hydrate aqueous solution, lysate are transferred in 25ml test tube, add 10ml water-saturated n-butanol solution in test tube,
Extraction, then plus 10ml water-saturated n-butanol solution extraction, take supernatant liquid, set in 100ml evaporating dish, be evaporated, it is molten with methanol
Solution, is settled in 10ml volumetric flask, with filtering with microporous membrane, takes subsequent filtrate as test solution.
The preparation of reference substance solution: icariin reference substance, Epimedin A reference substance, Epimedin B reference substance, epimedin C are taken
Reference substance, treasured leaves of pulse plants glycosides I reference substance, ellagic acid reference substance and Paeoniflorin reference substance, add methanol to dissolve, concentration, which is respectively prepared, is
Epimedin A reference substance solution that icariin reference substance solution, the concentration of 0.236mg/ml is 1.5mg/ml, concentration 1.2mg/
The treasured that epimedin C reference substance solution that Epimedin B reference substance solution, the concentration of ml is 0.05mg/ml, concentration are 0.026mg/ml
The Paeoniflorin control that ellagic acid reference substance solution that leaves of pulse plants glycosides I reference substance solution, concentration are 1.2mg/ml, concentration are 1.43mg/ml
Product solution, it is spare.
By the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the precious leaves of pulse plants
Glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be settled to 5ml respectively and is made into the mixed of 7 kinds of ingredients
Close reference substance solution.
The measurement of HPLC method: it is accurate respectively to draw each 10uL of test solution and control solution, inject high performance liquid chromatography
Instrument, measurement, calculate to get.
Testing result shows that detection baseline is steady, cannot detect ellagic acid, and target component is reduced in sample, is not achieved
The present invention detects the experiment effect of 7 kinds of effective component simultaneously.
2 Mobile phase B of test example is water, test separation situation
In accordance with the following methods detect spray agent for treating tendon and bone injury in effective component Paeoniflorin, icariin, ellagic acid, Epimedin A,
The content of Epimedin B, epimedin C, precious leaves of pulse plants glycosides I:
Chromatographic condition are as follows: Agilent ZORBA*S8~C18 column；Inner diameter size is 4.6*150mm, 5um, and Mobile phase B is
Water, mobile phase C be methanol, mobile phase D be acetonitrile, Detection wavelength 0~23min:230nm, 23~54min:254nm, 54~
137min:270nm, 35 DEG C of column temperature, gradient elution method is as follows:
| Time | Mobile phase B | Mobile phase C | Mobile phase D | Flow velocity |
| 0~14min | 92%~90% | —— | 8%~10% | 1ml/min |
| 14~17.5min | 90% | —— | 10% | 1ml/min |
| 17.5~22min | 90%~88% | —— | 10%~12% | 1ml/min |
| 22~33min | 88%~83% | —— | 12%~17% | 1ml/min |
| 33~50min | 83%~79% | —— | 17%~21% | 1ml/min |
| 50~51min | 79%~77.5% | —— | 21%~22.5% | 1~0.85ml/min |
| 51~80min | 77.5% | —— | 22.5% | 0.85ml/min |
| 80~80.01min | 77.5%~67% | 0~11% | 22.5%~22% | 0.85~1ml/min |
| 80.01~97min | 67%~65% | 11%~0% | 22%~35% | 1ml/min |
| 97~107min | 65% | —— | 35% | 1ml/min |
| 107~117min | 65%~50% | —— | 35%~50% | 1ml/min |
| 117~137min | 50%~35% | —— | 50~65% | 1ml/min |
The preparation of test solution: taking spray agent for treating tendon and bone injury 5ml, and precision is drawn, sets in 100ml evaporating dish, be evaporated, add
The dissolution of 5ml water, lysate are transferred in 25ml test tube, add 10ml water-saturated n-butanol solution in test tube, are extracted, then plus 10ml
The extraction of water-saturated n-butanol solution, takes supernatant liquid, sets in 100ml evaporating dish, be evaporated, dissolved with methanol, be settled to 10ml
In volumetric flask, with filtering with microporous membrane, take subsequent filtrate as test solution.
The preparation of reference substance solution: icariin reference substance, Epimedin A reference substance, Epimedin B reference substance, epimedin C are taken
Reference substance, treasured leaves of pulse plants glycosides I reference substance, ellagic acid reference substance and Paeoniflorin reference substance, add methanol to dissolve, concentration, which is respectively prepared, is
Epimedin A reference substance solution that icariin reference substance solution, the concentration of 0.236mg/ml is 1.5mg/ml, concentration 1.2mg/
The treasured that epimedin C reference substance solution that Epimedin B reference substance solution, the concentration of ml is 0.05mg/ml, concentration are 0.026mg/ml
The Paeoniflorin control that ellagic acid reference substance solution that leaves of pulse plants glycosides I reference substance solution, concentration are 1.2mg/ml, concentration are 1.43mg/ml
Product solution, it is spare.
By the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the precious leaves of pulse plants
Glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be settled to 5ml respectively and is made into the mixed of 7 kinds of ingredients
Close reference substance solution.
High effective liquid chromatography for measuring: accurate respectively to draw each 10uL of test solution and control solution, injection is efficient
Liquid chromatograph, measurement, calculate to get.
Testing result shows that detection baseline is steady, and each target component separating degree is poor in sample, and effect is not as good as embodiment.
Test example 3 measures spray agent for treating tendon and bone injury component content using art methods
According to detection method disclosed in the quality research for being permitted super winged seedling medicine " spray agent for treating tendon and bone injury " and quality standard promotion
Attempt the content of detection Paeoniflorin, ellagic acid, Epimedin A, Epimedin B, epimedin C, icariin, precious leaves of pulse plants glycosides I.
Instrument: (U.S.'s Agilent Technologies are limited for 1100~DAD of Agilent, Agilent~VWD high performance liquid chromatograph
Company), KQ~250B type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), Japan's Shimadzu AY~120 electronic balance,
The digital display of HH~4 thermostat water bath (Guo Hua Electrical Appliances Co., Ltd).
Chromatographic condition: chromatographic column is WONDASIL C18 (250X 4.6mm, 5um)；Detection wavelength: 230nm, 248nm,
270nm, wavelength convert method inspection, wavelength convert method check that design parameter is as follows:
| Time (minute) | Wavelength (nm) |
| 0 | 248 |
| 10 | 230 |
| 25 | 248 |
| 41 | 270 |
| 45 | 248 |
Flow velocity: 1ml/min；Column temperature: 25 DEG C, sample volume: 10ul；Mobile phase A~methanol, Mobile phase B~0.1% phosphoric acid are molten
Liquid, gradient elution method are as follows:
| Time (minute) | Mobile phase A | Mobile phase B |
| 0 | 70% | 30% |
| 20 | 60% | 40% |
| 30 | 45% | 55% |
| 50 | 35% | 65% |
| 60 | 20% | 80% |
Reagent, reagent: methanol (chromatographically pure), phosphoric acid, methanol (analysis alcohol), Robust drinking pure water；Muscles and bones wound is spraying
Agent.
The preparation of test solution: 5 bottles of muscles and bones spray of same batch are taken, after every bottle takes 50ml, precision to measure mixing
Test solution 5ml in evaporating dish, be evaporated, methanol dissolution is placed in 10ml volumetric flask, be ultrasonically treated 5min, methanol constant volume,
Shake up, take supernatant cross 0.45um miillpore filter to get.
The preparation of reference substance solution: taking icariin reference substance, and adding methanol to be dissolved to concentration is 0.2~0.3mg/ml, standby
With；Epimedin A reference substance is taken, adding methanol to be dissolved to concentration is 1~2mg/ml, spare；Epimedin B reference substance is taken, adds methanol molten
Solution to concentration is 1~2mg/ml, spare；Epimedin C reference substance is taken, adding methanol to be dissolved to concentration is 0.02~0.1mg/ml, standby
With；Precious leaves of pulse plants glycosides I reference substance is taken, adding methanol to be dissolved to concentration is 0.02~0.03mg/ml, spare；Ellagic acid reference substance is taken, first is added
It is 1~2mg/ml that alcohol, which is dissolved to concentration, spare；Paeoniflorin reference substance is taken, adding methanol to be dissolved to concentration is 1~1.8mg/ml, standby
With；
By the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, the precious leaves of pulse plants
Glycosides I takes 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul, 140ul to be made into the mixing reference substance of 7 kinds of ingredients respectively
Solution.
High effective liquid chromatography for measuring: accurate respectively to draw each 10uL of test solution and control solution, injection is efficient
Liquid chromatograph, measurement, calculate to get.
Testing result shows that detection baseline is steady, Paeoniflorin and icariin is only detected in sample, other compositions are unknown
Aobvious, effect is far away from embodiment.
To sum up the experimental result of comparative example and embodiment is shown, 1-7 of the embodiment of the present invention is used for spray agent for treating tendon and bone injury content
Measuring method could detect 7 kinds of effective component in spray agent for treating tendon and bone injury simultaneously, be more comprehensively to stable effective ingredients
Quantitative expedition method.
4 methodological study of test example and sample size measurement:
Test solution, reference substance solution, chromatographic condition etc. is handled according to the method for embodiment 1 to be tested as follows:
(1) linear relationship investigate: respectively the precision lower reference substance solution 2,5,10,15,20,25uL of extraction embodiment 1 into
Sample measurement.With peak area Y to x-ray return, obtain Paeoniflorin, ellagic acid, Epimedin A, Epimedin B, epimedin C, icariin,
The regression equation and the range of linearity such as table 1 of precious leaves of pulse plants glycosides I:
1 components regression equation of table and the range of linearity
| Ingredient | Regression equation | The range of linearity |
| Paeoniflorin | Y=73.856X~1.3175, r=0.9998 (n=6) | 1.058~13.225ug |
| Ellagic acid | Y=5387X~1149.5, r=0.9998 (n=6) | 0.644~8.05ug |
| Epimedin A | Y=1548.5X+8.1607, r=0.9998 (n=6) | 0.068~0.85ug |
| Epimedin B | Y=1640.7X+9.8463, r=0.9999 (n=6) | 0.044~0.55ug |
| Epimedin C | Y=2006.6X+27.168, r=0.9995 (n=6) | 0.1~1.25ug； |
| Icariin | Y=1499.5X~22.376, r=0.9998 (n=6) | 0.112~1.4ug |
| Precious leaves of pulse plants glycosides I | Y=5024.7X~29.95, r=0.9999 (n=6) | 0.058~0.725ug |
(2) Precision Experiment: the mixed reference substance solution prepared under accurate extraction embodiment 1 respectively, continuous sample introduction 6
It is secondary, measure the integrating peak areas value of Paeoniflorin, ellagic acid, Epimedin A, Epimedin B, epimedin C, icariin, precious leaves of pulse plants glycosides I
RSD such as table 2, shows that instrument precision is good.
The RSD of each Component peak area integrated value of table 2
| Ingredient | The RSD of integrating peak areas value |
| Paeoniflorin | 2.1% |
| Ellagic acid | 2.9% |
| Epimedin A | 0.8% |
| Epimedin B | 1.6% |
| Epimedin C | 0.7% |
| Icariin | 0.6% |
| Precious leaves of pulse plants glycosides I | 0.7% |
(3) repeated experiment: taking same batch sample, is prepared in parallel by 1 lower preparation method of test article of embodiment for examination
It 5 parts of product, is measured by embodiment 1 lower chromatographic condition, calculates content.It is Paeoniflorin in 6 parts of test solutions, ellagic acid, fixed towards the leaves of pulse plants
A, the average content and RSD such as table 3 of Epimedin B, epimedin C, icariin, precious leaves of pulse plants glycosides I, shows repeated good.
3 average content of table and RSD
| Ingredient | Average content | RSD |
| Paeoniflorin | 0.93mg/ml | 1.4% |
| Ellagic acid | 0.23mg/ml | 2.3% |
| Epimedin A | 0.011mg/ml | 2.8% |
| Epimedin B | 0.019mg/ml | 2.4% |
| Epimedin C | 0.012mg/ml | 2.4% |
| Icariin | 0.075mg/ml | 2.3% |
| Precious leaves of pulse plants glycosides I | 0.0051mg/ml | 2.3% |
(4) stability experiment: taking same batch sample, and it is molten to prepare test sample by 1 lower preparation method of test article of embodiment
Liquid, 0 after preparation, 2,4,8, sample introduction for 24 hours, measure Paeoniflorin in test sample, ellagic acid, Epimedin A, Epimedin B, epimedin C,
The RSD such as table 4 of the integrating peak areas value of icariin, precious leaves of pulse plants glycosides I shows that test solution is stablized interior for 24 hours.
The RSD of 4 integrating peak areas value of table
| Ingredient | The RSD of integrating peak areas value |
| Paeoniflorin | 2.2% |
| Ellagic acid | 2.7% |
| Epimedin A | 2.5% |
| Epimedin B | 1.8% |
| Epimedin C | 2.2% |
| Icariin | 1.6% |
| Precious leaves of pulse plants glycosides I | 0.9% |
(5) sample recovery rate is tested: test sample 2.5ml is taken, it is accurate respectively that Paeoniflorin, ellagic acid, Epimedin A, court is added
It is appropriate that the leaves of pulse plants determines B, epimedin C, icariin, precious leaves of pulse plants glycosides I reference substance, is prepared into confession by 1 lower preparation method of test article of embodiment
Test sample solution is measured by 1 lower chromatographic condition of embodiment, calculate the rate of recovery, as a result Paeoniflorin, ellagic acid, Epimedin A,
The rate of recovery (n=9) and RSD such as table 5 of Epimedin B, epimedin C, icariin, precious leaves of pulse plants glycosides I.
5 rate of recovery of table (n=9) and RSD
| Ingredient | The rate of recovery (n=9) | RSD |
| Paeoniflorin | 95.6% | 1.4% |
| Ellagic acid | 96.0% | 1.8% |
| Epimedin A | 97.8% | 2.3% |
| Epimedin B | 94.5% | 1.5% |
| Epimedin C | 93.5% | 2.7% |
| Icariin | 96.7% | 2.4% |
| Precious leaves of pulse plants glycosides I | 99.6% | 1.9% |
(6) sample size measures: taking three batches of spray agent for treating tendon and bone injury, by 1 lower section legal system available test sample solution of embodiment, surveys
Fixed 7 kinds of component contents, the results are shown in Table 6:
67 kinds of component content measurement result tables of table
From the above experimental results, we know that the present invention carries out assay using HPLC method, preferable peak shape, sample have been obtained
Target component separated with impurity preferably, reproducible, specificity is good, can carry out the detection of plurality of active ingredients simultaneously.
Summarize: 1, the quantitative approach that this experiment is established is measured by multipair batch sample, the results showed that the method letter
List, specificity are good and the rate of recovery is high, simple and direct feasible, can be used as spray agent for treating tendon and bone injury content assaying method；2, the present invention is built for the first time
The content assaying method for having found while having measured 7 kinds of effective component in spray agent for treating tendon and bone injury, measurement while realizing 7 kinds of ingredients,
Effectively have detected 7 kinds of most representative effective component Paeoniflorins in spray agent for treating tendon and bone injury in three main medicinal materials, excessive
The content of sheep leaves of pulse plants glycosides, ellagic acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I, is the liquid phase fingerprint of spray agent for treating tendon and bone injury
The foundation of spectrum library provides a kind of measurement scheme rapidly and efficiently；3, the present invention provides one kind have it is more comprehensive effectively at
The quantitative expedition method for the stability divided；Detection method of content of the invention, detection baseline is steady, separating degree is high, detection is accurate
Degree is high, stability is good.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
State, but on the basis of the present invention, it can be made it is some modify or improve, this is aobvious and easy to those skilled in the art
See.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of content assaying method for spray agent for treating tendon and bone injury, which is characterized in that right simultaneously using high performance liquid chromatography
7 kinds of effective components carry out assay, 7 kinds of effective components of the assay in spray agent for treating tendon and bone injury specifically: Paeoniflorin,
Icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I, the content assaying method the following steps are included:
The preparation of test solution, the preparation of reference substance solution, the measurement of HPLC method.
2. the content assaying method of spray agent for treating tendon and bone injury according to claim 1, it is characterised in that the test solution system
The specific steps of Preparation Method are as follows: stringing bone fracture 2~10ml of spray, precision are drawn, set in 100~200ml evaporating dish, be evaporated,
2~10ml water is added to dissolve, lysate is transferred in 25~50ml test tube, and add 5~20ml water-saturated n-butanol solution in test tube,
Extraction, then plus 5~20ml water-saturated n-butanol solution extraction, take supernatant liquid, set in 100~200ml evaporating dish, be evaporated,
It is dissolved, is settled in 2~10ml volumetric flask with methanol, with filtering with microporous membrane, take subsequent filtrate as test solution.
3. the method for spray agent for treating tendon and bone injury assay according to claim 2, which is characterized in that the test solution
The preparation method comprises the following steps: take 6~8ml of spray agent for treating tendon and bone injury, precision is drawn, and is set in 100ml evaporating dish, is evaporated, add 5ml water to dissolve,
Lysate is transferred in 25ml test tube, adds 10ml water-saturated n-butanol solution in test tube, extraction, then plus the positive fourth of 10ml water saturation
Alcoholic solution extraction, takes supernatant liquid, sets in 100ml evaporating dish, be evaporated, dissolved with methanol, be settled in 10ml volumetric flask, uses
Filtering with microporous membrane takes subsequent filtrate as test solution.
4. the content assaying method of spray agent for treating tendon and bone injury according to claim 1, it is characterised in that the reference substance solution system
The specific steps of Preparation Method are as follows: take icariin reference substance, adding methanol to be dissolved to concentration is 0.15~0.32mg/ml, spare；It takes
Epimedin A reference substance, adding methanol to be dissolved to concentration is 1~2mg/ml, spare；Epimedin B reference substance is taken, adds methanol to be dissolved to dense
Degree is 0.8~2mg/ml, spare；Epimedin C reference substance is taken, adding methanol to be dissolved to concentration is 0.01~0.1mg/ml, spare；It takes
Precious leaves of pulse plants glycosides I reference substance, adding methanol to be dissolved to concentration is 0.02~0.03mg/ml, spare；Ellagic acid reference substance is taken, adds methanol molten
Solution to concentration is 0.8~2mg/ml, spare；Paeoniflorin reference substance is taken, adding methanol to be dissolved to concentration is 0.8~2mg/ml, spare；
By the reference substance solution Paeoniflorin of preparation, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I, respectively
1~2ml, 1~1.5ml, 1~1.5ml, 100~150ul, 50~100ul, 250~300ul, 100~150ul is taken to be made into 7 kinds
The mixed reference substance solution of ingredient.
5. the content assaying method of spray agent for treating tendon and bone injury according to claim 4, it is characterised in that the reference substance solution system
The specific steps of Preparation Method are as follows: take icariin reference substance, adding methanol to be dissolved to concentration is 0.236mg/ml, spare；It takes towards the leaves of pulse plants
Determine A reference substance, adding methanol to be dissolved to concentration is 1.5mg/ml, spare；Epimedin B reference substance is taken, adds methanol to be dissolved to concentration to be
1.2mg/ml, it is spare；Epimedin C reference substance is taken, adding methanol to be dissolved to concentration is 0.05mg/ml, spare；Precious leaves of pulse plants glycosides I is taken to compare
Product, adding methanol to be dissolved to concentration is 0.026mg/ml, spare；Ellagic acid reference substance is taken, adding methanol to be dissolved to concentration is 1.2mg/
Ml, it is spare；Paeoniflorin reference substance is taken, adding methanol to be dissolved to concentration is 1.43mg/ml, spare；By the reference substance solution Chinese herbaceous peony of preparation
Medicine glycosides, icariin, ellagic acid, Epimedin A, Epimedin B, epimedin C, precious leaves of pulse plants glycosides I, take respectively 1.85ml, 1.19ml,
1.34ml, 110ul, 90ul, 280ul, 140ul are settled to the mixed reference substance solution that 5ml is made into 7 kinds of ingredients.
6. the content assaying method of spray agent for treating tendon and bone injury according to claim 1, it is characterised in that the HPLC method measurement
Chromatographic condition are as follows: Agilent ZORBA*S8~C18 column；Inner diameter size is 4.6*150mm, 5um；Mobile phase B is 0.05mol/L
Potassium dihydrogen phosphate, mobile phase C be methanol, mobile phase D be acetonitrile；0.85~1ml/min of flow velocity, Detection wavelength 0~
23min:230nm, 23~54min:254nm, 54~137min:270nm, carry out gradient elution by 35 DEG C of column temperature.
7. the content assaying method of spray agent for treating tendon and bone injury according to claim 6, it is characterised in that the gradient elution method
Are as follows:
。
8. the content assaying method of spray agent for treating tendon and bone injury according to claim 7, it is characterised in that the gradient elution method
Are as follows:
。
9. the content assaying method of spray agent for treating tendon and bone injury according to claim 1, it is characterised in that HPLC method measurement side
Method are as follows: it is accurate respectively to draw each 5~10uL of test solution and control solution, high performance liquid chromatograph is injected, is measured, meter
Calculate to get；The content standard is that the content of Paeoniflorin in spray agent for treating tendon and bone injury is 2000~2500ug/ml；Ellagic acid contains
Amount is 400~500ug/ml；The content of Epimedin A is 20~30ug/ml；The content of Epimedin B is 30~40ug/ml；Towards the leaves of pulse plants
The content for determining C is 20~30ug/ml；The content of icariin is 100~150ug/ml；The content of precious leaves of pulse plants glycosides I is 10~15ug/
ml。
10. a kind of content assaying method for spray agent for treating tendon and bone injury, which is characterized in that will be simultaneously in spray agent for treating tendon and bone injury 7
The stability study that the method that kind effective component carries out assay is applied to spray agent for treating tendon and bone injury is investigated.
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| CN110108814A (en) * | 2019-05-23 | 2019-08-09 | 贵州远程制药有限责任公司 | A kind of detection method for spray agent for treating tendon and bone injury |
| CN113456660A (en) * | 2021-08-02 | 2021-10-01 | 陕西师范大学 | Application of epimedin C in preventing and treating acute alcoholism |
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| CN1418650A (en) * | 2002-11-27 | 2003-05-21 | 贵州科顿制药有限责任公司 | Spray agent for treating tendon and bone injury |
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| CN1418650A (en) * | 2002-11-27 | 2003-05-21 | 贵州科顿制药有限责任公司 | Spray agent for treating tendon and bone injury |
| 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 |
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| CN110108814A (en) * | 2019-05-23 | 2019-08-09 | 贵州远程制药有限责任公司 | A kind of detection method for spray agent for treating tendon and bone injury |
| CN110108814B (en) * | 2019-05-23 | 2022-07-05 | 贵州远程制药有限责任公司 | Detection method of spray for tendon and bone injuries |
| CN113456660A (en) * | 2021-08-02 | 2021-10-01 | 陕西师范大学 | Application of epimedin C in preventing and treating acute alcoholism |
| CN113456660B (en) * | 2021-08-02 | 2022-10-25 | 陕西师范大学 | Application of epimedin C in preventing and treating acute alcoholism |
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