CN107271577B - Method for analyzing effective components of Qiling kidney-warming and bursa-eliminating preparation - Google Patents
Method for analyzing effective components of Qiling kidney-warming and bursa-eliminating preparation Download PDFInfo
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- CN107271577B CN107271577B CN201610221747.9A CN201610221747A CN107271577B CN 107271577 B CN107271577 B CN 107271577B CN 201610221747 A CN201610221747 A CN 201610221747A CN 107271577 B CN107271577 B CN 107271577B
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Abstract
The invention relates to an analysis method of active ingredients of a medicament, in particular to an analysis method of active ingredients of a Qiling kidney-warming and bursa-eliminating preparation. The method comprises the following steps: (1) preparation of control solution: (2) preparation of a test solution: (3) method for analyzing active ingredients: comprises flavonoid compounds, phenolic acid compounds, triterpenoid saponin compounds, sesquiterpene glycoside compounds and alkaloid compounds.
Description
The technical field is as follows:
the invention relates to an analysis method of effective components of a medicament, in particular to an analysis method of effective components of a Qiling kidney-warming and bursa-eliminating preparation.
Background art:
polycystic ovary syndrome (PCOS) is a common gynecological disease in adolescent and women of childbearing age, with an incidence rate of approximately 6% to 10% in women of childbearing age. The pathogenesis of PCOS is 'disorder of sex-stimulating essence of the heaven and the hair, phlegm is blocked in the uterus', and the PCOS is related to the dysfunction of spleen and kidney in traditional Chinese medicine. Modern medical research finds that PCOS is caused by abnormal regulation function of a hypothalamus-pituitary-ovary axis, so that endocrine disorder is caused, and the PCOS is mainly characterized by hyperandrogenism, insulin resistance and ovulation failure and clinically shows the quadruple symptoms of amenorrhea, infertility, obesity and hirsutism.
The astragalus root and Poria cocos prescription for warming the kidney and eliminating the sacs (original name prescription for tonifying the kidney and reducing the phlegm) is a multi-year clinical proved prescription for treating PCOS, consists of 5 medicinal materials of astragalus membranaceus Radix, epimedium epimidii Herba, Rhizoma Atractylodis Atractylodes rhizome Rhizoma, Poria cocos Poria and salvia miltiorrhiza Miltiorrhizae Radix, has the effects of tonifying the kidney and reducing the phlegm, strengthening the spleen and eliminating the dampness, regulating menstruation and relieving the pain and the like, is clinically used along with the increase and the decrease of symptoms, and has obvious curative effect. The specific prescription is as follows: 30-50 parts of astragalus root, 10-30 parts of epimedium, 10-30 parts of atractylodes rhizome, 30-50 parts of tuckahoe and 10-30 parts of salvia miltiorrhiza. The preparation method comprises weighing the raw materials at a certain weight ratio, decocting in water, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.20-1.35 at 55-60 deg.C, adding adjuvants or excipients for preparation, and forming.
Clinical observation of influence of the kidney tonifying and phlegm reducing formula on hyperandrogenism of patients with polycystic ovarian syndrome [ J ]. Shanghai J.J., 2015,49(8):53-55.) the kidney tonifying and phlegm reducing formula is used for treating the patients, and endocrine indexes are compared, so that the result shows that the curative effect of a treatment group is remarkable, the hyperandrogenism can be obviously improved, and ovulation is promoted. The kidney-tonifying and phlegm-reducing compound granules have the effects on reproductive endocrine of female offspring of pregnant rat with hyperandrogenism [ J ]. Chinese medicine J2011, 26(12):2872-2875.) the kidney-tonifying and phlegm-reducing formula is used for treating pregnant rat with hyperandrogenism, and the effects of lowering androgen level, reversing polycystic change of ovary and improving ovulation function are found. In the same morning (in the same morning, in the multiple crystal group, the traditional Chinese medicine and Western medicine, 2011,31(12): 1639) -1644.) experiments show that the kidney-tonifying and phlegm-resolving formula can improve the insulin resistance and the glycolipid metabolism, and is possibly related to the regulation and control of the protein expression of the insulin signal transduction molecule.
At present, the basic research of the materials of the formula of warming the kidney and eliminating the bursa is only reported. The invention adopts UPLC-ESI-IT-TOF/MS analysis method to carry out comprehensive research on chemical components, and identifies partial characteristic component sources, thereby providing guidance for substance basis and quality standard research.
Disclosure of Invention
In order to solve the technical problems, the invention provides an analysis method of effective components of a Qiling kidney-warming and bursa-eliminating preparation, which can quickly detect the effective components of the Qiling kidney-warming and bursa-eliminating preparation.
The invention is realized by the following technical scheme:
an analytical method of effective components of a Qiling kidney-warming and bursa-eliminating preparation comprises the following steps:
(1) preparation of control solution:
(2) preparation of a test solution:
(3) method for analyzing active ingredients: comprises flavonoid compounds, phenolic acid compounds, triterpenoid saponin compounds, sesquiterpene glycoside compounds and alkaloid compounds.
The step (1) preparation of a control solution: weighing appropriate amount of chlorogenic acid, calycosin glucoside, calycosin, hyperoside, rosmarinic acid, lithospermic acid, formononetin, salvianolic acid A, icariin, formononetin, astragaloside IV, baohuoside I, agastachoside B, atractyloside A, epimedin B and epimedin C reference substances in a same measuring bottle, adding appropriate amount of methanol, ultrasonically dissolving, cooling to room temperature, and fixing to constant volume to obtain mixed reference substance solution.
The preparation of the test solution in the step (2) comprises a sample test solution and a single medicinal material test solution. Preferably, the test solution in step (2) is prepared by weighing radix astragali and Poria, ultrasonically extracting with 30-50% methanol for 20-60min, cooling to room temperature, and filtering the supernatant with 0.22 μm filter membrane to obtain sample test solution; weighing single medicinal material or extract, ultrasonic extracting with 0-50% methanol for 20-60min, cooling to room temperature, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain radix astragali medicinal material test solution, rhizoma Atractylodis medicinal material test solution, Poria medicinal material test solution, Saviae Miltiorrhizae radix medicinal material test solution, and herba Epimedii medicinal material test solution.
The flavonoid compounds in the step (3) comprise hyperin, calycosin glucoside, wengowstrin E, icariin A, anhydroicaritin-3-O-rhamnose (1-2) -furoic acid-7-O-glucose, formononetin, baohuoside II, calycosin, wengowstrin F, epimedin A, epimedin B, epimedin C, icariin, anhydroicaritin-3-O-rhamnose (1-2) -furoic acid-7-O-glucose, large flower icariin F, icariside I, epimedin B and 2' -rhamnose icariside II; the phenolic acid compounds include tanshinol, salvianolic acid H or I, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, iso-salvianolic acid B or salvianolic acid E, salvianolic acid A, chlorogenic acid, iso-chlorogenic acid, and neochlorogenic acid; the triterpenoid saponin compounds comprise astragaloside I, astragaloside II, isoastragaloside I and astragaloside IV; the sesquiterpene glycoside compounds comprise atractyloside, and the alkaloid compounds comprise magnoline.
The method for analyzing the active ingredient in the step (3) comprises ultra high performance liquid chromatography, mass spectrometry and peak assignment analysis.
The chromatographic conditions of the ultra-high performance liquid chromatography are as follows:
using a PDA detector, C18Or T3 or HILIC chromatography column, mobile phase: adopts 0 to 0.5 percent formic acid water solution-acetonitrile with the volume flow of 0.1 to 0.6 mL/min. Preferably, a C18 chromatography column is used, mobile phase: adopting 0.1 percent formic acid water solution-acetonitrile, and carrying out gradient elution with the volume flow of 0.4 mL/min; the column temperature is 30 ℃; the gradient elution condition is
The mass spectrum conditions are as follows: an electrospray ionization ion source (ESI) is adopted, positive ions and negative ions are respectively detected, the scanning range is m/z 100-1200, the ion source temperature is 220-350 ℃, the capillary voltage negative ion mode is 3.0-4.0kV, the positive ion mode is 3.5-5.0kV, and the volume flow of atomized gas is 1.2-2.0L/min. Preferably, sodium trifluoroacetate is used as a correction fluid, the atomization gas is high-purity nitrogen, the collision gas is argon, the ion source temperature is 250 ℃, the capillary voltage negative ion mode is 3.5kV, the positive ion mode is 4.5kV, and the volume flow of the atomization gas is 1.5L/min;
the peak compound attribution method adopts an UPLC-ESI-IT-TOF/MS method, obtains a compound structure through comparison of a reference substance, a map, mass spectrum data and molecular weight and fragment information of a reference document, and indicates that the compound structure is derived from 36 chemical components of 4 medicinal materials including astragalus, epimedium, rhizoma atractylodis and salvia miltiorrhiza.
The invention relates to a formula for warming kidney and eliminating bursa, belonging to the prior art, which is a traditional Chinese medicine compound preparation prepared by extracting and processing 30-50 parts of astragalus root, 10-30 parts of epimedium, 10-30 parts of rhizoma atractylodis, 30-50 parts of tuckahoe and 10-30 parts of salvia miltiorrhiza as raw material medicines.
The method comprises the following steps: decocting the raw materials with water, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.20-1.35 at 55-60 deg.C, and drying; the extracts of the medicinal materials are prepared by decocting the raw materials with water, filtering, concentrating the filtrate into fluid extract with relative density of 1.20-1.35 at 55-60 deg.C, and drying.
In the above compositions, the weight of the medicine is taken as the proportion by weight, and can be increased or decreased according to the corresponding proportion during production, for example, the weight can be increased or decreased in units of kilogram in large-scale production or tons in small-scale production or milligrams in small-scale production, but the proportion of the raw medicine weight proportion among the compositions is not changed.
The proportion of the weight proportion is obtained by scientific screening, and for special patients, such as severe or mild patients, obese patients or lean patients, the proportion of the components can be correspondingly adjusted, the increase or decrease is not more than 100 percent, and the drug effect is not changed.
The single traditional Chinese medicine, especially ministerial medicine and adjuvant medicine, in the composition can also be replaced by proper traditional Chinese medicine with the same property, and the medicinal effect of the replaced traditional Chinese medicine preparation is unchanged.
The formulation of the invention may be in any pharmaceutically acceptable dosage form including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, pills, powders, ointments, pellets, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches. The formulations of the present invention, preferably oral dosage forms, are: capsule, tablet, oral liquid, granule, pill, powder, pellet, and unguent.
The Chinese medicinal preparation of the present invention, which is a preparation for oral administration, may contain conventional excipients such as binders, fillers, diluents, tabletting agents, lubricants, disintegrants, coloring agents, flavoring agents and wetting agents,
the preparation of the invention can be used for determining the usage amount according to the condition of a patient, and can be taken three times a day, 1-20 doses each time, such as: 1-20 bags or granules or tablets.
The detection method is obtained by screening, and the screening process is as follows:
1 Instrument and reagent
1.1 instruments
Ultra-high performance liquid-ion trap-time-of-flight tandem mass spectrometry (Shimadzu, Japan, including autosampler, binary gradient pump, column oven, on-line degasser, PDA detector, LC-solution3.6 chromatography workstation); electronic balance (XS205, METTLER TOLEDO, Switzerland), Milli-Q ultrapure water system (Millipore, USA), ultrasonic cleaner (KQ-500DV, ultrasound instruments, Inc. of Kunshan).
1.2 drugs and reagents
Comparison products: chlorogenic acid (batch No. 110753-200413), calycosin glucoside (batch No. 111920-201505), hyperin (batch No. 111521-201205), rosmarinic acid (batch No. 111871-201203), icariin (batch No. 110737-200415), formononetin (batch No. 111703-200602) and astragaloside (batch No. 110781781-201314) were purchased from China food and drug testing institute; calycosin (lot No. 201304), lithospermic acid (lot No. 201309), formononetin (lot No. 201306), salvianolic acid a (lot No. 201307), baohuoside I (lot No. 201102), hodoroside B (lot No. AW412S), atractyloside a (lot No. AW413A), zhuoding a (lot No. AM774E), zhuoding B (lot No. AM775E), zhuoding C (lot No. AM776E) were purchased from tianjin scientific and technological co; all reference substances are more than or equal to 98.0 percent by mass. Astragalus root and poria cocos wolf extract (batch number 20150701, provided by modern Chinese medicine development center of Tianshili research institute) for warming kidney and eliminating bursa. Astragalus membranaceus [ batch 20140425-01, Astragalus membranaceus (Fisch.) Bge. of Leguminosae (Leguminosae), Astragalus membranaceus (Bge.) Hsiao, dried root of Epimedium brevicornus (Bge.), Epimedium Epimedium [ batch 20150324, dried leaf of Epimedium brevicornum Maxim. of Berberidaceae (Berberidaceae)), Atractylodes lancea [ batch 20140430-01, dried sclerotium of Atractylodes lancea (Schw.) of Compositae, dried root and rhizome of Atractylodes lancea (Thunb.) DC., Poria [ batch 20141250 ], Polyporaceae (Polyporaceae), Poria cocos (Schw.) Wolf ], Salvia miltiorrhiza [ batch 20141201, dried root and rhizome of Salvia miltiorrhiza (Labiatae), dried root and rhizome of Salvia miltiorrhiza Bge, Bmila and root of Salvia miltiorrhiza, all of Leguminosae, and modern Lemnaceae, resource of Chinese medicine, California.
Methanol, acetonitrile (chromatographically pure, Merck, Germany), formic acid (chromatographically pure, Shanghai Runje).
2 method
2.1 preparation of control solutions
Weighing appropriate amount of chlorogenic acid, calycosin glucoside, calycosin, hyperoside, rosmarinic acid, lithospermic acid, formononetin, salvianolic acid A, icariin, formononetin, astragaloside IV, baohuoside I, agastachoside B, atractyloside A, epimedin B and epimedin C reference substances in a same measuring bottle, adding appropriate amount of methanol, ultrasonically dissolving, cooling to room temperature, and fixing to constant volume to obtain mixed reference substance solution.
2.2 preparation of test solutions of the extracts
Accurately weighing 0.5g of radix astragali and Poria extract of formula for warming kidney and eliminating bursa, placing in a conical flask with a stopper, accurately adding 10mL of 30-50% (preferably 40%) methanol, weighing, performing ultrasonic treatment (power 100W and frequency 40kHz) for 20-60min (preferably 40min), cooling to room temperature, supplementing loss mass, shaking, centrifuging, and filtering the supernatant with 0.22 μm filter membrane to obtain the final product.
2.3 preparation of test solution of Single herb
Weighing each single medicinal material in the formula for warming kidney and eliminating bursa, extracting each medicinal material separately according to the preparation process, weighing appropriate amount of each medicinal material extract dry powder according to the proportion and the extract yield of the Chinese medicinal materials in the formula, and preparing according to the method of '2.2' to obtain the test solution of each medicinal material.
2.4 chromatographic conditions
By C18Column, mobile phase: adopting 0-0.5% (preferably 0.1%) formic acid aqueous solution (A) -acetonitrile (B), and gradient elution conditions are 0-2 min and 2% of B; 2-10 min, 2% -16% of B; 10-12 min, 16% B; 12-13 min, 16% -18% of B; 13-35 min, 18% -40% of B; 35-38 min, 40% -60% B; 38-40 min, 60% -99% B. The volume flow rate is 0.1-0.6 (preferably 0.4) mL/min; the column temperature is 30 ℃; the sample size was 2. mu.L.
2.5 Mass Spectrometry conditions
Adopting an electrospray ionization ion source (ESI), respectively detecting positive and negative ions within a scanning range of m/z of 100-1200, taking sodium trifluoroacetate as a correction fluid, using high-purity nitrogen as atomizing gas, argon as collision gas, respectively detecting the positive and negative ions within a scanning range of m/z of 100-1200, using the ion source temperature of 220-; the ion source temperature is preferably 250 ℃, the capillary voltage negative ion mode is 3.5kV, the positive ion mode is 4.5kV, and the volume flow of the atomizing gas is 1.5L/min.
3 condition groping
3.1 examination of extraction solvent for sample solution of extract
Examining the extraction effect of methanol and water with different concentrations, 0.5g of the extract of QILINGGEN prescription for warming kidney and eliminating bursa is precisely weighed, and 10mL of water, 30% methanol-water (v/v), 40% methanol-water (v/v), 50% methanol-water (v/v), 70% methanol-water (v/v) and 100% methanol are respectively used as extraction solvents. As a result, it was found that 30-50% methanol-water (v/v) was the most effective as the extraction solvent because the peak shape of the chromatogram was good and the number of peaks was large, and thus 30-50% methanol-water (v/v) was selected as the extraction solvent. The results are shown in FIG. 1.
3.2 investigation of the column
The results of the investigation on the ACQUITY UPLC BEH C18 column, the ACQUITY UPLC HSS T3 column, the ACQUITY UPLCHSS HILIC column, the ZORBOX RRHD C18 column, the ZORBOX RRHT C18 column, the Kinetex C18 column and the Luna C18 column show that the chromatographic columns can separate a large number of chromatographic peaks and have obvious separation effects, and can be applied to the invention. The results are shown in FIG. 2
3.3 investigation of the mobile phase
3.3.1 different elution conditions
And (3) screening the influence of the UPLC gradient elution condition on the spectrum separation effect. Screening was performed with acetonitrile-water solution at different ratios and different elution procedures, and the representative chromatogram is shown in FIG. 3. As is clear from the results, the elution conditions of the third embodiment were selected because the number of chromatographic peaks separated in the fourth embodiment was large and the separation effect was most remarkable.
The first scheme is as follows:
scheme II:
the third scheme is as follows:
scheme four
3.3.2 whether or not formic acid is added to the mobile phase:
after addition of formic acid to the mobile phase, the number of peaks increased significantly and the peak shape and resolution improved. FIG. 4 shows the result of UPLC-ESI-IT-TOF/MS analysis
Analyzing the extractive solution and each medicinal solution of QILINGGANWENSHIXIAOCAO prescription by UPLC-ESI-IT-TOF/MS technology, and making total ion flow diagram of mixed reference solution in negative ion mode shown in figure 5 and that of QILINGGANWENSHIXIAOCAO prescription in positive and negative ion mode shown in figure 6. Attribution research is carried out on 5 medicinal materials in the prescription, and the chromatogram is shown in figure 7. A total of 36 compounds were identified based on their precise relative molecular masses, fragmentation information, controls and reference-related literature, and the results are shown in tables 1 and 2. The identified compound is derived from 4 medicinal materials of astragalus, epimedium, rhizoma atractylodis and salvia miltiorrhiza, and comprises 19 flavonoid components, 11 phenolic acid components, 4 triterpenoid saponin components, 1 sesquiterpene glycoside and 1 alkaloid component. The compounds of each component were characterized.
TABLE 1 data and compound attribution of Qiling extract in UPLC-ESI-IT-TOF/MS anion mode
*The confirmation of a reference substance is carried out; a-astragalus root b-epimedium c-atractylodes rhizome dPoria e-Danshen root, the following table is
TABLE 2 UPLC-ESI of Qiling formula extract for warming kidney and eliminating bursa+Data and compound assignment in positive ion mode for IT-TOF/MS
4.1 flavonoid analysis
Peak 7 (t)R14.695min) gives M/z 447[ M + H ] respectively]+And M/z 491[ M + HCOO]-The relative molecular mass 446 of the compound is determined, and the peak belongs to the astragalus root medicinal material. Comparing with the reference, the No. 7 peak is determined to be calycosin glucoside.
Peak number 16 (t)R20.267min) to give M/z 663[ M + H ] respectively]+And M/z 661[ M-H]-The relative molecular mass 662 of the compound is determined, and the peak is attributed to the epimedium medicinal material. The secondary mass spectrum takes M/z 663 as a parent ion to lose rhamnosyl group to generate M/z 517[ M + H-Rha ] M/z]+The fragments of (a); further loss of glucosyl groups to yield M/z 355[ M + H-Rha-Glc]+The fragment of (a), i.e. aglycone; aglycone loses C4H8Fragment ions of m/z 299 were obtained. Loss of rhamnosyl and glucosyl groups with M/z 661 as parent ion to yield M/z 353[ M-H-Rha-Glc]-The fragment ion of (3), and the peak No. 16 was determined to be icariin a.
4.2 resolution of phenolic acid Compounds
Peak No. 17 (t)R20.932min) gives M/z 717[ M-H)]-And belongs to the salvia miltiorrhiza medicinal material, and the No. 17 peak is presumed to be salvianolic acid B. Breaking with m/z 717 as parent ion, losing 1 molecule of danshensu to generate m/z 519, and continuing losing 1 molecule of danshensu to form m/z 321[13]The fragment ions of (a).
4.3 resolution of triterpenoid saponins
Peak No. 37 (t)R37.568min) and peak number 38 (t)R38.043min) belonging to radix astragali, and all having M/z 891[ M + Na ]]+Peak, M/z 867[ M-H ]]-Peak sum M/z 913[ M + HCOO]-And (3) determining that the relative molecular mass is 868, the two belong to isomers, and presuming that the No. 37 peak is astragalus saponin I and the No. 38 peak is isoastragalus saponin I according to the polarities of the two.
4.4 analysis of sesquiterpene glycoside Compounds
Peak No. 3 (t)R9.465min) to give M/z 471[ M + Na ] respectively]+Peak sum M/z 493[ M + HCOO]-Peak, calculated molecular formula C21H36O10Comparing the retention time of the ultraviolet chromatogram of each medicinal material, the peak belongs to the rhizoma atractylodis medicinal material. Comparing with reference substance, and determining that the peak 3 is atractyloside A.
4.5 alkaloid Compound resolution
Peak No. 6 (t)R11.909min) gives M/z 342[ M + H)]+Peak, its secondary fragment ion has M/z 297[ M- (CH)3)2NH]+And M/z 265[ M- (CH)3)2NH－CH3OH]+And the peak belongs to epimedium medicinal materials, and the No. 6 peak is presumed to be magnoline.
The invention is respectively examined aiming at different extraction solvents and different mobile phases, and finally the ultrasonic extraction effect of 30-50% methanol is determined to be the best, and the peak shape and the separation degree of each chromatographic peak are the best by taking 0-0.5% formic acid aqueous solution-acetonitrile as the mobile phase. The flavonoid components in the formula respond well in a positive ion mode, and the phenolic acid components in the salvia miltiorrhiza medicinal material respond well in a negative ion mode, so that the astragalus root and poria cocos kidney warming and bursa eliminating formula extract is detected simultaneously in the positive and negative ion modes, and comprehensive mass spectrum information of chemical components is obtained.
The traditional Chinese medicine compound generally comprises a plurality of medicinal materials, the chemical components are complex, and the price of a reference substance is high, so that the qualitative and quantitative research on the compound extract is difficult to realize. The research adopts a UPLC-ESI-IT-TOF/MS method to obtain the multi-level mass spectrum fragments of chemical components, so that the chemical composition of the fragments is conjectured, and a quick and effective method is provided for the chemical component analysis of the Chinese herbal compound. The method has been widely applied to various aspects of Chinese herbal compound research.
The research comprehensively analyzes the chemical components of the astragalus root and poria cocos prescription extract for warming the kidney and eliminating the cyst, and finds that the main components contained in the astragalus root and poria cocos prescription extract have good consistency with the related pharmacologically active components of polycystic ovarian syndrome reported in the literature, thereby reflecting the rationality of potential pharmacodynamic material bases and the prescription to a certain extent.
Description of the drawings:
FIG. 1 UPLC chromatograms of different extraction solvents, wherein water, 30% methanol-water, 40% methanol-water, 50% methanol-water, 70% methanol-water, and 100% methanol are sequentially arranged from bottom to top
FIG. 2 UPLC chromatograms of different columns, from bottom to top, an ACQUITY UPLC BEH C18 column, an ACQUITY UPLC HSS T3 column, an ACQUITY UPLC HSS HILIC column, a ZORBOX RRHD C18 column, a ZORBOX RRHTC18 column, a Kinetex C18 column, a Luna C18 column
Fig. 3 UPLC chromatograms of different elution systems.
FIG. 3 shows schemes one, two, three and four from bottom to top in sequence
FIG. 4 Mobile phase screening UPLC chromatogram in which 0.5% formic acid, 0.2% formic acid, 0.1% formic acid, no formic acid added are sequentially from top to bottom
3-atractyloside A4-chlorogenic acid 7-calycosin glucoside 8-hyperin 12-rosmarinic acid 14-lithospermic acid 15-formononetin 19-calycosin 20-salvianolic acid A22-epimedin A23-epimedin B24-epimedin C25-icariin 28-formononetin 31-astragaloside 34-hodeosin B36-baceosin I
FIG. 5 Total ion flow diagram in negative ion mode of mixed control solution
FIG. 6 is a schematic diagram of negative (A) and positive (B) ion flow diagrams of radix astragali and Poria extract UPLC-ESI-IT-TOF/MS
FIG. 7 UPLC chromatogram of radix astragali and Poria extract and its respective medicinal materials (254nm)
A-QILINGGEN prescription extract B-astragali radix extract C-Epimedii herba extract D-Atractylodes lancea rhizome extract E-Poria extract F-Saviae Miltiorrhizae radix extract
The specific implementation mode is as follows:
the invention is further illustrated by the following examples.
Example 1
Preparation of control solutions
Weighing appropriate amount of chlorogenic acid, calycosin glucoside, calycosin, hyperoside, rosmarinic acid, lithospermic acid, formononetin, salvianolic acid A, icariin, formononetin, astragaloside IV, baohuoside I, agastachoside B, atractyloside A, epimedin B and epimedin C reference substances in a same measuring bottle, adding appropriate amount of methanol, ultrasonically dissolving, cooling to room temperature, and fixing to constant volume to obtain mixed reference substance solution.
Preparation of test solution
Accurately weighing 0.5g of radix astragali and Poria extract of formula for warming kidney and eliminating bursa, placing in a conical flask with a stopper, accurately adding 10mL of 30-50% methanol, weighing, performing ultrasonic treatment (power 100W and frequency 40kHz) for 20-60min, cooling to room temperature, supplementing loss mass, shaking, centrifuging, and filtering the supernatant with 0.22 μm filter membrane to obtain the final product.
and 4, calculating the content of the effective components in the test sample according to the chromatogram.
Chromatographic conditions
Using a C18 column, mobile phase: adopting 0-0.5% formic acid water solution (A) -acetonitrile (B), and gradient elution conditions are 0-2 min and 2% B; 2-10 min, 2% -16% of B; 10-12 min, 16% B; 12-13 min, 16% -18% of B; 13-35 min, 18% -40% of B; 35-38 min, 40% -60% B; 38-40 min, 60% -99% B. The volume flow is 0.1-0.6 mL/min; the column temperature is 30 ℃; the sample size was 2. mu.L.
Example 2
The method comprises the following steps:
(1) preparation of control solution: weighing appropriate amount of chlorogenic acid, calycosin glucoside, calycosin, hyperoside, rosmarinic acid, lithospermic acid, formononetin, salvianolic acid A, icariin, formononetin, astragaloside IV, baohuoside I, agastachoside B, atractyloside A, epimedin B and epimedin C reference substances in a same measuring bottle, adding 40% methanol, ultrasonically dissolving, cooling to room temperature, and fixing to a certain volume to obtain a mixed reference substance solution. The concentration of each component in the obtained solution is as follows in sequence: 0.062, 0.057, 0.0106, 0.047, 0.063, 0.0195, 0.02, 0.059, 0.038, 0.0201, 0.0238, 0.0164, 0.0354, 0.0182, 0.075, 0.09, 0.084mg/ml
(2) Preparation of a test solution:
precisely weighing 0.5028g of QILINGGEN preparation for warming kidney and eliminating bursa, pulverizing, adding 40% methanol, ultrasonic treating for 20-60min, cooling to room temperature, shaking, centrifuging, collecting supernatant, filtering with 0.22 μm filter membrane, and diluting to 25ml with 40% methanol to obtain sample solution 20.11mg/ml
and 4, calculating the content of the effective components in the test sample according to the chromatogram.
Wherein the conditions of the ultra-high liquid chromatography are as follows: using a C18 column, mobile phase: adopting 0.1 percent formic acid water solution-acetonitrile, and carrying out gradient elution with the volume flow of 0.4 mL/min; the column temperature is 30 ℃;
Claims (7)
1. an analytical method of effective components of a astragalus root and poria cocos kidney warming and capsule eliminating preparation is characterized by comprising the following steps:
1) preparation of control solution:
2) preparation of a test solution:
3) method for analyzing active ingredients: comprises flavonoid compounds, phenolic acid compounds, triterpenoid saponin compounds, sesquiterpene glycoside compounds and alkaloid compounds,
wherein the flavonoids include hyperoside, calycosin glucoside, wengoxin E, icariin A, anhydroicaritin-3-O-rhamnose (1-2) -furoic acid-7-O-glucose, formononetin, baohuoside II, calycosin, wengoxin F, epimedin A, epimedin B, epimedin C, icariin, large flower icariin F, baohuoside I, icariside I, sagoroside B, and 2 "-rhamnose icariside II; the phenolic acid compounds include tanshinol, salvianolic acid H or I, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, iso-salvianolic acid B or salvianolic acid E, salvianolic acid A, chlorogenic acid, iso-chlorogenic acid, and neochlorogenic acid; the triterpenoid saponin compounds comprise astragaloside I, astragaloside II, isoastragaloside I and astragaloside IV; the sesquiterpene glycoside compounds comprise atractyloside, and alkaloid compounds comprise magnoline,
method for analyzing active ingredients: the method comprises the following steps of ultra-high performance liquid chromatography, mass spectrometry and peak compound assignment, wherein the conditions of the ultra-high performance liquid chromatography are as follows: using a C18 column, mobile phase: adopting 0.1 percent formic acid water solution-acetonitrile, and carrying out gradient elution with the volume flow of 0.4 mL/min; the column temperature is 30 ℃;
2. the assay of claim 1, wherein: the preparation of the test solution in the step 2) comprises a sample test solution and a single medicinal material test solution.
3. The analytical method of claim 1, wherein the mass spectrometry conditions are: an electrospray ionization ion source is adopted, positive ions and negative ions are respectively detected, the scanning range m/z is 100-1200, the ion source temperature is 220-350 ℃, the capillary tube voltage negative ion mode is 3.0-4.0kV, the positive ion mode is 3.5-5.0kV, and the volume flow of the atomized gas is 1.2-2.0L/min.
4. The analysis method of claim 1, wherein the peak compound assignment method adopts a UPLC-ESI-IT-TOF/MS method, and the compound structure is obtained by comparison of reference substances, spectrum, mass spectrum data and molecular weight and fragment information of reference documents, and the total 36 chemical components of 4 medicinal materials including radix astragali, herba Epimedii, rhizoma Atractylodis and radix Salviae Miltiorrhizae are identified.
5. The assay of claim 1, wherein: step 2) preparing a test solution, weighing the astragalus root and poria cocos kidney warming and sac removing preparation, carrying out ultrasonic extraction for 20-60min by 30-50% of methanol, cooling to room temperature, and filtering supernate with a 0.22 mu m filter membrane to obtain a sample test solution;
weighing single medicinal material or extract, ultrasonic extracting with 0-50% methanol for 20-60min, cooling to room temperature, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain radix astragali medicinal material test solution, rhizoma Atractylodis medicinal material test solution, Poria medicinal material test solution, Saviae Miltiorrhizae radix medicinal material test solution, and herba Epimedii medicinal material test solution.
6. The analytical method as in claim 5, wherein the kidney-warming and bursal-eliminating preparation is prepared from astragalus root 30-50 parts, epimedium 10-30 parts, atractylodes rhizome 10-30 parts, poria cocos wolf 30-50 parts, red sage root 10-30 parts; the method comprises the following steps: decocting the raw materials with water, mixing decoctions, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.20-1.35 at 55-60 deg.C, and drying; the extracts of the medicinal materials are prepared by decocting the raw materials with water, filtering, concentrating the filtrate into fluid extract with relative density of 1.20-1.35 at 55-60 deg.C, and drying.
7. The analytical method of claim 1, comprising the steps of:
1) preparation of control solution: weighing appropriate amount of chlorogenic acid, calycosin glucoside, calycosin, hyperoside, rosmarinic acid, lithospermic acid, formononetin, salvianolic acid A, icariin, formononetin, astragaloside IV, baohuoside I, agastachoside B, atractyloside A, epimedin B and epimedin C reference substances in a same measuring bottle, adding appropriate amount of methanol, ultrasonically dissolving, cooling to room temperature, and fixing to constant volume to obtain mixed reference substance solution;
2) preparation of a test solution:
precisely weighing the formula, precisely adding 30-50% methanol, weighing, ultrasonically treating for 20-60min, cooling to room temperature, supplementing loss, shaking, centrifuging, and filtering the supernatant with 0.22 μm filter membrane to obtain sample solution; decocting each single medicinal material in the formula respectively with water, concentrating, filtering, drying to obtain dry powder of each medicinal material extract, precisely weighing each dry powder of each medicinal material extract, adding 30-50% methanol, weighing for a certain mass, performing ultrasonic treatment for 20-60min, cooling to room temperature, supplementing loss mass, shaking, centrifuging, and filtering supernatant with 0.22 μm filter membrane to obtain test solution of each medicinal material;
3) method for analyzing active ingredients: the method comprises the following steps of ultra-high performance liquid chromatography, mass spectrometry and peak assignment, wherein the chromatographic conditions are as follows: by C18Column, mobile phase: adopting 0.1 percent formic acid water solution-acetonitrile, and carrying out gradient elution with the volume flow of 0.4 mL/min; the column temperature is 30 ℃;
mass spectrum conditions: adopting an electrospray ionization ion source ESI to respectively detect positive ions and negative ions, wherein the scanning range m/z is 100-1200, sodium trifluoroacetate is used as a correction liquid, the atomization gas is high-purity nitrogen, the collision gas is argon, the ion source temperature is 250 ℃, the capillary voltage negative ion mode is 3.5kV, the positive ion mode is 4.5kV, and the volume flow of the atomization gas is 1.5L/min;
peak assignment analysis method: analyzing the sample solution of the Qiling kidney-warming and bursa-eliminating prescription and each medicinal material solution by adopting an UPLC-ESI-IT-TOF/MS technology, mixing total ions of a reference substance solution in an anion mode and total ions of the Qiling kidney-warming and bursa-eliminating prescription in a positive and negative ion mode, performing attribution research on 5 medicinal material chromatographic peaks in the prescription, and identifying 36 compounds in total according to the accurate relative molecular mass, fragment information, a reference substance and reference related documents of the compounds.
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| CN107727772B (en) * | 2017-11-21 | 2020-04-10 | 匡海学 | Method for qualitatively and quantitatively detecting triterpenoid saponin in traditional Chinese medicine by using electrospray protonation cracking-mass spectrum multiple reaction detection mode |
| CN108037197B (en) * | 2017-11-27 | 2020-08-07 | 中山市中智药业集团有限公司 | Method for qualitatively and quantitatively analyzing chemical components of non-volatile secondary metabolites of houttuynia cordata wall-broken decoction pieces |
| CN108693289B (en) * | 2018-05-28 | 2020-07-14 | 贵阳中医学院 | Method for determining content of magnoflorine in herringbone fruit medicinal material |
| CN109342585A (en) * | 2018-10-10 | 2019-02-15 | 泓博元生命科技（深圳）有限公司 | The determination method of Herba Epimedii |
| CN110095524B (en) * | 2019-05-15 | 2022-04-22 | 山东省分析测试中心 | Triterpene saponin mass spectrum structure analysis method |
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| CN110579553B (en) * | 2019-09-29 | 2023-08-01 | 江苏省中医院 | Quality detection method of ginseng-sunflower vein-invigorating particles |
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