A method for detecting the medicinal effect substances of semen euryae and fructus lycii in plasma

Abstract

This invention belongs to the field of biomedical technology and provides a method for detecting the pharmacodynamic substances of Cuscuta-Lycium barbarum in plasma. This method can simultaneously and quantitatively detect six pharmacodynamic substances of Cuscuta-Lycium barbarum (hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine) in rat plasma. Furthermore, it performs pharmacodynamic analysis on the hormone levels (E2, FSH, LH, and AMH) in the serum of POI rats, constructs drug-time-effect curves, and performs pharmacokinetic-pharmacodynamic correlation analysis. The method provided by this invention not only enables efficient and accurate quantitative analysis of pharmacodynamic substances but also provides 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.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a method for detecting the medicinal substances of Cuscuta chinensis and Lycium barbarum in plasma. Background Technology

[0002] Cuscuta chinensis and Lycium barbarum are classic Chinese medicinal herbs commonly used to nourish kidney yin and regulate liver and kidney function. Traditional medicine believes that Cuscuta chinensis has the effects of "tonifying the kidney and replenishing essence, nourishing the liver and improving eyesight," while Lycium barbarum is known for "nourishing the liver and kidneys, nourishing blood and calming the mind." Modern pharmacological research shows that Cuscuta chinensis and Lycium barbarum contain various bioactive components, such as flavonoids, glycosides, and betaine, which have antioxidant, anti-inflammatory, endocrine-regulating, and ovarian-protective effects. However, due to their complex composition and the involvement of multiple pharmacodynamic components, traditional research methods struggle to clarify their pharmacokinetic characteristics and pharmacodynamic mechanisms. Establishing detection methods for the six pharmacodynamic substances in Cuscuta chinensis-Lycium barbarum (hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine) is crucial for pharmacokinetic and pharmacodynamic mechanism analysis. Inadequate detection methods will affect subsequent pharmacokinetic and pharmacodynamic mechanism analyses.

[0003] Primary ovarian insufficiency (POI) is a disease characterized by premature ovarian failure, often leading to decreased estrogen levels and reduced ovarian reserve, severely impacting women's reproductive health and quality of life. Current treatments mainly include hormone replacement therapy, ovulation-inducing drugs, and traditional Chinese medicine (TCM) therapy. However, these therapies have certain limitations. For example, long-term hormone use may cause metabolic disorders, increased risk of cardiovascular disease and breast cancer; while TCM therapy has a relatively high safety profile, its active ingredients are unclear, and its treatment mechanism remains ambiguous. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting the pharmacodynamic substances of Cuscuta-Lycium barbarum in plasma. This method can simultaneously quantify six pharmacodynamic substances of Cuscuta-Lycium barbarum (hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine) in rat plasma, and further perform pharmacodynamic analysis on hormone levels (E2, FSH, LH, and AMH) in the serum of POI rats, constructing drug-time-effect curves and performing pharmacokinetic-pharmacokinetic (PK-PD) correlation analysis. The method provided by this invention not only enables efficient and accurate quantitative analysis of pharmacodynamic substances, but also provides 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-mentioned technical problems is as follows:

[0006] This invention provides a method for detecting the medicinal substances of Cuscuta chinensis-Lycium barbarum in plasma, comprising the following steps:

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

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

[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℃.

[0010] Mass spectrometry conditions included: electrospray ionization (ESI), capillary voltage of 3.0 kV, orifice gas flow rate of 50 L / h, ion source temperature of 150 °C, desolvent gas temperature of 350 °C, desolvent gas flow rate of 650 L / h, and multiple reaction monitoring (MRM) mode.

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

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

[0013] Furthermore, the active ingredients of Cuscuta-Lycium barbarum include hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine.

[0014] Furthermore, in step (2), the quantitative ion pairs and parameter settings for hyperoside include: ion mode [MH]. - , t R The time was 1.75 min, the mother 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 pairs and parameter settings for astragaloside include: ion mode [MH]. - , t RThe time was 2.00 min, the mother 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 pairs and parameter settings for isoquercitrin include: ion mode [MH]. - , t R The time was 1.71 min, the mother 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 pairs and parameter settings for quercetin include: ion mode [MH]. - , t R The time was 3.18 min, the mother 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 pairs and parameter settings for kaempferol include: ion mode [MH]. - , t R The time was 4.02 min, the mother 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 pairs and parameter settings for betaine include: ion mode [MH]. - , t R The time was 0.57 min, the mother ion was 118.09, the daughter ion was 59.00, the declustering voltage was 45 V, and the collision energy was 20 V.

[0020] Furthermore, 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 that of the internal standard; X represents the betaine content in plasma (in ng / mL). The betaine content (X0 + X...) is calculated... a When calculating the endogenous betaine content (X0), the base-free plasma betaine content (X0) should be deducted and recorded. a For analytical purposes, when determining the betaine content in drug-containing plasma using pharmacokinetic methods, the pre-gavage endogenous betaine content should be deducted.

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

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

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

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

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

[0026] The detection method described above, employed in this invention, offers advantages such as high specificity, good linearity, high precision, high accuracy, and good stability in separation. The extraction recoveries range from 83.1% to 103.45%, and the matrix effects range from 86.97% to 113.62%, meeting the requirements for biological sample detection.

[0027] This invention provides a method for constructing a PK-PD model of pharmacodynamic substances from Cuscuta chinensis and Lycium barbarum, comprising the following steps:

[0028] (1) Set up a Cuscuta-Lycium barbarum treatment group and a POI blank group;

[0029] (2) The above method was used to detect the pharmacodynamic substances of Cuscuta chinensis-Lycium barbarum in plasma and to perform PK analysis;

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

[0031] (4) Construct the PK-PD model and perform analysis.

[0032] Further, step (1) includes the following steps: obtaining POI rats by modeling; administering Cuscuta-Lycium barbarum oral solution to the Cuscuta-Lycium barbarum group by gavage at a dose of 4.2 g / kg / d; and administering an equal volume of water to the POI blank group.

[0033] Furthermore, the preparation method of Cuscuta chinensis-Lycium barbarum gavage solution includes the following steps: Cuscuta chinensis and Lycium barbarum are mixed in a mass ratio of 1:1, pulverized, and then 95% ethanol is added at a material-to-liquid ratio of 1:8. The mixture is then soaked and extracted by reflux. The residue is dried and then decocted with water. The ethanol extract and water extract are combined and concentrated into an extract. Pure water is added to dissolve the extract and obtain the Cuscuta chinensis-Lycium barbarum gavage solution.

[0034] Furthermore, the Cuscuta-Lycium barbarum gavage solution contains hyperoside 3.9813 mg / g, astragaloside 1.3709 mg / g, isoquercitrin 1.02 mg / g, quercetin 1.2901 mg / g, kaempferol 1.4589 mg / g, and betaine 8.9721 mg / g.

[0035] Furthermore, in step (2), the content of dodder-goji berries as active ingredients in plasma samples taken at different time points was analyzed by using DAS2.0 software to perform non-compartmental model fitting analysis on the data, and the pharmacokinetic parameters Cmax, Tmax, t1 / 2z, AUC and MRT of each component were calculated.

[0036] Furthermore, in step (3), the effects of Cuscuta chinensis-Lycium barbarum on the levels of E2, FSH, LH and AMH in rat serum were fitted using log-linear, linear, Emax and S-type Emax pharmacodynamic models, respectively. The PD effect value was the difference between the pharmacodynamic values ​​at each time point between the POI blank group and the Cuscuta chinensis-Lycium barbarum administration group. 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 pharmacokinetic analysis is taken as the PK value, and the difference between the POI blank group and the treatment group (i.e., the Cuscuta chinensis-Lycium barbarum administration group) at each time point in the measured pharmacodynamic index is taken as the PD effect value. This is done sequentially according to time points to establish a one-to-one correspondence, creating a "concentration-time-effect" curve. The data is then imported into Phoenix 8.2 software and fitted according to AIC and BIC parameters to establish a PK-PD model to express the ratio change between the PK and PD indices. The K value is obtained based on the model fitting formula. e0 E max EC 50 The parameters n, AIC, and BIC describe the relationship between the components and the effects.

[0038] Furthermore, in step (4), the PK-PD model formula is E = E0 + E max *C n / (EC 50 n +C n In the formula, E is the effect value. max To produce the maximum effect, I max To produce the maximum inhibitory effect, EC 50 IC50 is the drug concentration that produces half of the maximum effect. 50 , where is the half-inhibitory concentration, C is the drug concentration, n is the shape factor, reflecting the slope of the middle segment of the shape curve; E0 is the pre-dose effect value.

[0039] The optimal PK-PD model fitting formula for hyperoside with E2 and AMH is E = E0 + Emax *C n / (EC 50 n +C n The AIC values ​​were 58.21 and 29.28, respectively, and the BIC values ​​were 61.04 and 30.62, respectively; the best-fit 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, respectively, and the BIC values ​​were 39.23 and 28.64, respectively.

[0040] The optimal PK-PD model fitting formula for astragaloside with E2 and AMH is E = E0 + E max *C n / (EC 50 n +C n The best-fit 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 for isoquercitrin with E2 and AMH is E = E0 + E max *C n / (EC 50 n +C n The best-fit formula for FSH and LH is E = E0 - I. max *C n / (IC 50 n +C n ).

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

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

[0045] This invention provides the use of one or a mixture of several of hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol and betaine in the preparation of a medicament for treating POI.

[0046] The beneficial effects of this invention include: This invention successfully established an analytical method for the simultaneous quantification of six pharmacodynamic substances (hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine) in rat plasma using UPLC-TQ-MS / MS technology. This method exhibits good specificity, no interference from endogenous substances, good linearity, high precision, high accuracy, good extraction and recovery rate, good stability, and no significant matrix effects. All of these indicators meet international analytical methodological standards, ensuring the scientific validity and reliability of the results and providing an efficient tool for pharmacokinetic studies of multi-component compound drugs.

[0047] This invention successfully established a method for quantifying six active ingredients of Cuscuta chinensis-Lycium barbarum in rat plasma for the treatment of POI, and applied it to in vivo pharmacokinetic studies in POI rats. The study found that Cuscuta chinensis-Lycium barbarum can be rapidly absorbed after oral administration, but is cleared slowly in vivo, has a long duration of action, and the peak concentrations of each component differ significantly.

[0048] PK-PD correlation analysis revealed that most drug concentrations and effects did not show a direct correlation after administration, exhibiting a certain lag. Furthermore, the correlations of different compounds with different efficacy indicators varied. Hyperoside, astragaloside, and kaempferol showed better retrograde effects and may be key active substances for Cuscuta-Lycium barbarum to exert their therapeutic effect on POI. Attached Figure Description

[0049] Figure 1 The extraction ion chromatograms of each component are shown below. Among them, 1 is hyperoside, 2 is astragaloside, 3 isoquercitrin, 4 is quercetin, 5 is kaempferol, 6 is betaine, and 7 is styracin (internal standard). From left to right, column A is blank plasma, column B is blank plasma + reference standard, and column C is drug-containing plasma.

[0050] Figure 2 Average blood concentration-time curves of six components after oral administration of Cuscuta chinensis-Lycium barbarum to rats ( n = 6);

[0051] Figure 3 Plasma concentration-time bar charts for E2, FSH, LH, and AMH (n=6);

[0052] Figure 4 Plasma concentration-time curves for E2, FSH, LH, and AMH;

[0053] Figure 5 The plasma concentration-time fitting curves for E2, FSH, LH, and AMH are shown.

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

[0055] Figure 7 The drug concentration-effect curve of astragaloside in POI rats (n=6);

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

[0057] Figure 9 The drug concentration-response curve of quercetin in POI rats (n=6);

[0058] Figure 10 The drug concentration-effect curve of kaempferol in POI rats (n=6);

[0059] Figure 11 The drug concentration-effect curve of betaine in POI rats (n=6). Detailed Implementation

[0060] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

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

[0062] Hyperoside (batch number: 22041804), astragaloside (batch number: 21101302), isoquercitrin (batch number: 20112404), betaine (batch number: DJ0615YA13), and styracin (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 Institutes for Food and Drug Control; vinylcyclohexene dioxide was purchased from the National Institutes for Food and Drug Control. The reagents (VCD, batch number: C14604065) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; the 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-Müllerian hormone (AMH) kit (batch number: 202205) were all purchased from Jiangsu Enzyme Immunoassay Co., Ltd.

[0063] Female SD rats (200±5g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd., Experimental Animal Production License No. SCXK(Liaoning)2020-0001. All rats used in each experiment were from the same batch, with free access to food and water, and were acclimatized for one week. The animal room temperature was 22±1℃, and the humidity was 45%-60%. All experimental procedures complied with the requirements of the Animal Ethics Committee of Harbin University of Commerce and were approved by the Experimental Animal Ethics Committee of Harbin University of Commerce (Review No. HSDYXY-2022031).

[0064] The preparation method of the gavage solution includes the following steps: Cuscuta chinensis and Lycium barbarum are mixed in a 1:1 (mass ratio) and pulverized. The mixture is then soaked in 95% ethanol (material-to-liquid ratio 1:8) for 12 hours, and extracted by reflux three times, 3 hours each time. The residue is dried and then decocted twice with 10 times its weight of water, 1 hour each time. The ethanol and water extracts are combined and concentrated to a paste. The paste is then dissolved in pure water to obtain the Cuscuta chinensis-Lycium barbarum gavage solution, which is set aside for later use. The contents of each component are as follows: hyperoside 3.9813 mg / g, astragaloside 1.3709 mg / g, isoquercitrin 1.02 mg / g, quercetin 1.2901 mg / g, kaempferol 1.4589 mg / g, and betaine 8.9721 mg / g.

[0065] The preparation method of the internal standard solution includes the following steps: Accurately weigh 1 mg of gentianin and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with methanol, and shake well to obtain the internal standard stock solution. Accurately pipette the internal standard stock solution into a volumetric flask, dilute to 10 mL with methanol, and shake well to obtain an internal standard solution with a gentianin concentration of 10 ng / mL. Store at 4℃ for later use.

[0066] The preparation method of the reference solution includes the following steps: Accurately weigh appropriate amounts of hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine reference standards, and add them separately to pure methanol to prepare 0.10 mg / mL hyperoside solution, 0.05 mg / mL astragaloside solution, 0.05 mg / mL isoquercitrin solution, 0.05 mg / mL quercetin solution, 0.05 mg / mL kaempferol solution, and 100.00 mg / mL betaine solution, respectively, as reference solutions.

[0067] Unless otherwise specified, all instruments involved in this invention are conventional instruments already existing in the prior art and can be obtained through legitimate commercial channels. All reagents and experimental materials involved in this invention, unless otherwise specified, are conventional reagents and materials already existing in the prior art and can be obtained through legitimate commercial channels or prepared using conventional methods in the field. Unless otherwise specified, all experimental methods involved in the following embodiments are conventional experimental methods already existing in the prior art.

[0068] The following is a description through specific embodiments.

[0069] Example 1

[0070] (1) Animal grouping and administration

[0071] The modeling method for POI rats includes the following steps: Take SD rats with normal estrous cycles (4-5 days), inject them intraperitoneally with VCD modeling drug solution for 15 days, the dose is 0.0015 mL per gram of rat body weight per day, and after 8 weeks of modeling, examine vaginal exfoliated cell smears for 14 consecutive days. If the estrous cycle is disordered, it proves that the modeling is successful.

[0072] Before administration of the drug to the model POI rats, the rats were weighed and their weights were recorded. They were then randomly divided into a Cuscuta-Lycium barbarum administration group (experimental group) and a POI blank group (i.e., model POI rats that were not administered the drug), with 6 rats in each group. The experimental group was administered Cuscuta-Lycium barbarum gavage solution at a dose of 4.2 g / kg / d (based on crude drug), while the POI blank group was given an equal volume of water.

[0073] The preparation method of the VCD modeling solution mentioned above includes: mixing the VCD and sesame oil thoroughly at a ratio of 1.5 mL VCD and 18.5 mL sesame oil.

[0074] (2) Collect plasma and preprocess it.

[0075] Rat plasma from the experimental group and the POI blank group was collected and pretreated, including the following steps: 100 μL of rat plasma was accurately pipetted, 10 μL of methanol was added, and the mixture was vortexed for 1 min. Then, 10 μL of internal standard solution was added, and the mixture was vortexed for 1 min. After that, 400 μL of methanol was added, and the mixture was vortexed for 4 min. After centrifugation at 4 °C and 14000 r / min for 10 min, 300 μL of supernatant was collected and dried under nitrogen at 37 °C. The residue was reconstituted with 50 μL of methanol, vortexed for 4 min, and centrifuged at 4 °C and 14000 r / min for 10 min. The supernatant was collected.

[0076] (3) The contents of hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol and betaine in the supernatant were determined by UPLC-TQ-MS / MS.

[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 in 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); all percentages are volume percentages. Injection volume: 2 μL; flow rate: 0.25 mL / min; column temperature: 30 °C.

[0078] Mass spectrometry conditions: electrospray ionization source (ES I); capillary voltage: 3.0 kV; cone gas flow rate: 50 L / h; ion source temperature: 150 ℃; desolvent gas temperature: 350 ℃; desolvent gas flow rate: 650 L / h; multiple reaction monitoring (MRM) mode was used for scanning. The quantitative ion pairs and parameter settings of the analytes are shown in Table 1.

[0079] Table 1. Quantitative Ion Pairs and Parameter Settings

[0080]

[0081] Based on the peak areas obtained from the detection, the contents of hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine were calculated using regression equations.

[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 that of the internal standard; X represents the betaine content in plasma (in ng / mL). The betaine content (X0 + X...) is calculated. a When calculating the endogenous betaine content (X0), the base-free plasma betaine content (X0) should be deducted and recorded. a For analytical purposes. When determining the betaine content in drug-containing plasma using pharmacokinetic methods, the pre-orally generated betaine content should be deducted.

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

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

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

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

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

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

[0089] All measured dynamic parameters are expressed as mean ± standard deviation. The results are presented. Statistical analysis was performed using SPSS 25.0 software. Mean drug-time curves were plotted using Origin.

[0090] Example 2: Methodology

[0091] (1) Method specificity

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

[0093] Group A was the POI blank group plasma. 100 μL of POI blank group rat plasma was accurately pipetted into a centrifuge tube, 10 μL of methanol was added, and the tube was vortexed for 1 min without internal standard. After vortexing for another 1 min, 400 μL of methanol was added, and the tube was vortexed for 4 min. The tube was then centrifuged at 4°C and 14000 rpm for 10 min. 300 μL of the supernatant was collected, dried under nitrogen at 37°C, and the residue was reconstituted with 50 μL of methanol. The tube was vortexed for 4 min, and then centrifuged at 4°C and 14000 rpm for 10 min. The supernatant was then used for analysis. The remaining experimental and detection methods were as described in Example 1.

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

[0095] Six reference standards were used: 0.10 mg / mL hyperoside solution, 0.05 mg / mL astragaloside solution, 0.05 mg / mL isoquercitrin 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 standard solution was added to each tube.

[0096] Vortex for 1 min, add 400 μL of methanol, vortex for 4 min, centrifuge at 14000 r / min for 10 min at 4 °C, collect 300 μL of supernatant, dry under nitrogen at 37 °C, redissolve the residue in 50 μL of methanol, vortex for 4 min, centrifuge at 14000 r / min for 10 min at 4 °C, and collect the supernatant for analysis. The remaining experimental and detection methods are the same as in Example 1.

[0097] Group C consisted of drug-containing plasma. POI rats were fasted for 12 hours, and plasma samples were collected 1 hour after administration of Cuscuta chinensis-Lycium barbarum gavage solution. The experimental and detection methods were as described in Example 1.

[0098] The experimental results are shown in Figure 1 The horizontal axis represents peak time, and the vertical axis represents abundance. Figure 1 The following components were identified: 1-hypericin; 2-astragalin; 3-isoquercetin; 4-quercetin; 5-kaempferol; 6-betaine; and 7-amyrin (internal standard). Hypericin, astragalin, isoquercetin, quercetin, kaempferol, and amyrin (IS) were not detected in rat blank plasma, indicating that endogenous components in plasma do not affect the detection of these components. Betaine is an endogenous component, and the pre-gavage endogenous betaine content should be deducted. Under the liquid phase gradient elution conditions used in this experiment, each component showed good separation and a good response signal. This method has good specificity and can be used for subsequent biological detection.

[0099] (2) Linear relationship

[0100] Preparation of standard curve: Add 100 μL of POI blank plasma to each tube, and add 10 μL each of hyperoside solution, astragaloside solution, isoquercitrin solution, quercetin solution, kaempferol solution, and betaine solution to each tube (one reference solution per tube). Vortex for 1 min, add 10 μL of internal standard solution to each tube, vortex for 1 min, add 400 μL of methanol to each tube, vortex for 4 min, centrifuge at 14000 r / min at 4℃ for 10 min, collect 300 μL of supernatant, dry under nitrogen at 37℃, redissolve the residue in 50 μL of methanol, vortex for 4 min, centrifuge at 14000 r / min at 4℃ for 10 min, and collect the supernatant.

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

[0102] The concentrations of astragaloside in the solutions were 0.1, 10, 20, 30, 40, and 50 ng / mL.

[0103] The concentrations of isoquercitrin in the isoquercitrin solutions were 0.1, 10, 20, 30, 40, and 50 ng / mL.

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

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

[0106] The betaine solutions contained betaine concentrations of 100, 1000, 5000, 10000, 50000, and 100000 ng / mL. The method described in Example 1 was used for detection. A standard curve was obtained by performing a partial least squares linear regression operation on the plasma concentration (X, in ng / mL) using the ratio of the area of ​​the analyte to the area of ​​the internal standard (Y). The limit of quantitation (LOQ) was determined using a standard with a signal-to-noise ratio (S / N) of 10; the limit of detection (LOD) was defined as S / N = 3.

[0107] The standard curve equations for hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine in rat plasma are shown in Table 2. Each compound showed good linearity within a certain range, with 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 plasma and add 10 μL of each of the high, medium, and low concentration reference solutions to obtain three final concentrations of blank spiked samples as quality control (QC) samples (one reference solution added to each tube). Perform six replicates, vortex for 1 min, add 10 μL of internal standard solution to each tube, vortex for 1 min, add 400 μL of methanol to each tube, vortex for 4 min, centrifuge at 14000 r / min at 4℃ for 10 min, take 300 μL of supernatant, blow dry with nitrogen at 37℃, reconstitute the residue with 50 μL of methanol, vortex for 4 min, centrifuge at 14000 r / min at 4℃ for 10 min, take the supernatant for determination, the detection method is the same as in Example 1, repeat the detection 6 times; continuously measure for 3 days, calculate the concentration of QC sample according to the accompanying standard curve of the day, precision is expressed as RSD, accuracy is expressed as relative error (RE).

[0112] The results are shown in Table 3. In rat plasma, the intra-day and inter-day precision of the six compounds—hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine—were all 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 both precision and accuracy did not exceed ±15%, they met the requirements for biological sample detection.

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

[0114]

[0115] (4) Extraction recovery rate and matrix effect

[0116] Take 100 μL of POI blank plasma and add 10 μL of each of the high, medium and low concentration reference solutions (one reference solution per tube) to obtain three final concentration blank spiked samples as quality control (QC) samples. Add 10 μL of internal standard solution to each tube, vortex for 1 min, add 400 μL of methanol to each tube, vortex for 4 min, centrifuge at 4℃ and 14000 r / min for 10 min, take 300 μL of supernatant, blow dry with nitrogen at 37℃, add 50 μL of methanol to the residue to redissolve, vortex for 4 min, centrifuge at 4℃ and 14000 r / min for 10 min, take the supernatant for measurement, and record the peak area as A.

[0117] Take 100 μL of POI blank plasma, add 20 μL of methanol, vortex for 1 min, centrifuge at 14000 r / min for 10 min at 4℃, collect 300 μL of supernatant, and dry under nitrogen at 37℃ to obtain residue. Prepare 6 tubes of residue using the above method, and add 10 μL each of the following solutions: 0.10 mg / mL hyperoside, 0.05 mg / mL astragaloside, 0.05 mg / mL isoquercitrin, 0.05 mg / mL quercetin, 0.05 mg / mL kaempferol, and 100.00 mg / mL betaine (one reference solution added to each tube); add 10 μL of the corresponding internal standard at a concentration of 10 ng / mL to each tube, then add 30 μL of methanol, vortex, centrifuge, and inject the supernatant for analysis. Record the peak area as B.

[0118] Take a reference solution of the corresponding mass concentration, inject it for analysis, and the peak area is C.

[0119] The above procedures were performed in parallel to prepare five samples for each concentration. The extraction recovery and matrix effect were calculated using the following formulas: 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, namely hyperoside, astragaloside, isoquercitrin, 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 meet the requirements for biological sample detection.

[0121] Table 4. Extraction and recovery rates and matrix effects of six compounds from rat plasma (n=6)

[0122]

[0123] (5) Stability

[0124] Prepare QC samples of low, medium, and high quality concentrations, with six samples prepared in parallel for each concentration, and examine the stability of the analyte under the following conditions.

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

[0126] After the quality control (QC) samples were placed untreated at 20℃±5℃ for 12 h, 10 μL of internal standard solution was added to each tube, and the tubes were vortexed for 1 min. Then, 400 μL of methanol was added to each tube, and the tubes were vortexed for 4 min. After centrifugation at 4℃ and 14000 r / min for 10 min, 300 μL of supernatant was collected and dried under nitrogen at 37℃. The residue was reconstituted with 50 μL of methanol, vortexed for 4 min, and centrifuged at 4℃ and 14000 r / min for 10 min. The supernatant was then used for determination, and the results were taken as short-term stability.

[0127] After the quality control (QC) samples were subjected to three cycles of freezing at -80°C and thawing at room temperature without any treatment, 10 μL of internal standard solution was added to each tube, and the tubes were vortexed for 1 min. Then, 400 μL of methanol was added to each tube, and the tubes were vortexed for 4 min. After centrifugation at 4°C and 14000 r / min for 10 min, 300 μL of supernatant was collected and dried under nitrogen at 37°C. The residue was reconstituted with 50 μL of methanol, vortexed for 4 min, and centrifuged at 4°C and 14000 r / min for 10 min. The supernatant was then used for testing, and the results were taken as the stability of repeated freeze-thaw cycles.

[0128] After the quality control (QC) samples were left untreated at -80℃ for one month, 10 μL of internal standard solution was added to each tube, and the mixture was vortexed for 1 min. Then, 400 μL of methanol was added to each tube, and the mixture was vortexed for 4 min. After centrifugation at 4℃ and 14000 r / min for 10 min, 300 μL of the supernatant was collected and dried under nitrogen at 37℃. The residue was reconstituted with 50 μL of methanol, vortexed for 4 min, and centrifuged at 4℃ and 14000 r / min for 10 min. The supernatant was then used for analysis, and the results were taken as the 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, including 12 hours at room temperature, three repeated freeze-thaw cycles, and one month at -80°C. The results showed that all RSD values ​​were less than 15%, and all Measured values ​​were between -80% and 120%, indicating that the compounds maintained good stability under the experimental conditions.

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

[0131]

[0132]

[0133] Example 3

[0134] The pharmacokinetic study of Cuscuta chinensis-Lycium barbarum was conducted using the method described in Example 1. The experimental results are as follows: the concentrations of the six components in the drug-containing plasma are listed in Table 6, and the pharmacokinetic curves are shown in Figure 1. Figure 2As shown, the main pharmacokinetic parameters are listed in Tables 7 and 8. The pharmacokinetic curves show that after a single dose, the components of Cuscuta chinensis-Lycium barbarum are absorbed rapidly in vivo, with peak concentrations ranging from 0.22 to 0.92 hours and half-lives from 4.43 to 7.97 hours, indicating slow elimination and a longer duration of action. The peak concentrations of the components varied considerably, ranging from 9.89 to 32060.15 ng / mL, with betaine showing the highest peak concentration. All components exhibited a clear trend of initial increase followed by decrease in concentration, indicating that the drug components are first absorbed into the bloodstream and then slowly metabolized and eliminated in the body, consistent with typical oral drug metabolism characteristics. The blood concentrations of flavonoids were relatively low, indicating low oral bioavailability. Betaine alkaloids were well absorbed, resulting in higher 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 of rats after gavage administration of Cuscuta chinensis and Lycium barbarum. n=6)

[0138]

[0139] Table 8. Pharmacokinetic parameters of six compounds in plasma of rats after gavage administration of Cuscuta chinensis and Lycium barbarum. n=6)

[0140]

[0141] Pharmacokinetics is a crucial aspect of modern research on traditional Chinese medicine (TCM). Based on kinetic principles and combined with mathematical models, it studies the absorption, distribution, metabolism, and excretion of TCM in the body. Pharmacokinetics can quantitatively analyze the dynamic changes of multiple components in vivo, providing a reliable theoretical basis for revealing and elucidating the pharmacodynamic material basis of TCM and guiding safe clinical use. This invention screened six active ingredients from Cuscuta chinensis-Lycium barbarum as pharmacokinetic indicators for subsequent pharmacokinetic analysis: hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine. This invention applied UPLC-TQ-MS / MS to conduct pharmacokinetic studies on these six active ingredients in rat plasma, laying the foundation for further research on the pharmacodynamic substances and mechanisms of action of Cuscuta chinensis-Lycium barbarum in treating pox irritation syndrome (POI).

[0142] This invention establishes a method with good linearity, specificity, precision, accuracy, stability, matrix-free properties, and good recovery within a certain range. It can simultaneously quantify six compounds (hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine) in rat plasma and was used in pharmacokinetic studies in POI rats. The study found that Cuscuta chinensis-Lycium barbarum is rapidly absorbed after gavage administration, but its clearance in vivo is slow, its duration of action is long, and the peak concentrations of each component differ significantly.

[0143] Example 4

[0144] PK-PD correlation analysis can simultaneously elucidate the relationship between blood drug concentration, effect, and time, contributing to a comprehensive and systematic understanding of the pharmacodynamic material basis and mechanism of action of traditional Chinese medicine compound prescriptions in treating diseases. This invention, based on the pharmacokinetic process of six active components of Cuscuta chinensis and Lycium barbarum in POI rat plasma and combined with pharmacodynamic studies, established a PK-PD model. The correlation between the blood concentrations of hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine in Cuscuta chinensis and Lycium barbarum and E2, FSH, LH, and AMH was analyzed, and mathematical modeling was used for fitting, providing experimental evidence for the study of the pharmacodynamic material basis of this drug.

[0145] Animals were grouped, administered medication, and plasma samples were collected using the method described in Example 1. Serum was obtained after plasma processing and used to detect E2, FSH, LH, and AMH levels. Rat estradiol (E2) kits, rat follicle-stimulating hormone (FSH) kits, rat luteinizing hormone (LH) kits, and anti-Müllerian hormone (AMH) kits were used to detect rat E2, FSH, LH, and AMH levels at different blood collection time points, respectively, following the specific methods outlined in the kit instructions. One-way ANOVA was performed using SPSS 25.0 software.

[0146] 4.1 PD Model Analysis

[0147] The effects of Cuscuta chinensis-Lycium barbarum on serum E2, FSH, LH, and AMH levels in rats were fitted using log-linear, linear, Emax, and S-type Emax pharmacodynamic models, respectively. The PD effect value was the difference in pharmacodynamic values ​​at each time point between the POI blank group and the Cuscuta chinensis-Lycium barbarum treatment group. The optimal PD model was determined. This process may include the following steps:

[0148] (1) Data preparation and import

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

[0150] Importing data: Open Phoenix and create a new project; click File → Import → select the data file in the menu bar and follow the wizard to complete the import; check the data table structure and confirm that the column names and data types are correct (such as numeric and categorical variables).

[0151] (2) Create a pharmacodynamic analysis workflow

[0152] To create a new Workflow: Right-click "Workflows" in the project → Add Workflow; drag and drop the required modules (such as Model, Effect Site, etc.) onto 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 the Emax model or the Sigmoid Emax model) from the model library, or define a custom 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-maximum 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] Algorithm settings: In the model settings, select the parameter estimation algorithm (such as nonlinear least squares method, maximum likelihood method). Specify the error model (additive error, proportional error, etc.).

[0158] Initial value setting: Initial values ​​are automatically generated using Phoenix's "Initial Fit" feature.

[0159] (5) Run the fitting analysis

[0160] Perform the calculation: Click the Run button in the workflow to start the fitting process. Check the log window to ensure there are no errors (such as convergence failure or data errors).

[0161] (6) Result diagnosis and evaluation

[0162] Key Output: K e0 E max EC 50The parameters are n, AIC, and BIC.

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

[0164] Goodness of fit: The model quality is evaluated through residual plots, AIC / BIC values, and R².

[0165] Graphical validation: Generate a scatter plot of "observed values ​​vs. predicted values" in the results. Plot the effect vs. concentration curve to check if the model fits the data trend.

[0166] (7) Model optimization and validation

[0167] Adjust the model: If the fit is poor, try modifying the model structure (such as adding the Hill coefficient or using a compartmentalized model).

[0168] PD model analysis results: The OD values ​​measured from E2, FSH, LH, and AMH standards were plotted on the ordinate, and the blood drug concentration (ng / mL) on the abscissa. A standard curve was fitted using Phoenix software, 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 based on the standard curve, are shown in Table 10. The concentration-time trend is as follows: Figure 3 and Figure 4 As shown; the concentration-time trends of ΔE2, ΔFSH, ΔLH and ΔAMH are as follows. Figure 5 As shown.

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

[0170]

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

[0172]

[0173] After modeling, the levels of E2, FSH, LH, and AMH in rats changed. E2 and AMH levels initially increased and then decreased; FSH and LH levels initially decreased, then increased, then decreased again, and then increased again. E2 levels reached their maximum concentration at 12 hours, then gradually decreased; AMH levels reached their maximum concentration at 24 hours, then gradually decreased; FSH levels reached their minimum concentration at 0.75 hours, then gradually increased, then decreased, and then increased again; LH levels reached their minimum concentration at 0.5 hours, then gradually increased, then decreased, and then increased again. As shown in Table 10, all indicators changed significantly after drug administration to the model rats. Compared with the POI blank group, the E2 level in rats in the Cuscuta chinensis-Lycium barbarum treatment group increased significantly from 8 to 12 h (P < 0.05), and there was no significant difference between the two groups after 12 h (P > 0.05); the FSH level decreased significantly at 0.75 h and 4 h 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 0.5 h (P < 0.01) and 6 h after administration (P < 0.05), and there was no significant difference between the two groups at other times (P > 0.05); the AMH level began to increase significantly at 12 h (P < 0.05), increased significantly at 24 h (P < 0.01), and thereafter there was no significant difference between the two groups (P > 0.05).

[0174] 4.2 PK-PD Correlation Analysis of Cuscuta-Lycium barbarum in POI Rats

[0175] The concentrations of each component at each time point in the pharmacokinetic data of Table 6 in Example 3 were used as PK values. The difference between the measured pharmacodynamic indicators at each time point (POI blank group - treatment group) was used as PD effect values. The treatment group was the Cuscuta chinensis-Lycium barbarum administration group. The data were processed sequentially to establish a one-to-one correspondence between the time points, creating a "concentration-time-effect" curve. The data was then imported into Phoenix 8.2 software and fitted using AIC and BIC parameters to establish the optimal PK-PD model, expressing the ratio change between the PK and PD indicators. The K value obtained from the model fitting formula was... e0 E max EC 50 The parameters n, AIC, and BIC describe the relationship between the components and the effects.

[0176] Results of PK-PD correlation analysis in POI rats treated with Cuscuta chinensis-Lycium barbarum:

[0177] (1) Selecting the fitting formula for the PK-PD model

[0178] Using AIC and BIC values ​​as the main criteria for measuring goodness of fit, the PK-PD model with smaller AIC and BIC values ​​was selected as the better fit. The results showed that the best PK-PD model fit 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, respectively, and the BIC values ​​were 61.04 and 30.62, respectively; the best-fit 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, respectively, and the BIC values ​​were 39.23 and 28.64, respectively. Similarly, the optimal PK-PD model fitting formula for astragaloside with E2 and AMH is E = E0 + E max *C n / (EC 50 n +C n The best-fit formula for FSH and LH is E = E0 - I. max *C n / (IC 50 n +C n The optimal PK-PD model fit formula for isoquercitrin with E2 and AMH is E = E0 + E max *C n / (EC 50 n +C n The best-fit formula for FSH and LH is E = E0 - I. max *C n / (IC 50 n +C n The optimal PK-PD model fit formula for quercetin with E2 and AMH is E = E0 + E max *C n / (EC 50 n +C n The best-fit formula for FSH and LH is E = E0 - I. max *C n / (IC 50 n +C n The optimal PK-PD model fitting formula for kaempferol with E2 and AMH is E = E0 + E max *C n / (EC 50 n +C n The best-fit formula for FSH and LH is E = E0 - I. max *C n / (IC 50 n +C n The optimal PK-PD model fit formula for betaine with E2 and AMH is E = E0 + E max *C n / (EC 50 n +C n The best-fit 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 To produce the maximum effect, I max To produce the maximum inhibitory effect, EC 50 IC50 is the drug concentration that produces half of the maximum effect. 50 , where is the half-inhibitory concentration, C is the drug concentration, n is the shape factor, reflecting the slope of the middle segment of the shape curve; E0 is the pre-dose effect value.

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

[0181] Table 11 shows the relevant parameters of hyperoside fitted by PK-PD. A concentration-response curve was then established based on this, with the horizontal axis representing plasma concentration and the vertical axis representing the effect value. Figure 6 According to EC 50 The values ​​suggest that the therapeutic effect of hyperoside on POI may be related to E2 and AMH levels, with the specific correlation order being: E2 > AMH > FSH > LH. e0 Let be the rate constant, representing the speed at which the chemical component's PD-reversal index is adjusted. Therefore, the order of speed at which hyperoside reverses the PD index is: AMH > E2 > FSH > LH. The value of n reflects the linear relationship of the curve; results show that in most models, if the value of n 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 for 6 compounds (n=6)

[0183]

[0184]

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

[0186] The relevant parameters of astragaloside fitted by PK-PD are shown in Table 11, and its blood concentration-effect curves are shown in Table 11. Figure 7 According to EC 50 The values ​​suggest that the therapeutic effect of astragaloside on POI may be related to AMH and E2 levels, with the specific order of correlation being: AMH > E2 > LH > FSH. (Based on K...) e0 It can be seen that the order of the speed at which astragaloside corrects PD indicators is: AMH > E2 > LH > FSH. When E2, FSH, LH and AMH are used as effect indicators, the concentration-response curves also show a counterclockwise hysteresis loop, indicating that there is a significant hysteresis between the maximum effect value and the maximum plasma concentration.

[0187] (4) Fitting results of the PK-PD model of isoquercitrin

[0188] The relevant parameters of isoquercitrin fitted by PK-PD are shown in Table 11, and its blood concentration-effect curve is shown in Table 11. Figure 8 According to EC 50 The values ​​suggest that the therapeutic effect of isoquercitrin on POIs may be related to E2 and LH levels, with the specific order of correlation being: E2 > LH > AMH > FSH. (Based on K...) e0 It can be seen that the order of speed at which isoquercitrin corrects PD indicators is: AMH > E2 > FSH > LH. From... Figure 8 It can be seen that when E2, FSH and AMH are used as effect indicators, the concentration-response curves all have a counterclockwise hysteresis loop, indicating that there is a significant hysteresis between the maximum effect value and the maximum blood drug concentration. When LH is used as the effect indicator, the concentration-response curve shows a clockwise correlation and no hysteresis.

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

[0190] The relevant parameters of quercetin fitted by PK-PD are shown in Table 11, and its blood concentration-effect curves are shown in Table 11. Figure 9 According to EC 50 The values ​​suggest that the therapeutic effect of quercetin on POIs may be related to E2 and AMH levels, with the specific order of correlation being: AMH > E2 > LH > FSH. (Based on K...) e0 It can be seen that the order of quercetin's PD index correction speed is: AMH > E2 > FSH > LH. From... Figure 9It can be seen that when E2, FSH, LH and AMH are used as effect indicators, the concentration-effect curves also have a counterclockwise hysteresis loop, indicating that there is a significant hysteresis between the maximum effect value and the maximum blood drug concentration.

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

[0192] The relevant parameters of kaempferol fitted by PK-PD are shown in Table 11, and its blood concentration-effect curves are shown in Table 11. Figure 10 According to EC 50 The values ​​suggest that the therapeutic effect of kaempferol on point of injury (POI) may be related to E2 and AMH levels, with the specific order of correlation being: AMH > E2 > FSH = LH. (Based on K...) e0 It can be seen that the order of the speed of kaempferol's PD indicator correction is: AMH > E2 > FSH > LH. From... Figure 10 It can be seen that when E2, FSH, LH and AMH are used as effect indicators, the concentration-effect curves also have a counterclockwise hysteresis loop, indicating that there is a significant hysteresis between the maximum effect value and the maximum blood drug concentration.

[0193] (7) Fitting results of the PK-PD model for betaine

[0194] The relevant parameters of betaine fitted by PK-PD are shown in Table 11, and its blood concentration-effect curves are shown in Table 11. Figure 11 According to EC 50 The values ​​suggest that the therapeutic effect of betaine on POI may be related to AMH and LH levels, with the specific order of correlation being: AMH > LH > E2 > FSH. (Based on K...) e0 It can be seen that the order of the speed of betaine correction using the PD indicator is: AMH > E2 > FSH > LH. From... Figure 11 It can be seen that when E2 and AMH are used as effect indicators, the concentration-response curves also have counterclockwise hysteresis loops, indicating that there is a significant hysteresis between the maximum effect value and the maximum blood drug concentration. When FSH and LH are used as effect indicators, the concentration-response curves show a clockwise correlation and no hysteresis occurs.

[0195] The Emax results for each component are shown in Table 11. The order of the effects of the six components on E2 is: hyperoside > astragaloside > kaempferol > isoquercitrin > quercetin > betaine; the order of the effects on FSH is: hyperoside > quercetin > astragaloside > kaempferol > isoquercitrin > betaine; the order of the effects on LH is: kaempferol > quercetin > astragaloside > betaine > hyperoside > isoquercitrin; and the order of the effects on AMH is: betaine > hyperoside > astragaloside > quercetin > kaempferol > isoquercitrin. In summary, hyperoside, astragaloside, and kaempferol exhibit superior effects under these four efficacy indicators and may be the key active substances for Cuscuta chinensis-Lycium barbarum to exert their therapeutic effects on POI.

[0196] Six compounds were analyzed by pharmacokinetic (PK) assay after oral administration of Cuscuta chinensis and Lycium barbarum to POI rats using UPLC-TQ-MS / MS. Simultaneously, serum E2, FSH, LH, and AMH levels in POI rats were analyzed by a kit-based pharmacodynamic assay. Finally, PK-PD correlation analysis was performed on the pharmacokinetic and pharmacodynamic data, and drug-time-effect curves were constructed. The fitting results for each component indicated that the components may have a synergistic effect, working together to treat POI. The overall PK-PD fitting results showed that hyperoside, astragaloside, and kaempferol had similar efficacy to these pharmacodynamic indicators and exhibited the best effects. Hyperoside is a pharmacopoeia-controlled component of Cuscuta chinensis and is also one of its main active ingredients.

[0197] Example 5

[0198] This invention established and optimized the mass spectrometry conditions for six compounds. The experiments investigated both positive and negative ion scanning modes. The results showed that hyperoside, astragaloside, isoquercitrin, quercetin, and kaempferol showed better responses in the negative ion scanning mode, while betaine showed a better response in the positive ion scanning mode. The results also indicated that Cuscuta chinensis-Lycium barbarum var. sarcodactylis was rapidly absorbed after oral administration, but its clearance in vivo was slow and its duration of action was prolonged. The peak concentrations of the various components differed significantly, and the flavonoid components all exhibited bimodal peaks. Flavonoids are characterized by rapid absorption, slow clearance, and long duration of action in vivo, but their absorption capacity and bioavailability are relatively poor. Furthermore, the pharmacokinetic plasma concentration-time curves of most flavonoids exhibit a bimodal pattern, which is speculated to be a result of enterohepatic circulation. Additionally, some flavonoid aglycones are also metabolites of some flavonoid glycosides; when these are reabsorbed, their plasma concentration-time curves also show a bimodal pattern. Analysis of experimental results suggests that the combination of Cuscuta chinensis and Lycium barbarum may promote their absorption, and this combination may enhance the efficacy of the drugs by promoting the absorption of these components, demonstrating the scientific basis of the compound formulation.

[0199] In summary, this invention employed UPLC-TQ-MS / MS to perform pharmacokinetic (PK) analysis on six compounds obtained from oral administration of Cuscuta chinensis-Lycium barbarum to POI rats. Simultaneously, pharmacodynamic (PD) analysis was performed on serum E2, FSH, LH, and AMH levels in POI rats using a kit-based method. Finally, PK-PD correlation analysis was conducted between pharmacokinetic and pharmacodynamic data, and drug-time-effect curves were constructed. The fitting results for each component indicate that the components may have a synergistic effect, working together to treat POI. The overall PK-PD fitting results show that hyperoside, astragaloside, and kaempferol have similar efficacy to these pharmacodynamic indicators and exhibit the best effects. This experiment is the first to establish a PK-PD model for Cuscuta chinensis-Lycium barbarum, providing a reference for elucidating the pharmacodynamic material basis, clinical application, and subsequent development and utilization of Cuscuta chinensis-Lycium barbarum, and also providing experimental evidence for the scientific and rational use of Cuscuta chinensis-Lycium barbarum.

[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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting the medicinal substances of Cuscuta chinensis-Lycium barbarum in plasma, characterized in that, The active ingredients of Cuscuta-Lycium barbarum include hyperoside, astragaloside, isoquercitrin, quercetin, kaempferol, and betaine; the method for detecting the active ingredients of Cuscuta-Lycium barbarum in plasma includes the following steps: (1) Collection of plasma samples and pretreatment: Add 10 μL of methanol to the plasma, vortex for 1 min, add 10 μL of internal standard solution, vortex for 1 min, add 400 μL of methanol, vortex for 4 min, centrifuge at 4℃ and 14000 r / min for 10 min, take 300 μL of supernatant, blow dry with nitrogen at 37℃, add 50 μL of methanol to the residue to redissolve, vortex for 4 min, centrifuge at 4℃ and 14000 r / min for 10 min, take the supernatant for UPLC-TQ-MS / MS detection in step (2); (2) UPLC-TQ-MS / MS detection was performed; 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℃. Mass spectrometry conditions included: electrospray ionization (ESI), capillary voltage of 3.0 kV, orifice gas flow rate of 50 L / h, ion source temperature of 150 °C, desolvent gas temperature of 350 °C, desolvent gas flow rate of 650 L / h, and multiple reaction monitoring (MRM) mode.

2. The method for detecting the pharmacologically active substances of Cuscuta chinensis-Lycium barbarum in plasma according to claim 1, characterized in that, The internal standard solution was a methanol solution of gentianin, and the concentration of gentianin in the internal standard solution was 10 ng / mL.

3. A method for constructing a PK-PD model of pharmacodynamic substances from Cuscuta chinensis and Lycium barbarum, characterized in that, Includes the following steps: (1) Set up a Cuscuta-Lycium barbarum treatment group and a POI blank group; (2) The method described in claim 1 or 2 is used to detect the pharmacodynamic substances of Cuscuta chinensis-Lycium barbarum in plasma and to perform PK analysis; (3) Serum levels of E2, FSH, LH and AMH were measured and PD analysis was performed; (4) Construct the PK-PD model and perform analysis.

4. The method for constructing the PK-PD model of the pharmacodynamic substances of Cuscuta chinensis-Lycium barbarum according to claim 3, characterized in that, In step (2), the content of dodder-goji berries in plasma samples taken at different time points was analyzed by non-compartmental model fitting using DAS2.0 software, and the pharmacokinetic parameters Cmax, Tmax, t1 / 2z, AUC and MRT of each dodder-goji berry active ingredient were calculated.

5. The method for constructing the PK-PD model of the pharmacodynamic substances of Cuscuta chinensis-Lycium barbarum according to claim 3 or 4, characterized in that, In step (3), the effects of Cuscuta chinensis-Lycium barbarum on the levels of E2, FSH, LH and AMH in rat serum were fitted using log-linear, linear, Emax and S-type Emax pharmacodynamic models, respectively. The PD effect value was determined by the difference in pharmacodynamic values ​​at each time point between the POI blank group and the Cuscuta chinensis-Lycium barbarum administration group.

6. The method for constructing the PK-PD model of dodder-wolfberry pharmacodynamic substances according to claim 3 or 4, characterized in that, In step (4), the concentration of each component at each time point in pharmacokinetic analysis is taken as the PK value, and the difference between the POI blank group and the Cuscuta-Lycium barbarum administration group at each time point in the measured pharmacodynamic index is taken as the PD effect value. This is done sequentially according to time points to establish a one-to-one correspondence, creating a "concentration-time-effect" curve. The data is then imported into Phoenix 8.2 software and fitted using AIC and BIC parameters to establish a PK-PD model to express the ratio change between the PK and PD indices. The K value is obtained based on the model fitting formula. e0 E max EC 50 The parameters n, AIC, and BIC describe the relationships between components and effects; the PK-PD model formula is E=E0+E max *C n / (EC 50 n +C n In the formula, E is the effect value. max To produce the maximum effect, I max To produce the maximum inhibitory effect, EC 50 IC50 is the drug concentration that produces half of the maximum effect. 50 , where is the half-inhibitory concentration, C is the drug concentration, n is the shape factor, reflecting the slope of the middle segment of the shape curve; E0 is the pre-dose effect value.

7. The method for constructing the PK-PD model of the pharmacodynamic substances of Cuscuta chinensis-Lycium barbarum according to claim 3 or 4, characterized in that, In step (1), the Cuscuta-Lycium barbarum administration group was given Cuscuta-Lycium barbarum gavage solution by gavage at a dose of 4.2 g / kg / d; the POI blank group was given an equal volume of water; the Cuscuta-Lycium barbarum gavage solution contained hyperoside 3.9813 mg / g, astragaloside 1.3709 mg / g, isoquercitrin 1.02 mg / g, quercetin 1.2901 mg / g, kaempferol 1.4589 mg / g and betaine 8.9721 mg / g.

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