Method for detecting pharmacodynamic substances of semen cuscutae-fructus lycii in plasma

Abstract

The invention belongs to the technical field of biological medicines, and provides a method for detecting semen cuscutae-fructus lycii pharmacodynamic substances in blood plasma, which can be used for simultaneously and quantitatively detecting six semen cuscutae-fructus lycii pharmacodynamic substances (hyperoside, astragalus smicus glycoside, isoquercitrin, quercetin, kaempferol and betaine) in rat blood plasma. And further performing pharmacodynamic analysis on hormone levels (E2, FSH, LH and AMH) in PO I rat serum, constructing a drug-time-effect curve, and performing PK-PD correlation analysis. The method provided by the invention not only can efficiently, accurately and quantitatively analyze pharmacodynamic substances, but also can provide comprehensive pharmacokinetic and pharmacodynamic data support for clinical treatment of PO I, thereby providing a scientific basis for optimization and application of related drugs.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a method for detecting dodder seed-wolfberry fruit medicinal substances in plasma. Background Art

[0002] Cuscuta and Lycium barbarum are classic Chinese medicines commonly used to nourish kidney yin and regulate liver and kidney. Traditional medicine believes that Cuscuta has the effect of "tonifying kidney and essence, nourishing liver and improving eyesight", while Lycium barbarum is known for "tonifying liver and kidney, nourishing blood and calming nerves". Modern pharmacological studies have shown that Cuscuta and Lycium barbarum contain a variety of bioactive ingredients, such as flavonoids, glycosides and betaine, which have antioxidant, anti-inflammatory, endocrine regulating and ovarian function protecting effects. However, due to its complex composition and the involvement of multiple active ingredients, traditional research methods are difficult to clarify its pharmacokinetic properties and pharmacodynamic mechanisms. When conducting pharmacokinetic properties and pharmacodynamic mechanism analysis, it is crucial to establish a detection method for six Cuscuta and Lycium barbarum active substances (hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol and betaine). If the detection method is not good, it will affect the subsequent analysis of pharmacokinetic properties and pharmacodynamic mechanisms.

[0003] Primary ovarian insufficiency (POI) is a disease characterized by premature ovarian failure, which often leads to decreased estrogen levels and ovarian reserve function in patients, seriously affecting women's reproductive health and quality of life. Existing treatments mainly include hormone replacement therapy, ovulation-inducing drugs, and traditional Chinese medicine conditioning, but these therapies have certain limitations. For example, long-term use of hormones may cause metabolic disorders, cardiovascular disease, and increased risk of breast cancer; and although traditional Chinese medicine therapy is relatively safe, its active ingredients are unclear and its treatment mechanism is not yet clear. Summary of the invention

[0004] The object of the present invention is to provide a method for detecting the pharmacological substances of Cuscuta-Wolfberry in plasma. The method can simultaneously quantify six pharmacological substances of Cuscuta-Wolfberry (hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol and betaine) in rat plasma, and further perform pharmacodynamic analysis on the hormone levels (E2, FSH, LH and AMH) in the serum of POI rats, construct drug-time-effect curves and perform PK-PD correlation analysis. The method provided by the present invention can not only efficiently and accurately quantify the pharmacological substances, but also provide comprehensive pharmacokinetic and pharmacodynamic data support for the clinical treatment of POI, thereby providing a scientific basis for the optimization and application of related drugs.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides a method for detecting dodder seed-wolfberry fruit medicinal substances in plasma, comprising the following steps:

[0007] (1) Collection and pretreatment of plasma samples;

[0008] (2) UPLC-TQ-MS / MS detection;

[0009] Chromatographic conditions included: Waters ACQUITY UPLC BEH C18 column; mobile phase A was acetonitrile, mobile phase B was 0.1% formic acid in water; gradient elution, 0-1 min, 15%-25% A, 1-4 min, 25%-40% A, 4-5 min, 40%-25% A, 5-6 min, 25%-15% A; injection volume: 2 μL; flow rate 0.25 mL / min; column temperature 30°C;

[0010] The mass spectrometry conditions included: electrospray ionization source ESI, capillary voltage of 3.0 kV, cone gas flow rate of 50 L / h, ion source temperature of 150°C, desolvation gas temperature of 350°C, desolvation gas flow rate of 650 L / h, and scanning in multiple reaction monitoring mode.

[0011] Furthermore, in step (1), 100 μL of plasma was added with 10 μL of methanol, vortexed for 1 min, and then 10 μL of internal standard solution was added. After vortexing for 1 min, 400 μL of methanol was added, vortexed for 4 min, and then centrifuged at 4°C and 14000 r / min for 10 min. 300 μL of supernatant was taken, dried with nitrogen at 37°C, 50 μL of methanol was added to the residue for re-dissolution, vortexed for 4 min, and centrifuged at 4°C and 14000 r / min for 10 min. The supernatant was used for UPLC-TQ-MS / MS detection in step (2).

[0012] Furthermore, the internal standard solution is a methanol solution of formononetin, and the concentration of formononetin in the internal standard solution is 10 ng / mL.

[0013] Furthermore, the medicinal substances of Cuscuta seeds and wolfberry fruits include hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine.

[0014] Further, in step (2), the quantitative ion pair and parameter settings of hyperoside include: the ion mode is [MH] - , t R The reaction time was 1.75 min, the parent ion was 463.09, the daughter ion was 300.03, the declustering voltage was 60 V, and the collision energy was 28 V.

[0015] The quantitative ion pair and parameter settings of astragalus glycosides include: the ion mode is [MH] - , t RThe reaction time was 2.00 min, the parent ion was 447.09, the daughter ion was 284.03, the declustering voltage was 58 V, and the collision energy was 28 V.

[0016] The quantitative ion pair and parameter settings of isoquercetin include: the ion mode is [MH] - , t R The reaction time was 1.71 min, the parent ion was 463.09, the daughter ion was 301.05, the declustering voltage was 60 V, and the collision energy was 27 V.

[0017] The quantitative ion pair and parameter settings for quercetin include: the ion mode is [MH] - , t R The reaction time was 3.18 min, the parent ion was 301.03, the daughter ion was 151.00, the declustering voltage was 45 V, and the collision energy was 25 V.

[0018] The quantitative ion pair and parameter settings for kaempferol include: the ion mode is [MH] - , t R The reaction time was 4.02 min, the parent ion was 285.04, the daughter ion was 151.03, the declustering voltage was 55 V, and the collision energy was 27 V.

[0019] The quantitative ion pair and parameter settings for betaine include: ion mode is [MH] - , t R The reaction time was 0.57 min, the parent ion was 118.09, the daughter ion was 59.00, the declustering voltage was 45 V, and the collision energy was 20 V.

[0020] Further, the regression equation for detecting the betaine content is Y=0.001X+177.19, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; X represents the content of betaine in plasma (in ng / mL), and the betaine content is calculated as (X0+X a ), the endogenous betaine content (X0) should be deducted and the depleted plasma betaine content (X a ) is for analysis. When the pharmacokinetic determination of betaine content in drug-containing plasma is performed, the endogenous betaine content before oral administration is deducted accordingly.

[0021] The regression equation for detecting the content of hyperoside is Y=0.047X+0.3297, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the content of hyperoside in plasma, in ng / mL.

[0022] The regression equation for detecting the content of astragaloside is Y=0.0278X+0.8997, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the content of astragaloside in plasma, in ng / mL.

[0023] The regression equation for detecting the isoquercetin content is Y=0.0163X+0.56, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the isoquercetin content in plasma, in ng / mL.

[0024] The regression equation for detecting the quercetin content is Y=0.0101X+0.1721, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the content of quercetin in plasma, in ng / mL.

[0025] The regression equation for detecting the kaempferol content is Y=0.0916X+0.1505, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the kaempferol content in plasma, in ng / mL.

[0026] The present invention adopts the above detection method, which has the advantages of good separation effect, good linearity, high precision, high accuracy, good stability, etc. The extraction recovery rate is between 83.1% and 103.45%, and the matrix effect is between 86.97% and 113.62%, which meets the requirements for biological sample detection.

[0027] The present invention provides a method for constructing a PK-PD model of a dodder seed-wolfberry fruit medicinal substance, comprising the following steps:

[0028] (1) Set up a dodder seed-wolfberry drug group and a POI blank group;

[0029] (2) Using the above method, the active substances of Cuscuta australis and Lycium barbarum in plasma were detected and PK analysis was performed;

[0030] (3) Serum E2, FSH, LH, and AMH levels were measured and PD analysis was performed;

[0031] (4) Construct a PK-PD model and conduct analysis.

[0032] Furthermore, step (1) includes the following steps: modeling POI rats; gavage of 4.2 g / kg / d of cuscuta seed-wolfberry gavage solution to the cuscuta seed-wolfberry administration group; and administration of an equal volume of water to the POI blank group.

[0033] Furthermore, the preparation method of the dodder seed-wolfberry intragastric solution includes the following steps: mixing dodder seed and wolfberry in a mass ratio of 1:1, crushing and adding 95% ethanol, with a solid-liquid ratio of 1:8, soaking, and reflux extraction; drying the residue and then decocting with water, combining the alcohol extract and the water extract to concentrate into an extract, adding pure water, and dissolving to obtain the dodder seed-wolfberry intragastric solution.

[0034] Furthermore, the Cuscuta seeds-Wolfberry fruit intragastric administration solution contained 3.9813 mg / g hyperoside, 1.3709 mg / g astragaloside, 1.02 mg / g isoquercetin, 1.2901 mg / g quercetin, 1.4589 mg / g kaempferol, and 8.9721 mg / g betaine.

[0035] Furthermore, in step (2), the content of the active substances of Cuscuta australis-Lycium barbarum detected in the plasma samples taken at different time points is subjected to non-compartmental model fitting analysis using DAS2.0 software to calculate the pharmacokinetic parameters Cmax, Tmax, t1 / 2z, AUC and MRT of each component.

[0036] Furthermore, in step (3), the log-linear, linear, Emax and S-type Emax pharmacodynamic models were used to fit the effects of Cuscuta-Wolfberry on the levels of E2, FSH, LH and AMH in rat serum, respectively, where the PD effect value was the difference between the efficacy values ​​of the POI blank group and the Cuscuta-Wolfberry administration group at each time point, and the PD model with the best fitting value was determined.

[0037] Further, in step (4), the concentration of each component at each time point in the pharmacokinetics is taken as the PK value, and the difference between the POI blank group and the treatment group (i.e., the dodder seed-wolfberry administration group) at each time point in the measured pharmacodynamic index is taken as the PD effect value. The "concentration-time-effect" curve is established in a one-to-one correspondence according to the time point sequence, and the data is imported into Phoenix 8.2 software for fitting according to the AIC and BIC parameters to establish a PK-PD model to express the ratio change between the PK index and the PD index. The K obtained according to the model fitting formula e0 、E max ,EC 50 , n, AIC and BIC parameters describe the relationship between each component and effect.

[0038] Further, the PK-PD model formula in step (4) is E=E0+E max *C n / (EC 50 n +C n ), where E is the effect value, E max is the maximum effect that can be produced, I max The maximum inhibitory effect that can be produced is EC 50 IC is the drug concentration that causes half of the maximal effect. 50 is the half-inhibitory concentration, C is the drug concentration, n is the shape factor, which reflects the slope of the middle section of the shape curve; E0 is the effect value before administration.

[0039] The optimal PK-PD model fitting formula of hyperoside, E2 and AMH is E=E0+Emax *C n / (EC 50 n +C n ), the AIC values ​​were 58.21 and 29.28, and the BIC values ​​were 61.04 and 30.62; the best fitting formula for FSH and LH was E=E0-I max *C n / (IC 50 n +C n ), the AIC values ​​were 37.69 and 27.38, and the BIC values ​​were 39.23 and 28.64, respectively.

[0040] The optimal PK-PD model fitting formula of astragalus glycosides, E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n ).

[0041] The optimal PK-PD model fitting formula of isoquercetin, E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n ).

[0042] The optimal PK-PD model fitting formula of quercetin, E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n ).

[0043] The optimal PK-PD model fitting formula of kaempferol, E2 and AMH is E=E0+E max *Cn / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n ).

[0044] The optimal PK-PD model fitting formula of betaine, E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n ).

[0045] The present invention provides the use of one or a mixture of hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine in preparing a medicine for treating POI.

[0046] The beneficial effects of the present invention include: the present invention adopts UPLC-TQ-MS / MS technology, and successfully establishes an analytical method for simultaneously quantifying 6 kinds of Cuscuta-Lycium barbarum pharmacodynamic substances (hyperoside, astragaloside, isoquercetin, quercetin, kaempferol, betaine) in rat plasma. The method has good specificity, no endogenous substance interference, good linear relationship, high precision, high accuracy, good extraction recovery rate, good stability, and no obvious matrix effect. The above indicators all meet the international analytical methodology standards, ensuring the scientificity and reliability of the results, and providing an efficient tool for the pharmacokinetic study of multi-component compound drugs.

[0047] The present invention successfully established a method for quantifying six active ingredients of Cuscuta chinensis-Wolfberry fruit for treating POI in rat plasma, and applied it to the in vivo pharmacokinetic study of POI rats; the study found that Cuscuta chinensis-Wolfberry fruit can be rapidly absorbed after oral administration, but is slowly cleared from the body, has a longer duration of action, and has a large difference in the peak concentrations of each ingredient.

[0048] Through PK-PD correlation analysis, it was found that there was no direct correspondence between the concentration and effect of most drugs after administration, and there was a certain lag phenomenon; and the correlation of different compounds to different efficacy indicators was different. Among them, hyperoside, astragaloside, and kaempferol had better callback effects, which may be the key pharmacological substances of Cuscuta australis-lycium barbarum to play a role in treating POI. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The ion current chromatograms of each component are extracted, where 1 is hyperoside, 2 is astragaloside, 3 is isoquercetin, 4 is quercetin, 5 is kaempferol, 6 is betaine, and 7 is formononetin (internal standard); from left to right, column A is blank plasma, column B is blank plasma + reference substance, and column C is drug-containing plasma;

[0050] Figure 2 The average blood drug concentration-time curve of the six components after rats were gavaged with Cuscuta australis and Lycium barbarum ( n=6);

[0051] Figure 3 is the plasma concentration-time bar graph of E2, FSH, LH, and AMH (n=6);

[0052] Figure 4 It is the plasma concentration-time curve of E2, FSH, LH and AMH;

[0053] Figure 5 It is the plasma concentration-time fitting curve diagram of E2, FSH, LH and AMH;

[0054] Figure 6 is the drug concentration-effect curve of hyperoside in POI rats (n=6);

[0055] Figure 7 is the drug concentration-effect curve of astragalus glycoside in POI rats (n=6);

[0056] Figure 8 is the drug concentration-effect curve of isoquercetin in POI rats (n=6);

[0057] Fig. 9 is the drug concentration-effect curve of quercetin in POI rats (n=6);

[0058] Fig.10 is the drug concentration-effect curve of kaempferol in POI rats (n=6);

[0059] Fig.11 This is the drug concentration-effect curve of betaine in POI rats (n=6). DETAILED DESCRIPTION

[0060] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0061] Xevo TQ-XS mass spectrometer was purchased from Waters, USA.

[0062] Hyperoside (batch number: 22041804), astragaloside (batch number: 21101302), isoquercetin (batch number: 20112404), betaine (batch number: DJ0615YA13), formononetin (batch number: H06S9Z69494) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; quercetin (batch number: Y29A10Y779806) and kaempferol (batch number: A01HJ190000) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products; vinylcyclohexene dioxide (vinylcyclohexene Dioxide, VCD, batch number: C14604065) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; rat estradiol (E2) kit (batch number: 202205), rat follicle-stimulating hormone (FSH) kit (batch number: 202205), rat luteinizing hormone (LH) kit (batch number: 202205), and anti-Mullerian hormone (AMH) kit (batch number: 202205) were all purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.

[0063] Female SD rats (200 ± 5 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with the experimental animal production license number SCXK (Liao) 2020-0001. The rats used in a single experiment were from the same batch, with free access to food and water, and were adaptively raised for 1 week. The room temperature of the animal room was 22 ± 1 ° C and the humidity was 45%-60%. The experimental operations were in accordance with the requirements of the Animal Ethics Committee of Harbin University of Commerce and were reviewed and approved by the Experimental Animal Ethics Committee of Harbin University of Commerce (review number HSDYXY-2022031).

[0064] The preparation method of the intragastric drug solution comprises the following steps: mixing Cuscuta seeds and Lycium barbarum in a 1:1 (mass ratio), crushing, adding 95% ethanol (solid-liquid ratio 1:8) and soaking for 12 hours, heating and refluxing extraction 3 times, each time for 3 hours; drying the drug residue and then decocting it with 10 times the mass of water 2 times, each time for 1 hour, combining the alcohol extract and the water extract to concentrate into an extract, adding pure water to dissolve to obtain a Cuscuta seeds-Lycium barbarum drug pair intragastric drug solution, and setting aside. The content of each component is: hyperoside 3.9813mg / g, astragaloside 1.3709mg / g, isoquercitrin 1.02mg / g, quercetin 1.2901mg / g, kaempferol 1.4589mg / g, betaine 8.9721mg / g.

[0065] The preparation method of the internal standard solution comprises the following steps: accurately weighing 1 mg of formononetin, placing it in a 10 mL volumetric flask, dissolving it with methanol and diluting it to the mark, shaking it well, and obtaining an internal standard stock solution. Accurately pipetting the internal standard stock solution into a volumetric flask, diluting it to 10 mL with methanol, shaking it well, and obtaining an internal standard solution with a formononetin concentration of 10 ng / mL. Store it in a refrigerator at 4°C for future use.

[0066] The preparation method of the reference solution comprises the following steps: accurately weighing appropriate amounts of hyperoside, astragalus glycoside, isoquercetin, quercetin, kaempferol, and betaine reference substances, respectively adding pure methanol to prepare 0.10 mg / mL hyperoside solution, 0.05 mg / mL astragalus glycoside solution, 0.05 mg / mL isoquercetin solution, 0.05 mg / mL quercetin solution, 0.05 mg / mL kaempferol solution, and 100.00 mg / mL betaine solution as reference solutions.

[0067] The instruments involved in the present invention, unless otherwise specified, are all conventional instruments already in the prior art and can be obtained through regular commercial channels. The reagents, experimental materials, etc. involved in the present invention, unless otherwise specified, are all conventional reagents and materials already in the prior art and can be obtained through regular commercial channels or prepared by conventional methods in the art. The experimental methods involved in the following embodiments, unless otherwise specified, are all conventional experimental methods already in the prior art.

[0068] The following is an introduction through specific embodiments.

[0069] Example 1

[0070] (1) Animal grouping and drug administration

[0071] The modeling method of POI rats includes the following steps: taking SD rats with normal estrous cycle (4-5 days), intraperitoneally injecting VCD modeling solution for 15 days, with a daily dose of 0.0015 mL per gram of rat body weight, and performing vaginal exfoliated cell smear examination for 14 consecutive days after the modeling is stopped for 8 weeks. If the estrous cycle is disordered, it proves that the modeling is successful.

[0072] The model POI rats were weighed and recorded before administration, and randomly divided into a dossier-wolfberry administration group (experimental group) and a POI blank group (i.e., model POI rats without administration), with 6 rats in each group. The experimental group was intragastrically administered with dossier-wolfberry gavage at a dose of 4.2 g / kg / d (in terms of crude drug), and the POI blank group was given an equal volume of water.

[0073] The preparation method of the VCD modeling liquid mentioned above comprises: fully mixing VCD and sesame oil in a ratio of 1.5 mL VCD to 18.5 mL sesame oil.

[0074] (2) Collecting plasma and pre-treating it

[0075] The rat plasma of the experimental group and the POI blank group was taken respectively, and pretreatment was performed, including the following steps: 100 μL of rat plasma was accurately aspirated, 10 μL of methanol was added, vortexed for 1 min, 10 μL of internal standard solution was added, vortexed for 1 min, 400 μL of methanol was added, vortexed for 4 min, centrifuged at 4°C and 14000 r / min for 10 min, 300 μL of supernatant was taken, dried with nitrogen at 37°C, 50 μL of methanol was added to the residue for re-dissolution, vortexed for 4 min, centrifuged at 4°C and 14000 r / min for 10 min, and the supernatant was taken.

[0076] (3) The supernatant was detected by UPLC-TQ-MS / MS for the contents of hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine.

[0077] Chromatographic conditions: Waters ACQUITY UPLC BEH C18 (1.7 μm, 50 mm × 2.1 mm) column; mobile phase: acetonitrile (A)-0.1% formic acid water (B); gradient elution (0-1 min, 15%-25% A; 1-4 min, 25%-40% A; 4-5 min, 40%-25% A; 5-6 min, 25%-15% A); the above percentages are all volume percentages. Injection volume: 2 μL; flow rate: 0.25 mL / min; column temperature: 30°C.

[0078] Mass spectrometry conditions: electrospray ion source (ESI); capillary voltage: 3.0 kV; cone gas flow rate: 50 L / h; ion source temperature: 150 °C; desolvation gas temperature: 350 °C; desolvation gas flow rate: 650 L / h; multiple reaction monitoring mode (MRM) scanning was adopted, and the quantitative ion pairs and parameter settings of the components to be measured are shown in Table 1.

[0079] Table 1 Quantitative ion pairs and parameter settings

[0080]

[0081] According to the peak areas obtained by detection, the contents of hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine were calculated respectively using regression equation.

[0082] The regression equation for detecting betaine content is Y=0.001X+177.19, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; X represents the content of betaine in plasma (in ng / mL). The betaine content (X0+X a ), the endogenous betaine content (X0) should be deducted and the depleted plasma betaine content (X a ) for analysis. When the pharmacokinetic determination of betaine content in drug-containing plasma is performed, the endogenous betaine content before oral administration is deducted accordingly.

[0083] The regression equation for detecting the content of hyperoside is Y=0.047X+0.3297, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the content of hyperoside in plasma (in ng / mL).

[0084] The regression equation for detecting the content of astragaloside is Y=0.0278X+0.8997, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the content of astragaloside in plasma (in ng / mL).

[0085] The regression equation for detecting the isoquercetin content is Y=0.0163X+0.56, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the content of isoquercetin in plasma (in ng / mL).

[0086] The regression equation for detecting the quercetin content is Y=0.0101X+0.1721, where Y represents the ratio of the peak area of ​​the analyte to the internal standard; and X represents the content of quercetin in plasma (in ng / mL).

[0087] The regression equation for detecting the kaempferol content is Y=0.0916X+0.1505, where Y represents the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard; and X represents the content of kaempferol in plasma (in ng / mL).

[0088] (4) According to the contents of hyperoside, astragaloside, isoquercetin, quercetin, kaempferol, and betaine detected in step (3), the data were fitted with a non-compartmental model using DAS2.0 software to calculate the pharmacokinetic parameters Cmax, Tmax, t1 / 2z, AUC, and MRT of each component.

[0089] The measured kinetic parameters are expressed as mean ± standard deviation. SPSS 25.0 software was used for statistical analysis. Origin was used to draw the average drug-time curve.

[0090] Example 2 Investigation Methodology

[0091] (1) Method specificity

[0092] Set up three groups: A, B and C.

[0093] Group A is the plasma of the POI blank group. 100 μL of plasma from the POI blank group rats was accurately drawn into a centrifuge tube, 10 μL of methanol was added, vortexed for 1 min, no internal standard was added, vortexed for 1 min, 400 μL of methanol was added, vortexed for 4 min, centrifuged at 4°C, 14000 r / min for 10 min, 300 μL of supernatant was taken, dried with nitrogen at 37°C, 50 μL of methanol was added to the residue for redissolution, vortexed for 4 min, centrifuged at 4°C, 14000 r / min for 10 min, and the supernatant was taken for determination. The rest of the experimental methods and detection methods refer to Example 1.

[0094] Group B consisted of POI blank group plasma + control substance. In each centrifuge tube, the POI blank group rat plasma was 100 μL, 10 μL of methanol was added to each tube, and vortexed for 1 min. Seven tubes were set up, and the control substance was added to the 1st to 6th tubes, and 10 μL of internal standard solution was added to the 7th tube.

[0095] There were 6 kinds of reference substances, namely 0.10 mg / mL hyperoside solution, 0.05 mg / mL astragaloside solution, 0.05 mg / mL isoquercetin solution, 0.05 mg / mL quercetin solution, 0.05 mg / mL kaempferol solution, and 100.00 mg / mL betaine solution. 10 μL of one reference substance solution was added to each tube.

[0096] Vortex for 1 min, add 400 μL methanol, vortex for 4 min, centrifuge at 4°C, 14000 r / min for 10 min, take 300 μL supernatant, blow dry with nitrogen at 37°C, add 50 μL methanol to the residue, vortex for 4 min, centrifuge at 4°C, 14000 r / min for 10 min, and take the supernatant for determination. The rest of the experimental methods and detection methods refer to Example 1.

[0097] Group C is drug-containing plasma. POI rats were fasted for 12 hours, and plasma samples were collected 1 hour after intragastric administration of Cuscuta australis-Wolfberry solution. The experimental methods and detection methods were referred to Example 1.

[0098] The experimental results are shown in Figure 1 , the horizontal axis is the peak time, and the vertical axis is the abundance. Figure 1 1-Hyperoside; 2-Astragaloside; 3-Isoquercitrin; 4-Quercetin; 5-Kaempferol; 6-Betaine; 7-Formononetin (internal standard). Hyperoside, Astragaloside, Isoquercitrin, Quercetin, Kaempferol, Formononetin (IS) were not detected in the blank plasma of rats, indicating that the endogenous components in the plasma did not affect the detection of the above components; betaine is an endogenous component, and the endogenous betaine content before gavage should be deducted; under the liquid phase gradient elution conditions of this experiment, each component has a good separation effect and has a good response signal. This method has good specificity and can be used for later biological detection.

[0099] (2) Linear relationship

[0100] Preparation of standard curve: 100 μL of POI blank group plasma was added to each tube, and 10 μL of each of hyperoside solution, astragalus glycoside solution, isoquercetin solution, quercetin solution, kaempferol solution, and betaine solution were added to each tube (one reference solution was added to each tube), vortexed for 1 min, 10 μL of internal standard solution was added to each tube, vortexed for 1 min, 400 μL of methanol was added to each tube, vortexed for 4 min, centrifuged at 4°C and 14000 r / min for 10 min, 300 μL of supernatant was taken, dried with nitrogen at 37°C, 50 μL of methanol was added to the residue to re-dissolve, vortexed for 4 min, centrifuged at 4°C and 14000 r / min for 10 min, and the supernatant was taken.

[0101] In the hyperoside solutions, the concentrations of hyperoside were 1, 20, 40, 60, 80, and 100 ng / mL, respectively.

[0102] In the astragaloside solution, the concentrations of astragaloside were 0.1, 10, 20, 30, 40, and 50 ng / mL, respectively.

[0103] In the isoquercetin solutions, the concentrations of isoquercetin are 0.1, 10, 20, 30, 40, and 50 ng / mL, respectively.

[0104] In the quercetin solutions, the concentrations of quercetin were 0.1, 10, 20, 30, 40, and 50 ng / mL, respectively.

[0105] In the kaempferol solutions, the concentrations of kaempferol were 0.1, 10, 20, 30, 40, and 50 ng / mL, respectively.

[0106] In the betaine solution, the concentration of betaine is 100, 1000, 5000, 10000, 50000, 100000 ng / mL respectively. The method in Example 1 is used for detection. The ratio of the area of ​​the analyte to the internal standard (Y) is used for the concentration of the analyte in plasma (X, in ng / mL) by partial least squares method for linear regression calculation to obtain a standard curve. The limit of quantification (LOQ) is measured with a standard signal-to-noise ratio (S / N) of 10, and the limit of detection (LOD) of the analyte is 3.

[0107] The standard curve equations of hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine in rat plasma are shown in Table 2. Each compound has good linearity within a certain range, r>0.99.

[0108] Table 2 Linear relationships of six compounds in rat plasma

[0109]

[0110] (3) Precision and accuracy

[0111] Take 100 μL of POI blank group plasma and add 10 μL of high, medium and low concentration reference solution respectively to obtain three blank spiked samples with final concentrations as quality control (QC) samples (one reference solution is added to each tube), and 6 parallel copies are vortexed for 1 min. 10 μL of internal standard solution is added to each tube, vortexed for 1 min, 400 μL of methanol is added to each tube, vortexed for 4 min, centrifuged at 4°C and 14000 r / min for 10 min, 300 μL of supernatant is taken, dried with nitrogen at 37°C, 50 μL of methanol is added to the residue for re-dissolution, vortexed for 4 min, centrifuged at 4°C and 14000 r / min for 10 min, and the supernatant is taken for determination. The detection method refers to Example 1, and the detection is repeated 6 times; the measurement is continuous for 3 days, and the concentration measured by the QC sample is calculated according to the accompanying standard curve of the day. The precision is expressed as RSD, and the accuracy is expressed as relative error (RE).

[0112] The results are shown in Table 3. The intra-day and inter-day precisions of the six compounds, hyperoside, astragaloside, isoquercetin, quercetin, kaempferol, and betaine in rat plasma were less than 15%; the intra-day accuracy ranged from -9.00% to 12.11%, and the inter-day accuracy ranged from -5.39% to 13.38%. Since the precision and accuracy did not exceed ±15%, they all met the requirements for biological sample testing.

[0113] Table 3 Precision and accuracy of 6 compounds in rat plasma (n=6)

[0114]

[0115] (4) Extraction recovery and matrix effect

[0116] Take 100 μL of POI blank group plasma and add 10 μL of high, medium and low concentration reference solution respectively (one reference solution is added to each tube) to obtain three blank spiked samples with final concentrations as quality control (QC) samples. Add 10 μL of internal standard solution to each tube and vortex for 1 min. Add 400 μL of methanol to each tube and vortex for 4 min. Centrifuge at 4°C and 14000 r / min for 10 min. Take 300 μL of supernatant and blow dry with nitrogen at 37°C. Add 50 μL of methanol to the residue for re-dissolution. Vortex for 4 min. Centrifuge at 4°C and 14000 r / min for 10 min. Take the supernatant for determination, and the peak area is recorded as A.

[0117] Take 100 μL of POI blank group plasma, add 20 μL of methanol, vortex for 1 min, centrifuge at 4°C and 14000 r / min for 10 min, take 300 μL of supernatant, blow dry with nitrogen at 37°C to obtain residue. Use the above method to set up 6 tubes of residue, add 10 μL of 0.10 mg / mL hyperoside solution, 0.05 mg / mL astragaloside solution, 0.05 mg / mL isoquercetin solution, 0.05 mg / mL quercetin solution, 0.05 mg / mL kaempferol solution, 100.00 mg / mL betaine solution (add 1 reference solution to each tube); add 10 μL of the corresponding internal standard with a concentration of 10 ng / mL to each tube, add 30 μL of methanol, vortex, centrifuge, take the supernatant for sampling and analysis, and record the peak area as B.

[0118] Take the reference solution of corresponding mass concentration, inject and analyze, the peak area is C.

[0119] The above operation was repeated for 5 times for each concentration. The extraction recovery and matrix effect were calculated according to the following formula: extraction recovery = A / B × 100%, matrix effect = B / C × 100%.

[0120] The experimental results are shown in Table 4. The extraction recoveries of six compounds, including hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine in rat plasma, were all between 83.1% and 103.45%, and the matrix effects were all between 86.97% and 113.62%, which met the requirements for biological sample detection.

[0121] Table 4 Extraction recovery and matrix effect of 6 compounds in rat plasma (n=6)

[0122]

[0123] (5) Stability

[0124] QC samples of low, medium and high mass concentrations were prepared, and 6 samples were prepared in parallel for each concentration to investigate the stability of the components to be tested under the following conditions.

[0125] Take 100 μL of POI blank group plasma and add 10 μL of high, medium and low concentration reference solution respectively (one reference solution is added to each tube) to obtain three final concentrations of blank spiked samples as quality control (QC) samples.

[0126] After the quality control (QC) samples were placed at 20℃±5℃ for 12h without treatment, 10μL of internal standard solution was added to each tube, vortexed for 1min, 400μL of methanol was added to each tube, vortexed for 4min, centrifuged at 4℃ and 14000r / min for 10min, 300μL of supernatant was taken, dried with nitrogen at 37℃, 50μL of methanol was added to the residue for re-dissolution, vortexed for 4min, centrifuged at 4℃ and 14000r / min for 10min, and the supernatant was taken for measurement. The measurement result was used as the short-term stability.

[0127] The quality control (QC) samples were untreated and frozen at -80°C and thawed at room temperature for 3 times. Then, 10 μL of internal standard solution was added to each tube and vortexed for 1 min. Then, 400 μL of methanol was added to each tube and vortexed for 4 min. The samples were centrifuged at 4°C and 14000 r / min for 10 min. 300 μL of supernatant was taken and dried with nitrogen at 37°C. 50 μL of methanol was added to the residue to re-dissolve it, vortexed for 4 min, and centrifuged at 4°C and 14000 r / min for 10 min. The supernatant was taken for measurement, and the measurement results were used as the stability of repeated freezing and thawing.

[0128] After the quality control (QC) samples were placed at -80°C without treatment for 1 month, 10 μL of internal standard solution was added to each tube and vortexed for 1 min. 400 μL of methanol was added to each tube and vortexed for 4 min. The tubes were centrifuged at 4°C and 14000 r / min for 10 min. 300 μL of supernatant was taken and dried with nitrogen at 37°C. 50 μL of methanol was added to the residue to re-dissolve it, vortexed for 4 min, and centrifuged at 4°C and 14000 r / min for 10 min. The supernatant was taken for measurement, and the measurement results were used as long-term stability.

[0129] The experimental results are shown in Table 5. This experiment investigated the stability of six compounds in plasma under different storage conditions: room temperature for 12 hours, repeated freeze-thaw cycles for three times, and storage at -80°C for one month. The results showed that all RSD values ​​were less than 15%, and all Measured values ​​were between -80% and 120%, indicating that they could maintain good stability under the experimental conditions.

[0130] Table 5 Stability of 6 compounds in rat plasma (n=6)

[0131]

[0132]

[0133] Example 3

[0134] The pharmacokinetics of Cuscuta Semen and Lycium Barbarum were studied by the method of Example 1. The experimental results are as follows: The concentrations of the six components in drug-containing plasma are listed in Table 6. The drug-time curves are shown in Table 6. Figure 2As shown in Table 7 and Table 8, the main pharmacokinetic parameters are listed in Table 7 and Table 8. It can be seen from the drug-time curve that after a single dose, the components of Cuscuta-Lycium barbarum are absorbed quickly in the body, the peak time of all components in the body is between 0.22-0.92h, and the half-life is between 4.43-7.97h, indicating that the components are slowly cleared in the body and have a long duration of action; the peak concentrations of the components vary greatly, ranging from 9.89-32060.15ng / mL, and the peak concentration of betaine is higher. All components show an obvious trend of increasing concentration first and then decreasing, indicating that the drug components are first absorbed into the blood and then slowly metabolized and eliminated in the body, which is in line with the typical oral metabolism characteristics of drugs. The blood concentration of flavonoid components is relatively low, indicating that the oral bioavailability of flavonoid components is low. Betaine alkaloids are well absorbed and have high blood concentrations.

[0135] Table 6 Concentrations of the six components in drug-containing plasma ( n=6)

[0136]

[0137] Table 7 Pharmacokinetic parameters of six compounds in plasma after oral administration of Cuscuta australis-Lycium barbarum to rats ( n=6)

[0138]

[0139] Table 8 Pharmacokinetic parameters of six compounds in plasma after oral administration of Cuscuta australis-Lycium barbarum to rats ( n=6)

[0140]

[0141] Pharmacokinetics is an important part of the modernization of traditional Chinese medicine research; it is based on the principle of kinetics and combines mathematical models to study the absorption, distribution, metabolism and excretion of traditional Chinese medicine in the body. Pharmacokinetics can quantitatively analyze the dynamic changes of multiple components in the body, providing a reliable theoretical basis for revealing and clarifying the material basis of the efficacy of traditional Chinese medicine and guiding clinical safe drug use. The present invention screened 6 active ingredients in Cuscuta chinensis-Lycium barbarum as PK indicators for subsequent pharmacokinetic analysis, namely hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine. The present invention uses UPLC-TQ-MS / MS to study the pharmacokinetic of these 6 active ingredients in rat plasma, laying the foundation for further studying the pharmacodynamic substances and mechanism of action of Cuscuta chinensis-Lycium barbarum in the treatment of POI.

[0142] The present invention has established a method with good linear relationship within a certain range, good specificity, high precision and accuracy, good stability, no matrix effect and good recovery rate, which can simultaneously quantify 6 compounds (hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine) in rat plasma, and used it for pharmacokinetic study of POI rats. The study found that Cuscuta chinensis-Lycium barbarum can be rapidly absorbed after oral administration, but its clearance in the body is slow, the action time is long, and the peak concentrations of each component vary greatly.

[0143] Example 4

[0144] PK-PD correlation analysis can simultaneously clarify the relationship between blood drug concentration, effect and time, and is helpful to comprehensively and systematically clarify scientific issues such as the pharmacodynamic material basis and mechanism of action of traditional Chinese medicine compound for treating diseases. The present invention combines the pharmacokinetic process study of the six active ingredients of Cuscuta seeds-Lycium barbarum in POI rat plasma with the pharmacodynamic study, establishes a PK-PD model, analyzes the correlation between the blood drug concentrations of hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine in Cuscuta seeds-Lycium barbarum and E2, FSH, LH, and AMH, and uses a mathematical model method for fitting, providing an experimental basis for the study of the pharmacodynamic material basis of the drug.

[0145] Animals were grouped, administered, and plasma samples were collected using the method in Example 1. Serum was obtained after plasma treatment for detection of E2, FSH, LH, and AMH levels. Rat estradiol (E2) kit, rat follicle-stimulating hormone (FSH) kit, rat luteinizing hormone (LH) kit, and anti-Mullerian hormone (AMH) kit were used to detect rat E2, FSH, LH, and AMH levels in serum at different blood collection time points, respectively. The specific method was referred to the kit instructions. SPSS25.0 software was used for single-factor analysis of variance.

[0146] 4.1 PD model analysis

[0147] The log-linear, linear, Emax and S-type Emax pharmacodynamic models were used to fit the levels of E2, FSH, LH and AMH in rat serum affected by Cuscuta-Lycium barbarum, respectively, where the PD effect value was the difference between the pharmacodynamic values ​​of the POI blank group and the Cuscuta-Lycium barbarum administration group at each time point, and the PD model with the best fitting value was determined. The following steps may be included:

[0148] (1) Data preparation and import

[0149] Data format: Ensure that the data file (Excel) contains pharmacodynamic-related variables (such as time, drug concentration, effect size, individual ID, etc.).

[0150] Import data: Open Phoenix and create a new project; click File → Import → select the data file and follow the wizard to complete the import; check the data table structure and confirm that the column name and data type are correct (such as numeric type, categorical variable).

[0151] (2) Create a pharmacodynamic analysis workflow

[0152] Create a new Workflow: Right-click "Workflows" in the project → `Add Workflow`; drag the required modules (such as Model, Effect Site, etc.) to the workflow canvas.

[0153] (3) Define the model structure

[0154] Select or customize a model: Double-click the Model module in the workflow. Select a preset pharmacodynamic model (such as Emax model, Sigmoid Emax model) in the model library, or customize the equation through Structural Model.

[0155] For example: \[E=E_0+\frac{E_{\text{max}}\cdotC^n}{EC_{50}^n+C^n}\]. Where \(E_0\) is the baseline effect, \(E_{\text{max}}\) is the maximum effect, \(EC_{50}\) is the half-maximal effect concentration, and \(n\) is the Hill coefficient. Define the input variables (such as concentration C, effect E) and the parameters to be estimated (\(E_0,E_{\text{max}},EC_{50},n\).

[0156] (4) Configuration parameter estimation method

[0157] Set the algorithm: In the model setting, select the parameter estimation algorithm (such as nonlinear least squares, maximum likelihood). Specify the error model (additive error, proportional error, etc.).

[0158] Initial value setting: Use Phoenix's "Preliminary Fit" function to automatically generate initial values.

[0159] (5) Run the fitting analysis

[0160] Execute the calculation: Click the Run button in the workflow to start the fit. Check the log window to make sure there are no errors (such as convergence failure, data errors).

[0161] (6) Result diagnosis and evaluation

[0162] Key output: K e0 、E max ,EC 50, n, AIC and BIC parameters.

[0163] Parameter estimates: View the estimated values ​​of parameters such as \(E_{\text{max}},EC_{50}\) and their confidence intervals.

[0164] Goodness of fit: Evaluate model quality through residual plots, AIC / BIC values, \(R^2\), etc.

[0165] Graphical verification: Generate a scatter plot of "observed value vs. predicted value" in the results. Draw the effect-concentration curve (Effect vs. Concentration) to check whether the model fits the data trend.

[0166] (7) Model optimization and verification

[0167] Adjust the model: If the fit is poor, try to modify the model structure (such as increasing the Hill coefficient, compartmental model).

[0168] PD model analysis results: The OD values ​​measured by the E2, FSH, LH, and AMH standards are the ordinate, and the blood drug concentration (ng / mL) is the abscissa. Phoenix software fits the standard curve, and the results are shown in Table 9. The concentration data of E2, FSH, LH, and AMH in serum collected at different time points calculated according to the standard curve are shown in Table 10. The concentration-time change trend is shown in Figure 3 and Figure 4 As shown; the concentration-time trend of ΔE2, ΔFSH, ΔLH and ΔAMH is shown Figure 5 shown.

[0169] Table 9 ELISA standard curves of E2, FSH, LH and AMH

[0170]

[0171] Table 10E2, FSH, LH and AMH concentrations in drug-containing serum ( n=6)

[0172]

[0173] After modeling, the levels of E2, FSH, LH and AMH in rats will change. The levels of E2 and AMH in rats will rise first and then fall after modeling; the levels of FSH and LH will fall first, then rise, then fall and then rise after modeling. The E2 level reaches the maximum concentration at 12h, then gradually declines, the AMH level reaches the maximum concentration at 24h, then gradually declines; the FSH level reaches the lowest concentration at 0.75h, then gradually rises, then gradually declines and then rises; the LH level reaches the lowest concentration at 0.5h, then gradually rises, then gradually declines and then rises. As shown in Table 10, all indicators of the model rats changed significantly after administration. Compared with the POI blank group, the E2 level of rats in the Cuscuta-Wolfberry group increased significantly at 8-12h (P<0.05), and there was no significant difference between the two groups after 12h (P>0.05); the FSH level decreased significantly at 0.75h and 4h after administration (P<0.05), and there was no significant difference between the two groups at other times (P>0.05); the LH level decreased significantly at 0.5h (P<0.01) and 6h (P<0.05) after administration, and there was no significant difference between the two groups at other times (P>0.05); the AMH level began to increase significantly at 12h (P<0.05), and increased significantly at 24h (P<0.01), and there was no significant difference between the two groups thereafter (P>0.05).

[0174] 4.2 PK-PD correlation analysis of Cuscuta australis-Lycium barbarum treatment of POI rats

[0175] The concentration of each component at each time point in the pharmacokinetics in Table 6 of Example 3 was taken as the PK value, and the difference between each time point (POI blank group-treatment group) in the measured efficacy index was taken as the PD effect value. The treatment group was the Cuscuta chinensis-Lycium barbarum administration group. The "concentration-time-effect" curve was established in a one-to-one correspondence according to the time point sequence, and the data was imported into Phoenix 8.2 software for fitting according to the AIC and BIC parameters to establish the optimal PK-PD model to express the ratio change between the PK index and the PD index. According to the model fitting formula, K e0 、E max ,EC 50 , n, AIC and BIC parameters describe the relationship between each component and effect.

[0176] Results of PK-PD correlation analysis of Cuscuta australis-Lycium barbarum in the treatment of POI rats:

[0177] (1) Screening of PK-PD model fitting formula

[0178] The AIC value and BIC value were used as the main criteria for measuring goodness of fit. The PK-PD model with smaller AIC value and BIC value was selected as the better fitting result. The results showed that the best PK-PD model fitting formula for hyperoside, E2 and AMH was E=E0+E max *C n / (EC 50 n +C n ), the AIC values ​​were 58.21 and 29.28, and the BIC values ​​were 61.04 and 30.62; the best fitting formula for FSH and LH was E=E0-I max *C n / (IC 50 n +C n ), the AIC values ​​were 37.69 and 27.38, and the BIC values ​​were 39.23 and 28.64, respectively. Similarly, the best PK-PD model fitting formula for astragalus glycosides with E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n The optimal PK-PD model fitting formula of isoquercetin, E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n The optimal PK-PD model fitting formula of quercetin, E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n The optimal PK-PD model fitting formula of kaempferol, E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n The best PK-PD model fitting formula of betaine, E2 and AMH is E=E0+E max *C n / (EC 50 n +C n ); The best fitting formula for FSH and LH is E=E0-I max *C n / (IC 50 n +C n ).

[0179] Where E is the effect size, E max is the maximum effect that can be produced, I max The maximum inhibitory effect that can be produced is EC 50 IC is the drug concentration that causes half of the maximal effect. 50 is the half-inhibitory concentration, C is the drug concentration, n is the shape factor, which reflects the slope of the middle section of the shape curve; E0 is the effect value before administration.

[0180] (2) PK-PD model fitting results of hyperoside

[0181] The parameters related to hyperoside after PK-PD fitting are shown in Table 11, and the concentration-effect curve is established, where the horizontal axis is the blood concentration and the vertical axis is the effect value. Figure 6 According to EC 50 The value can be inferred that the pharmacological effect of hyperoside in treating POI may be related to the levels of E2 and AMH, and the specific order of correlation is: E2>AMH>FSH>LH. e0 is the rate constant, which indicates the speed of the chemical component callback effect index. It can be seen that the order of hyperoside callback PD index is: AMH>E2>FSH>LH. The n value can reflect the linear relationship of the curve. The results show that in most models, if the n value is not 1, the curve is not a simple linear relationship. Figure 6 It can be seen that when E2, FSH, LH and AMH are used as effect indicators, the concentration-effect curves all show a counterclockwise hysteresis loop. This result indicates that the maximum drug effect lags behind the maximum blood drug concentration.

[0182] Table 11 PK-PD model parameters of 6 compounds (n=6)

[0183]

[0184]

[0185] (3) PK-PD model fitting results of astragaloside

[0186] The parameters related to astragalus glycoside after PK-PD fitting are shown in Table 11, and the blood drug concentration-effect curve is shown in Figure 7 According to EC 50 The value can be inferred that the efficacy of astragalus glycosides in treating POI may be related to the levels of AMH and E2, and the specific order of correlation is: AMH>E2>LH>FSH. e0 It can be seen that the order of the speed of astragalus glycosides to correct PD indicators is: AMH>E2>LH>FSH. When E2, FSH, LH and AMH are used as effect indicators, the concentration-effect curves also have counterclockwise hysteresis loops, indicating that there is an obvious hysteresis phenomenon between the maximum effect value and the maximum blood drug concentration.

[0187] (4) PK-PD model fitting results of isoquercetin

[0188] The relevant parameters of isoquercetin after PK-PD fitting are shown in Table 11, and its blood drug concentration-effect curve is shown in Figure 8 According to EC 50 The value can be inferred that the pharmacological effect of isoquercetin in treating POI may be related to the levels of E2 and LH, and the specific order of correlation is: E2>LH>AMH>FSH. e0 It can be seen that the order of the speed of isoquercetin correction of PD indicators is: AMH>E2>FSH>LH. Figure 8 It can be seen that when E2, FSH and AMH are used as effect indicators, the concentration-effect curves also have counterclockwise hysteresis loops, indicating that the maximum effect value and the maximum blood drug concentration have obvious hysteresis phenomena. When LH is used as the effect indicator, the concentration-effect curve shows a clockwise correlation without hysteresis phenomena.

[0189] (5) PK-PD model fitting results of quercetin

[0190] The relevant parameters of quercetin after PK-PD fitting are shown in Table 11, and its blood concentration concentration-effect curve is shown in Fig. 9 According to EC 50 The value can be inferred that the pharmacological effect of quercetin in treating POI may be related to the levels of E2 and AMH, and the specific order of correlation is: AMH>E2>LH>FSH. e0 It can be seen that the order of quercetin's callback to PD index is: AMH>E2>FSH>LH. Fig. 9It can be seen that when E2, FSH, LH and AMH are used as effect indicators, the concentration-effect curves also have counterclockwise hysteresis loops, indicating that there is an obvious hysteresis phenomenon between the maximum effect value and the maximum blood drug concentration.

[0191] (6) PK-PD model fitting results of kaempferol

[0192] The relevant parameters of kaempferol after PK-PD fitting are shown in Table 11, and its blood concentration-effect curve is shown in Fig.10 According to EC 50 The value can be inferred that the pharmacological effect of kaempferol in treating POI may be related to the levels of E2 and AMH, and the specific order of correlation is: AMH>E2>FSH=LH. e0 It can be seen that the order of the speed of kaempferol's callback to PD index is: AMH>E2>FSH>LH. Fig.10 It can be seen that when E2, FSH, LH and AMH are used as effect indicators, the concentration-effect curves also have counterclockwise hysteresis loops, indicating that there is an obvious hysteresis phenomenon between the maximum effect value and the maximum blood drug concentration.

[0193] (7) PK-PD model fitting results of betaine

[0194] The relevant parameters of betaine after PK-PD fitting are shown in Table 11, and its blood concentration-effect curve is shown in Fig.11 According to EC 50 The value can be inferred that the pharmacological effect of betaine in treating POI may be related to the levels of AMH and LH, and the specific order of correlation is: AMH>LH>E2>FSH. e0 It can be seen that the order of betaine callback PD index is: AMH>E2>FSH>LH. Fig.11 It can be seen that when E2 and AMH are used as effect indicators, the concentration-effect curves also have counterclockwise hysteresis loops, indicating that the maximum effect value and the maximum blood drug concentration have obvious hysteresis phenomena. When FSH and LH are used as effect indicators, the concentration-effect curves show a clockwise correlation and no hysteresis phenomenon occurs.

[0195] The Emax results of each component are shown in Table 11. The effect sizes of the six components on E2 are: hyperoside> astragaloside> kaempferol> isoquercetin> quercetin> betaine; the effect size of FSH is: hyperoside> quercetin> astragaloside> kaempferol> isoquercetin> betaine; the effect size of LH is: kaempferol> quercetin> astragaloside> betaine> hyperoside> isoquercetin; the effect size of AMH is: betaine> hyperoside> astragaloside> quercetin> kaempferol> isoquercetin. In summary, hyperoside, astragaloside, and kaempferol have better effects under the four efficacy indicators, and may be the key pharmacodynamic substances of Cuscuta australis-wolfberry in the treatment of POI.

[0196] The UPLC-TQ-MS / MS method was used to analyze the PK of six compounds after oral administration of Cuscuta chinensis-Lycium barbarum to POI rats. At the same time, the kit method was used to analyze the levels of E2, FSH, LH and AMH in the serum of POI rats. Finally, the pharmacokinetic data and pharmacodynamic data were analyzed by PK-PD correlation, and the drug-time-effect curve was constructed. The fitting results of each component showed that the components may work synergistically and work together to treat POI. The overall fitting results of PK-PD showed that hyperoside, astragaloside and kaempferol had similar efficacy to these efficacy indicators and had the best effect. Hyperoside is the pharmacopoeial quality control component of Cuscuta chinensis and is also one of its main active ingredients.

[0197] Example 5

[0198] The present invention establishes and optimizes the mass spectrometry conditions of 6 compounds. In the experiment, positive and negative ion scanning modes were investigated. The results show that 5 compounds, including hyperoside, astragaloside, isoquercetin, quercetin and kaempferol, have better responses in the negative ion scanning mode, and betaine has better responses in the positive ion scanning mode. The results show that Cuscuta chinensis-wolfberry fruit can be rapidly absorbed after oral administration, but the clearance in the body is slow, the action time is long, and the peak concentrations of various components are quite different. In addition, the flavonoid components all have certain double peaks. Flavonoids are characterized by rapid absorption, slow clearance and long duration of action in the body, but poor absorption capacity and low bioavailability; and the in vivo kinetic blood concentration-time curves of most flavonoids show a bimodal feature, which is speculated to be the result of flavonoids undergoing enterohepatic circulation in the body. At the same time, some flavonoid aglycones are also metabolites of some flavonoid glycosides. When they are absorbed again, the blood concentration-time curve will also show a bimodal curve. Through the analysis of experimental results, it was found that the combination of Cuscuta chinensis and Lycium barbarum may promote their absorption, and the combination of Cuscuta chinensis and Lycium barbarum may enhance the efficacy by promoting these components, reflecting the scientific nature of the compound compatibility.

[0199] In summary, the present invention adopts UPLC-TQ-MS / MS method, and PK analysis is performed on 6 compounds after POI rats are gavaged with Cuscuta chinensis-Wolfberry. At the same time, the kit determination method is used to perform PD analysis on the levels of E2, FSH, LH and AMH in the serum of POI rats. Finally, the pharmacokinetic data and pharmacodynamic data are subjected to PK-PD correlation analysis, and the drug-time-effect curve is constructed. The fitting results of each component show that the components may be synergistic and work together to treat POI. The overall fitting results of PK-PD show that hyperoside, astragaloside, and kaempferol are similar to these efficacy indicators and have the best effect. This experiment established the PK-PD model of Cuscuta chinensis-Wolfberry for the first time, which provides a reference for clarifying the material basis of the efficacy of Cuscuta chinensis-Wolfberry, clinical application and subsequent development and utilization, and also provides an experimental basis for the scientific and rational use of Cuscuta chinensis-Wolfberry.

[0200] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the pharmacological substances of Cuscuta seeds and Lycium barbarum in plasma, characterized in that: The following steps are involved: (1) Collection and pretreatment of plasma samples; (2) UPLC-TQ-MS / MS detection; Chromatographic conditions included: Waters ACQUITY UPLC BEH C18 column; mobile phase A was acetonitrile, mobile phase B was 0.1% formic acid in water; gradient elution, 0-1 min, 15%-25% A, 1-4 min, 25%-40% A, 4-5 min, 40%-25% A, 5-6 min, 25%-15% A; injection volume 2 μL; flow rate 0.25 mL / min; column temperature 30°C; The mass spectrometry conditions included: electrospray ion source ESI, capillary voltage of 3.0 kV, cone gas flow rate of 50 L / h, ion source temperature of 150°C, desolvation gas temperature of 350°C, desolvation gas flow rate of 650 L / h, and scanning in multiple reaction monitoring mode.

2. The method for detecting the medicinal substances of Cuscuta seeds and Lycium barbarum in plasma according to claim 1, characterized in that: In step (1), 10 μL of methanol was added to the plasma, vortexed for 1 min, and then 10 μL of the internal standard solution was added. After vortexing for 1 min, 400 μL of methanol was added, vortexed for 4 min, and then centrifuged at 4°C and 14000 r / min for 10 min. 300 μL of the supernatant was taken, dried with nitrogen at 37°C, 50 μL of methanol was added to the residue for re-dissolution, vortexed for 4 min, and centrifuged at 4°C and 14000 r / min for 10 min. The supernatant was used for UPLC-TQ-MS / MS detection in step (2).

3. The method for detecting the medicinal substances of Cuscuta australis-Wolfberry in plasma according to claim 2, characterized in that: The internal standard solution is a methanol solution of formononetin, and the concentration of formononetin in the internal standard solution is 10 ng / mL.

4. The method for detecting the medicinal substance of Cuscuta australis-Wolfberry in plasma according to any one of claims 1 to 3, characterized in that: The active ingredients of Cuscuta australis-wolfberry include hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine.

5. A method for constructing a PK-PD model of Cuscuta seeds and wolfberry fruit active substances, characterized in that: The following steps are involved: (1) Set up a dodder seed-wolfberry drug group and a POI blank group; (2) using the method according to any one of claims 1 to 4 to detect the active ingredients of Cuscuta australis-Lycium barbarum in plasma, and performing PK analysis; (3) Serum E2, FSH, LH, and AMH levels were measured and PD analysis was performed; (4) Construct a PK-PD model and conduct analysis.

6. The method for constructing the PK-PD model of the Cuscuta seeds and Lycium barbarum medicinal substances according to claim 5, characterized in that: In step (2), the content of Cuscuta australis-Wolfberry active substances detected in plasma samples taken at different time points is subjected to non-compartmental model fitting analysis using DAS2.0 software to calculate the pharmacokinetic parameters Cmax, Tmax, t1 / 2z, AUC and MRT of each Cuscuta australis-Wolfberry active substance.

7. The method for constructing a PK-PD model of the dodder seed-wolfberry fruit pharmacodynamic substance according to claim 5 or 6, characterized in that: In step (3), the log-linear, linear, Emax and S-type Emax pharmacodynamic models were used to fit the effects of Cuscuta-Wolfberry on the levels of E2, FSH, LH and AMH in rat serum, respectively. The PD effect value was the difference between the efficacy values ​​of the POI blank group and the Cuscuta-Wolfberry treatment group at each time point, and the PD model was determined.

8. The method for constructing a PK-PD model of the dodder seed-wolfberry fruit pharmacodynamic substance according to claim 5 or 6, characterized in that: In step (4), the concentration of each component at each time point in the pharmacokinetics is taken as the PK value, and the difference between the POI blank group and the Cuscuta australis-wolfberry administration group at each time point in the measured pharmacodynamic index is taken as the PD effect value. The "concentration-time-effect" curve is established in a one-to-one correspondence according to the time point sequence, and the data is imported into Phoenix 8.2 software for fitting according to the AIC and BIC parameters to establish a PK-PD model to express the ratio change between the PK index and the PD index. The K obtained according to the model fitting formula e0 、E max ,EC 50 , n, AIC and BIC parameters describe the relationship between each component and effect; the PK-PD model formula is E = E0 + E max *C n / (EC 50 n +C n ), where E is the effect value, E max is the maximum effect that can be produced, I max The maximum inhibitory effect that can be produced is EC 50 IC is the drug concentration that causes half of the maximal effect. 50 is the half-inhibitory concentration, C is the drug concentration, n is the shape factor, which reflects the slope of the middle section of the shape curve; E0 is the effect value before administration.

9. The method for constructing a PK-PD model of the dodder seed-wolfberry fruit pharmacodynamic substance according to claim 5 or 6, characterized in that: In step (1), the dodder seed-wolfberry administration group was gavaged with dodder seed-wolfberry gavage solution at a dosage of 4.2 g / kg / d; the POI blank group was given an equal volume of water; the dodder seed-wolfberry gavage solution contained 3.9813 mg / g hyperoside, 1.3709 mg / g astragaloside, 1.02 mg / g isoquercitrin, 1.2901 mg / g quercetin, 1.4589 mg / g kaempferol and 8.9721 mg / g betaine.

10. Use of one or a mixture of hyperoside, astragaloside, isoquercetin, quercetin, kaempferol and betaine in the preparation of a drug for treating POI.

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