CN109239224B - Method for simultaneously and quantitatively measuring 9 blood-entering components in spina date seed water extract - Google Patents

Method for simultaneously and quantitatively measuring 9 blood-entering components in spina date seed water extract Download PDF

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CN109239224B
CN109239224B CN201811209768.4A CN201811209768A CN109239224B CN 109239224 B CN109239224 B CN 109239224B CN 201811209768 A CN201811209768 A CN 201811209768A CN 109239224 B CN109239224 B CN 109239224B
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spina date
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CN109239224A (en
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闫艳
张敏
杜晨晖
张福生
秦雪梅
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Kampo Extract Biotechnology Hainan Co ltd
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Shanxi University
Shanxi University of Chinese Mediciine
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Abstract

The invention belongs to the technical field of research on in vivo metabolism of Chinese patent medicines, and aims to solve the problems that the HPLC-UV sensitivity is low and the time is too long in quantitative analysis of blood components of spina date seeds at present, and the analysis of trace components in blood plasma is not suitable; the LC-MS/MS is only used for determining the pharmacokinetics of a single component, is not beneficial to the research of the pharmacokinetics of the traditional Chinese medicine, and provides a method for simultaneously and quantitatively determining 9 blood-entering components in the wild jujube seed aqueous extract. Simultaneously and quantitatively measuring 9 blood-entering components in the spina date seed aqueous extract by using UHPLC-Q active Orbitrap HR/MS; the detection sensitivity is improved, and the analysis time is shortened; the simultaneous quantitative analysis of three polar compounds of flavone, saponin and alkaloid is realized; provides analysis technical guarantee for the pharmacokinetics research of the spina date seeds in normal and PCPA insomnia model rats, and provides reference for simultaneous quantitative analysis of multiple components of other oral traditional Chinese medicines.

Description

Method for simultaneously and quantitatively measuring 9 blood-entering components in spina date seed water extract
Technical Field
The invention belongs to the technical field of research on in vivo metabolism of Chinese patent medicines, and particularly relates to a method for simultaneously and quantitatively determining 9 blood-entering components in a spina date seed water extract.
Background
The semen Ziziphi Spinosae is Rhamnaceae plant cortex ChoerospondiatisZiziphus jujuba Mill. var. spinosaDried mature seeds of (Bunge) Hu ex H.F. Chou have the effects of nourishing heart and liver, calming heart and tranquilizing, arresting sweating and promoting the production of body fluid, and are the first choice medicament for clinically treating insomnia. Modern pharmacological research shows that the total saponins, total flavones and total alkaloids in the spina date seeds are main active ingredients for playing a role in tranquilizing and hypnotizing.
As is well known, the absorption of effective components of Chinese medicines into blood is a prerequisite for the exertion of the curative effect, and the multi-component, multi-target and multi-action approaches are the characteristics of clinical effect. The existing phytochemistry research shows that the chemical components in the spina date seeds are rich and diverse in structure, and saponins, flavonoids and alkaloids are used as main characteristic components. At present, the pharmacokinetic research on the spina date seeds mainly focuses on the research of administering a certain monomer component or spina date seed flavone extract and the like in the spina date seeds of rats through oral administration or intravenous injection. However, the oral administration mode of the spina date seed aqueous extract is closer to the clinical administration mode. The spina date seed is rich in chemical components with various structural types such as flavonoids, saponins, alkaloids, triterpenic acids and the like, and the pharmacokinetic characteristics of the components with different types are greatly different. The pharmacokinetic behavior of representing one traditional Chinese medicine by one component is not feasible in the research of the traditional Chinese medicine. In addition, the quantitative analysis of the blood-entering components of the spina date seeds at the present stage is mainly to determine the spinosad and the derivative 6 thereof by HPLC-UV and LC-MS/MS methods'''Pharmacokinetics of several compounds like feruloylscinolone and jujuboside A, jujuboside B, etc. The analysis sensitivity of HPLC-UV is low, so that the HPLC-UV is not suitable for analyzing trace components in plasma, and the analysis time is too long; LC-MS/MS has high measurement sensitivity, but is applied to pharmacokinetics in which only a single component is measured.
Disclosure of Invention
The invention aims to solve the problems that the HPLC-UV analysis sensitivity is low, the method is not suitable for analyzing the trace components in the blood plasma, and the analysis time is too long in the quantitative analysis of the blood-entering components of the spina date seeds at the present stage; the LC-MS/MS is high in determination sensitivity, but is only applied to the pharmacokinetics of single component determination, and is not beneficial to the research of the pharmacokinetics of traditional Chinese medicines, and provides a method for simultaneously and quantitatively determining 9 blood-entering components in a spina date seed aqueous extract and application in the pharmacokinetics of orally taken normal and PCPA insomnia model rats of the spina date seed aqueous extract.
The invention is realized by the following technical scheme: a simultaneous quantitative determination method for 9 blood-entering components in a wild jujube seed aqueous extract adopts a UHPLC-Q active Orbitrap HR/MS method, and simultaneously quantitatively determines 9 active components in the wild jujube seed aqueous extract: linderane, magnoflorine, viscapine, spilanthol, swertisin, kaempferol-3-O-rutinoside, 6'''-feruloylscinolone, jujuboside a and jujuboside B; the method comprises the following specific steps:
(1) preparation of single control stock solutions: precisely weighing reference substances, respectively, adding initial mobile phase of chromatography analyzed by UHPLC-Q active Orbitrap HR/MS to obtain single reference substance stock solution containing 2.032 mg/mL of lindera aggregate alkali and 1.130 mg/mL of magnoflorine, and adding methanol to obtain single reference substance stock solution containing 2.184 mg/mL of Vienningin, 2.016 mg/mL of spinosin, 1.972 mg/mL of swertisin and kaempferol-3-O2.058 mg/mL of-rutinoside, 6'''-single control stock solutions of 2.012 mg/mL of feruloylscinolone, 2.040 mg/mL of jujuboside a, 2.064 mg/mL of jujuboside B;
preparation of working solution: precisely measuring the single reference substance stock solution respectively, and adding methanol to prepare a mixed reference substance solution, wherein: 325.12 ng/mL of linderane, 18080 ng/mL of magnoflorine, viscapine, 2419.2 ng/mL of spinosin, 94.66 ng/mL of swertisin, kaempferol-3-O-rutinoside 246.96 ng/mL, 6'''-128.77 ng/mL of feruloylscinolone acetonide, 652.8 ng/mL of jujuboside a, 412.8 ng/mL of jujuboside B; precisely measuring the mixed reference substance solution, respectively adding methanol to gradually dilute the mixed reference substance solution to 4/5, 1/2, 1/4, 1/8, 1/40 and 1/80 times of the concentration of the mixed reference substance solution, and shaking up to obtain a working solution with mass concentration gradient;
(2) preparation of internal standard solution: selecting palmatine hydrochloride, daidzin and astragaloside IV as internal standard substances for measuring the content of alkaloid, flavonoid and saponin compounds respectively, precisely weighing the internal standard substances respectively, and adding methanol to prepare a single internal standard solution of palmatine hydrochloride 0.984 mg/mL, daidzin 1.080 mg/mL and astragaloside IV 1.392 mg/mL; precisely absorbing single internal standard solution, and diluting with methanol to obtain mixed internal standard solution containing palmatine hydrochloride 78.72 ng/mL, daidzin 216.00 ng/mL, and astragaloside 556.80 ng/mL;
(3) establishment of a standard curve: precisely absorbing 100 muL of blank plasma, adding 10 muL of internal standard, swirling for 30 s, then respectively adding 10 muL of mixed working solution, swirling for 30 s, then adding a solvent, swirling for 5 min, centrifuging for 10 min at 13000 rpm and 4 ℃, taking 350 muL of supernatant, adding the supernatant into a clean EP (EP) tube, carrying out vacuum centrifugation at 1400 rpm, carrying out air blow-drying at 40 ℃, adding 100 muL of initial mobile phase to residues, swirling for 2 min, centrifuging for 5 min at 13000 rpm and 4 ℃, taking the supernatant, and carrying out UHPLC-Q active Orbitrap HR/MS (ultra high performance reactor/mass spectrometry) analysis; taking the ratio of the peak area of the component to be detected to the peak area of the internal standard as the ordinate (y), taking the concentration of the component to be detected added into the plasma as the abscissa (x), and adopting 1/c2Performing linear regression by a weighted least square method to establish a curve;
(4) and (3) detection of the sample:
(a) preparing a spina date seed water extract: crushing 500 g of spina date seed decoction pieces, sieving the crushed mixture to 60 percent, sieving the crushed mixture by using a No. 1 pharmacopoeia sieve, adding 10 times of water, soaking the mixture for 30 min, heating the mixture to boil, performing reflux extraction for 2 h, filtering the mixture, adding 8 times of water into filter residues, performing reflux extraction for 2 h, filtering the mixture, and combining the filtrate; concentrating under reduced pressure to crude drug concentration of 0.5 g/mL, freeze drying, and dissolving with water;
(b) collection of plasma samples: the concentration of the orally-taken spina date seed aqueous extract for the rat is 40 g/Kg based on crude drug amount, blood is collected to the rat by 300 muL within 0.083-24 h after administration, the rat is placed in a centrifugal tube containing 0.1% heparin sodium anticoagulant, the solution is kept still for 30 min, the solution is centrifuged at 4 ℃ and 3500 rpm for 10 min, blood plasma is separated, and the solution is stored at-20 ℃ for later use;
(c) and (3) detection of the sample: accurately sucking 100 mu L of rat plasma, and treating by adopting a blank plasma sample treatment method, wherein acetonitrile is adopted as a solvent in the plasma sample treatment, and the dosage of the acetonitrile is 3 times that of the plasma sample; the treated plasma samples containing the drug are subjected to UHPLC-Q active Orbitrap HR/MS analysis, and the concentration of 9 components in the plasma samples is calculated according to a standard curve.
The chromatographic conditions of the UHPLC-Q active Orbitrap HR/MS analysis are as follows: the chromatographic column is ACQUITY UPLC HST 3 column, 150 mm x 2.1 mm, 1.8 mu m, mobile phase A: b is acetonitrile: 0.1% formic acid water or a: b is 0.1% formic acid acetonitrile: 0.1% formic acid water, gradient elution program is 0-1.5 min, 17% A; 1.5-3 min, 17 → 19% A; 3-7 min, 19 → 33% A; 7-12 min, 33 → 98% A; 12-14 min, 98 → 17% A; 14-19 min, 17% A; the flow rate is 0.3 mL/min; the column temperature was 40 ℃; the sample introduction amount is 3 muL;
mass spectrum conditions: an ion source: a source of HESI; positive and negative ions are switched and scanned simultaneously; the working mode is as follows: Full-MS; mass spectrum parameters: flow rate of sheath gas: 35 arb; flow rate of auxiliary gas: 10 arb; spraying voltage: 3.2 KV (+), 2.5 KV (-); capillary temperature: 320 ℃; ion source temperature: 300 ℃; resolution 70000; the scanning range m/z is 150-1500 Da.
The initial chromatographic mobile phase of the UHPLC-Q active Orbitrap HR/MS analysis in the step (1) is 17% of 0.1% of formic acid acetonitrile-83% of 0.1% of formic acid water.
In the step (b), the rats are subjected to blood collection at 0.083, 0.167, 0.333, 0.5, 0.75, 1, 2, 4, 6, 10, 12 and 24 hours after administration of the rats respectively.
The invention selects an internal standard method to simultaneously quantify 9 components, 3 different types of internal standards are respectively selected because the components to be measured belong to 3 different types, and the palmatine hydrochloride, the daidzin and the astragaloside are respectively used as the internal standards for measuring the content of alkaloid, flavonoid and saponin compounds.
The solvent adopted in the blank plasma sample treatment is any one of methanol, acetonitrile or ethyl acetate, and the dosage of the solvent is 3-5 times of that of the blank plasma sample. Preferably, the solvent used in the processing of the blank plasma sample is acetonitrile, and the amount of the solvent is 3 times that of the blank plasma sample.
Mobile phase investigation, mobile phase a: b is preferably 0.1% formic acid acetonitrile: 0.1% formic acid water.
The invention reasonably sets the chromatographic conditions, the selection of the internal standard substance and the pretreatment conditions of the sample, improves the detection sensitivity and shortens the analysis time; the simultaneous quantitative analysis of three different polar compounds of flavonoids, saponins and alkaloids is realized; can be used for pharmacokinetic study of normal and 9 components in plasma of PCPA insomnia model rats after oral administration of spina date seed aqueous extract; provides technical reference for simultaneous quantitative analysis of multiple components of other oral traditional Chinese medicines.
In conclusion, 9 kinds of spina date seed aqueous extracts (linderane, magnoflorine, viscapine, spilanthol, swertisin and kaempferol-3-O-rutinoside, 6'''Active ingredients of feruloylscinolone, spina date seed saponin A and spina date seed saponin B) are taken as research objects, and a novel method for analyzing 9 active ingredients in the spina date seed water extract in a quantitative mode at the same time is established by adopting an UHPLC-Q active Orbitrap HR/MS technology. The 9 selected components of the invention cover 3 active components with different polarities in the spina date seed, so that the pharmacokinetics research of the spina date seed is more comprehensive. The Full-MS scanning mode takes the first-class accurate mass number of high-performance parent ions as quantitative ions, and realizes that 9 blood-entering components of the wild jujube seed aqueous extract are accurately and quantitatively analyzed in a single analysis after the wild jujube seed aqueous extract is orally taken to a rat.
Drawings
FIG. 1 is a diagram of an extracted ion flow of 9 components, in which: a is blank plasma, B is blank plasma + reference substance, C is plasma containing medicine; FIG. 2 is a graph showing the mean drug concentration-time curves of 9 ingredients in the normal group (C) and model group (M) rats after oral administration of the aqueous extract of Zizyphi Spinosae semen.
Detailed Description
The present invention will be described more fully hereinafter with reference to the following examples. The reagents and equipment used in the examples were as follows:
1. the instrument comprises the following steps: a Thermo Fisher U3000 ultra-high performance liquid chromatograph which is provided with an online degasser, a quaternary gradient pump, a column incubator, an ultraviolet detector and an automatic sample injector (Thermo Fisher Scientific company, USA)TM Q ExactiveTMOrbitrap mass spectrometer (Germany); one hundred thousand analytical balances model CPA225D, Sartorius beijing instrument systems ltd, germany; vacuum centrifuge concentrator, Eppendorf, Germany.
2. Drugs and reagents: comparison products: linderane (batch No. HC 225036198), magnoflorine (batch No. HA 061308198), viscapine (batch No. HV 187847198), spinosin (batch No. 20160314), swertisin (batch No. HA 062908198), kaempferol-3-O-Rutinosides (batch Nos. HK 201623198), 6'''Feruloyl pinocembrin (batch No. 20160313), spina date seed saponin A (batch No. 20160315A), daidzin (batch No. HD 031189198), astragaloside IV (batch No. HA 012079198) were purchased from Chengniao Biotechnology Co., Ltd, spina date seed saponin B (batch No. 20170210) was purchased from Nanjing spring and autumn bioengineering Co., Ltd, palmatine hydrochloride (batch No. 130427) was purchased from Vickgi Biotechnology Co., Ltd, Sichuan province, and the mass fraction of the reference substance was greater than 98% as determined by HPLC normalization method. Para-chlorophenylalanine (PCPA) (batch GK 01-JBQH) available from Daiheiai (chemical) industries, Inc. and having a purity of greater than 98%. Acetonitrile and formic acid were both obtained from Fisher, and methanol was obtained from merck. Other reagents were analytically pure.
Example 1: a simultaneous quantitative determination method for 9 blood-entering components in a wild jujube seed aqueous extract adopts a UHPLC-Q active Orbitrap HR/MS method, and simultaneously quantitatively determines 9 active components in the wild jujube seed aqueous extract: linderane, magnoflorine, viscapine, spilanthol, swertisin, kaempferol-3-O-rutinoside, 6'''-feruloylscinolone, jujuboside a and jujuboside B; the method comprises the following specific steps:
(1) preparation of single control stock solutions: precisely weighing reference substances, respectively, adding initial mobile phase of chromatography analyzed by UHPLC-Q active Orbitrap HR/MS to obtain single reference substance stock solution containing 2.032 mg/mL of lindera aggregate alkali and 1.130 mg/mL of magnoflorine, and adding methanol to obtain single reference substance stock solution containing 2.184 mg/mL of Vienningin, 2.016 mg/mL of spinosin, 1.972 mg/mL of swertisin and kaempferol-3-O2.058 mg/mL of-rutinoside, 6'''-single control stock solutions of 2.012 mg/mL of feruloylscinolone, 2.040 mg/mL of jujuboside a, 2.064 mg/mL of jujuboside B;
preparation of working solution: are respectively precisely measuredAdding methanol into the single reference substance stock solution to prepare a mixed reference substance solution, wherein: 325.12 ng/mL of linderane, 18080 ng/mL of magnoflorine, viscapine, 2419.2 ng/mL of spinosin, 94.66 ng/mL of swertisin, kaempferol-3-O-rutinoside 246.96 ng/mL, 6'''-128.77 ng/mL of feruloylscinolone acetonide, 652.8 ng/mL of jujuboside a, 412.8 ng/mL of jujuboside B; precisely measuring the mixed reference substance solution, respectively adding methanol to gradually dilute the mixed reference substance solution to 4/5, 1/2, 1/4, 1/8, 1/40 and 1/80 times of the concentration of the mixed reference substance solution, and shaking up to obtain a working solution with mass concentration gradient;
(2) preparation of internal standard solution: selecting palmatine hydrochloride, daidzin and astragaloside IV as internal standard substances for measuring the content of alkaloid, flavonoid and saponin compounds respectively, precisely weighing the internal standard substances respectively, and adding methanol to prepare a single internal standard solution of palmatine hydrochloride 0.984 mg/mL, daidzin 1.080 mg/mL and astragaloside IV 1.392 mg/mL; precisely absorbing single internal standard solution, and diluting with methanol to obtain mixed internal standard solution containing palmatine hydrochloride 78.72 ng/mL, daidzin 216.00 ng/mL, and astragaloside 556.80 ng/mL;
(3) chromatographic and mass spectrometric conditions:
chromatographic conditions are as follows: the chromatographic column is ACQUITY UPLC HST 3 columns (150 mm x 2.1 mm, 1.8 μm), mobile phase A: b is acetonitrile: 0.1% formic acid water or a: b is 0.1% formic acid acetonitrile: 0.1% formic acid water, preferably 0.1% formic acid acetonitrile: 0.1% formic acid water, gradient elution program is 0-1.5 min, 17% A; 1.5-3 min, 17 → 19% A; 3-7 min, 19 → 33% A; 7-12 min, 33 → 98% A; 12-14 min, 98 → 17% A; 14-19 min, 17% A. The flow rate is 0.3 mL/min; the column temperature was 40 ℃; the sample size is 3 muL.
Mass spectrum conditions: an ion source: a source of HESI; positive and negative ions are switched and scanned simultaneously; the working mode is as follows: Full-MS; mass spectrum parameters: flow rate of sheath gas: 35 arb; flow rate of auxiliary gas: 10 arb; spraying voltage: 3.2 KV (+), 2.5 KV (-); capillary temperature: 320 ℃; ion source temperature: 300 ℃; resolution 70000; the scanning range m/z 150-.
Table 112 Mass Spectrometry parameters of the Components
Figure DEST_PATH_IMAGE001
(4) Treatment of plasma samples: the effect of protein precipitation (methanol and acetonitrile) and liquid-liquid extraction (ethyl acetate) on the recovery and matrix effect of 9 components was first examined during plasma sample processing. The results show that kaempferol-3-OThe recovery of rutinoside is low and extraction with ethyl acetate results in magnoflorine, viscapine, spilanthol, swertisin and 6'''The recovery rate of feruloylscinonide is extremely low, and finally acetonitrile with higher recovery rate and low matrix effect on the component to be detected is selected as a reliable solvent for protein precipitation. Secondly, the precipitation effects of acetonitrile with different proportions (3 times, 4 times and 5 times) are compared, and the result shows that the extraction recovery rate of protein tretinoin precipitated by using the acetonitrile with 3 times of volume is highest, the recovery rates of other components are equivalent, so that the acetonitrile with 3 times of volume is selected to precipitate the plasma protein.
Precisely absorbing 100 muL of blank plasma, adding 10 muL of internal standard, swirling for 30 s, then respectively adding 10 muL of working solution, swirling for 30 s, then adding 300 muL of acetonitrile, swirling for 5 min, centrifuging (13000 rpm, 10 min, 4 ℃), taking 350 muL of supernatant, adding the supernatant into a clean EP tube, centrifuging at 1400 rpm in vacuum, drying air at 40 ℃, adding 100 muL of initial mobile phase into residues, swirling for 2 min, centrifuging (13000 rpm, 5 min, 4 ℃), taking supernatant, and carrying out UHPLC-MS analysis.
Example 2: methodology investigation
1. Specificity test: blank plasma, processed solution of blank plasma and drug-containing plasma from 6 rats of different sources were processed and analyzed under the conditions of example 1. Comparing the chromatograms of the three, and observing whether interference exists in the blank plasma at the peak positions of the component to be detected and the internal standard. The results are shown in FIG. 1, and the retention times of the components to be measured and the internal standard are respectively: 3.23 min of linderane, 4.12 min of magnoflorine, 2.85 min of viscapine, 6.13 min of spinosin, 6.87 min of swertisin, 3-side-effect of kaempferolO7.37 min, 6 of-rutinoside'''-feruloylscinolone acetonide 8.14 min, jujuboside A10.76 min, jujuboside B11.14 min, palmatine hydrochloride 10.22 min, daidzin 4.60 min, and astragaloside IV 11.10 min. And the blank plasma corresponding to the peak positions of the component to be detected and the internal standard has no interference, which indicates that the specificity of the method is good.
2. Linear range and quantitative lower limit: taking the ratio of the peak area of the component to be detected to the peak area of the internal standard as the ordinate (y), taking the concentration of the component to be detected added into the plasma as the abscissa (x), and adopting 1/c2And performing linear regression by using a weighted least square method to obtain a regression curve equation. Measurements of 3 standard curves were completed within a day. The lower limit of quantitation refers to a signal-to-noise ratio (S/N) greater than or equal to 10. The standard curve, correlation coefficient, linear range and LLOQ results of the 9 components to be measured are shown in Table 2, and the results show that the components to be measured have good linear relationship in the respective concentration ranges, and the correlation coefficientr 20.980 to 0.999 percent.
TABLE 29 linearity and range of ingredients,r 2 And LLOQ
Figure 193798DEST_PATH_IMAGE002
3. And (3) testing precision and accuracy: precision and accuracy were determined using 4 concentrations of QC samples. The precision in the day adopts the samples of the same analysis batch to carry out continuous sample injection for 5 times, the precision in the day adopts the samples of 3 analysis batches to carry out continuous sample injection for 3 days, each analysis day carries out continuous sample injection for 5 times, and the precision RSD% and the accuracy RE% are calculated. The results of the daily and daytime precision and accuracy of the 9 components to be tested are shown in Table 3, the daily precision RSD is less than 19.96 percent, and the accuracy is-15.27 to 17.31 percent; the precision RSD% in daytime is less than 19.62%, and the accuracy% is-14.33% -17.92%. The data show that the precision and the accuracy are both in the standard range, and the result shows that the method has good precision and accuracy and can be used for analyzing biological samples.
Precision and accuracy within and during the day of the 39 ingredients in Table 39
Figure DEST_PATH_IMAGE003
4. Matrix effect and extraction recovery test: matrix effects and extraction recovery were determined using 4 concentrations of QC samples. Matrix effect: plasma samples (n = 5) were processed according to the "processing of plasma samples" method of example 1, and the peak areas of the component to be measured and the internal standard were each calculated as a. And (3) replacing blank plasma with a blank solvent (acetonitrile) of 100 mu L, wherein other operations are consistent with the processing method of the plasma sample treatment in the example 1 (n = 5), and the peak areas of the component to be detected and the internal standard are respectively calculated as B. Matrix effect = a/B100%, and RSD values were calculated. Extraction recovery rate: blank plasma was treated as a solution by the method of "treatment of plasma sample" in example 1 (n = 5) after precipitation of proteins with acetonitrile, and the peak area of the component to be measured and the internal standard was calculated as C. The recovery rate was extracted as a/C100% and RSD values were calculated. The extraction recovery rate range of 9 components to be detected in the QC sample is 80.75-113.75%, the matrix effect range is 77.67-114.10%, RSD% <15%, the extraction recovery rates of internal standard palmatine hydrochloride, daidzin and astragaloside are 91.92%, 97.98% and 96.76%, respectively, and the matrix effects are 108.93%, 101.38% and 98.49%, respectively, the results show that the extraction recovery rate of the sample preparation method of the experiment is good, no obvious matrix effect influence exists, and the results are shown in Table 4.
TABLE 49 matrix Effect and recovery from extraction of ingredients
Figure 110939DEST_PATH_IMAGE004
5. And (3) stability test: and (3) carrying out stability examination on the QC samples with 4 concentrations, wherein the short-term stability of the samples placed at the room temperature of 25 ℃ for 4 h, the long-term stability of the samples placed at the temperature of 20 ℃ for 30 days, the stability after 3 freeze-thaw cycles (-20 ℃ to 20 ℃), and the stability of the samples placed in an automatic sample injector for 12 h after treatment are analyzed. Calculate precision RSD% and accuracy RE%. The stability of the 9 components to be tested was investigated using 4 concentrations of QC samples under different conditions (samples placed at 25 ℃ for 4 h, -20 ℃ for 30 days, 3 freeze-thaw cycles, 12 h in an autosampler) and the results are given in Table 5. The results show that all analytes are stable in the whole analysis process, the RE percent is between-15.13 and 19.78 percent, and the RSD percent is between 0.38 and 16.23 percent.
TABLE 59 stability of ingredients
Figure DEST_PATH_IMAGE005
Example 3: pharmacokinetic determination of 9 components in rat plasma after oral administration of spina date seed aqueous extract to rats of normal and PCPA insomnia models
(1) Preparing a spina date seed water extract: taking about 500 g of spina date seed tablets, crushing (60% passing through a No. 1 pharmacopoeia sieve), precisely weighing, adding 10 times of water, soaking for 30 min, heating to boil, performing reflux extraction for 2 h, filtering, adding 8 times of water into filter residues, performing heating reflux extraction for 2 h, filtering, and combining filtrates; concentrating under reduced pressure to 0.5 g/mL (crude drug amount), freeze drying, and dissolving with water.
(2) Animal experiments and plasma sample collection: healthy male SD rats, weighing 280 + -20 g, were provided by the laboratory animal technology, Inc., Viton, Beijing. Rats were randomly divided into 2 groups, a normal group and a model group, with 6 rats per group. An insomnia model was prepared with PCPA. The PCPA solution is injected into the abdominal cavity of a model group rat for 400 mg/kg for three consecutive days, after the last administration, whether the circadian rhythm of the rat disappears or not is observed, and if the circadian rhythm of the rat disappears, the model building is successful. The rats in the normal group and the model group are fasted for 12 hours before administration, water is freely drunk, and 40 g/Kg (crude drug amount) of spina date seed water extract is orally taken. After administration, taking 300 muL of blood from 0.083, 0.167, 0.333, 0.5, 0.75, 1, 2, 4, 6, 10, 12 and 24 h retroorbital venous plexus, placing the blood in a centrifuge tube containing 0.1% heparin sodium anticoagulant, standing for 30 min, centrifuging (3500 rpm, 10 min and 4 ℃), separating plasma, and storing at-20 ℃ for later use.
(3) The sample pretreatment method comprises the following steps: precisely sucking 100 mu L of rat plasma, and treating the rat plasma with the plasma sample treatment method in the example 1.
(4) Content determination of 9 kinds of seeds in blood plasma after oral administration of spina date seed aqueous extract by normal and PCPA insomnia model rats:
the treated plasma samples containing drug were subjected to UHPLC-Q active Orbitrap HR/MS analysis under the same conditions as in example 1, and plasma sample concentrations were calculated from the standard curve. The mean drug concentration-time curves of the 9 components in the plasma after the wild jujube seed aqueous extract is orally taken by the rats in the normal group and the model group are shown in figure 2; the pharmacokinetic parameters were calculated using DAS 3.2.8 data processing software using a non-compartmental model, and the results are shown in table 6.
TABLE 6 pharmacokinetic parameters of 9 components after oral administration of spina date seed aqueous extract to rats in Normal group (C) and model group (M)
Figure 766042DEST_PATH_IMAGE006
Note: ND: the blood concentration is lower than LLOQ.
As shown in figure 2, the blood concentration of the lindera root alkali, the magnoline and the vefuninine in the blood plasma of normal rats after the spina date seed aqueous extract is orally taken for 6 hours is lower than that of the LLOQ, and when the blood concentration of the flavin is lower than that of the LLOQ, the kaempferol-3-O-rutinosides at plasma levels below LLOQ within 24 h of administration; after the model rat orally takes the spina date seed aqueous extract for 6 hours, the blood concentration of magnoflorine and vefuninine in plasma is lower than LLOQ.
The content ratio of 9 components to be detected in the spina date seed aqueous extract to C in blood plasmamaxAnd AUC0-tNot completely consistent, indicating different levels of entry of different components into the body. As shown in the study, 6'''The peak reaching time of the feruloylscinolone, the spinosin and the jujuboside A and B is relatively long, but 9 components to be detected of the invention all reach the peak in half an hour, which indicates that other components in the jujuba seeds aqueous extract can promote the absorption of the components.
As shown in Table 6, T of linderane and magnoflorine after oral administration of aqueous extract of Zizyphi Spinosae semen in normal ratsmax0.2 h and 0.46 h respectively, Cmax4.66 ng/mL and 140.29 ng/mL, respectively, T1/2The absorption rates of the two alkaloids are higher, but the elimination rate of the magnoflorine is higher than that of the linderane, namely the absorption rate is 2.87 h and 1.15 h respectively. Spirosin and 6'''-T of feruloylscinonidemax0.28 h and 0.36 h respectively, Cmax106.54 ng/mL and 48.45 ng/mL, respectively, T1/2Respectively 4.74 h and 2.21 h. Compared with spinosin, 6'''Feruloylscinonide is absorbed slowly, and has low exposure and rapid elimination. We speculate that this result is due to 6'''The feruloyl group exists in the structure of feruloylscinonide, so that the fat solubility of the feruloylscinonide is increased, and the feruloylserosin is easier to distribute in tissues. It is noteworthy that when the drug flavin exhibits a bimodal appearance, this may be due to two reasons: 1. 6. mu.l of'''-feruloylscinolone and spinosin are continuously hydrolyzed in the gut to convert to swertisin; 2. when the drug flavin is likely to have liver and intestine circulation. In addition, the absorption of viscapine in normal rats is fast (T)max0.5 h), slow elimination (T)1/2Was 3.17 h). Under the detection condition, kaempferol-3-O-plasma concentrations of rutinoside are lower than LLOQ. T of jujuboside A and Bmax3.46 h and 0.96 h respectively, Cmax57.81 ng/mL and 14.02 ng/mL, respectively, T1/22.44 h and 8.28 h respectively. It can be seen that the absorption of jujuboside B is faster than that of jujuboside A, and the elimination is very slow.
For alkaloid components, the C of the lindera aggregate is compared with the C of the normal rat after the model rat orally takes the spina date seed aqueous extractmax、AUC0-tAnd T1/2Increase, TmaxDecrease indicates that the model rat absorbs the linderane faster and eliminates the drug more slowly (P)<0.05), the exposure in vivo is obviously improved; magnolia alkaloid CmaxAnd AUC0-tDecrease of TmaxThe reduction and the obvious increase of CL/F indicate that the model rat has accelerated absorption and elimination speed of magnoflorine but has reduced exposure in vivo.
For flavone C-sugar components, compared with normal rats, model rats orally take spina date seed aqueous extract 6 times'''-T of feruloylscinonidemaxIs remarkably reduced (P)<0.05), indicating that the model rat absorbs the protein quickly; t is1/2Is remarkably improved (P)<0.05), indicating a slow elimination rate; AUC0-tA decrease indicates a decrease in vivo exposure. For higher content of spinosyns in spina date seeds, model rat CmaxAnd AUC0-tIncrease, TmaxA decrease, indicating an accelerated absorption and an increased exposure in vivo; the CL/F decreases, indicating a slower rate of elimination. Model swertisin Cmax、AUC0-¥And T1/2Significantly increased (P)<0.05), the CL/F is obviously reduced, which indicates that the absorption of the model rat is enhanced, the exposure in vivo is obviously increased, and the elimination speed is reduced. Model rat Wei-caining CmaxIncrease, TmaxAnd T1/2The decrease indicates that the absorption and elimination of vefuninine by the model rat are accelerated. Thus, it can be seen that 6 takes apigenin as a parent nucleus'''The absorption of feruloylscinolone, spinosin and swertisin in the model rat is accelerated and the elimination rate is slowed down. Wherein the in vivo exposure of the spinosyns and swertisin is increased. The Virginine which is also carbon sugar shows a tendency of accelerating absorption in a model rat body. But T thereof1/2Shortening and eliminating speed. Compared with normal rats, flavone oxysaccharide kaempferol-3-OThe exposure of rutinoside in model rats was significantly increased.
For spina date seed saponin A and B with higher content of saponins in spina date seed, compared with normal rat, T of spina date seed saponin A in model rat body1/2Increase, TmaxAnd CL/F are obviously reduced, which shows that the absorption of the model rat spina date seed saponin A is accelerated (P)<0.05), slow elimination (P)<0.05) and there is a tendency for the exposure to decrease in vivo. AUC of jujuboside B in model rat0-tAnd CL/F remained essentially the same as normal rats. However, TmaxShowing a decreasing trend, indicating a faster peak arrival. Therefore, the absorption of the jujuboside A and the jujuboside B in a model rat is accelerated; the elimination speed of the jujuboside A is obviously reduced, and the elimination speed of the jujuboside B is basically unchanged.
The method for simultaneously quantifying 9 active ingredients in the spina date seed aqueous extract can be applied to pharmacokinetic research of the spina date seed aqueous extract after oral administration of the spina date seed aqueous extract to normal and PCPA insomnia rats.

Claims (2)

1. A blood-entering component of 9 kinds in semen Ziziphi Spinosae water extractSimultaneously, the quantitative determination method adopts a UHPLC-Q active Orbitrap HR/MS method, and simultaneously quantitatively determines 9 active ingredients in the wild jujube seed aqueous extract: linderane, magnoflorine, viscapine, spilanthol, swertisin, kaempferol-3-O-rutinoside, 6'''-feruloylscinolone, jujuboside a and jujuboside B; the method is characterized in that: the method comprises the following specific steps:
(1) preparation of single control stock solutions: precisely weighing reference substances, respectively, adding initial mobile phase of chromatography analyzed by UHPLC-Q active Orbitrap HR/MS to obtain single reference substance stock solution containing 2.032 mg/mL of lindera aggregate alkali and 1.130 mg/mL of magnoflorine, and adding methanol to obtain single reference substance stock solution containing 2.184 mg/mL of Vienningin, 2.016 mg/mL of spinosin, 1.972 mg/mL of swertisin and kaempferol-3-O2.058 mg/mL of-rutinoside, 6'''-single control stock solutions of 2.012 mg/mL of feruloylscinolone, 2.040 mg/mL of jujuboside a, 2.064 mg/mL of jujuboside B;
preparation of working solution: precisely measuring the single reference substance stock solution respectively, and adding methanol to prepare a mixed reference substance solution, wherein: 325.12 ng/mL of linderane, 18080 ng/mL of magnoflorine, viscapine, 2419.2 ng/mL of spinosin, 94.66 ng/mL of swertisin, kaempferol-3-O-rutinoside 246.96 ng/mL, 6'''-128.77 ng/mL of feruloylscinolone acetonide, 652.8 ng/mL of jujuboside a, 412.8 ng/mL of jujuboside B; precisely measuring the mixed reference substance solution, respectively adding methanol to gradually dilute the mixed reference substance solution to 4/5, 1/2, 1/4, 1/8, 1/40 and 1/80 times of the concentration of the mixed reference substance solution, and shaking up to obtain a working solution with mass concentration gradient;
(2) preparation of internal standard solution: selecting palmatine hydrochloride, daidzin and astragaloside IV as internal standard substances for measuring the content of alkaloid, flavonoid and saponin compounds respectively, precisely weighing the internal standard substances respectively, and adding methanol to prepare a single internal standard solution of palmatine hydrochloride 0.984 mg/mL, daidzin 1.080 mg/mL and astragaloside IV 1.392 mg/mL; precisely absorbing single internal standard solution, and diluting with methanol to obtain mixed internal standard solution containing palmatine hydrochloride 78.72 ng/mL, daidzin 216.00 ng/mL, and astragaloside 556.80 ng/mL;
(3) establishment of a standard curve: precisely absorbing 100 muL of blank plasma, adding 10 muL of internal standard, swirling for 30 s, then respectively adding 10 muL of mixed working solution, swirling for 30 s, then adding 300 muL of acetonitrile, swirling for 5 min, centrifuging at 13000 rpm and 4 ℃ for 10 min, taking 350 muL of supernatant, adding the supernatant into a clean EP (EP) tube, centrifuging at 1400 rpm in vacuum, drying air at 40 ℃, adding 100 muL of initial mobile phase, swirling for 2 min, centrifuging at 13000 rpm and 4 ℃ for 5 min, taking the supernatant, and carrying out UHPLC-Q active Orbitrap HR/MS (ultra high performance reactor/mass spectrometry) analysis; taking the ratio of the peak area of the component to be detected to the peak area of the internal standard as the ordinate (y), taking the concentration of the component to be detected added into the plasma as the abscissa (x), and adopting 1/c2Performing linear regression by a weighted least square method to establish a curve; the results of the standard curve, correlation coefficient, linear range and LLOQ of the 9 components to be measured are shown in the following table, and the results show that the components to be measured have good linear relationship in the respective concentration ranges, and the correlation coefficientr 20.980-0.999 percent;
Figure 983334DEST_PATH_IMAGE001
；
(4) and (3) detection of the sample:
(a) preparing a spina date seed water extract: crushing 500 g of spina date seed decoction pieces, sieving the crushed mixture to 60 percent, sieving the crushed mixture by using a No. 1 pharmacopoeia sieve, adding 10 times of water, soaking the mixture for 30 min, heating the mixture to boil, performing reflux extraction for 2 h, filtering the mixture, adding 8 times of water into filter residues, performing reflux extraction for 2 h, filtering the mixture, and combining the filtrate; concentrating under reduced pressure to crude drug concentration of 0.5 g/mL, freeze drying, and dissolving with water;
(b) collection of plasma samples: the concentration of the orally-taken spina date seed aqueous extract for the rat is 40 g/Kg based on crude drug amount, blood is collected to the rat by 300 muL within 0.083-24 h after administration, the rat is placed in a centrifugal tube containing 0.1% heparin sodium anticoagulant, the solution is kept still for 30 min, the solution is centrifuged at 4 ℃ and 3500 rpm for 10 min, blood plasma is separated, and the solution is stored at-20 ℃ for later use;
(c) and (3) detection of the sample: accurately sucking 100 mu L of rat plasma, and treating by adopting a blank plasma sample treatment method, wherein acetonitrile is adopted as a solvent in the plasma sample treatment, and the dosage of the acetonitrile is 3 times that of the plasma sample; carrying out UHPLC-Q active Orbitrap HR/MS analysis on the processed plasma sample containing the medicine, and calculating the concentration of 9 components in the plasma sample according to a standard curve;
the chromatographic conditions of the UHPLC-Q active Orbitrap HR/MS analysis are as follows: the chromatographic column is ACQUITY UPLC HST 3 column, 150 mm x 2.1 mm, 1.8 mu m, mobile phase A: b is acetonitrile: 0.1% formic acid water or a: b is 0.1% formic acid acetonitrile: 0.1% formic acid water, gradient elution program is 0-1.5 min, 17% A; 1.5-3 min, 17 → 19% A; 3-7 min, 19 → 33% A; 7-12 min, 33 → 98% A; 12-14 min, 98 → 17% A; 14-19 min, 17% A; the flow rate is 0.3 mL/min; the column temperature was 40 ℃; the sample introduction amount is 3 muL;
mass spectrum conditions: an ion source: a source of HESI; positive and negative ions are switched and scanned simultaneously; the working mode is as follows: Full-MS; mass spectrum parameters: flow rate of sheath gas: 35 arb; flow rate of auxiliary gas: 10 arb; spraying voltage: + 3.2 KV, -2.5 KV; capillary temperature: 320 ℃; ion source temperature: 300 ℃; resolution 70000; the scanning range m/z is 150-1500 Da.
2. The method for simultaneously and quantitatively determining 9 blood-entering components in the wild jujube seed aqueous extract according to claim 1, wherein the method comprises the following steps: in the step (b), the rats are subjected to blood collection at 0.083, 0.167, 0.333, 0.5, 0.75, 1, 2, 4, 6, 10, 12 and 24 hours after administration of the rats respectively.
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