CN114609308B - Gamboge bone strengthening pill fingerprint spectrum and construction method and application thereof - Google Patents
Gamboge bone strengthening pill fingerprint spectrum and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a gamboge bone strengthening pill fingerprint spectrum and a construction method and application thereof, and belongs to the technical field of medicines. The construction method of the gamboge bone strengthening pill fingerprint spectrum comprises the following steps: extracting resina garciniae bone-invigorating pill with methanol, and taking the extractive solution as sample solution; taking methanol solutions of echinacoside, acteoside, naringin, jojodine A, jojodine B, jodine C and icariine as reference solutions; and respectively performing high performance liquid chromatography analysis on the reference substance solution and the test substance solution, and taking a chromatographic peak of the reference substance solution as a benchmark to obtain the gamboge bone strengthening pill fingerprint. The fingerprint spectrum of the gamboge bone-strengthening pill is established by adopting the high performance liquid chromatography, so that the quality of the gamboge bone-strengthening pill can be comprehensively reflected, the feeding and strict standard production operation can be conveniently and effectively guided, and the safety and the effectiveness of clinical medication of the gamboge bone-strengthening pill are ensured.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a gamboge bone strengthening pill fingerprint spectrum and a construction method and application thereof.
Background
Various senile rheumatic and orthopedic diseases such as hypertrophic spondylitis, cervical spondylosis, calcaneal spur, hypertrophic arthritis, and Kaschin-Beck disease are all "arthromyodynia" in traditional Chinese medicine. Hypertrophic spondylitis is a systemic disease, which is commonly called osteoarthropathy and is chronic arthritis caused by joint degeneration and damaged articular cartilage, and the cause is degeneration of bones, namely, the disease is bone degeneration caused by accumulation of daily traumas, wind-cold and the like by taking the intrinsic factor of kidney qi deficiency as the root. At present, analgesic, 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 strengthening pill has the main functions of tonifying kidney, promoting blood circulation and relieving pain. The traditional Chinese medicine composition takes kidney tonifying as the main ingredient, treats bones as the secondary ingredient, treats both principal and secondary aspect of disease, and is suitable for treating osteoarthritis, ankylosing spondylitis, cervical spondylosis, lumbar disc herniation, scapulohumeral periarthritis and sciatica which are diagnosed by Western medicine. The gamboge bone strengthening pill is prepared from radix rehmanniae Preparata, herba Pyrolae, rhizoma Drynariae, cistanchis herba, herba Epimedii, caulis Spatholobi and parched Raphani semen. The disease causes pathogenesis of the disease is caused by deficiency of liver and kidney and obstruction of collaterals by blood stasis. Liver stores blood and tendons, such as liver blood deficiency, blood failing to nourish tendons, tendons and bones, joints failing to nourish them, and long-term illness; kidney governs bone marrow generation, and kidney essence deficiency causes deficiency of marrow formation source, and long-term and deep years, bone paralysis is enough. For liver and kidney tonifying, tendons and bones strengthening, blood circulation promoting, blood stasis dispelling, qi circulation promoting, and pain relieving.
The gamboge bone strengthening pill is prepared with prepared rehmannia root and through mixing with other materials, and has sweet taste, mild nature, capacity of nourishing liver and kidney meridian, nourishing Yin and blood, replenishing essence, nourishing marrow, nourishing liver and kidney.
Herba Epimedii in the Gu-Gang Jian-Gu Wan formula is pungent and sweet in taste, warm in nature, enter liver and kidney meridians, tonify kidney and strengthen yang, dispel wind and remove dampness; cistanche is sweet and salty in taste, warm in nature, enters kidney and large intestine meridians, tonifies kidney yang, benefits essence and blood, enters kidney and generates marrow. The two drugs of epimedium and cistanche share the principal yang of tonifying kidney and the monarch drug of auxiliary yin, and the principal drug of yin middle energizer to evaluate yang and qi with little fire, yin and yang are combined for tonifying. The hot drynaria rhizome has bitter taste and warm nature, enters kidney and liver meridians, tonifies kidney and bone, and relieves pain. Herba Pyrolae is sweet in taste, bitter and warm in nature, and has the effects of invigorating liver and kidney, expelling wind-damp, strengthening tendons and bones, stopping bleeding, and tonifying bones to relieve pain. The four medicines are auxiliary monarch medicines for tonifying liver and kidney, strengthening tendons and bones and are ministerial medicines.
The gamboge bone strengthening pill has warm nature, bitter and sweet taste, can tonify kidney, replenish essence and add marrow, also can smooth channels and collaterals, promote qi and blood circulation, promote blood circulation, remove meridian obstruction, tonify bone and relieve pain, and is an adjuvant drug.
The garcinia bone-invigorating pill is prepared by stir-frying radish seeds, and has the effects of invigorating bones, promoting digestion, regulating qi, and being mild in nature, pungent and sweet in taste, so as to prevent the defect of nourishing and greasiness.
Because of the characteristics of clear prescription, reasonable compatibility and the like of the gamboge bone strengthening pill, the gamboge bone strengthening pill needs to be internally controlled for quality control in order to ensure the drug effect. At present, related scholars also do some researches, such as the research of using high performance liquid chromatography to measure the icariin content in the gamboge bone-strengthening pills (Cheng Guo, RP-HPLC method to measure the icariin content in the gamboge bone-strengthening pills, straits pharmacy, 2013, seventh stage, pages 118-119); zhao Yong the content of icariin and naringin in the pill is determined by high performance liquid chromatography (Zhao Yong, quality standard research of pill, chinese pharmacist, 2013, 1 st stage, 76-80 pages); the method for measuring the icariin content in the gamboge bone-strengthening pills by using high performance liquid chromatography is studied in the Wenzhen and the like (the method for measuring the icariin content in the gamboge bone-strengthening pills by using high performance liquid chromatography is carried out in the Wenzhen and the like, chinese medicine standard, 2009, 3 rd phase, pages 210-212); jiang Xiangzhi and the like have studied the measurement of the content of echinacoside in gamboge bone-invigorating pills by high performance liquid chromatography (Jiang Xiang, HPLC method for measuring the content of echinacoside in gamboge bone-invigorating pills, chinese pharmacist, 2009, 4 th, pages 481-482). However, the above methods all use one or two ingredients to reflect the quality of the gamboge bone-strengthening pill, and the gamboge bone-strengthening pill has certain unilateral property and cannot be comprehensively reflected.
Disclosure of Invention
The invention aims to provide a gamboge bone-strengthening pill fingerprint spectrum, 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 construction method of a gamboge bone strengthening pill fingerprint spectrum, which comprises the following steps:
extracting resina garciniae bone-invigorating pill with methanol, and taking the extractive solution as sample solution; taking methanol solutions of echinacoside, acteoside, naringin, jojodine A, jojodine B, jodine C and icariine as reference solutions;
and respectively performing high performance liquid chromatography analysis on the reference substance solution and the test substance solution, and taking a chromatographic peak of the reference substance solution as a benchmark to obtain the gamboge bone strengthening pill fingerprint.
Preferably, the dosage ratio of the gamboge bone strengthening pill to the methanol is (1-3) g: (10-50) mL.
Preferably, the extraction is carried out under ultrasonic conditions, the power of the ultrasonic wave is 70-90W, and the frequency is 200-300 kHz; the extraction time is 20-40 min.
Preferably, when the high performance liquid chromatography analysis 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 phosphoric acid aqueous solution with the volume fraction of 0.01-0.3%; the flow rate of the mobile phase system is 0.5-1.5 mL/min;
the gradient elution method is adopted, and the gradient elution program is as follows: 0-15 min, the volume fraction of the mobile phase A is increased from 8% to 10%; 15-55 min, the volume fraction of the mobile phase A is increased from 10% to 30%; 55-65 min, the volume fraction of the mobile phase A is increased from 30% to 45%; 65-75 min, and the volume fraction of the mobile phase A is maintained at 45%; 75-90 min, the volume fraction of the mobile phase A is increased from 45% to 80%.
Preferably, when the high performance liquid chromatography is performed, the filler in the chromatographic column is octadecylsilane chemically bonded silica gel, 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 sample injection volume is 5-15 mu L.
Preferably, the theoretical plate number is not less than 4000 as calculated from icariine peak when the high performance liquid chromatography analysis is performed.
Preferably, the fingerprint of the gamboge bone strengthening pill contains 20 common peaks, wherein the peaks No. 5, no. 8, no. 10, no. 13, no. 14, no. 15 and No. 16 are respectively identified as echinacoside, acteoside, naringin, jojodine A, jojodine B, jodine C and icariine.
Preferably, the 20 common peaks respectively belong to epimedium herb, suberect spatholobus stem, pyrola herb, stir-fried radish seed, prepared rehmannia root, cistanche deserticola and hot drynaria rhizome; wherein, the No. 1 peak belongs to the rhizoma drynariae, the pyrola and the suberect spatholobus stem, the No. 2 peak belongs to the rhizoma drynariae and the suberect spatholobus stem, the No. 3 peak belongs to the rhizoma drynariae, the No. 4 peak belongs to the herba cistanches, the No. 5 peak belongs to the herba cistanches, the No. 6 peak belongs to the herba epimedii and the pyrola, the No. 7 peak belongs to the stir-fried radish seeds and the rhizoma drynariae, the No. 8 peak belongs to the herba cistanches and the prepared rehmannia root, the No. 9 peak belongs to the herba cistanches and the prepared rehmannia root, the No. 10 peak belongs to the rhizoma drynariae, the No. 11 peak belongs to the rhizoma drynariae and the herba epimedii, the No. 12 peak belongs to the herba epimedii, the No. 13 peak belongs to the herba epimedii, the No. 14 peak belongs to the herba epimedii, the No. 15 peak belongs to the herba epimedii, the No. 16 peak belongs to the herba epimedii, the No. 17 peak belongs to the prepared herba epimedii, the No. 19 peak belongs to the herba epimedii, and the No. 20 peak belongs to the herba epimedii.
The invention provides the gamboge bone-strengthening pill fingerprint spectrum obtained by the construction method of the gamboge bone-strengthening pill fingerprint spectrum.
The invention provides application of the fingerprint spectrum of the gamboge bone-strengthening pill in quality control of gamboge bone-strengthening pills.
The invention provides a construction method of a gamboge bone strengthening pill fingerprint spectrum, which comprises the following steps: extracting resina garciniae bone-invigorating pill with methanol, and taking the extractive solution as sample solution; taking methanol solutions of echinacoside, acteoside, naringin, jojodine A, jojodine B, jodine C and icariine as reference solutions; and respectively performing high performance liquid chromatography analysis on the reference substance solution and the test substance solution, and taking a chromatographic peak of the reference substance solution as a benchmark to obtain the gamboge bone strengthening pill fingerprint. The fingerprint spectrum of the gamboge bone-strengthening pill is established by adopting the high performance liquid chromatography, so that the integral chemical components of the gamboge bone-strengthening pill can be comprehensively reflected, the single compound or medicinal materials are not identified, the problems of singleness and unilateralness of the existing quality control method are overcome, the feeding and strict standard production operation can be more effectively guided, an effective means is provided for the integral quality control and evaluation of the gamboge bone-strengthening pill, the quality stability, consistency and controllability of the gamboge bone-strengthening pill are better ensured, and the safety and effectiveness of clinical medication of the gamboge bone-strengthening pill are further ensured.
Furthermore, the fingerprint spectrum of the gamboge bone-strengthening pill contains 20 common peaks, 7 components are identified, and meanwhile, the 20 common peaks are attributed to single medicinal materials, so that the current situation of each component in the gamboge bone-strengthening pill is comprehensively reflected, and a reference basis can be provided for quality control of the gamboge bone-strengthening pill.
The fingerprint spectrum of the gamboge bone-strengthening pill constructed by the method has rich chromatographic peaks, contains the effective components of prepared rehmannia root, pyrola herb, hot drynaria rhizome, cistanche salsa, epimedium herb, suberect spatholobus stem and stir-fried radish seed, can better monitor and evaluate the quality of the gamboge bone-strengthening pill, guides standardized production and has higher application value.
Drawings
FIG. 1 is an HPLC chromatogram of a mixed control solution;
FIG. 2 is a pattern diagram of common mode gamboge bone strengthening pills;
FIG. 3 is a superposition of fingerprint patterns of 11 batches of gamboge bone-strengthening pills;
FIG. 4 is a diagram showing the comparison between a sample fingerprint and each of the chromatograms of the medicinal materials;
FIG. 5 is a chart of a gamboge bone-strengthening pill at a wavelength of 360nm;
FIG. 6 is a chart of a gamboge bone-strengthening pill at a wavelength of 320 nm;
FIG. 7 is a chart of a gamboge bone-strengthening pill at a wavelength of 280nm;
FIG. 8 is a chart of a gamboge bone-strengthening pill at 254nm wavelength;
FIG. 9 is a chart of a gamboge bone-strengthening pill at a wavelength of 230 nm;
FIG. 10 is a chart of a gamboge bone-strengthening pill at a wavelength of 210 nm;
FIG. 11 is a chart of a gamboge bone-strengthening pill under the condition of 203nm wavelength.
Detailed Description
The invention provides a construction method of a gamboge bone strengthening pill fingerprint spectrum, which comprises the following steps:
extracting resina garciniae bone-invigorating pill with methanol, and taking the extractive solution as sample solution; taking methanol solutions of echinacoside, acteoside, naringin, jojodine A, jojodine B, jodine C and icariine as reference solutions;
and respectively performing high performance liquid chromatography analysis on the reference substance solution and the test substance solution, and taking a chromatographic peak of the reference substance solution as a benchmark to obtain the gamboge bone strengthening pill fingerprint.
In the invention, the gamboge bone strengthening pill is specifically prepared from prepared rehmannia root, pyrola herb, scalded drynaria rhizome, cistanche deserticola, epimedium herb, suberect spatholobus stem and stir-fried radish seed; the source of the gamboge bone strengthening pill is not particularly limited, and commercial products which are well known to the person skilled in the art, such as gamboge bone strengthening pills produced by Jilin Jichun pharmaceutical Co., ltd, can be adopted; can also be prepared by methods well known to those skilled in the art. In the invention, the preparation method of the gamboge bone strengthening pill preferably comprises the following steps: taking 750g of prepared rehmannia root, 500g of pyrola herb, 500g of hot drynaria rhizome, 500g of cistanche salsa, 500g of epimedium herb, 500g of suberect spatholobus stem and 250g of stir-fried radish seed, and crushing the pyrola herb and the epimedium herb into fine powder to obtain a powder raw material; decocting the other five medicinal materials with water for 2h and 1h respectively, mixing the decoctions, filtering, concentrating the filtrate into soft extract, mixing with the powder materials, drying, pulverizing, sieving, adding 80-100 g refined honey into 100g of the obtained powder materials, and making into big honeyed pill.
The invention adopts methanol to extract gamboge bone-strengthening pills, and the obtained gamboge bone-strengthening pill extract is used as a sample solution. The invention preferably mixes the gamboge bone-strengthening pill with methanol for extraction to obtain gamboge bone-strengthening pill extract. The gamboge bone strengthening pill is preferably ground and then extracted. In the invention, the dosage ratio of the gamboge bone strengthening pill to the methanol is preferably (1-3) g: (10-50) mL, more preferably (1-2) g: (15-25) mL. In the present invention, the extraction is preferably performed under ultrasonic conditions, and the power of the ultrasonic wave is preferably 70 to 90W, more preferably 80W; the frequency of the ultrasonic wave is preferably 200-300 kHz, more preferably 250kHz; the extraction time is preferably 20 to 40min, more preferably 30min. In the present invention, the post-extraction preferably further comprises filtration, and the filtration membrane used for the filtration is preferably a microporous membrane of 0.45 μm. In the embodiment of the invention, gamboge bone strengthening pills are taken, ground, precisely weighed 1g, placed in a conical bottle 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 ℃) to weigh again, the reduced weight is complemented by methanol, shaken uniformly, and filtered through a microporous membrane of 0.45 mu m to obtain a subsequent filtrate as a sample solution.
The invention takes methanol solution of echinacoside, acteoside, naringin, jojodine A, jodine B, jodine C and icariine as reference substance solution. In the invention, the concentration of echinacoside in the reference substance solution is preferably 0.13-0.15 mg/mL, the concentration of acteoside is preferably 0.05-0.06 mg/mL, the concentration of naringin is preferably 0.05-0.06 mg/mL, the concentration of hodgkin A is preferably 0.009-0.010 mg/mL, the concentration of hodgkin B is preferably 0.025-0.029 mg/mL, the concentration of hodgkin C is preferably 0.05-0.06 mg/mL, and the concentration of icariine is preferably 0.10-0.11 mg/mL. The invention preferably takes methanol as a solvent, and respectively prepares single reference substance stock solutions of echinacoside, acteoside, naringin, jojodine A, jojodine B, jodine C and icariine reference substances, then takes a proper amount of each single reference substance stock solution to mix and dilute with methanol, filters, and takes the subsequent filtrate as a reference substance solution; in the invention, the concentrations of echinacoside, acteoside, naringin, jojodine A, jojodine B, jodine C and icariine in the single reference stock solution are independently preferably 0.4-0.6 mg/mL. In the embodiment of the invention, a proper amount of echinacoside, acteoside, naringin, jojodine A, jojodine B, jojodine C and icariine reference substances are precisely weighed respectively, put into a 10mL measuring flask, and are respectively prepared into 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 0.5140mg/mL by taking methanol as a solvent; then respectively precisely sucking 3mL, 1mL, 0.2mL, 0.5mL, 1mL and 2mL of each single reference stock solution, placing into another 10mL measuring flask, adding methanol to fix the volume, shaking uniformly, and preparing 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, and passing through a microporous filter membrane with the concentration of 0.45 mu m, thereby obtaining a continuous filtrate as a reference solution.
After the reference substance solution and the test substance solution are obtained, the reference substance solution and the test substance solution are respectively subjected to high performance liquid chromatography analysis, and the fingerprint of the gamboge bone strengthening pill is obtained by taking the chromatographic peak of the reference substance solution as a benchmark. In the invention, when the high performance liquid chromatography analysis 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 phosphoric acid aqueous solution with the 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 further preferably 0.1%; the flow rate of the mobile phase system is preferably 0.5 to 1.5mL/min, more preferably 0.9mL/min. The invention adopts a gradient elution mode, and the gradient elution program is preferably as follows: 0-15 min, the volume fraction of the mobile phase A is increased from 8% to 10%; 15-55 min, the volume fraction of the mobile phase A is increased from 10% to 30%; 55-65 min, the volume fraction of the mobile phase A is increased from 30% to 45%; 65-75 min, and the volume fraction of the mobile phase A is maintained at 45%; 75-90 min, the volume fraction of mobile phase A increases from 45% to 80% (i.e. the present invention specifically records chromatograms within 90 min).
In the present invention, in the case of performing the high performance liquid chromatography, the filler in the column is preferably octadecylsilane chemically bonded silica, and the column is preferably Shimadzu C 18 The specification of the chromatographic column is preferably: the length is 250mm, the inner diameter is 4.60mm, and the filling is carried outThe particle size of the filling agent is 5 mu m; the column temperature is preferably 25 to 45 ℃, more preferably 30 ℃; the detector is preferably a VWD detector, the detection wavelength preferably being 210-360 nm, more preferably 280nm; the sample volume is preferably 5 to 15. Mu.L, more preferably 10. Mu.L.
In the present invention, when the HPLC analysis is performed, the theoretical plate number is preferably not less than 4000, more preferably 5000 to 8000, as calculated on icariine peaks.
After the high performance liquid chromatography analysis is carried out on the reference substance solution and the test substance solution respectively, the fingerprint spectrum of the gamboge bone strengthening pill is obtained by taking the chromatographic peak of the reference substance solution as a benchmark. In the invention, the control substance solution is subjected to high performance liquid chromatography analysis to obtain a control substance chromatogram; carrying out high performance liquid chromatography on the sample solution to obtain a sample chromatogram; the sample chromatogram is preferably guided into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to perform similarity analysis, so as to obtain the gamboge bone strengthening pill fingerprint. In the invention, the traditional Chinese medicine chromatographic fingerprint similarity evaluation system is preferably 2012A edition of the traditional Chinese medicine chromatographic fingerprint similarity evaluation system; the similarity analysis is preferably performed by a median method, and the time window width is preferably set to be 0.1min, and 20 common peaks are calibrated in total. In the embodiment of the invention, the high performance liquid chromatography is carried out on 11 batches of gamboge bone-strengthening pills, and the obtained sample chromatograms are guided into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis, wherein the similarity of each batch of gamboge bone-strengthening pills is more than 0.90. The sample chromatogram is preferably compared with the chromatogram of the reference substance, and chromatographic peaks at the same retention time as each reference substance are identified, and the result shows that the No. 5 peak, the No. 8 peak, the No. 10 peak, the No. 13 peak, the No. 14 peak, the No. 15 peak and the No. 16 peak in the 20 common peaks are respectively identified as echinacoside, verbascoside, naringin, hederagenin A, hederagenin B, hederagenin C and icariine. The invention preferably carries out component attribution on 20 common peaks according to standard fingerprint spectrum conditions, wherein the 20 common peaks are respectively attributed to seven medicinal materials of epimedium herb, suberect spatholobus stem, pyrola herb, stir-fried radish seed, prepared rehmannia root, cistanche deserticola and hot drynaria rhizome; wherein, the No. 1 peak belongs to the rhizoma drynariae, the pyrola and the suberect spatholobus stem, the No. 2 peak belongs to the rhizoma drynariae and the suberect spatholobus stem, the No. 3 peak belongs to the rhizoma drynariae, the No. 4 peak belongs to the herba cistanches, the No. 5 peak belongs to the herba cistanches, the No. 6 peak belongs to the herba epimedii and the pyrola, the No. 7 peak belongs to the stir-fried radish seeds and the rhizoma drynariae, the No. 8 peak belongs to the herba cistanches and the prepared rehmannia root, the No. 9 peak belongs to the herba cistanches and the prepared rehmannia root, the No. 10 peak belongs to the rhizoma drynariae, the No. 11 peak belongs to the rhizoma drynariae and the herba epimedii, the No. 12 peak belongs to the herba epimedii, the No. 13 peak belongs to the herba epimedii, the No. 14 peak belongs to the herba epimedii, the No. 15 peak belongs to the herba epimedii, the No. 16 peak belongs to the herba epimedii, the No. 17 peak belongs to the prepared herba epimedii, the No. 19 peak belongs to the herba epimedii, and the No. 20 peak belongs to the herba epimedii.
The invention provides the gamboge bone-strengthening pill fingerprint spectrum obtained by adopting the construction method of the gamboge bone-strengthening pill fingerprint spectrum.
The invention provides application of the fingerprint spectrum of the gamboge bone-strengthening pill in quality control of gamboge bone-strengthening pills.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: fingerprint spectrum of gamboge bone strengthening pills in different batches is detected
1. Instrument and reagent
1.1Agilent 1220 high performance liquid chromatograph (Agilent, USA); KQ-250B ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); BSA 124S electronic balance (certolis); QUINTIX35-1CN electronic balance (Sidoris).
1.2 purified water (Waha), acetonitrile (chromatographic purity), other reagents were all analytically pure. Echinacoside, acteoside, naringin, and reference icariin (lot numbers 111670-201706, 111530-201914, 110722-201815, 110623-72-8, 1106-23-9, 111780-201905, and 110737-202017, respectively) with purities of 89.7%, 95.2%, 91.7%, 98.0%, 94.3%, 98.1%) respectively. Gamboge bone strengthening pill (lot numbers 20200201, 20200202, 20200203, 20200401, 20200402, 20200801, 20200802, 20210101, 20210401, 20210404, 20210801, jilin Jichun pharmaceutical Co., ltd.).
2. Fingerprint measurement
2.1 chromatographic conditions: the filler in the chromatographic column is octadecylsilane chemically bonded silica gel, and the chromatographic column is Shimadzu C 18 The specification of the 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 30 ℃; the detector is a VWD detector, and the detection wavelength is 280nm; 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 phosphoric acid aqueous solution with the volume fraction of 0.1%, and the flow rate of the mobile phase system is 0.9mL/min; the gradient elution method is adopted, and the gradient elution program is as follows: 0-15 min, the volume fraction of the mobile phase A is increased from 8% to 10%; 15-55 min, the volume fraction of the mobile phase A is increased from 10% to 30%; 55-65 min, the volume fraction of the mobile phase A is increased from 30% to 45%; 65-75 min, and the volume fraction of the mobile phase A is maintained at 45%; 75-90 min, the volume fraction of the mobile phase A is increased from 45% to 80%; the theoretical plate number is 6000-7000 calculated according to icariine peak.
2.2 preparation of test solutions: taking gamboge bone strengthening pills, grinding, taking about 1g, precisely weighing, placing into a conical bottle with a plug, precisely adding 25mL of methanol, weighing, performing ultrasonic treatment for 30min under the conditions of 80W power and 250kHz frequency, cooling to room temperature (25 ℃) to weigh again, supplementing the lost weight with methanol, shaking uniformly, passing through a microporous filter membrane of 0.45 mu m, and taking the subsequent filtrate as a sample solution.
2.3 preparation of control drug and control extract: weighing control extract or control medicinal material according to prescription proportion and preparation method, and preparing according to test sample preparation method.
2.4 preparation of a mixed control solution: respectively precisely weighing appropriate amounts of echinacoside, acteoside, naringin, jojodine A, jojodine B, jojodine C and icariine reference substances, placing 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 taking methanol as a solvent; then respectively precisely sucking 3mL, 1mL, 0.2mL, 0.5mL, 1mL and 2mL of the reference stock solution, placing into another 10mL measuring flask, adding methanol to constant volume, shaking uniformly 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 microporous filter membrane with the thickness of 0.45 mu m, and taking the subsequent filtrate as a mixed reference solution.
2.5 determination: respectively sucking the mixed reference solution and the sample solution at a precise concentration of 10 μl, injecting into high performance liquid chromatograph, and recording chromatogram within 90min, respectively shown in fig. 1 and fig. 2 (details of each peak identification in the figures will be described later).
Example 2:11 batches of gamboge bone-strengthening pills fingerprint analysis
1. Fingerprint similarity analysis
Taking 11 batches of gamboge bone-strengthening pills, preparing a sample according to the preparation method of the sample solution in the embodiment 1, carrying out sample injection analysis according to the chromatographic conditions in the embodiment 1, recording the fingerprint of the gamboge bone-strengthening pills, carrying out similarity analysis on the fingerprint of the 11 batches of gamboge bone-strengthening pills by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012A edition issued by the national formulary Committee, setting S1 as a reference map, and calibrating 20 common peaks in total by using a median method, wherein the similarity of the 11 batches of gamboge bone-strengthening pills is greater than 0.90, and the similarity evaluation result of the 11 batches of gamboge bone-strengthening pills sample is shown in the table 1. The relative retention times of the 20 common peaks were substantially identical, while there was a large difference in relative peak areas, and standard fingerprint data are shown in table 2. Fig. 3 is a fingerprint spectrum superposition diagram of 11 batches of gamboge bone-strengthening pills, and S1-S11 in fig. 3 correspond to lot numbers 20200201, 20200202, 20200203, 20200401, 20200402, 20200801, 20200802, 20210101, 20210401, 20210404 and 20210801 respectively.
TABLE 111 evaluation results of similarity of samples of Garcinia cambogia bone-invigorating pills
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. Common peak assignments: comparing HPLC chromatogram of the mixed reference substance solution (figure 1) with sample fingerprint (figure 3), and identifying chromatographic peak at the same retention time as each reference substance. The results showed that peaks No. 5, no. 8, no. 10, no. 13, no. 14, no. 15, and No. 16 of the 20 common peaks were designated as echinacoside, acteoside, naringin, hodgkin a, hodgkin B, hodgkin C, and icariine, respectively.
3. Belonging to the medicinal materials: carrying out chromatographic peak attribution according to standard fingerprint conditions, wherein S1-S7 in FIG. 4 correspond to seven medicinal materials of herba Epimedii, caulis Spatholobi, herba Pyrolae, parched Raphani semen, radix rehmanniae Preparata, herba cistanches and rhizoma Drynariae; s8 is a sample; s9 is a mixed reference substance. The 20 common peaks respectively belong to seven medicinal materials of epimedium herb, suberect spatholobus stem, pyrola herb, stir-fried radish seed, prepared rehmannia root, cistanche deserticola and hot drynaria rhizome; wherein, the No. 1 peak belongs to the rhizoma drynariae, the pyrola and the suberect spatholobus stem, the No. 2 peak belongs to the rhizoma drynariae and the suberect spatholobus stem, the No. 3 peak belongs to the rhizoma drynariae, the No. 4 peak belongs to the herba cistanches, the No. 5 peak belongs to the herba cistanches, the No. 6 peak belongs to the herba epimedii and the pyrola, the No. 7 peak belongs to the stir-fried radish seeds and the rhizoma drynariae, the No. 8 peak belongs to the herba cistanches and the prepared rehmannia root, the No. 9 peak belongs to the herba cistanches and the prepared rehmannia root, the No. 10 peak belongs to the rhizoma drynariae, the No. 11 peak belongs to the rhizoma drynariae and the herba epimedii, the No. 12 peak belongs to the herba epimedii, the No. 13 peak belongs to the herba epimedii, the No. 14 peak belongs to the herba epimedii, the No. 15 peak belongs to the herba epimedii, the No. 16 peak belongs to the herba epimedii, the No. 17 peak belongs to the prepared herba epimedii, the No. 19 peak belongs to the herba epimedii, and the No. 20 peak belongs to the herba epimedii.
Example 3: wavelength selection
20210101 batches of samples are taken for testing, and the overall effect of the chromatograms under the detection wavelength streets of 360nm, 320nm, 280nm, 254nm, 230nm, 210nm and 203nm is respectively examined, and other chromatographic conditions are the same as those of the example 1, and the chromatograms are 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 is low and the number of peaks is relatively small (FIG. 6); the detection wavelength is 280nm, the chromatogram baseline is flat, the peak shape is better, the number of peaks is more, the response value is highest, and the condition is the best (figure 7); at a detection wavelength of 254nm, the chromatogram has a flatter baseline and a better peak shape, but has a slightly poorer separation effect than 280nm (FIG. 8); when the detection wavelength is 230nm, the base line of the chromatogram is flatter, the peak shape is better, the number of peaks is more, and the separation effect is poorer (figure 9); at a detection wavelength of 210nm, the chromatogram has uneven baseline and huge fluctuation of chromatographic peaks (FIG. 10); at a detection wavelength of 203nm, the chromatogram baseline is not at the standard position (FIG. 11).
Because the fingerprint is not for measuring the accurate content of a certain component, but fully reflects the information of chemical components, the fingerprint with 280nm as the detection wavelength has more peaks, the reflected information is more complete, the peak shape is best, the absorption value of each peak is good, the base line is stable, and the condition of larger absorption of near ultraviolet impurity peaks is avoided. Thus 280nm is chosen as the optimal detection wavelength.
Example 4: methodology investigation
20210101 batches were taken for testing, and chromatographic conditions and test solutions were prepared in the same manner as in example 1.
(1) Precision test
Taking gamboge bone strengthening pill, preparing test solution, continuously sampling for 6 times, and recording 20 main chromatographic peak retention times and peak areas. Icariin is used as a reference peak, the relative peak area RSD is less than 3.0% (n=6), and the relative retention time RSD is less than 1.0% (n=6), so that the instrument precision is proved to be good. The experimental results are shown in Table 3.
(2) Stability test
Taking gamboge bone strengthening pills, preparing test sample solutions, testing at 0h, 2h, 4h, 8h, 12h and 24h respectively, and recording 20 main chromatographic peak retention times and peak areas. Icariin is used as a reference peak, the relative peak area RSD is less than 3.0% (n=6), and the relative retention time RSD is less than 1.0% (n=6), so that the sample has good stability within 24 hours. The experimental results are shown in Table 3.
(3) Repeatability test
6 parts of gamboge bone strengthening pills are prepared into a test solution, the test is carried out, and 20 main chromatographic peak retention times and peak areas are recorded. Icariin was used as a reference peak, the relative peak area RSD was less than 3.0% (n=6), and the relative retention time RSD was less than 1.0% (n=6), demonstrating good stability. The experimental results are shown in Table 3.
TABLE 3 methodological investigation of experimental results
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. A construction method of a gamboge bone strengthening pill fingerprint spectrum comprises the following steps:
extracting resina garciniae bone-invigorating pill with methanol, and taking the extractive solution as sample solution; taking methanol solutions of echinacoside, acteoside, naringin, jojodine A, jojodine B, jodine C and icariine as reference solutions;
respectively performing high performance liquid chromatography analysis on the reference substance solution and the test substance solution, and taking a chromatographic peak of the reference substance solution as a benchmark to obtain a gamboge bone strengthening pill fingerprint;
when the high performance liquid chromatography analysis is carried out, 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 the volume fraction of 0.01-0.3%; the flow speed of the mobile phase system is 0.5-1.5 mL/min; the filler in the chromatographic column is octadecylsilane chemically bonded silica gel;
the gradient elution method is adopted, and the gradient elution program is as follows: 0-15 min, wherein the volume fraction of the mobile phase A is increased from 8% to 10%; 15-55 min, wherein the volume fraction of the mobile phase A is increased from 10% to 30%; 55-65 min, wherein the volume fraction of the mobile phase A is increased from 30% to 45%; 65-75 min, and maintaining the volume fraction of the mobile phase A at 45%; 75-90 min, wherein the volume fraction of the mobile phase A is increased from 45% to 80%;
the gamboge bone strengthening pill fingerprint contains 20 common peaks, wherein the No. 5 peak, the No. 8 peak, the No. 10 peak, the No. 13 peak, the No. 14 peak, the No. 15 peak and the No. 16 peak are respectively identified as echinacoside, acteoside, naringin, hederadin A, hederadin B, hederadin C and icariine;
the 20 common peaks respectively belong to epimedium herb, suberect spatholobus stem, pyrola herb, stir-fried radish seed, prepared rehmannia root, cistanche deserticola and hot drynaria rhizome; wherein, the No. 1 peak belongs to the rhizoma drynariae, the pyrola and the suberect spatholobus stem, the No. 2 peak belongs to the rhizoma drynariae and the suberect spatholobus stem, the No. 3 peak belongs to the rhizoma drynariae, the No. 4 peak belongs to the herba cistanches, the No. 5 peak belongs to the herba cistanches, the No. 6 peak belongs to the herba epimedii and the pyrola, the No. 7 peak belongs to the stir-fried radish seeds and the rhizoma drynariae, the No. 8 peak belongs to the herba cistanches and the prepared rehmannia root, the No. 9 peak belongs to the herba cistanches and the prepared rehmannia root, the No. 10 peak belongs to the rhizoma drynariae, the No. 11 peak belongs to the rhizoma drynariae and the herba epimedii, the No. 12 peak belongs to the herba epimedii, the No. 13 peak belongs to the herba epimedii, the No. 14 peak belongs to the herba epimedii, the No. 15 peak belongs to the herba epimedii, the No. 16 peak belongs to the herba epimedii, the No. 17 peak belongs to the prepared herba epimedii, the No. 19 peak belongs to the herba epimedii, and the No. 20 peak belongs to the herba epimedii.
2. The method for constructing the fingerprint spectrum of the gamboge bone-strengthening pill according to claim 1, which is characterized in that the dosage ratio of the gamboge bone-strengthening pill to the methanol is (1-3) g: (10-50) mL.
3. The method for constructing the fingerprint spectrum of the gamboge bone strengthening pill according to claim 2, wherein the extraction is carried out under an ultrasonic condition, the power of the ultrasonic wave is 70-90W, and the frequency is 200-300 kHz; the extraction time is 20-40 min.
4. The method for constructing a fingerprint spectrum of a gamboge bone strengthening pill according to claim 1, wherein the specification of the chromatographic column is as follows when the high performance liquid chromatography analysis is performed: 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 sample injection volume is 5-15 mu L.
5. The method for constructing a fingerprint spectrum of a gamboge bone strengthening pill according to claim 1, wherein the theoretical plate number is not lower than 4000 calculated according to icariine peaks when the high performance liquid chromatography analysis is performed.
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