CN114609308A - Finger print of gamboge bone-invigorating pill and construction method and application thereof - Google Patents
Finger print of gamboge bone-invigorating pill and construction method and application thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The invention provides a gamboge bone-invigorating pill fingerprint spectrum, a construction method and application thereof, and belongs to the technical field of medicines. The method for constructing the finger print of the gamboge bone strengthening pill comprises the following steps: extracting the gamboge bone-invigorating pills by adopting methanol, and taking the obtained gamboge bone-invigorating pill extracting solution as a test solution; methanol solution of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin is used as control solution; and respectively carrying out high performance liquid chromatography analysis on the reference substance solution and the test solution, and taking the chromatographic peak of the reference substance solution as a reference to obtain the finger print of the gamboge bone-invigorating pill. The invention adopts the high performance liquid chromatography to establish the fingerprint of the gamboge bone-invigorating pill, can comprehensively reflect the quality of the gamboge bone-invigorating pill, is convenient for more effectively guiding feeding and strictly standardizing production operation, and is beneficial to ensuring the safety and the effectiveness of clinical medication of the gamboge bone-invigorating pill.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a gamboge bone-invigorating pill fingerprint and a construction method and application thereof.
Background
Various senile rheumatic bone diseases such as hypertrophic spondylitis, cervical spondylosis, calcaneal spur, proliferative arthritis, Kaschin-Beck disease and the like are all called as 'arthralgia syndrome' in traditional Chinese medicine. Hypertrophic spondylitis is a systemic disease, which is called osteoarthropathy, and is a chronic arthritis caused by joint degeneration and articular cartilage destruction, the cause of the systemic disease is the degeneration of bones, namely, the disease is the bone degeneration caused by the intrinsic factor of kidney qi deficiency and the accumulation of daily small trauma, wind cold and the like. At present, analgesics, non-steroidal anti-inflammatory drugs, adrenocortical hormone, hyaluronic acid, superoxide dismutase (SOD) and D-glucosamine are mainly adopted for treating the diseases at home and abroad, but most of the six drugs have obvious side effects.
The gamboge bone-invigorating pill has the main functions of tonifying kidney, promoting blood circulation and relieving pain. Treats both principal and secondary aspects of diseases by taking kidney tonifying as the basis and treating bones, and is good at treating osteoarthritis, ankylosing spondylitis, cervical spondylosis, lumbar disc herniation, scapulohumeral periarthritis and sciatica in western medicine diagnosis. The gamboge bone-invigorating pill is prepared from radix rehmanniae Preparata, herba Pyrolae, scalded rhizoma Drynariae, Cistanchis herba, herba Epimedii, caulis Spatholobi and parched Raphani semen. The pathogenesis of the disease is caused by liver and kidney deficiency and blood stasis blocking collaterals. The liver stores blood and governs tendons, for example, liver blood deficiency, blood failing to nourish tendons, muscles and joints failing to nourish them can lead to long-term illness; kidneys govern bones and produce marrow, and if kidney essence is deficient, the source of marrow generation is insufficient, so long-term bone Bi-syndrome is produced . For treatment, it is good at tonifying liver and kidney, strengthening tendons and bones, promoting blood circulation, removing blood stasis, activating qi to alleviate pain.
Radix rehmanniae Preparata is added in the prescription of the gamboge bone-strengthening pill, has sweet taste and slightly warm nature, enters liver and kidney channels, nourishes yin and blood, supplements essence and fills marrow, and tonifies true yin of liver and kidney, so that the gamboge bone-strengthening pill is a monarch drug.
In the gamboge bone-strengthening pill formula, the epimedium herb is pungent and sweet in taste and warm in nature, enters liver and kidney channels, tonifies kidney and strengthens yang, and dispels wind and removes dampness; cistanchis herba is sweet, salty and warm in nature, enters kidney and large intestine channels, tonifies kidney yang, benefits essence and blood, enters kidney and produces marrow. The epimedium and the cistanche deserticola share the kidney-tonifying yang-invigorating effect and the yin-tonifying monarch drug, and the yin-middle yang-invigorating effect and the qi-generating effect of the shaggy fire are combined to tonify the yin and the yang. Scalded rhizoma Drynariae is bitter in taste and warm in nature, enters kidney and liver meridians, tonifies kidney and strengthens bone, and continues to hurt and relieve pain. Herba Pyrolae is sweet, bitter and warm in nature, enters liver and kidney meridians, dispels wind-damp, strengthens tendons and bones, stops bleeding, tonifies bone and relieves pain. The four ingredients are used as ministerial drugs for assisting the monarch drugs in tonifying liver and kidney and strengthening tendons and bones.
The gamboge bone-invigorating pill, JIXUETENG, has warm nature, bitter and sweet taste, can tonify kidney, replenish vital essence, nourish marrow, dredge channels and collaterals, promote qi and blood circulation, relieve pain when the circulation is stopped, promote blood circulation, dredge collaterals, tonify bone and alleviate pain, and is an adjuvant drug.
In the gamboge bone-invigorating pill formula, the fried radish seed has the advantages of bone invigorating, digestion promoting and qi regulating, has mild property, pungent and sweet taste, and is used as a guiding drug for preventing the disadvantages of tonifying and greasy taste.
Because the gamboge bone-invigorating pill has the characteristics of clear prescription, reasonable compatibility and the like, the internal control quality of the gamboge bone-invigorating pill needs to be kept in order to ensure the drug effect. At present, some researches are also made by related scholars, such as those in the department of Dermatology and the like, which research the determination of the icariin content in the gamboge bone-invigorating pill by using high performance liquid chromatography (the content of icariin in the gamboge bone-invigorating pill in the department of Dermatology and the determination of the icariin content in the gamboge bone-invigorating pill by using an RP-HPLC method, the year 2013, the seventh stage 118 and 119 pages); content of icariin and naringin in the gamboge bone-strengthening pill is determined by high performance liquid chromatography in Zhaoyong (Zhaoyong, gamboge bone-strengthening pill quality standard research, Chinese pharmacist, 2013, No. 1, page 76-80); the determination of the icariin content in the gamboge bone-invigorating pills by using the high performance liquid chromatography is researched in Wenzhi and the like (the determination of the icariin content in the gamboge bone-invigorating pills by using the high performance liquid chromatography in Wenzhi, Chinese medicine standard, 2009, 3 rd stage, 210 and 212 pages); research on determining the content of echinacoside in gamboge bone-invigorating pills by using high performance liquid chromatography (determination of echinacoside content in gamboge bone-invigorating pills by using HPLC method in Zingiberaceae, 2009, 4 th, 481-482 pages). However, the above methods reflect the quality of the gamboge bone-invigorating pill by using one or two ingredients, and have certain sheet properties, which cannot comprehensively reflect the quality of the gamboge bone-invigorating pill.
Disclosure of Invention
The invention aims to provide a gamboge bone-invigorating pill fingerprint spectrum and a construction method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for constructing a finger print of a gamboge bone-invigorating pill, which comprises the following steps:
extracting the gamboge bone-invigorating pills by adopting methanol, and taking the obtained gamboge bone-invigorating pill extracting solution as a test solution; methanol solution of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin is used as control solution;
and respectively carrying out high performance liquid chromatography analysis on the reference substance solution and the test solution, and taking the chromatographic peak of the reference substance solution as a reference to obtain the finger print of the gamboge bone-invigorating pill.
Preferably, the dosage ratio of the gamboge bone-invigorating pill to the methanol is (1-3) g: (10-50) mL.
Preferably, the extraction is carried out under the ultrasonic condition, the power of the ultrasonic is 70-90W, and the frequency is 200-300 kHz; the extraction time is 20-40 min.
Preferably, when the high performance liquid chromatography is performed, the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid water solution with a volume fraction of 0.01-0.3%; the flow rate of the mobile phase system is 0.5-1.5 mL/min;
adopting a gradient elution mode, wherein the gradient elution program comprises the following steps: the volume fraction of the mobile phase A is increased from 8% to 10% in 0-15 min; the volume fraction of the mobile phase A is increased from 10% to 30% in 15-55 min; the volume fraction of the mobile phase A is increased from 30% to 45% in 55-65 min; 65-75 min, and maintaining the volume fraction of the mobile phase A at 45%; and (3) increasing the volume fraction of the mobile phase A from 45% to 80% in 75-90 min.
Preferably, when the high performance liquid chromatography is performed, a filler in a chromatographic column is octadecylsilane chemically bonded silica, and the specification of the chromatographic column is as follows: the length is 250mm, the inner diameter is 4.60mm, and the particle size of the filler is 5 mu m; the column temperature is 25-45 ℃; the detector is a VWD detector, and the detection wavelength is 210-360 nm; the injection volume is 5-15 mu L.
Preferably, the high performance liquid chromatography is performed with a theoretical plate number of not less than 4000 as calculated from the icariin peak.
Preferably, the gamboge bone-strengthening pill fingerprint contains 20 common peaks, wherein the peak No. 5, the peak No. 8, the peak No. 10, the peak No. 13, the peak No. 14, the peak No. 15 and the peak No. 16 are respectively identified as echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin.
Preferably, the 20 common peaks are respectively classified into epimedium, caulis spatholobi, pyrola, stir-fried radish seed, prepared rehmannia root, cistanche deserticola and scalded rhizoma drynariae; wherein, the No. 1 peak belongs to the scalded drynaria rhizome, the Chinese pyrola herb and the suberect spatholobus stem, the No. 2 peak belongs to the scalded drynaria rhizome and the suberect spatholobus stem, the No. 3 peak belongs to the scalded drynaria rhizome, the No. 4 peak belongs to the desertliving cistanche, the No. 5 peak belongs to the desertliving cistanche, the No. 6 peak belongs to the epimedium herb and the Chinese pyrola herb, the No. 7 peak belongs to the fried radish seed and the scalded drynaria rhizome, the No. 8 peak belongs to the desertliving cistanche and the prepared rehmannia root, the No. 9 peak belongs to the desertliving cistanche and the prepared rehmannia root, the No. 10 peak belongs to the scalded drynaria rhizome, the No. 11 peak belongs to the scalded drynaria rhizome and the epimedium, the No. 12 peak belongs to the epimedium herb, the No. 13 peak belongs to the epimedium herb, the No. 14 peak belongs to the epimedium, the No. 15 peak belongs to the epimedium, the No. 16 peak belongs to the epimedium, the 17 peak belongs to the prepared rehmannia root, the No. 18 peak belongs to the epimedium, the No. 19 peak belongs to the epimedium, and the No. 20 peak belongs to the epimedium.
The invention provides a gamboge bone-invigorating pill fingerprint obtained by the method for constructing the gamboge bone-invigorating pill fingerprint in the technical scheme.
The invention provides application of the gamboge bone-invigorating pill fingerprint spectrum in the technical scheme in the quality control of the gamboge bone-invigorating pill.
The invention provides a method for constructing a finger print of a gamboge bone-invigorating pill, which comprises the following steps: extracting the gamboge bone-invigorating pills by adopting methanol, and taking the obtained gamboge bone-invigorating pill extracting solution as a test solution; methanol solution of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin is used as control solution; and respectively carrying out high performance liquid chromatography analysis on the reference substance solution and the test solution, and taking the chromatographic peak of the reference substance solution as a reference to obtain the finger print of the gamboge bone-invigorating pill. The finger print of the gamboge bone-invigorating pill is established by adopting the high performance liquid chromatography, the whole chemical components of the gamboge bone-invigorating pill can be comprehensively reflected, a single compound or a medicinal material is not identified, the problems of singleness and one-sidedness of the existing quality control method are solved, the feeding can be more effectively guided, the production operation is strictly regulated, and an effective means is provided for the whole quality control and evaluation of the gamboge bone-invigorating pill, so that the quality stability, consistency and controllability of the gamboge bone-invigorating pill are better ensured, and the safety and effectiveness of the clinical medication of the gamboge bone-invigorating pill are further ensured.
Furthermore, the gamboge bone-invigorating pill fingerprint contains 20 common peaks, 7 ingredients of the gamboge bone-invigorating pill are identified, and single medicinal material attribution is carried out on the 20 common peaks, so that the current situation of each ingredient in the gamboge bone-invigorating pill is comprehensively reflected, and a reference basis can be provided for quality control of the gamboge bone-invigorating pill.
The gamboge bone-invigorating pill constructed by the method has rich chromatographic peaks, contains effective components belonging to prepared rehmannia root, pyrola, scalded rhizoma drynariae, cistanche, epimedium, caulis spatholobi and fried radish seed, can better monitor and evaluate the quality of the gamboge bone-invigorating pill, guides the standardized production and has higher application value.
Drawings
FIG. 1 is an HPLC chromatogram of a mixed control solution;
FIG. 2 is a common pattern map of gamboge bone-invigorating pills;
FIG. 3 is a fingerprint map overlay of 11 batches of gamboge bone-invigorating pills;
FIG. 4 is a chart of sample fingerprint and chromatogram comparison attribution;
FIG. 5 is a spectrum of Garcinia cambogia bone-invigorating pill at a wavelength of 360 nm;
FIG. 6 is a spectrum of Garcinia cambogia bone-invigorating pill at a wavelength of 320 nm;
FIG. 7 is a graph of Garcinia cambogia bone-invigorating pill at a wavelength of 280 nm;
FIG. 8 is a spectrum of a gamboge bone-invigorating pill at a wavelength of 254 nm;
FIG. 9 is a spectrum of Garcinia cambogia bone-invigorating pill at a wavelength of 230 nm;
FIG. 10 is a spectrum of Garcinia cambogia bone-invigorating pill at a wavelength of 210 nm;
FIG. 11 is a spectrum of Garcinia cambogia bone-invigorating pill at a wavelength of 203 nm.
Detailed Description
The invention provides a method for constructing a finger print of a gamboge bone-invigorating pill, which comprises the following steps:
extracting the gamboge bone-invigorating pills by adopting methanol, and taking the obtained gamboge bone-invigorating pill extracting solution as a test solution; methanol solution of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin is used as control solution;
and respectively carrying out high performance liquid chromatography analysis on the reference substance solution and the test solution, and taking the chromatographic peak of the reference substance solution as a reference to obtain the finger print of the gamboge bone-invigorating pill.
In the invention, the gamboge bone-invigorating pill is prepared from prepared rehmannia root, pyrola, scalded drynaria rhizome, cistanche, epimedium, suberect spatholobus stem and stir-fried radish seed; the source of the gamboge bone-invigorating pill is not particularly limited, and commercially available products well known to those skilled in the art, such as gamboge bone-invigorating pills produced by Jilin Jichun pharmaceutical GmbH, Inc., can be used; can also be prepared by methods well known to those skilled in the art. In the present invention, the preparation method of the gamboge bone-invigorating pill preferably comprises the following steps: taking 750g of prepared rehmannia root, 500g of pyrola, 500g of scalded rhizoma drynariae, 500g of cistanche, 500g of epimedium herb, 500g of suberect spatholobus stem and 250g of stir-fried radish seed, and crushing the pyrola and the epimedium herb into fine powder to obtain a powdery raw material; and (2) adding water into the other five medicinal materials, decocting for two times, wherein the first decocting time is 2 hours, and the second decocting time is 1 hour, combining the obtained decoctions, filtering, concentrating the filtrate into thick paste, then uniformly mixing the thick paste with the powder raw materials, drying, crushing and sieving the thick paste in sequence, and adding 80-100 g of refined honey into every 100g of the obtained powder raw materials to prepare big honeyed pills, namely the gamboge bone-strengthening pills.
The gamboge bone-invigorating pill is extracted by adopting methanol, and the obtained gamboge bone-invigorating pill extracting solution is used as a test solution. The gamboge bone-invigorating pill is preferably mixed with methanol for extraction to obtain the gamboge bone-invigorating pill extracting solution. The gamboge bone-invigorating pill is preferably extracted after being ground into fine powder. In the invention, the preferable dosage ratio of the gamboge bone-invigorating pill to the methanol is (1-3) g: (10-50) mL, more preferably (1-2) g: (15-25) mL. In the invention, the extraction is preferably carried out under an ultrasonic condition, and the power of the ultrasonic is preferably 70-90W, and more preferably 80W; the frequency of the ultrasonic wave is preferably 200-300 kHz, and more preferably 250 kHz; the extraction time is preferably 20-40 min, and more preferably 30 min. In the invention, the extraction process preferably further comprises filtration, and the filtration membrane used for the filtration is preferably a 0.45 μm microporous filtration membrane. In the embodiment of the invention, the gamboge bone-invigorating pill is taken, ground, precisely weighed to be 1g, placed in a conical flask with a plug, precisely added with 25mL of methanol, weighed, extracted for 30min under the ultrasonic condition of 80W and 250kHz, cooled to room temperature (25 ℃) and weighed again, the weight loss is compensated by methanol, shaken evenly, filtered through a 0.45 mu m microporous filter membrane, and the subsequent filtrate is taken as the sample solution.
The invention takes methanol solution of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin as reference solution. In the invention, the concentration of echinacoside in the control solution is preferably 0.13-0.15 mg/mL, the concentration of verbascoside is preferably 0.05-0.06 mg/mL, the concentration of naringin is preferably 0.05-0.06 mg/mL, the concentration of epimedin A is preferably 0.009-0.010 mg/mL, the concentration of epimedin B is preferably 0.025-0.029 mg/mL, the concentration of epimedin C is preferably 0.05-0.06 mg/mL, and the concentration of icariin is preferably 0.10-0.11 mg/mL. The method preferably takes methanol as a solvent, single reference substance stock solutions are prepared by respectively taking echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin as reference substances, then an appropriate amount of each single reference substance stock solution is mixed and diluted by the methanol, and the mixture is filtered, and a subsequent filtrate is taken as a reference substance solution; in the invention, the concentration of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin in the single control stock solution is independently and preferably 0.4-0.6 mg/mL. In the embodiment of the invention, a proper amount of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin reference substances are respectively and precisely weighed and placed in a 10mL measuring flask, and methanol is used as a solvent to respectively prepare single reference substance stock solutions with the concentrations of 0.4637mg/mL, 0.5664mg/mL, 0.5309mg/mL, 0.4547mg/mL, 0.5713mg/mL, 0.5177mg/mL and 0.5140 mg/mL; then precisely sucking 3mL, 1mL, 0.2mL, 0.5mL, 1mL and 2mL of each single reference substance stock solution respectively, putting the single reference substance stock solution into another 10mL measuring flask, adding methanol to fix the volume, shaking up to prepare methanol solutions with the concentrations of 0.13911mg/mL, 0.05664mg/mL, 0.05309mg/mL, 0.00909mg/mL, 0.02857mg/mL, 0.05177mg/mL and 0.10280mg/mL respectively, passing through a 0.45-micrometer microporous filter membrane, and taking the subsequent filtrate as the reference substance solution.
After obtaining a reference substance solution and a test substance solution, respectively carrying out high performance liquid chromatography analysis on the reference substance solution and the test substance solution, and obtaining the finger print of the gamboge bone-invigorating pill by taking the chromatographic peak of the reference substance solution as a reference. In the invention, when the high performance liquid chromatography is performed, the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is preferably acetonitrile, the mobile phase B is preferably a phosphoric acid aqueous solution with a volume fraction of 0.01-0.3%, and the volume fraction of the phosphoric acid aqueous solution is more preferably 0.05-0.2%, and is further preferably 0.1%; the flow rate of the mobile phase system is preferably 0.5-1.5 mL/min, and more preferably 0.9 mL/min. The invention preferably adopts a gradient elution mode, and the gradient elution program is preferably as follows: the volume fraction of the mobile phase A is increased from 8% to 10% in 0-15 min; the volume fraction of the mobile phase A is increased from 10% to 30% in 15-55 min; the volume fraction of the mobile phase A is increased from 30% to 45% in 55-65 min; 65-75 min, and maintaining the volume fraction of the mobile phase A at 45%; and (3) 75-90 min, increasing the volume fraction of the mobile phase A from 45% to 80% (namely recording the chromatogram in 90min in the invention).
In the present invention, when the high performance liquid chromatography is performed, the filler in the column is preferably octadecylsilane chemically bonded silica, and the column is preferably Shimadzu C18A column, preferably having a specification of: the length is 250mm, the inner diameter is 4.60mm, and the particle size of the filler is 5 mu m; the preferred column temperature is 25-45 ℃, and the more preferred column temperature is 30 ℃; the detector is preferably a VWD detector, and the detection wavelength is preferably 210-360 nm, more preferably 280 nm; the injection volume is preferably 5-15 μ L, and more preferably 10 μ L.
In the present invention, the number of theoretical plates is preferably not less than 4000, more preferably 5000 to 8000, calculated from the icariin peak, in the high performance liquid chromatography.
After the reference substance solution and the test solution are respectively subjected to high performance liquid chromatography analysis, the gamboge bone-invigorating pill fingerprint spectrum is obtained by taking the chromatographic peak of the reference substance solution as the reference. In the invention, after the high performance liquid chromatography analysis is carried out on the reference solution, a reference chromatogram is obtained; carrying out high performance liquid chromatography analysis on the test solution to obtain a sample chromatogram; preferably, the sample chromatogram is introduced into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for similarity analysis, so that the gamboge bone-invigorating pill fingerprint is obtained. In the invention, the traditional Chinese medicine chromatogram fingerprint similarity evaluation system is preferably a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012A version; the invention preferably utilizes a median method to carry out the similarity analysis, the time window width is preferably set to be 0.1min, and 20 common peaks are calibrated. In the embodiment of the invention, high performance liquid chromatography is carried out on 11 batches of gamboge bone-strengthening pills, and the obtained sample chromatogram is introduced into a traditional Chinese medicine chromatography fingerprint similarity evaluation system for similarity analysis, wherein the similarity of each batch of gamboge bone-strengthening pills is greater than 0.90. In the invention, the sample chromatogram and the reference chromatogram are preferably compared, chromatographic peaks at the same retention time as that of each reference are identified, and the results show that the peaks 5, 8, 10, 13, 14, 15 and 16 in the 20 common peaks are respectively identified as echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin. The invention preferably belongs 20 common peaks according to standard fingerprint conditions, wherein the 20 common peaks are respectively classified into seven medicinal materials of epimedium, suberect spatholobus stem, Chinese pyrola herb, stir-fried radish seed, prepared rehmannia root, desertliving cistanche and scalded rhizoma drynariae; wherein, the No. 1 peak belongs to the scalded drynaria rhizome, the Chinese pyrola herb and the suberect spatholobus stem, the No. 2 peak belongs to the scalded drynaria rhizome and the suberect spatholobus stem, the No. 3 peak belongs to the scalded drynaria rhizome, the No. 4 peak belongs to the desertliving cistanche, the No. 5 peak belongs to the desertliving cistanche, the No. 6 peak belongs to the epimedium herb and the Chinese pyrola herb, the No. 7 peak belongs to the fried radish seed and the scalded drynaria rhizome, the No. 8 peak belongs to the desertliving cistanche and the prepared rehmannia root, the No. 9 peak belongs to the desertliving cistanche and the prepared rehmannia root, the No. 10 peak belongs to the scalded drynaria rhizome, the No. 11 peak belongs to the scalded drynaria rhizome and the epimedium, the No. 12 peak belongs to the epimedium herb, the No. 13 peak belongs to the epimedium herb, the No. 14 peak belongs to the epimedium, the No. 15 peak belongs to the epimedium, the No. 16 peak belongs to the epimedium, the 17 peak belongs to the prepared rehmannia root, the No. 18 peak belongs to the epimedium, the No. 19 peak belongs to the epimedium, and the No. 20 peak belongs to the epimedium.
The invention provides a gamboge bone-invigorating pill fingerprint obtained by the method for constructing the gamboge bone-invigorating pill fingerprint in the technical scheme.
The invention provides application of the gamboge bone-invigorating pill fingerprint spectrum in the technical scheme in the quality control of the gamboge bone-invigorating pill.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: detecting fingerprint of gamboge bone-invigorating pills in different batches
1. Instrument and reagent
1.1Agilent model 1220 high performance liquid chromatograph (Agilent, usa); KQ-250B ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); BSA 124S electronic balance (sardolis); QUINTIX35-1CN electronic balance (Sadolis).
1.2 pure water (Wahaha), acetonitrile (chromatographic purity), and other reagents are analytically pure. Echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin reference substances (the batches are 111670-. Gamboge bone-strengthening pills (batch numbers 20200201, 20200202, 20200203, 20200401, 20200402, 20200801, 20200802, 20210101, 20210401, 20210404, 20210801, Jilin Jichun pharmaceutical Co., Ltd.).
2. Fingerprint determination
2.1 chromatographic conditions: the filler in the chromatographic column is octadecylsilane chemically bonded silica, and the chromatographic column is Shimadzu C18Column, specification: the length is 250mm, the inner diameter is 4.60mm, and the particle size of the filler is 5 mu m; the column temperature is 30 ℃; the detector is a VWD detector, and the detection wavelength is 280 nm; the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution with the volume fraction of 0.1%, and the flow rate of the mobile phase system is 0.9 mL/min; adopting a gradient elution mode, wherein the gradient elution program comprises the following steps: the volume fraction of the mobile phase A is increased from 8 percent for 0-15 minTo 10%; the volume fraction of the mobile phase A is increased from 10% to 30% in 15-55 min; the volume fraction of the mobile phase A is increased from 30% to 45% in 55-65 min; 65-75 min, and maintaining the volume fraction of the mobile phase A at 45%; the volume fraction of the mobile phase A is increased from 45% to 80% in 75-90 min; the theoretical plate number is 6000 to 7000 by icariin peak.
2.2 preparation of test solution: grinding resina Garciniae bone-invigorating pill, collecting about 1g, precisely weighing, placing into conical flask with plug, precisely adding 25mL of methanol, weighing, ultrasonically treating at 80W and 250kHz for 30min, cooling to room temperature (25 deg.C), weighing again, supplementing lost weight with methanol, shaking, passing through 0.45 μm microporous membrane, and collecting filtrate as sample solution.
2.3 preparation of reference medicinal materials and reference extracts: weighing the reference extract or the reference medicinal material according to the prescription proportion and the preparation method, and preparing according to the preparation method of the test sample.
2.4 preparation of mixed control solutions: respectively precisely weighing appropriate amount of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin reference substances, putting into a 10mL measuring flask, and respectively preparing reference substance stock solutions with mass concentrations of 0.4637mg/mL, 0.5664mg/mL, 0.5309mg/mL, 0.4547mg/mL, 0.5713mg/mL, 0.5177mg/mL and 0.5140mg/mL by using methanol as a solvent; then precisely sucking 3mL, 1mL, 0.2mL, 0.5mL, 1mL and 2mL of the reference stock solution into another 10mL measuring flask, adding methanol to fix the volume, shaking up to prepare methanol solutions with the concentrations of 0.13911mg/mL, 0.05664mg/mL, 0.05309mg/mL, 0.00909mg/mL, 0.02857mg/mL, 0.05177mg/mL and 0.10280mg/mL respectively, filtering the methanol solutions through a 0.45-micrometer microporous filter membrane, and taking the subsequent filtrate as a mixed reference solution.
2.5 determination: precisely sucking 10 μ L of each of the mixed reference solution and the test solution, injecting into a high performance liquid chromatograph, and recording chromatogram within 90min, as shown in FIG. 1 and FIG. 2 (the identification of each peak in the figure will be described in detail later).
Example 2: 11 batches of gamboge bone-invigorating pill fingerprint analysis
1. Fingerprint similarity analysis
Taking 11 batches of gamboge bone-invigorating pills, preparing samples according to the preparation method of the test solution in the embodiment 1, carrying out sample injection analysis according to the chromatographic conditions in the embodiment 1, recording the fingerprint spectrum of the gamboge bone-invigorating pills, carrying out similarity analysis on the fingerprint spectrum of the 11 batches of gamboge bone-invigorating pills by using 'traditional Chinese medicine chromatography fingerprint spectrum similarity evaluation system 2012A edition' issued by the State pharmacopoeia Committee, setting S1 as a reference spectrum, calibrating 20 common peaks in total by using a median method and setting the time window width to be 0.1min, wherein the similarity of the 11 batches of gamboge bone-invigorating pills is greater than 0.90, and the similarity evaluation result of the 11 batches of gamboge bone-invigorating pills is shown in Table 1. The relative retention times of the 20 common peaks were substantially consistent, while the relative peak areas were greatly different, and the standard fingerprint data are shown in table 2. Fig. 3 is a superimposed graph of fingerprint spectra of 11 batches of gamboge bone-invigorating pills, wherein S1-S11 in fig. 3 correspond to batches 20200201, 20200202, 20200203, 20200401, 20200402, 20200801, 20200802, 20210101, 20210401, 20210404 and 20210801 respectively.
Table 111 gamboge bone-invigorating pill sample similarity evaluation results
TABLE 2 Standard fingerprint data
| Peak number | Average Retention time (min) | Peak area (S1) | Retention time RSD (%) | Peak area RSD (%) |
| 1 | 11.232 | 214.243 | 0.1 | 25.91 |
| 2 | 21.206 | 101.090 | 0.15 | 6.09 |
| 3 | 23.493 | 71.733 | 0.12 | 18.27 |
| 4 | 32.866 | 508.699 | 0.11 | 7.9 |
| 5 | 33.514 | 704.334 | 0.08 | 9.83 |
| 6 | 34.002 | 115.128 | 0.06 | 20.1 |
| 7 | 40.250 | 68.457 | 0.05 | 9.95 |
| 8 | 40.911 | 181.610 | 0.05 | 11.09 |
| 9 | 43.668 | 144.183 | 0.05 | 27.88 |
| 10 | 45.335 | 349.189 | 0.04 | 14.35 |
| 11 | 50.497 | 60.139 | 0.05 | 11.06 |
| 12 | 56.874 | 56.400 | 0.05 | 8.74 |
| 13 | 58.099 | 69.874 | 0.04 | 4.4 |
| 14 | 59.019 | 145.624 | 0.06 | 3.78 |
| 15 | 59.928 | 268.494 | 0.06 | 3.96 |
| 16 | 60.996 | 547.803 | 0.05 | 4.81 |
| 17 | 68.492 | 33.707 | 0.05 | 8.56 |
| 18 | 72.028 | 94.610 | 0.04 | 10.21 |
| 19 | 72.390 | 200.161 | 0.02 | 13.4 |
| 20 | 77.094 | 358.918 | 0.03 | 7.39 |
2. Consensus peak assignment: the HPLC chromatogram of the mixed control solution (fig. 1) was compared to the sample fingerprint (fig. 3) and the peak of the chromatogram at the same retention time as the control was assigned. The results showed that among the 20 common peaks, peak No. 5, peak No. 8, peak No. 10, peak No. 13, peak No. 14, peak No. 15, and peak No. 16 were respectively identified as echinacoside, verbascoside, naringin, epimedin a, epimedin B, epimedin C, and icariin.
3. Medicinal material attribution: performing chromatogram peak assignment according to standard fingerprint conditions, as shown in FIG. 4, wherein S1-S7 in FIG. 4 correspond to seven medicinal materials of herba Epimedii, caulis Spatholobi, herba Pyrolae, parched Raphani semen, radix rehmanniae Preparata, Cistanchis herba, and scalded rhizoma Drynariae, respectively; s8 is a sample; s9 is a mixed control. 20 common peaks are respectively attributed to the seven medicinal materials of epimedium, suberect spatholobus stem, Chinese pyrola herb, stir-fried radish seed, prepared rehmannia root, desertliving cistanche and scalded rhizoma drynariae; wherein, the No. 1 peak belongs to the scalded drynaria rhizome, the Chinese pyrola herb and the suberect spatholobus stem, the No. 2 peak belongs to the scalded drynaria rhizome and the suberect spatholobus stem, the No. 3 peak belongs to the scalded drynaria rhizome, the No. 4 peak belongs to the desertliving cistanche, the No. 5 peak belongs to the desertliving cistanche, the No. 6 peak belongs to the epimedium herb and the Chinese pyrola herb, the No. 7 peak belongs to the fried radish seed and the scalded drynaria rhizome, the No. 8 peak belongs to the desertliving cistanche and the prepared rehmannia root, the No. 9 peak belongs to the desertliving cistanche and the prepared rehmannia root, the No. 10 peak belongs to the scalded drynaria rhizome, the No. 11 peak belongs to the scalded drynaria rhizome and the epimedium, the No. 12 peak belongs to the epimedium herb, the No. 13 peak belongs to the epimedium herb, the No. 14 peak belongs to the epimedium, the No. 15 peak belongs to the epimedium, the No. 16 peak belongs to the epimedium, the 17 peak belongs to the prepared rehmannia root, the No. 18 peak belongs to the epimedium, the No. 19 peak belongs to the epimedium, and the No. 20 peak belongs to the epimedium.
Example 3: selection of wavelength
20210101 batches of samples are taken for testing, the overall effect of the chromatogram under the detection wavebands street of 360nm, 320nm, 280nm, 254nm, 230nm, 210nm and 203nm is respectively considered, other chromatographic conditions are the same as example 1, and each chromatogram is shown in figures 5-11. The results showed that the peak value was small, the response value was low and the number of peaks was small at a detection wavelength of 360nm (FIG. 5); at a detection wavelength of 320nm, the response value was low and the number of peaks was relatively small (FIG. 6); when the detection wavelength is 280nm, the chromatogram base line is relatively flat, the peak shape is relatively good, the number of peaks is relatively large, the response value is the highest, and the condition is the optimal condition (figure 7); when the detection wavelength is 254nm, the chromatogram base line is relatively flat, the peak shape is relatively good, but the separation effect is slightly poor compared with that of 280nm (figure 8); when the detection wavelength is 230nm, the chromatogram base line is flatter, the peak shape is better, the peak number is more, and the separation effect is poorer (figure 9); when the detection wavelength is 210nm, the base line of the chromatogram is uneven, and the fluctuation of the chromatographic peak is huge (figure 10); at a detection wavelength of 203nm, the chromatogram baseline was not in the standard position (FIG. 11).
Because the fingerprint is not used for measuring the accurate content of a certain component, but is required to fully reflect the information of chemical components, the fingerprint taking 280nm as the detection wavelength has more peaks, more complete reflected information, the best peak shape, good absorption value of each peak and stable base line, and avoids the condition of large absorption of near-ultraviolet impurity peaks. Therefore 280nm was chosen as the optimal detection wavelength.
Example 4: methodology investigation
20210101 samples were taken for testing, and the chromatographic conditions and the test solution were prepared in the same manner as in example 1.
(1) Precision test
Preparing a test solution from the gamboge bone-invigorating pill, continuously injecting samples for 6 times, and recording the retention time of 20 main chromatographic peaks and the peak area of each main chromatographic peak. The relative peak area RSD is less than 3.0% (n is 6) and the relative retention time RSD is less than 1.0% (n is 6) by using the icariin as a reference peak, which proves that the precision of the instrument is good. The results are shown in Table 3.
(2) Stability test
Preparing a test solution from the gamboge bone-invigorating pills, testing for 0h, 2h, 4h, 8h, 12h and 24h respectively, and recording the retention time of 20 main chromatographic peaks and the peak area. The relative peak area RSD is less than 3.0% (n is 6) and the relative retention time RSD is less than 1.0% (n is 6) by taking the icariin as a reference peak, and the stability of the sample in 24h is proved to be good. The results are shown in Table 3.
(3) Repeatability test
Taking the gamboge bone-invigorating pills, preparing 6 parts of test solution, testing, and recording the retention time of 20 main chromatographic peaks and the peak area of each peak. Good stability was demonstrated by using icariin as a reference peak, a relative peak area RSD of < 3.0% (n 6), and a relative retention time RSD of < 1.0% (n 6). The results are shown in Table 3.
TABLE 3 methodological investigation of the experimental results
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for constructing a finger print of a gamboge bone-invigorating pill comprises the following steps:
extracting the gamboge bone-invigorating pills by adopting methanol, and taking the obtained gamboge bone-invigorating pill extracting solution as a test solution; methanol solution of echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin is used as control solution;
and respectively carrying out high performance liquid chromatography analysis on the reference substance solution and the test solution, and taking the chromatographic peak of the reference substance solution as a reference to obtain the finger print of the gamboge bone-invigorating pill.
2. The method for constructing the gamboge bone-invigorating pill fingerprint spectrum according to claim 1, wherein the dosage ratio of the gamboge bone-invigorating pill to the methanol is (1-3) g: (10-50) mL.
3. The method for constructing the gamboge bone-invigorating pill fingerprint as claimed in claim 2, wherein the extraction is performed under ultrasonic conditions, the power of the ultrasonic is 70-90W, and the frequency is 200-300 kHz; the extraction time is 20-40 min.
4. The method for constructing the finger-print of gamboge bone-invigorating pill according to claim 1, wherein when the high performance liquid chromatography is performed, the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with a volume fraction of 0.01-0.3%; the flow rate of the mobile phase system is 0.5-1.5 mL/min;
adopting a gradient elution mode, wherein the gradient elution program comprises the following steps: the volume fraction of the mobile phase A is increased from 8% to 10% in 0-15 min; the volume fraction of the mobile phase A is increased from 10% to 30% in 15-55 min; the volume fraction of the mobile phase A is increased from 30% to 45% in 55-65 min; 65-75 min, and maintaining the volume fraction of the mobile phase A at 45%; and (3) increasing the volume fraction of the mobile phase A from 45% to 80% in 75-90 min.
5. The method for constructing finger-print of gamboge bone-invigorating pill according to claim 1, wherein the filler in the chromatographic column is octadecylsilane chemically bonded silica when performing the high performance liquid chromatography, and the specification of the chromatographic column is as follows: the length is 250mm, the inner diameter is 4.60mm, and the particle size of the filler is 5 mu m; the column temperature is 25-45 ℃; the detector is a VWD detector, and the detection wavelength is 210-360 nm; the injection volume is 5-15 mu L.
6. The method for constructing fingerprint of gamboge bone-invigorating pill according to claim 1 wherein the number of theoretical plates is not less than 4000 on the basis of icariin peak in the HPLC analysis.
7. The method for constructing the fingerprint of gamboge bone-invigorating pill according to any one of claims 1 to 6, wherein the fingerprint of gamboge bone-invigorating pill contains 20 common peaks, wherein peaks No. 5, 8, 10, 13, 14, 15 and 16 are respectively identified as echinacoside, verbascoside, naringin, epimedin A, epimedin B, epimedin C and icariin.
8. The method for constructing finger-print of gamboge bone-invigorating pill according to claim 7, wherein the 20 common peaks are respectively attributed to epimedium, spatholobus stem, pyrola, stir-fried radish seed, prepared rehmannia root, cistanche deserticola and scalded drynaria rhizome; wherein, the No. 1 peak belongs to the scalded drynaria rhizome, the Chinese pyrola herb and the suberect spatholobus stem, the No. 2 peak belongs to the scalded drynaria rhizome and the suberect spatholobus stem, the No. 3 peak belongs to the scalded drynaria rhizome, the No. 4 peak belongs to the desertliving cistanche, the No. 5 peak belongs to the desertliving cistanche, the No. 6 peak belongs to the epimedium herb and the Chinese pyrola herb, the No. 7 peak belongs to the fried radish seed and the scalded drynaria rhizome, the No. 8 peak belongs to the desertliving cistanche and the prepared rehmannia root, the No. 9 peak belongs to the desertliving cistanche and the prepared rehmannia root, the No. 10 peak belongs to the scalded drynaria rhizome, the No. 11 peak belongs to the scalded drynaria rhizome and the epimedium, the No. 12 peak belongs to the epimedium herb, the No. 13 peak belongs to the epimedium herb, the No. 14 peak belongs to the epimedium, the No. 15 peak belongs to the epimedium, the No. 16 peak belongs to the epimedium, the 17 peak belongs to the prepared rehmannia root, the No. 18 peak belongs to the epimedium, the No. 19 peak belongs to the epimedium, and the No. 20 peak belongs to the epimedium.
9. The gamboge bone-invigorating pill fingerprint obtained by the method for constructing the gamboge bone-invigorating pill fingerprint according to any one of claims 1 to 8.
10. The use of the finger print of gamboge bone-invigorating pill of claim 9 in the quality control of the gamboge bone-invigorating pill.
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| US20090130237A1 (en) * | 2007-11-19 | 2009-05-21 | Bionovo, Inc. | Process of making purified extract of scutellaria barbata d. don |
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