A characteristic spectrum construction method and characteristic spectrum of a bone health preparation

Abstract

The present invention relates to the field of drug detection technology, specifically a method for constructing a characteristic spectrum of a bone health preparation and a characteristic spectrum thereof. After the present invention prepares a test sample and a reference sample respectively, suitable chromatographic conditions are established: 0.1% phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, a specific mobile phase system is established, and gradient elution is performed according to a specific procedure, and HPLC is used for determination, and a characteristic spectrum suitable for quality detection of bone health preparations is finally obtained. The present invention obtains 36 common peaks, and identifies 8-18 characteristic peaks therein, and the source of the identified chemical components is attributed, so that the quality of bone health capsules and other dosage forms can be detected and evaluated more comprehensively, objectively and accurately.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a method for constructing a characteristic spectrum of a bone health preparation and a characteristic spectrum thereof. Background Art

[0002] Gukang Capsule is a national exclusive product independently developed by Guizhou Weikang Zifan Pharmaceutical Co., Ltd. for the treatment of orthopedic diseases. It is an exclusive new ethnic medicine developed based on the folk prescription of the Miao people using modern scientific and technological pharmaceutical means. It is included in the Orthopedic Volume of the National Collection of Traditional Chinese Medicine Standards and the Single-page Standard of the State Food and Drug Administration (2005). The current standard number is: WS-10464 (ZD-0464)-2005-2012Z. The selected medicinal materials for this prescription are all the medicinal materials commonly used by the Miao people in Guizhou. According to the theory of "gang, meridian, symptom, and disease" in Miao medicine, based on the folk prescriptions of banana root, oxalis, and oxalis that have the effects of reducing swelling and relieving pain, promoting blood circulation and removing blood stasis, relaxing muscles and tendons, and strengthening kidneys and bones, the company's scientific researchers have conducted clinical verification and screening, and the formula is determined to be banana root, oxalis, oxalis, psoralea corylifolia, and Panax notoginseng. It has the effects of nourishing the liver and kidneys, strengthening the tendons and bones, dredging meridians and relieving pain. It is used to treat fractures, osteoarthritis, osteoporosis caused by liver and kidney deficiency and meridian stasis.

[0003] At present, there is no characteristic spectrum established in the quality control of Gukang Capsules. The research on Gukang Capsules focuses on clinical drug use research and content determination research. Most of them are to determine the content of a single component in Gukang Capsules through different methods and parameters. For example, ① High performance liquid chromatography method to determine the content of icariin in Gukang Capsules, Li Meiqin et al., Strait Pharmacy, 2007-02-28; the method is: octadecyl bonded silica gel as filler; mobile phase: methanol-2% glacial acetic acid (1:1); detection wavelength: 246nm; column temperature: room temperature; flow rate: 1.0m L / min; ② Reverse phase high performance liquid chromatography (RP-HPLC) method to determine the content of tanshinone ⅡA in Gukang Capsules, Sun Jianyu et al., Medical Guide, 2007-04-01; the method is: use Symmetry C18 chromatographic column (4.6mm×250mm, 5μm), methanol-water (82:18) as mobile phase, flow rate of 1.0mL / min, detection wavelength of 270nm; ③LC-MS method for simultaneous determination of the contents of 6 main components (notoginseng saponin R1, ginsenoside Rg1, ginsenoside Rb1, chuanxiong saponin VI, psoralen and isopsoralen) in Gukang capsules, Yang Jiefang, China Pharmacy, 2012-9-12; The method is: using liquid chromatography-mass spectrometry technology. LC conditions: chromatographic column is Agilent ZorbaxSB-C18 (150mm×4.6mm, 5μm), mobile phase is methanol-water (gradient elution), column temperature is 25℃, flow rate is 0.25mL / min, and analysis time is 30min.

[0004] Or it may be the characteristic spectrum of a single medicinal material used in Gukang Capsule, for example, ① Digital research on fingerprint spectrum of Panax notoginseng from different sources, Tong Xiangwei et al., Medical Information, 2021-07-15; the method is: high performance liquid chromatography is used to establish the fingerprint spectrum of Panax notoginseng medicinal materials, the chromatographic column is InertSustainTMAQ-C18, the mobile phase is acetonitrile-0.1% phosphoric acid solution, gradient elution, flow rate 1.0ml / min, detection wavelength 203nm, column temperature 25℃, injection volume 10μl, recording time 70min, and methodological investigation is carried out; ② HPLC characteristic spectrum of Dipsacus asper and content determination of three active ingredients, Chen Pu et al., Chinese Medicinal Materials, 2017-07-04; the method is: Thermo BDS Hypersil C18 (250mm×4.6mm, 5μm) chromatographic column is used, acetonitrile-0.1% phosphoric acid solution is used as the mobile phase for gradient elution, the flow rate is 1.0mL / min, the column temperature is 30℃, and the detection wavelength is 220nm. Results: The HPLC spectra of 10 batches of Radix Dipsaci samples from different origins had good consistency, and 12 common peaks were calibrated; ③ Establishment of UPLC fingerprint of Psoralea corylifolia and determination of the contents of 12 main components, Wang Juan et al., Chinese Herbal Medicine, 2021-01-20; The method is: using Shim-pack GIST-HP chromatographic column (100mm×2.1mm, 1.9μm); using water and acetonitrile as mobile phases, gradient elution, and simultaneously determining the contents of 12 components in Psoralea corylifolia. Results: The UPLC fingerprint of Psoralea corylifolia was established, and the similarity of 29 batches of medicinal materials was between 0.827 and 0.989. A total of 21 common peaks were calibrated, and 12 of the chromatographic peaks were identified.

[0005] At present, the map and detection method for Gukang products (CN201310382486.5 Quality Control Method for Gukang Drugs) only targets acaciatin-6-C-β-D-glucoside in Oxalis, using it as an indicator component for content determination in Gukang Capsules. However, there is no overall characteristic map and construction method for multiple drugs in Gukang Capsules. The analysis direction is relatively single, and it is unable to provide a basis for comprehensive quality control of Gukang Capsules.

[0006] Therefore, in order to objectively and truly reflect the types and quantities of chemical components contained in Gukang Capsules and oral dosage forms such as tablets, granules, oral liquids made from the same medicinal materials, and to be able to more comprehensively, objectively and accurately detect and evaluate the quality of Gukang Capsules and other dosage forms, there is an urgent need to provide a comprehensive detection method for the characteristic spectrum of Gukang preparations, which should preferably have the advantages of simple method, good stability, high precision, etc., and can effectively provide a reference for the quality control of Gukang Capsules and other preparations. Summary of the invention

[0007] In order to solve the above technical problems existing in the prior art, the present invention provides a method for constructing a characteristic spectrum of a bone health preparation and a characteristic spectrum thereof, which are as follows:

[0008] A method for constructing a characteristic spectrum of a bone health preparation, wherein the bone health preparation is made of five medicinal materials, namely, banana root, oxalis, dipsaci, psoralea corylifolia and notoginseng. The characteristic spectrum is a process of constructing a characteristic spectrum of an HPLC method by gradient elution of dipsaci, psoralea corylifolia, notoginseng and oxalis in the preparation, using octadecylsilane bonded silica gel as a filler, 0.1% phosphoric acid aqueous solution as a mobile phase A, and acetonitrile as a mobile phase B; wherein the gradient elution conditions are as follows:

[0009]

[0010] The method for constructing the characteristic spectrum of the above-mentioned Gukang preparation comprises the following steps:

[0011] (1) Testing conditions

[0012] Octadecylsilane bonded silica gel is used as filler, 0.1% phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed; the theoretical plate number calculated based on the isopsoralen peak should be no less than 100,000;

[0013] The gradient elution conditions are:

[0014]

[0015] (2) Preparation of reference solution

[0016] Take neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, psoralen, isopsoralen, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, notoginseng saponin R1, ginsenoside Rg1 and ginsenoside Re, psoralen, isopsoralen, dipsaponin VI, ginsenoside Rb1, ginsenoside Rd, bakuchiol and loganin reference substances, add 70% methanol to prepare a mixed reference substance solution containing 0.25 mg of each substance per 1 ml, as the reference substance solution;

[0017] (3) Preparation of test solution

[0018] Take the contents of the Gukang preparation, put them into a container, add 70% methanol, seal them and let them stand, then ultrasonicate them 1-3 times, let them cool, add methanol to make up the lost weight, shake them well, filter them, and take the filtrate to obtain the product;

[0019] (4) Determination method

[0020] Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the HPLC, and measure;

[0021] (5) Establishment of characteristic spectrum: Conduct multiple batches of test samples, record chromatograms, examine the characteristic peaks of the chromatograms of each test sample solution, select representative compound peaks in each medicinal material from the common peaks of the test sample solutions of the Gukang preparation as characteristic peaks, and identify and determine the chemical components of the chromatographic peaks to obtain the characteristic spectrum of Gukang Capsules.

[0022] Furthermore, the characteristic map construction method comprises the following specific steps:

[0023] (1) Detection conditions - chromatographic conditions and system suitability test

[0024] Octadecylsilane bonded silica gel is used as a filler, the column length is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.6 μm; 0.1% phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed; the flow rate is 0.3 ml per minute; the column temperature is 45° C.; the detection wavelength is 203 nm; the theoretical plate number calculated based on the isopsoralen peak should be no less than 100,000;

[0025] The gradient elution conditions are:

[0026]

[0027]

[0028] (2) Preparation of reference solution

[0029] Take neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, psoralen, isopsoralen, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, notoginseng saponin R1, ginsenoside Rg1 and ginsenoside Re, psoralen, isopsoralen, dipsaponin VI, ginsenoside Rb1, ginsenoside Rd, bakuchiol and loganin reference substances, add 70% methanol to prepare a mixed reference substance solution containing 0.25 mg of each substance per 1 ml, as the reference substance solution;

[0030] (3) Preparation of test solution

[0031] Take the contents of the bone health preparation, put them in a container, add 70% methanol, seal them and let them stand for 10-15 hours, the mass volume ratio of the contents of the bone health preparation and 70% methanol is 1:12.5; then take the test solution after standing, ultrasonically treat it for 25-40 minutes at a power of 200W and a frequency of 40kHz, let it stand for 40-80 minutes, and then ultrasonically treat it for 25-40 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filter to obtain it;

[0032] (4) Determination method

[0033] Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the HPLC, and measure;

[0034] (5) Establishment of characteristic spectrum: Conduct multiple batches of test samples, record chromatograms, examine the characteristic peaks of the chromatograms of each test sample solution, select representative compound peaks in each medicinal material from the common peaks of the test sample solutions of the Gukang preparation as characteristic peaks, and identify and determine the chemical components of the chromatographic peaks to obtain the characteristic spectrum of Gukang Capsules.

[0035] The bone health preparation can be a bone health capsule prepared according to the national drug standard (standard number: WS-10464 (ZD-0464)-2005-2012Z), or a tablet, granule, oral liquid or other oral dosage form prepared according to the following method: 600g of banana root, 400g of oxalis, 600g of psoralea corylifolia, 300g of radix dipsaci, and 150g of notoginseng. Among the above five flavors, 30g of banana root and notoginseng are respectively crushed into fine powder, and the remaining banana root and the remaining psoralea corylifolia, oxalis and radix dipsaci are decocted in water for three times, each time for 1 hour, the decoction is combined, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.31 to 1.35 at 60°C, the above fine powder is added, mixed, dried, crushed into fine powder, mixed, and then the auxiliary materials required for pharmaceutically acceptable oral dosage forms are added to make tablets, granules, oral liquid or other oral dosage forms.

[0036] The preparation of the test solution in step (3) also includes a centrifugation step, which is to place the filtrate after ultrasound in a centrifuge tube and centrifuge it at 12,000 rpm for 5-20 minutes.

[0037] When establishing the characteristic spectrum in step (5), it also includes the preparation of negative test solution and the preparation of single medicinal material test solution, so as to identify and determine the attribution of chromatographic peaks: Preparation of negative test solution: According to the preparation method of bone health preparation, prepare negative control samples lacking banana root, Oxalis, Psoralea, Dipsacus asper and Panax notoginseng respectively, and then prepare negative sample solutions of each medicinal material according to the method of step (3); Preparation of single medicinal material test solution: According to the preparation method of bone health preparation, prepare single medicinal samples of banana root, Oxalis, Psoralea, Dipsacus asper and Panax notoginseng respectively, and then prepare single medicinal material sample solutions according to the method of step (3).

[0038] The characteristic spectrum of step (5) is established by respectively examining the characteristic peaks of the chromatograms of multiple batches of test sample solutions, obtaining a total of 36 common peaks, and formulating 8-18 characteristic peaks from the 36 common peaks, and identifying and determining the chemical components of the chromatographic peaks to obtain the characteristic spectrum of the bone health preparation.

[0039] Furthermore, in the step (5), the characteristic peaks are proposed by selecting 16 peaks of representative compounds in each medicinal material and 1 peak in Oxalis from the 36 common peaks, and a total of 17 characteristic peaks are proposed; the chemical composition identification and attribution of the chromatographic peaks are determined by comparing the chromatographic behavior and spectrum of the chemical reference substance in the Gukang test solution, the test solution of each medicine alone, the negative control solution lacking each medicine, and the blank solution of 70% methanol with the reference solution, and 18 peaks are identified, peak 1: neochlorogenic acid; peak 2: chlorogenic acid; peak 3: cryptochlorogenic acid; peak A: loganin; peak 4: psoralen; peak 5: isochlorogenic acid Psoralen; Peak 7: isochlorogenic acid B; Peak 8: isochlorogenic acid A; Peak 9: isochlorogenic acid C; Peak 10: notoginseng saponin R1; Peak 11: ginsenoside Rg1 and ginsenoside Re; Peak 12: psoralen; Peak 13 (S): isopsoralen; Peak 14: dipsaponin VI; Peak 15: ginsenoside Rb1; Peak 16: ginsenoside Rd; Peak 17: bakuchiol; the eight characteristic peaks, namely peaks 1 to 3, peak A, peaks 7 to 9 and peak 14, are derived from dipsacus; five peaks, namely peaks 4, peak 5, peak 12, peak 13 (S) and peak 17, are derived from psoralea corylifolia; four peaks, namely peaks 10, peak 11, peak 15 and peak 16, are derived from notoginseng.

[0040] The characteristic spectrum constructed by the characteristic spectrum construction method of the above-mentioned bone health preparation is as follows: Figure 2 shown.

[0041] The above-mentioned characteristic spectrum construction method of Gukang preparation or the characteristic spectrum of Gukang preparation is used in the identification and quality control of Gukang capsule products.

[0042] The above-mentioned characteristic spectrum construction method and characteristic spectrum of Gukang Capsule can be used to detect and evaluate the quality of Gukang Capsule in a relatively comprehensive, objective and accurate manner, and effectively provide a reference for the quality control of Gukang Capsule.

[0043] Before determining the chromatographic conditions for this study, the applicant conducted a number of experiments. The specific conditions and parameters in this application were selected after comprehensive comparison of the experimental results. The specific experiments and results during the exploration process are as follows:

[0044] 1. Selection of HPLC and UHPLC

[0045] The experiment used high performance liquid chromatography and ultra high performance liquid chromatography to study the characteristic spectrum of Gukang Capsules. The results showed that ultra high performance liquid chromatography took less time and had better separation than high performance liquid chromatography, so ultra high performance liquid chromatography was used for the study.

[0046] 2. Investigation of chromatographic columns

[0047] The chromatographic columns ①CORTECSTM C18 (4.6mm×150mm, 2.7μm), ②CORTECSTM C18+ (4.6mm×150mm, 2.7μm), ③Agilent Eclipse Plus C18 RRHD (2.1mm×150mm, 1.8μm), and ④CORTECSUPLC T3 (2.1mm×100mm, 1.6μm) were investigated. Compared with columns ③ and ④, the separation effect of chromatographic peaks of columns ① and ② was poor. There were slight differences in the detection and separation of chromatographic peaks between columns ③ and ④. The chromatographic peak separation effect, peak shape, and baseline of column ④ were better, and therefore column ④ CORTECS UPLC T3 (2.1mm×100mm, 1.6μm) was selected for analysis.

[0048] 3. Investigation of mobile phase

[0049] The experiment used the following mobile phase systems for investigation, and the best mobile phase system was selected based on the number of chromatographic peaks, separation effect, baseline stability, and chromatographic peak shape as evaluation indicators.

[0050] a Phase A: 0.1% formic acid in water, Phase B: 0.1% formic acid in acetonitrile;

[0051] b Phase A: 0.1% formic acid in water, Phase B: 0.1% formic acid in methanol;

[0052] cA phase: 0.1% formic acid aqueous solution, B phase: acetonitrile;

[0053] dA phase: 0.1% phosphoric acid aqueous solution, B phase: acetonitrile;

[0054] The experimental results show that, compared with conditions a and b, some chromatographic peak components were not detected under condition b, while the number of chromatographic peaks under condition a was larger, so acetonitrile was selected as the organic phase. Compared with conditions a, c, and d, when 0.1% phosphoric acid water was used as the aqueous phase and acetonitrile was used as the organic phase, the number of chromatographic peaks was the largest, the chromatographic peak separation effect was better, and the baseline was also relatively stable, so condition d was selected for analysis.

[0055] 4. Investigation of flow rate and column temperature

[0056] The experiment used different flow rates (0.25ml / min, 0.3ml / min, 0.35ml / min) and different column temperatures (35℃, 40℃, 45℃) for investigation. The results showed that when the flow rate was 0.25ml / min, 0.35ml / min and the column temperature was 35℃ and 40℃, the separation between individual chromatographic peaks did not meet the requirements. When the flow rate was 0.3ml / min and the column temperature was 45℃, the separation of the chromatographic peaks reached the best. Therefore, a flow rate of 0.3ml / min and a column temperature of 45℃ were selected for analysis.

[0057] 5. Wavelength Investigation

[0058] The experiment conducted a full-wavelength UV scan, selecting 203, 210, 230, 254, 280, 300, 330, and 350 nm for comparison. The results showed that the number of chromatographic peaks was the largest at a wavelength of 203 nm, which could more comprehensively reflect the information of each herbal medicine in the Gukang Capsule formula. The response values ​​of each chromatographic peak were high and the baseline was stable. Therefore, the wavelength of 203 nm was selected for detection.

[0059] 6. Optimization of Chromatography Time

[0060] The experiment optimized the chromatographic analysis time and adopted the gradient elution program in the table below. The separation effect, peak shape and baseline stability of each chromatographic peak were comprehensively considered. The information of each single medicinal material in the Gukang Capsule formula was taken into account more comprehensively, and there was no interference from the negative sample. The gradient elution program of the characteristic spectrum of Gukang Capsule was determined as method four. Finally, the chromatographic conditions of the characteristic spectrum of Gukang Capsule were determined as follows: the chromatographic column was CORTECS UPLC T3 (2.1mm×100mm, 1.6μm); 0.1% phosphoric acid aqueous solution was used as mobile phase A; acetonitrile was used as mobile phase B; the flow rate was 0.3mL / min; the column temperature was 45℃; the detection wavelength was 203nm; and gradient elution was performed according to method four.

[0061] Table 1 Gradient elution program

[0062]

[0063] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0064] The present invention has fully studied the characteristic spectrum of the Gukang test solution and obtained a method for constructing the characteristic spectrum of the Gukang preparation. Through the method, 36 common peaks of the Gukang preparation are clarified, and the chemical components of 8-18 characteristic peaks are identified, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, chuanxiong saponin VI, psoralen, isopsoralen, psoralen, isopsoralen, bakuchiol, notoginseng saponin R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rd, etc., and the sources of the identified components are attributed.

[0065] The characteristic spectrum construction method and characteristic spectrum of a bone-conserving preparation of the present application objectively and truly reflect the types and quantities of chemical components contained in bone-conserving capsules and other oral dosage forms, and can comprehensively, objectively and accurately detect and evaluate the quality of bone-conserving preparations, especially bone-conserving capsules. The detection method of the characteristic spectrum of bone-conserving preparations provided by the present application has the advantages of simple method, good stability and high precision, and multiple batches of bone-conserving capsules have been investigated, and it is found that the preparation process is stable and the product quality is reliable. It can effectively provide a reference for the quality control of bone-conserving capsules and other oral dosage forms. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a typical chromatogram of 35 batches of Gukang Capsules, in which 36 common peaks were observed;

[0067] Figure 2 It is the characteristic spectrum of 35 batches of Gukang Capsules, in which 17 characteristic peaks were proposed;

[0068] Figure 3 It is the characteristic spectrum of the reference solution, where Peak 1: Neochlorogenic acid; Peak 2: Chlorogenic acid; Peak 3: Cryptochlorogenic acid; Peak A: Loganin Peak 4: Psoralen; Peak 5: Isopsoralen; Peak 7: Isopsoralen B; Peak 8: Isopsoralen A; Peak 9: Isopsoralen C; Peak 10: Notoginsenoside R1; Peak 11: Ginsenoside Rg1 and Ginsenoside Re; Peak 12: Psoralen; Peak 13 (S): Isopsoralen; Peak 14: Dipsaconin VI; Peak 15: Ginsenoside Rb1; Peak 16: Ginsenoside Rd; Peak 17: Bakuchiol;

[0069] Figure 4 is the chromatogram of blank solution (70% methanol);

[0070] Figure 5 This is the UPLC spectrum of Gukang capsule and single herb;

[0071] Figure 6 This is the negative UPLC spectrum of Gukang Capsule and each single herb;

[0072] Figure 7 This is the chromatogram of Gukang Capsules obtained by the method of the present application;

[0073] Figure 8 This is the chromatogram of Gukang Capsules obtained by the method of Comparative Example 1;

[0074] Fig. 9 This is the chromatogram of Gukang Capsules obtained by the method of Comparative Example 2;

[0075] Fig.10 This is the chromatogram of Gukang Capsule obtained by the method of Comparative Example 3;

[0076] Fig.11This is the chromatogram of Gukang Capsules obtained according to the method of Comparative Example 4. DETAILED DESCRIPTION

[0077] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0078] Example:

[0079] Method for constructing characteristic map of Gukang capsule

[0080] 1 Materials

[0081] 1.1 Instrument

[0082] Agilent 1290 InfinityⅡ ultra high performance liquid chromatograph (Agilent Technologies, USA, including infusion pump, automatic sample injector, column oven, DAD detector); Mettler AE-240 electronic balance (Mettler-Toledo International Trading (Shanghai) Instrument Co., Ltd.), KQ5200E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0083] 1.2 Drug testing

[0084] Psoralen (batch number AF21031504), psoralen (batch number AF21042858), isopsoralen (batch number AF21031505), isopsoralen (batch number AF21071801), and ginsenoside Re (batch number AF20042104) were purchased from Chengdu Aifa Biotechnology Co., Ltd.; bakuchiol (batch number wkq21041202), dipsaponin VI (batch number wkq21010703), loganin (batch number wkq21071907), notoginsenoside R1 (batch number wkq21030407), ginsenoside Rg1 (batch number wkq21020401), and ginsenoside Rb1 (batch number wkq21041202). 1010701), ginsenoside Rd (batch number wkq21042607), neochlorogenic acid (3-caffeoylquinic acid) (batch number wkq21010802), cryptochlorogenic acid (5-caffeoylquinic acid) (batch number wkq20082705), isochlorogenic acid A (3,5-O-dicaffeoylquinic acid) (batch number wkq21020306), isochlorogenic acid B (3,4-O-dicaffeoylquinic acid) (batch number wkq21022206), isochlorogenic acid C (4,5-O-dicaffeoylquinic acid) (batch number wkq21012501) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd., and the purity of all reference substances was greater than 98%. Methanol and acetonitrile were chromatographically pure (Sinopharm Chemical Reagent Co., Ltd.), and water was ultrapure water.

[0085] Gukang Capsules are provided by Guizhou Weican Pharmaceutical Co., Ltd., with a total of 35 batches, including products from 2018 to 2021, see Table 2.

[0086] Table 2 Gukang Capsule Sample Information

[0087]

[0088] 2 Methods

[0089] 2.1 Solution preparation

[0090] 2.1.1 Preparation of test solution

[0091] Gukang Capsule is composed of five ingredients: banana root, Oxalis sorrel, Psoralea corylifolia, Dipsacus asper and Panax notoginseng. The main ingredients of banana root include steroidal and flavonoid compounds such as stigmasterol and 2,3,4-trihydroxyflavone. The main chemical components of Oxalis sorrel include flavonoid compounds such as quercetin and luteolin. The main chemical components of Dipsacus asper include triterpenoid saponins and iridoid compounds such as Dipsacus asper saponin VI, strychnine and loganic acid. The main chemical components of Psoralea corylifolia include coumarins, monoterpene phenols and flavonoid compounds such as psoralen, psoralen, bakuchiol and psoralen isoflavones. The main chemical components of Panax notoginseng include saponin compounds such as ginsenoside Rb, ginsenoside Rg1 and notoginsenoside R1. The chemical components contained in the above medicinal materials are soluble in methanol, ethanol and other solvents according to their physicochemical properties. Different concentrations of methanol, methanol reflux and ultrasonic treatment were used to extract the above chemical components in the preparation. The experimental results showed that ultrasonic extraction with 70% methanol had a better effect and was simple. Based on the investigation of the amount of the test sample, the extraction time, etc., the preparation method of the test sample solution was determined, namely: 2g of the content of Gukang Capsule was taken, placed in a stoppered conical flask, accurately weighed, 25mL of 70% methanol was accurately added, the weight was weighed, sealed, allowed to stand for 12 hours, ultrasonically treated for 30 minutes, allowed to stand for 1 hour, ultrasonically treated for 30 minutes, cooled, weighed again, and the lost weight was supplemented with 70% methanol. The weight was shaken well, filtered, an appropriate amount of the filtrate was taken, placed in a centrifuge tube and centrifuged at 12000 rpm for 10 minutes, and the supernatant was obtained.

[0092] 2.1.2 Preparation of negative test solution

[0093] According to the preparation method of Gukang Capsules, negative control samples lacking banana root, oxalis, psoralea, dipsaccharum and Panax notoginseng were prepared respectively, and negative sample solutions of each medicinal material were prepared according to the method under 2.1.1.

[0094] 2.1.3 Preparation of single herbal medicine test solution

[0095] Prepare single herb samples of banana root, oxalis, psoralea corylifolia, dipsoralea corylifolia and Panax notoginseng respectively according to the preparation method of Gukang Capsules, and prepare single herb sample solutions according to the method under 2.1.1.

[0096] 2.1.4 Preparation of reference substance solution

[0097] Precisely take 9.60 mg of neochlorogenic acid, 9.78 mg of chlorogenic acid, 10.13 mg of cryptochlorogenic acid, 9.86 mg of psoralen, 9.61 mg of isopsoralen, 9.61 mg of isochlorogenic acid B, 9.66 mg of isochlorogenic acid A, 9.99 mg of isochlorogenic acid C, 10.18 mg of notoginseng saponin R1, 10.02 mg of ginsenoside Rg, 10.00 mg of ginsenoside Re, 10.69 mg of psoralen, 10.14 mg of isopsoralen, 10.00 mg of chuanxiong saponin VI, 19.98 mg of ginsenoside Rb, and 10.20 mg of ginsenoside Rd. mg, bakuchiol 10.00 mg and loganin 10.56 mg were placed in 2 ml volumetric flasks respectively, dissolved and diluted to the scale with 70% methanol to obtain the above 18 reference substance stock solutions, accurately measured 100 μL of each of the above reference substance stock solutions, placed in a 2 ml volumetric flask, added 70% methanol to dilute to the scale to obtain 0.25 mg / mL mixed reference substance solutions, which were used as reference substance solutions.

[0098] 2.2 Chromatographic conditions

[0099] The experiment used high performance liquid chromatography and ultra-high performance liquid chromatography, and investigated CORTECSTM C18 (4.6mm×150mm, 2.7μm), CORTECSTM C18+ (4.6mm×150mm, 2.7μm), Agilent EclipsePlus C18 RRHD (2.1mm×150mm, 1.8μm), CORTECS UPLC T3 (2.1mm×100mm, 1.6μm) and other chromatographic columns. The results showed that the separation effect of Waters' CORTECS UPLC T3 (2.1mm×100mm, 1.6μm) chromatographic column was the best. After selecting the chromatographic column, the chromatographic conditions were optimized. The optimized chromatographic conditions are as follows:

[0100] The chromatographic column was CORTECS UPLC T3 (2.1 mm×100 mm, 1.6 μm); 0.1% phosphoric acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, the flow rate was 0.3 mL / min, and gradient elution was performed as specified in Table 3; the column temperature was 45° C.; and the detection wavelength was 203 nm.

[0101] Table 3 Gradient elution program of Gukang capsule characteristic spectrum

[0102]

[0103]

[0104] 2.3 Study on the characteristic spectrum of Gukang Capsule

[0105] 2.3.1 Precision test

[0106] According to the method proposed in 2.1.1, take the contents of Gukang Capsules with batch number 20210403 to prepare a test solution. According to the chromatographic conditions determined under 2.2, inject 5 times continuously and record the chromatogram. Taking peak 13 (isopsoralen) as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak. The relative retention time RSD is less than 0.068%, and the relative peak area RSD is less than 2.6%. The results show that the instrument has good precision, Table 4, Table 5.

[0107] 2.3.2 Repeatability test

[0108] According to the method proposed in 2.1.1, the contents of Gukang Capsules with batch number 20210403 were taken to prepare 5 test solutions, and the samples were injected according to the chromatographic conditions determined under 2.2, and the chromatograms were recorded. Taking peak 13 (isopsoralen) as the reference peak, the relative retention time and relative peak area of ​​each characteristic peak were calculated. The relative retention time RSD was less than 0.23%, and the relative peak area RSD was less than 3.2%. The results showed that the method had good repeatability, Table 6, Table 7.

[0109] 2.3.3 Stability study

[0110] According to the method proposed in 2.1.1, take the contents of Gukang Capsules with batch number 20210403 to prepare a test solution. According to the chromatographic conditions determined under 2.2, inject samples at 0, 2, 4, 6, 8, 12, and 24 hours, record the chromatogram, and use Peak 13 (isopsoralen) as the reference peak to calculate the relative retention time and relative peak area of ​​each characteristic peak. The RSD of the relative retention time was less than 0.4%, and the RSD of the relative peak area was less than 2.4%, indicating that the test solution had good stability within 24 hours, as shown in Tables 8 and 9.

[0111] Table 4 Precision test (relative retention time)

[0112]

[0113] Table 5 Precision test (relative peak area)

[0114]

[0115]

[0116] Table 6 Repeatability test (relative retention time)

[0117]

[0118] Table 7 Repeatability test (relative peak area)

[0119]

[0120] Table 8 Stability test (relative retention time)

[0121]

[0122] Table 9 Stability test (relative peak area)

[0123]

[0124] 2.3.4 Establishment of feature maps

[0125] Take the contents of 35 batches of Gukang Capsules and prepare the test solution according to the test solution preparation method proposed in 2.1.1. Separately, take 1 mL of reference substance solution containing about 0.25 mg of each of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganin, psoralen, isopsoralen, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, notoginseng saponin R1, ginsenoside Rg1 and ginsenoside Re, psoralen, isopsoralen, chuanxiong saponin VI, ginsenoside Rb1, ginsenoside Rd, and bakuchiol, the test solution of each drug alone, the negative control solution lacking each drug, and the blank solution (70% methanol). According to the chromatographic conditions proposed in 2.2, inject 1 μL of each sample, record the chromatogram, and examine the characteristic peaks of the chromatogram of each test solution. The typical chromatogram is shown in Figure 1 , among which the theoretical plate number of isopsoralen is 183,000, so it is stipulated that the theoretical plate number of isopsoralen shall not be less than 100,000. From the characteristic spectra of 35 batches of Gukang Capsules, 36 common peaks can be observed ( Figure 1 ), in order to simplify the judgment of the characteristic spectrum, 16 peaks of representative compounds in each medicinal material and 1 peak in Oxalis were selected as characteristic peaks from the 36 common peaks, and a total of 17 characteristic peaks were proposed ( Figure 2 ), the chromatograms of the reference substance solution and blank solution (70% methanol) are shown in Figure 3 , Figure 4 .

[0126] 2.3.5 Identification and attribution of chemical components of chromatographic peaks in the characteristic spectrum of Gukang Capsules

[0127] The chromatographic behavior and spectrum of the chemical reference substance in the Gukang test solution, the test solution of each drug alone, the negative control solution lacking each drug, and the blank solution (70% methanol) were compared with the reference substance solution. Figure 5 , Figure 6, 18 peaks were identified, including Peak 1: neochlorogenic acid; Peak 2: chlorogenic acid; Peak 3: cryptochlorogenic acid; Peak A: loganin; Peak 4: psoralen; Peak 5: isopsoralen; Peak 7: isochlorogenic acid B; Peak 8: isochlorogenic acid A; Peak 9: isochlorogenic acid C; Peak 10: notoginseng saponin R1; Peak 11: ginsenoside Rg1 and ginsenoside Re; Peak 12: psoralen; Peak 13 (S): isopsoralen; Peak 14: dipsaponin VI; Peak 15: ginsenoside Rb1; Peak 16: ginsenoside Rd; Peak 17: bakuchiol.

[0128] Among them, eight characteristic peaks, including peak 1 (neochlorogenic acid), peak 2 (chlorogenic acid), peak 3 (cryptochlorogenic acid), peak A (loganin), peak 7 (isochlorogenic acid B), peak 8 (isochlorogenic acid A), peak 9 (isochlorogenic acid C) and peak 14 (Dipsaconin VI), are derived from Dipsacus asper; five peaks, including peak 4 (psoralen), peak 5 (isopsoralen), peak 12 (psoralen), peak 13 (S) (isopsoralen) and peak 17 (bakuchiol), are derived from Psoralea corylifolia; four peaks, including peak 10 (notoginseng saponin R1), peak 11 (ginsenoside Rg1 and ginsenoside Re), peak 15 (ginsenoside Rb1) and peak 16 (ginsenoside Rd), are derived from Panax notoginseng. Since Peak A (loganin) in the preparation is small, poorly separated and difficult to identify, it is not included in the common peaks of the characteristic spectrum. Therefore, it is stipulated that Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 7, Peak 8, Peak 9, Peak 10, Peak 11, Peak 12, Peak 13, Peak 14, Peak 15, Peak 16, and Peak 17 in the characteristic spectrum of Gukang Capsules should correspond to the retention times of the peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, psoralen, isopsoralen, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, notoginseng saponin R1, ginsenoside Rg1 and ginsenoside Re, psoralen, isopsoralen, dipsaenoside VI, ginsenoside Rb1, ginsenoside Rd, and bakuchiol reference substance, respectively.

[0129] Peak 6 originates from Oxalis. Due to the lack of corresponding reference substances for Oxalis and the small amount of information under the chromatographic conditions, all 35 batches of Gukang Capsules have the characteristic (Peak 6) from Oxalis and are stable. The isopsoralen with the characteristic peak retention time in the middle of the characteristic spectrum and good separation and stability is the S peak. The relative retention time of Oxalis peak 6 is calculated to be 0.645 for control. According to the relative retention time range of peak 6 of 35 batches of 0.6437-0.6454, it is stipulated that its relative retention time should be within ±10% of the specified value.

[0130] Since there is a lack of corresponding reference materials for banana roots and the characteristic peaks in the test sample chromatogram contain very little information under various chromatographic conditions, the test sample solution preparation method and chromatographic conditions cannot reflect its characteristics and need further study.

[0131] During the research process, the researchers explored the chromatographic conditions reported in relevant literature or patents in the early stage to obtain chromatographic conditions that are more suitable for the construction of the fingerprint of Gukang Capsule. Some of the research processes are listed as follows:

[0132] Study Example 1: The chromatographic conditions in "Digital Research on Fingerprints of Panax Notoginseng from Different Sources" (Tong Xiangwei, Shi Kunlian, Liang Yeting, Shen Ying, Zhou Bei, Yi Yankui. Digital Research on Fingerprints of Panax Notoginseng from Different Sources [J]. Medical Information, 2021, 34(14): 106-109+120.) were used to prepare the test solution and perform HPLC analysis on the Gukang Capsule preparation.

[0133] Study Example 2: The chromatographic conditions in "HPLC Characteristic Spectrum of Dipsacus and Content Determination of Three Active Ingredients" (Chen Pu, Zhao Hua, He Yaqin, Wan Dingrong. HPLC Characteristic Spectrum of Dipsacus and Content Determination of Three Active Ingredients [J]. Chinese Medicinal Materials, 2017, 40(06): 1373-1376.) were used to prepare the test solution and perform HPLC analysis on the Gukang Capsule preparation.

[0134] Study Example 3: The chromatographic conditions in "Establishment of UPLC fingerprint of Psoralea corylifolia and determination of contents of 12 main components" (Wang Juan, Zhou Zhixing, Yang Li, Zhang Yue, Zhou Kun, Zhang Junxia. Establishment of UPLC fingerprint of Psoralea corylifolia and determination of contents of 12 main components [J]. Chinese Herbal Medicine, 2021, 52(02): 552-557.) were used to prepare the test solution and perform HPLC analysis on the Gukang capsule preparation.

[0135] Study Example 4: Using the chromatographic conditions in "LC-MS method for simultaneous determination of the contents of six main components in Gukang capsules" (Yang Jiefang. LC-MS method for simultaneous determination of the contents of six main components in Gukang capsules [J]. Chinese Pharmacy, 2012, 23 (35): 3344-3346.), the test solution of Gukang capsule preparation was prepared and analyzed by HPLC.

[0136] The results show:

[0137] 1. According to the methods and conditions in Comparative Examples 1-3, the results are as follows:

[0138] The chromatographic separation is poor, and it is impossible to identify the chemical components of the banana root, Oxalis sorrel, Psoralea corylifolia, Dipsacus asper, and Panax notoginseng contained in the prescription, which cannot fully reflect the quality of the Gukang Capsule preparation.

[0139] Figure 8-10 It is the chromatogram of Gukang Capsule obtained by the method of Comparative Examples 1-3.

[0140] 2. According to the method and conditions in Comparative Example 4, the situation is as follows:

[0141] Comparative Example 4 uses liquid chromatography-mass spectrometry technology to determine the content of ingredients in Gukang Capsules, which is to conduct quantitative quality control on Gukang Capsules, and analyzes the compound preparation of Gukang Capsules according to the literature method. Only the ginsenoside Rb1 component is well separated, and other components are not well separated.

[0142] Fig.11 This is the chromatogram of Gukang Capsules obtained according to the method of Comparative Example 4.

[0143] In addition, according to the preparation idea of ​​Gukang Capsule, 5 medicinal materials with the same weight ratio were selected, and the dried and mixed fine powder was obtained according to the preparation method before loading into the capsule in the quality standard of Gukang Capsule, and excipients such as compressible starch, microcrystalline cellulose, dextrin, sucrose, water, etc. were added to make tablets, granules, and oral liquid dosage forms respectively. The fingerprint map construction was studied in the same way. The results showed that each test solution was prepared according to the technical scheme of the present invention, and after HPLC determination under the same chromatographic conditions, 36 or more chromatographic peaks could be obtained, and 8 characteristic peaks such as new chlorogenic acid could be identified as coming from Dipsacus asper, 5 characteristic peaks such as psoralen came from Psoralea corylifolia, and 4 characteristic peaks such as ginsenoside Rg1 came from Panax notoginseng. This may be because the raw material composition of each oral dosage form is the same, and other excipients and impurities have less interference with its quality assessment.

[0144] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a bone health preparation, wherein the bone health preparation is made from five medicinal materials, namely, banana root, oxalis, dipsaccharum officinale, psoralea corylifolia and Panax notoginseng, characterized in that: The characteristic spectrum is a process of constructing a characteristic spectrum of HPLC method for gradient elution of Radix Dipsaci, Fructus Psoraleae, Radix Notoginseng and Oxalis Herba in the preparation, using CORTECS UPLC T3 specification: 2.1mm×100mm, 1.6μm as a chromatographic column, 0.1% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B; the flow rate is 0.3ml per minute; the column temperature is 45°C; the detection wavelength is 203nm; wherein the gradient elution conditions are as follows:

2. The method for constructing a characteristic spectrum of a bone health preparation according to claim 1, characterized in that: The steps of feature map construction are as follows: (1) Testing conditions The chromatographic column was CORTECS UPLC T3 with specifications of 2.1 mm × 100 mm, 1.6 μm, 0.1% phosphoric acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution; the theoretical plate number calculated based on the isopsoralen peak should be no less than 100,000; The gradient elution conditions are: (2) Preparation of reference solution Take neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, psoralen, isopsoralen, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, notoginseng saponin R1, ginsenoside Rg1 and ginsenoside Re, psoralen, isopsoralen, dipsaponin VI, ginsenoside Rb1, ginsenoside Rd, bakuchiol and loganin reference substances, add 70% methanol to prepare a mixed reference substance solution containing 0.25 mg of each substance per 1 ml, as the reference substance solution; (3) Preparation of test solution Take the contents of the Gukang preparation, put them into a container, add 70% methanol, seal them and let them stand, then ultrasonicate them 1-3 times, let them cool, add methanol to make up the lost weight, shake them well, filter them, and take the filtrate to obtain the product; (4) Determination method Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the HPLC, and measure; (5) Establishment of characteristic spectrum: Conduct multiple batches of test samples, record chromatograms, examine the characteristic peaks of the chromatograms of each test sample solution, select representative compound peaks in each medicinal material from the common peaks of the test sample solutions of the Gukang preparation as characteristic peaks, and identify and determine the chemical components of the chromatographic peaks to obtain the characteristic spectrum of Gukang Capsules.

3. The method for constructing the characteristic spectrum of the bone health preparation according to claim 2, characterized in that: The steps of feature map construction are as follows: (1) Detection conditions - chromatographic conditions and system suitability test The chromatographic column was CORTECS UPLC T3 with specifications of 2.1 mm × 100 mm, 1.6 μm; 0.1% phosphoric acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed; the flow rate was 0.3 ml per minute; the column temperature was 45° C.; the detection wavelength was 203 nm; the theoretical plate number calculated based on the isopsoralen peak should be no less than 100,000; The gradient elution conditions are: (2) Preparation of reference solution Take neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, psoralen, isopsoralen, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, notoginseng saponin R1, ginsenoside Rg1 and ginsenoside Re, psoralen, isopsoralen, dipsaponin VI, ginsenoside Rb1, ginsenoside Rd, bakuchiol and loganin reference substances, add 70% methanol to prepare a mixed reference substance solution containing 0.25 mg of each substance per 1 ml, as the reference substance solution; (3) Preparation of test solution Take the contents of the bone health preparation, put them in a container, add 70% methanol, seal them and let them stand for 10-15 hours, the mass volume ratio of the contents of the bone health preparation and 70% methanol is 1:12.5; then take the test solution after standing, ultrasonically treat it for 25-40 minutes at a power of 200W and a frequency of 40kHz, let it stand for 40-80 minutes, and then ultrasonically treat it for 25-40 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filter to obtain it; (4) Determination method Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the HPLC, and measure; (5) Establishment of characteristic spectrum: Conduct multiple batches of test samples, record chromatograms, examine the characteristic peaks of the chromatograms of each test sample solution, select representative compound peaks in each medicinal material from the common peaks of the test sample solutions of the Gukang preparation as characteristic peaks, and identify and determine the chemical components of the chromatographic peaks to obtain the characteristic spectrum of Gukang Capsules.

4. The method for constructing a characteristic spectrum of a bone health preparation according to any one of claims 1 to 3, characterized in that: The Bone Health preparation is in accordance with the national drug standards, the standard number is: Gukang capsules prepared by WS-10464 (ZD-0464)-2005-2012Z, or tablets, granules, oral liquid prepared by the following method: 600g of banana root, 400g of oxalis, 600g of psoralea corylifolia, 300g of dipsacus root, and 150g of panax notoginseng. Take 30g of banana root and panax notoginseng and grind them into fine powder respectively. Add water to the remaining banana root and the other psoralea corylifolia, oxalis and dipsacus root and boil them three times for 1 hour each time. Combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.31 to 1.35 at 60°C. Add the above fine powder, mix well, dry, grind into fine powder, mix well, and then add the necessary excipients for pharmaceutically acceptable oral dosage forms to make tablets, granules, oral liquid or other oral dosage forms.

5. The method for constructing a characteristic spectrum of a bone health preparation according to any one of claims 1 to 3, characterized in that: The preparation of the test solution in step (3) also includes a centrifugation step, which is to place the filtrate after ultrasound in a centrifuge tube and centrifuge it at 12,000 rpm for 5-20 minutes.

6. The method for constructing a characteristic spectrum of a bone health preparation according to any one of claims 1 to 3, characterized in that: When establishing the characteristic spectrum in step (5), it also includes the preparation of negative test solution and single herbal medicine test solution, so as to identify and determine the attribution of chromatographic peaks: Preparation of negative test solution: According to the preparation method of Gukang preparation, prepare negative control samples lacking banana root, Oxalis sorrel, Psoralea corylifolia, Dipsacus asper and Panax notoginseng respectively, and then prepare negative sample solutions of each medicinal material according to the method of step (3); Preparation of single herb test solution: According to the preparation method of Gukang preparation, prepare single herb samples of banana root, Oxalis sorrel, Psoralea corylifolia, Dipsacus asper and Panax notoginseng respectively, and then prepare single herb sample solution according to the method of step (3).

7. The method for constructing a characteristic spectrum of a bone health preparation according to any one of claims 1 to 3, characterized in that: The characteristic spectrum of step (5) is established by respectively examining the characteristic peaks of the chromatograms of multiple batches of test sample solutions, obtaining a total of 36 common peaks, and formulating 8-18 characteristic peaks from the 36 common peaks, and identifying and determining the chemical components of the chromatographic peaks to obtain the characteristic spectrum of the bone health preparation.

8. The method for constructing a characteristic spectrum of a bone health preparation according to claim 7, characterized in that: The characteristic peaks in step (5) are prepared by selecting 16 peaks of representative compounds in each medicinal material and 1 peak in Oxalis oxyphylla from the 36 common peaks as characteristic peaks, and a total of 17 characteristic peaks are prepared; The identification and attribution of the chemical components of the chromatographic peaks are determined by comparing the chromatographic behaviors and spectra of the chemical reference substances in the Gukang test solution, the test solutions of each herb alone, the negative control solutions lacking each herb, and the blank solution of 70% methanol with the reference solution, and 18 peaks are identified, including peak 1: neochlorogenic acid; peak 2: chlorogenic acid; peak 3: cryptochlorogenic acid; peak A: loganin; peak 4: psoralen; peak 5: isopsoralen; peak 7: isochlorogenic acid B; peak 8: isochlorogenic acid A; peak 9: isochlorogenic acid C; peak 10: notoginseng saponin R1; peak 11: ginsenoside Rg1 and ginsenoside Re; peak 12: psoralen; peak 13 (S): isopsoralen; peak 14: chuanxiong saponin VI; peak 15: ginsenoside Rb1; peak 16: ginsenoside Rd; peak 17: bakuchiol; The eight characteristic peaks, peak 1 to peak 3, peak A, peak 7 to peak 9 and peak 14, are derived from Dipsacus asper; five peaks, peak 4, peak 5, peak 12, peak 13 (S) and peak 17, are derived from Psoralea corylifolia; and four peaks, peak 10, peak 11, peak 15 and peak 16, are derived from Panax notoginseng.

9. The method for constructing the characteristic spectrum of the Gukang preparation according to any one of claims 1 to 8, and its application in the identification and quality control of Gukang capsule products.

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