A self-microemulsion of angelica and peony root and its preparation method and application

Abstract

The invention provides an angelica-peony self-microemulsion and a preparation method and application thereof. The angelica-peony self-microemulsion consists of 15.90% angelica-peony powder extract, a mixed emulsifier, an auxiliary emulsifier and an oil phase, has obvious advantages in improving solubility, drug loading and the like, lays a foundation for improving bioavailability, and has important application value in preparing hypoglycemic and / or lipid-lowering drugs.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, in particular to an angelica and peony self-microemulsion and a preparation method and application thereof. Background Art

[0002] Danggui Shaoyao Powder has the effects of nourishing blood and regulating liver, strengthening spleen and removing dampness. Animal pharmacokinetic studies have shown that Danggui Shaoyao Powder includes 7 chemical components, including paeoniflorin, paeoniflorin, ligustilide A, n-butylphthalide, ligustilide, atractylodes lactone III, and Tokinolide B, which can enter the blood after administration and have a certain exposure level in the body.

[0003] However, paeoniflorin and paeoniflorin have certain solubility in water, but their lipophilicity and biomembrane permeability are poor, resulting in an absolute oral bioavailability of only about 2%. Cnidium lactone A, n-butylphthalide, ligustilide, Tokinolide B, etc. are volatile oil substances, and Atractylodes lactone III is a sesquiterpenoid substance, which is easily soluble in chloroform and ethyl acetate, has strong fat solubility but poor water solubility, which affects the dissolution in the body and further affects the absorption in the body. Therefore, it is of great significance to improve the lipophilicity or solubility of the above compounds through formulation technology, thereby improving the absorption in the body and oral bioavailability.

[0004] Self-microemulsifying drug delivery systems (SMEDDS) are composed of surfactants (also known as emulsifiers), oil phases and / or co-emulsifiers. The drug is completely dissolved in the above-mentioned mixture to form a uniform solution. After entering the body, it spontaneously emulsifies under the action of gastrointestinal peristalsis to form nanoemulsions with a particle size between 10 and 100 nm.

[0005] Self-microemulsifying drug delivery system is a formulation technology that has been widely studied at home and abroad and is still developing in recent years. It can improve the bioavailability of drugs. The mechanism mainly includes increasing the solubility of poorly soluble drugs to improve the solubility of drugs, increasing the lipophilicity of drugs by dissolving hydrophilic drugs in nanoemulsions containing surfactants and oil phases, or forming nanoemulsions to reduce the Zeta potential value to improve the permeability of biomembranes. In recent years, there have been many reports on self-microemulsifying drug delivery systems of traditional Chinese medicine, but most of them are Chinese medicine monomers, single medicinal material extracts, etc. There are few reports on self-microemulsifying drug delivery systems of compound Chinese medicine extracts due to the large number of complex ingredients and low drug loading. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an angelica and peony root self-microemulsion.

[0007] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned Angelica and Paeonia lactiflora self-microemulsion.

[0008] Another technical problem to be solved by the present invention is to provide application of the above-mentioned Angelica sinensis and Paeonia lactiflora self-microemulsion.

[0009] The technical solution adopted by the present invention is:

[0010] A self-microemulsion of angelica and peony root, comprising 15.90% angelica and peony root powder extract, a mixed emulsifier, an auxiliary emulsifier and an oil phase, wherein:

[0011] The mixed emulsifier is 18.92%-34.69% Tween 80 and 6.31%-11.56% Kolliphor RH40; the auxiliary emulsifier is 11.21%-36.44% propylene glycol and 5.61%-18.22% glycerol; the oil phase is 4.21%-21.03% caprylic acid capric acid triglyceride; or

[0012] The mixed emulsifier is 16.82%-33.64% Tween 80 and 8.41%-16.82% Kolliphor HS15; the auxiliary emulsifier is 8.41%-36.44% propylene glycol and 4.21%-18.22% glycerol; and the oil phase is 4.21%-21.03% caprylic acid capric acid triglyceride.

[0013] Preferably, the above-mentioned Angelica and Peony self-microemulsion is composed of 15.90% Angelica and Peony powder extract, a mixed emulsifier, an emulsifier and an oil phase, wherein the mixed emulsifier is 18.92%-33.64% Tween 80 and 8.41%-11.56% Kolliphor RH40 / HS15; the emulsifier is 11.21%-36.44% propylene glycol and 5.61%-18.22% glycerol; and the oil phase is 4.21%-21.03% caprylic and capric triglyceride.

[0014] Preferably, the above-mentioned Angelica and Peony self-microemulsion is composed of 15.9% Angelica and Peony powder extract, 31.54% Tween 80, 10.51% Kolliphor RH40, 19.62% propylene glycol, 9.81% glycerol, and 12.62% caprylic and capric triglyceride.

[0015] The percentages of the above components are all weight percentages.

[0016] Preferably, the above-mentioned Angelica and Peony Self-microemulsion, the Angelica and Peony Powder extract is prepared by the following method: Angelica, Peony, Chuanxiong, Atractylodes, Alisma, and Poria are mixed and crushed, extracted by heating and refluxing with methanol solution, filtered, concentrated and then freeze-dried to obtain the Angelica and Peony Powder extract freeze-dried powder.

[0017] Preferably, in the above-mentioned Angelica and Peony Self-microemulsion, the weight ratio of Angelica, Peony, Chuanxiong, Atractylodes, Alisma, and Poria is 3:10:5:4:5:4.

[0018] Preferably, the above-mentioned Angelica and Peony Self-Microemulsion, the Angelica and Peony Powder extract is prepared by the following method: Angelica, Peony, Chuanxiong, Atractylodes, Alisma, and Poria are mixed and crushed according to a weight ratio, and 50% methanol solution is used for heating and reflux extraction twice at a solid-liquid ratio of 1:10, each time for 2 hours, filtering, concentrating and then freeze-drying to obtain the Angelica and Peony Powder extract freeze-dried powder.

[0019] The preparation method of the above-mentioned Angelica and Paeonia lactiflora self-microemulsion comprises the following specific steps:

[0020] (1) Weigh the Danggui Shaoyao Powder extract according to the prescribed amount, grind it into powder, and add propylene glycol and glycerin;

[0021] (2) After constant temperature oscillation in a water bath, ultrasonic heating is performed until the drug solution turns into a transparent reddish brown color;

[0022] (3) After cooling, add Tween 80, Kolliphor RH40 / HS15, and caprylic / capric triglyceride, stir evenly, and centrifuge to obtain the supernatant, which is the self-microemulsion.

[0023] Preferably, in the preparation method of the Angelica and Paeonia lactiflora self-microemulsion, in step (2), after oscillating in a water bath constant temperature oscillator at 37°C for 24 hours, ultrasonic heating at 50°C is performed until the drug solution turns into a transparent reddish brown color.

[0024] Preferably, in the method for preparing the Angelica and Paeonia lactiflora self-microemulsion, the centrifugal speed in step (3) is 10,000 rpm and the centrifugation is performed for 30 min.

[0025] Application of the angelica and peony self-microemulsion in the preparation of hypoglycemic and / or lipid-lowering drugs.

[0026] The beneficial effects of the present invention are:

[0027] The above-mentioned Angelicae Sinensis and Peony Root Self-Microemulsion uses 7 blood-entering components with a certain exposure level in Angelicae Sinensis and Peony Root Powder, including paeoniflorin, paeoniflorin, ligustilide A, n-butylphthalide, ligustilide, atractylodes lactone III, and Tokinolide B, as research objects, obtains a better self-microemulsion prescription through screening, and finally determines the Angelicae Sinensis and Peony Root Self-Microemulsion containing the drug. The Angelicae Sinensis and Peony Root Self-Microemulsion is compared with the commercially available Angelicae Sinensis and Peony Root Pills in terms of drug content, and a multi-media dissolution curve comparison study is conducted with Angelicae Sinensis and Peony Root Powder, Angelicae Sinensis and Peony Root Pills, etc. The results indicate that the Angelicae Sinensis and Peony Root Self-Microemulsion has obvious advantages in improving dissolution and drug loading, laying a foundation for improving bioavailability, and has important application value in the preparation of hypoglycemic and / or lipid-lowering drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a control experiment diagram of Angelica and Paeonia lactiflora self-microemulsion, where *P<0.05, **P<0.01, ***P<0.001;

[0029] Figure 2 This is the liquid chromatography-mass spectrometry of the validation of the pharmacokinetic analysis methodology-specificity study, where A is blank rat plasma, B is blank rat plasma plus reference substance, and C is rat plasma sample 45 minutes after oral administration. 1 Paeoniflorin 7.18min, 2 Paeoniflorin 8.18min, 3 Chuanxiong lactone A 17.18min, 4 n-butylphthalide 17.55min, 5 Ligustifolia lactone 19.01min, 6 Atractylodes lactone III 19.55min, 7 Tokinolide B 22.40min, 8 Clarithromycin (IS) 17.88min;

[0030] Figure 3 The average drug-time curves of each analyte in Danggui Shaoyao Powder and its oral self-microemulsion in rats (n=6), where P is Danggui Shaoyao Powder group, S is self-microemulsion group. 1 Paeoniflorin, 2 Paeoniflorin, 3 Chuanxiong lactone A, 4 n-butylphenyl peptide, 5 Ligustilide, 6 Atractylodes lactone III, 7 Tokinolide B. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] The Danggui Shaoyao Powder extract used is prepared by the following method: 450g of Angelica sinensis, 1500g of Peony, 750g of Chuanxiong, 600g of Atractylodes macrocephala, 750g of Alisma orientalis, and 600g of Poria are mixed and crushed, and 50% methanol solution is used for heating and reflux extraction twice at a solid-liquid ratio of 1:10, each time for 2 hours, filtered, concentrated and then freeze-dried to obtain the Danggui Shaoyao Powder extract freeze-dried powder.

[0034] Example 1

[0035] The invention discloses a self-microemulsion of angelica and peony root, which is composed of 15.9% angelica and peony root powder extract, 31.54% Tween 80, 10.51% Kolliphor RH40, 19.62% propylene glycol, 9.81% glycerol and 12.62% caprylic and capric triglyceride.

[0036] The preparation method of the above-mentioned Angelica and Peony self-microemulsion is as follows: weigh 15.9% of the Angelica and Peony powder extract in a prescription amount, grind it into powder, add 19.6% propylene glycol and 9.8% glycerol, oscillate in a 37°C water bath constant temperature oscillator for 24 hours, then switch to ultrasonic 50°C ultrasonic heating until the liquid is transparent reddish brown, add 31.5% Tween 80, 10.5% Kolliphor RH40, and 12.6% caprylic and capric triglyceride after cooling, stir evenly, centrifuge at 10,000 rpm for 30 minutes, and the supernatant after centrifugation is the self-microemulsion.

[0037] Example 2

[0038] The invention discloses a self-microemulsion of angelica and peony root, which is composed of 15.9% angelica and peony root powder extract, 18.92% Tween 80, 11.56% Kolliphor RH40, 36.44% propylene glycol, 5.61% glycerol and 14.87% caprylic and capric triglyceride.

[0039] The preparation method is the same as Example 1.

[0040] Example 3

[0041] The invention discloses a self-microemulsion of angelica and peony root, which is composed of 15.9% angelica and peony root powder extract, 34.69% Tween 80, 6.31% Kolliphor RH40, 11.21% propylene glycol, 10.86% glycerol and 21.03% caprylic and capric triglyceride.

[0042] The preparation method is the same as Example 1.

[0043] Example 4

[0044] The invention discloses a self-microemulsion of angelica and peony root, which is composed of 15.9% angelica and peony root powder extract, 25.87% Tween 80, 8.41% Kolliphor RH40, 23.16% propylene glycol, 18.22% glycerol and 8.44% caprylic and capric triglyceride.

[0045] The preparation method is the same as Example 1.

[0046] Example 5

[0047] The invention discloses a self-microemulsion of angelica and peony root, which is composed of 15.9% angelica and peony root powder extract, 28.35% Tween 80, 16.82% Kolliphor HS15, 15.96% propylene glycol, 4.21% glycerol and 18.76% caprylic and capric triglyceride.

[0048] The preparation method is the same as Example 1.

[0049] Application Example 1

[0050] Forty SPF male Wistar rats were randomly divided into a blank group (ig 0.5%

[0051] CMC-Na solution), model group (ig 0.5% CMC-Na solution), Angelica and Paeonia lactiflora self-microemulsion administration group described in Example 1 (ig 0.88g / mL crude drug equivalent self-microemulsion), blank self-microemulsion control group (ig equal amount of blank self-microemulsion prescription), fed for 8 weeks, during which, except for the blank group drinking ordinary water, the other groups freely drank 15% fructose aqueous solution to induce non-alcoholic fatty liver in rats. After the experimental period, the rats were weighed, the fasting blood glucose and postprandial blood glucose of the rats were measured, and the serum of the rats was collected.

[0052] Experimental results: Figure 1 As shown in the results, compared with the model group and the blank self-microemulsion group, the administration of Angelica and Peony Self-Microemulsion can significantly reduce the body weight of rats (P<0.01) and reduce their fasting blood glucose and postprandial blood glucose levels (P<0.001). In addition, the levels of insulin, total cholesterol, triglycerides and alanine aminotransferase were significantly reduced (P<0.05). This shows that the administration of Angelica and Peony Self-Microemulsion can regulate the islet resistance and lipid metabolism disorders caused by fructose aqueous solution, thereby playing a certain protective role on the liver.

[0053] Experimental Example 1 Study on the pharmacokinetics of Angelica and Paeonia lactiflora self-microemulsion in rats

[0054] Paeoniflorin, paeoniflorin, ligustilide A, n-butylphenyl peptide, ligustilide, atractylodes lactone III and tokinolide B were used as the component (analyte) indicators for the pharmacokinetic study of the angelica and peony self-microemulsion in rats described in Example 1.

[0055] 1 Experimental materials and instruments

[0056] 1.1 Experimental animals and feed formulation

[0057] SPF male SD rats, 150-180 g, 12 rats, were purchased from Beijing Weitonglihua Laboratory Animal Biotechnology Co., Ltd., license number: SCXK (Beijing) 2021-0011, and were kept in the laboratory SPF standard animal breeding room. Breeding conditions: temperature in the range of 23±2℃, humidity in the range of 50±10%, noise less than 60 decibels, artificial light for 12h / d, and ventilation rate of about 15 times / h. Animal feed was purchased from Beijing Keao Xieli Feed Co., Ltd., and the formula of each 1kg feed consisted of crude protein ≥180g, crude fat ≥40g, crude fiber ≤50g, lysine ≥8.2g, methionine + cystine ≥5.3g, arginine ≥9.9g, tryptophan ≥1.9g, histidine ≥4.0g, phenylalanine + tyrosine ≥11.0g, threonine ≥6.5g, leucine ≥14.4g, isoleucine ≥7.0g, valine ≥8.4g and various trace elements and vitamins. According to the National Guidelines for the Care and Use of Laboratory Animals, the animal ethics approval number for the experiment was N2022038.

[0058] 1.2 Reference substances, see Table 1

[0059] Table 1 Antibiotics and reference substances information list

[0060]

[0061] 1.3 Instruments, see Table 2

[0062] Table 2 Instrument information list

[0063]

[0064] 2 Experimental methods

[0065] 2.1 Method for determination of blood drug concentration in rats

[0066] 2.1.1 Chromatographic conditions and mass spectrometry conditions

[0067] Chromatographic conditions: ultra-high performance liquid mass spectrometer Waters Xevo TQD / PDA AcquityUPLC, chromatographic column Waters Acquity UPLC BEH C18 (2.1×100 mm, 1.7 μm), mobile phase A: 0.1% formic acid aqueous solution, phase B: methanol, column temperature 30°C, injection volume 3 μl, mobile phase gradient elution program Table 3.

[0068] Table 3 ULTRA HPLC mobile phase gradient elution program

[0069]

[0070] Mass spectrometry conditions: triple quadrupole mass spectrometer, ion source: electrospray ionization source ESI, positive and negative ion scanning detection mode, multiple reaction detection mode (MRM). In positive ion mode, the capillary voltage (Capillary) is 3.0 kv, and the cone voltage (Cone) is 30 V; in negative ion mode, the capillary voltage (Capillary) is 2.0 kv, and the cone voltage (Cone) is 37 V. The desolvation gas (N2) temperature is 350 ° C, the desolvation gas (N2) flow rate is 1000 L / h, and the cone gas flow rate is 70 L / h. The MRM parameters of all compounds are shown in Table 4.

[0071] Table 4 MRM parameter values ​​of various chemical components

[0072]

[0073]

[0074] 2.1.2 Preparation of internal standard solution

[0075] Accurately weigh an appropriate amount of clarithromycin reference substance, dissolve it in methanol and quantitatively dilute it to a concentration of approximately 50 ng / ml internal standard solution.

[0076] 2.1.3 Processing of calibration curve and test plasma samples

[0077] Preparation of calibration standards and establishment of calibration curves: Accurately weigh appropriate amounts of peony lactone glycosides, paeoniflorin, ligustilide A, n-butylphthalide, ligustilide, atractylodes lactone III, and tokinolide B reference substances, dissolve them in methanol, and quantitatively dilute the reference substance stock solution with a concentration of about 0.2 mg / ml. Take appropriate amounts of the above-mentioned various stock solutions and dilute the 7 concentration level linear working solutions step by step with 50% methanol water. Accurately measure 50 μL of blank plasma, add 200 μL of methanol, 10 μL of linear working solution, and 10 μL of internal standard solution, vortex for 5 minutes, centrifuge at 12,000 rpm for 15 minutes, and place the supernatant in a liquid phase vial. Analyze the sample through the LC-MS / MS system to obtain the mass spectrum and the peak areas of each analyte and internal standard. Based on the ratio of the peak area of ​​each analyte to the internal standard and the concentration, the weighted least squares method (w = 1 / x 2 ) was used for linear regression to obtain the linear equation of the calibration curve.

[0078] Plasma sample analysis: Accurately measure 50 μL of plasma, add 200 μL of methanol, 10 μL of 50% methanol water, and 10 μL of internal standard solution, vortex for 5 min, and centrifuge at 12,000 rpm for 15 min. Place the supernatant in a liquid phase vial and analyze it through the LC-MS / MS system. Substitute the ratio of the peak area of ​​each analyte to the peak area of ​​the internal standard into the linear equation to calculate the concentration of each analyte in the plasma sample.

[0079] 2.2 Validation of pharmacokinetic analysis methodology

[0080] The experiment was carried out in accordance with the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0081] 2.2.1 Method specificity investigation

[0082] 50 μL of blank plasma was taken from 3 rats (6 in total) in each group, and the internal standard solution was replaced with methanol, and processed according to the item "2.1.3 Analysis of plasma samples". The plasma samples of rats 45 minutes after administration were processed according to the item "2.1.3 Analysis of plasma samples", 50 μL of blank plasma was taken, a certain concentration of linear working solution was added, and processed according to the item "2.1.3 Preparation of calibration standard and establishment of calibration curve", and the samples were injected and analyzed by LC-MS / MS system to investigate whether the endogenous substances and other components of blank plasma interfered with the determination of each analyte.

[0083] 2.2.2 Linear equation, range, and LLOQ investigation

[0084] Take 50 μL of blank plasma, add a series of linear working solutions, and process according to "2.1.3 Preparation of calibration standards and establishment of calibration curves" to obtain the linear equations of each analyte. The correlation coefficient (R 2 ) were all greater than 0.99, and the linear concentration range was determined, and the minimum linear concentration was the minimum quantitative detection concentration (LLOQ).

[0085] 2.2.3 Precision and Accuracy

[0086] Take 50 μL of blank plasma, add certain concentrations of each analyte reference solution, prepare quality control samples at four concentration levels, namely LLOQ, low concentration, medium concentration and high concentration, and prepare 6 parallel samples for each concentration level. Process according to "2.1.3 Preparation of calibration standard samples and establishment of calibration curve", inject and analyze through LC-MS / MS system, obtain mass spectrum and peak area ratio of analyte to internal standard, calculate the concentration of each quality control sample based on the daily standard curve, calculate accuracy by taking theoretical concentration as reference, and evaluate intra-batch accuracy RE, inter-batch accuracy RE and intra-batch precision RSD, inter-batch precision RSD. The accuracy at LLOQ level should be between 80% and 120%, and the accuracy at other concentration levels should be between 85% and 115%. The accuracy RE and precision RSD at LLOQ level should not exceed 20%, and the accuracy RE and precision RSD at other levels should not exceed 15%.

[0087] 2.2.4 Extraction recovery and matrix effect

[0088] Take 50 μL of blank plasma, add certain concentrations of each analyte reference solution, prepare low-concentration, medium-concentration, and high-concentration quality control samples, prepare 6 copies of each concentration in parallel, process according to the item "2.1.3 Preparation of calibration standard samples and establishment of calibration curve", inject and analyze through LC-MS / MS system, obtain mass spectrum and analyte to internal standard peak area ratio R1, take 50 μL of blank plasma, except not adding 10 μL of 50% methanol aqueous solution, and then centrifuge according to the item "2.1.3 Plasma sample analysis", add 10 μL of low-concentration, medium-concentration, and high-concentration reference solution respectively, prepare 6 copies of each concentration in parallel, and inject and analyze through LC-MS / MS system, obtain mass spectrum and analyte to internal standard peak area ratio R2, replace 50 μL of blank plasma with water, except not adding 10 μL of 50% methanol aqueous solution Except for the 50% methanol aqueous solution, the others were processed as in "2.1.3 Plasma Sample Analysis", and 10 μL of low-concentration, medium-concentration, and high-concentration reference solutions were added respectively. Six copies of each concentration were prepared in parallel and analyzed by LC-MS / MS system to obtain the mass spectrum and the peak area ratio of the analyte to the internal standard R3. The percentage of R1 to R2 is the extraction recovery rate, and the percentage of R2 to R3 is the matrix effect, which should be between 85% and 115%, and the RSD should not exceed 15%.

[0089] 2.2.5 Residue

[0090] Take 50 μL of blank plasma, add the highest concentration of linear working solution, and process according to "2.1.3 Preparation of calibration standard and establishment of calibration curve". Then, inject and analyze the solution of blank plasma treated according to "2.1.3 Plasma sample analysis" through LC-MS / MS system in succession, and examine the residual amount of each analyte and internal standard in the mass spectrum of the solution after subsequent injection of blank plasma. Each analyte should not exceed 20% of the peak area of ​​LLOQ level, and the internal standard should not exceed 5%.

[0091] 2.2.6 Stability

[0092] Take 50 μL of blank plasma, add certain concentrations of each analyte reference solution, prepare low-concentration, medium-concentration and high-concentration quality control samples, and examine the short-term stability of the test solution in the sample plate (4°C) within 8 hours after the quality control sample is processed, the long-term stability of the quality control sample in the ultra-low temperature refrigerator at -80°C for 30 days, and the repeated freeze-thaw stability of the quality control sample between -80°C and room temperature (repeated 3 times, each interval of not less than 12 hours). Under each condition, the concentrations of the quality control samples of low, medium and high concentrations are measured respectively, and 6 replicates of each concentration are prepared in parallel. The accuracy RE should not exceed 15%.

[0093] 2.2.7 Dilution reliability

[0094] Accurately pipette 10 μL of a mixed reference solution with a paeoniflorin concentration of 100 μg / ml, paeoniflorin, ligustilide, and Tokinolide B concentrations of 50 μg / ml, n-butylphthalide, ligustilide A concentrations of 10 μg / ml, and atractylodes lactone III concentrations of 5 μg / ml, and add it to 90 μL of blank plasma to prepare plasma samples with concentrations of 10 μg / ml paeoniflorin, 5 μg / ml paeoniflorin, ligustilide, and Tokinolide B, 1 μg / ml n-butylphthalide, atractylodes lactone A concentrations, and 0.5 μg / ml. Dilute the above blood samples 10 times and 50 times with blank plasma, and make 6 replicates. Process according to "2.1.3 Plasma Sample Analysis", and perform sample analysis using the LC-MS / MS system. Calculate the accuracy RE and precision RSD of the concentration of each analyte, which should not exceed 15%.

[0095] 2.3 Dosage regimen and blood sample collection

[0096] 12 SD male rats, weighing 150-180g, were raised in a laboratory SPF-grade standard animal breeding room, and the rats were adapted for one week. The rats were randomly divided into 2 groups, 6 in each group, one group was administered with a suspension prepared from the extract of Danggui Shaoyao San (Group P), and the other group was administered with a self-microemulsion (Group S). They were fasted for 12 hours before taking the medicine, and the administration volume was 20ml / kg rat body weight, and each 1ml administration volume contained 180mg of Danggui Shaoyao San extract. Blank blood was taken before administration, and 0.083h, 0.167h, 0.333h, 0.5h, 0.75h, 1h, 1.5h, 2h, 3h, 5h, 7h, 12h, and 24h after administration. 0.5ml of blood was taken from the eye sockets and placed in a 1.5ml centrifuge tube coated with sodium heparin. Immediately centrifuged at 12000rpm for 15min, the upper plasma was aspirated, and stored in a -80℃ refrigerator for analysis.

[0097] 2.4 Pharmacokinetic studies and statistical analysis

[0098] Plasma samples at different time points after administration of rats were processed according to the item "2.1.3 Plasma sample analysis", and the blood drug concentration of each analyte at different time points was calculated by the standard curve equation. If the concentration of the analyte in the plasma sample exceeds the concentration range of the linear equation, blank plasma can be used for dilution according to the situation and then measured. The measured blood drug concentration data were calculated using the statistical moment method of DAS3.3.1 software (Drug and statistics 3.3.1, Medical College of Wannan, China) to calculate various pharmacokinetic parameters, mainly including AUC, T 1 / 2 etc., C. max 、T maxThe measured values ​​were used. The pharmacokinetic parameters of the Danggui Shaoyao self-microemulsion group (S) and the Danggui Shaoyao powder group (P) were tested by t test to examine whether there were significant differences between the parameters. P < 0.05 was considered statistically significant. The relative bioavailability (F) between the self-microemulsion group (S) and the Danggui Shaoyao powder group (P) was measured by the AUC between the S group and the P group. 0-24 Divide by percentage to calculate.

[0099] 3 Experimental results

[0100] 3.1 Validation of pharmacokinetic analysis methodology

[0101] 3.1.1 Specificity results

[0102] Depend on Figure 2 It can be seen that after treatment, endogenous substances and other components in blank plasma do not interfere with the determination of analytes, indicating that the method has good specificity.

[0103] 3.1.2 Linear equation and range, LLOQ results

[0104] Linear equations, concentration ranges, and correlation coefficients R for various analytes 2 The results of the minimum quantification limit LLOQ are detailed in Table 5. The R 2 All of them were greater than 0.99, and the LLOQs of various analytes were: 3.1 ng / ml for paeoniflorin, 15.6 ng / ml for paeoniflorin, 0.8 ng / ml for ligustilide A, 0.3 ng / ml for n-butylphthalide, 2.5 ng / ml for ligustilide, 0.6 ng / ml for atractylodes lactone III, and 3.1 ng / ml for Tokinolide B.

[0105] Table 5 Pharmacokinetic Methodology Validation - Linearity and Minimum Limit of Quantitation Results (Concentration Unit ng / ml)

[0106]

[0107]

[0108] 3.1.3 Accuracy and precision

[0109] The maximum absolute value of the intra-batch and inter-batch accuracy RE of various analytes in the quality control samples at various levels such as LLOQ, low concentration, medium concentration, and high concentration was 13.93%, and the maximum value of the precision RSD was 13.77%, both of which met the requirements. The specific results are shown in Table 6.

[0110] Table 6 Pharmacokinetic Methodology Validation - Accuracy and Precision Results (n = 6)

[0111]

[0112]

[0113] 3.1.4 Extraction recovery and matrix effect

[0114] For the quality control samples at low, medium and high concentration levels, the extraction recoveries and matrix effects of various analytes at each concentration level were between 85% and 115%, and the maximum RSDs were 9.66% and 9.34%, respectively. All of them were within 15% and met the requirements. The specific results are shown in Table 7.

[0115] Table 7 Pharmacokinetic Methodology Verification - Extraction Recovery and Matrix Effect Results (n = 6)

[0116]

[0117]

[0118] 3.1.5 Residue

[0119] Blank plasma to which the highest concentration of linear working solution was added was processed and then sampled for analysis. The maximum residues accounted for 3.9%, 4.3%, 8.3%, 3.9%, 1.2%, 5.5% and 3.6% of LLOQ for paeoniflorin, paeoniflorin, ligustilide A, n-butylphthalide, ligustilide, atractylodes lactone III and Tokinolide B, respectively, all of which were less than 20%, meeting the residue regulations.

[0120] 3.1.6 Solution stability

[0121] From the data in Table 8, it can be seen that the short-term stability of the test liquid sample plate (4°C) within 8 hours, the long-term stability of the refrigerator at ultra-low temperature of -80°C for 30 days, and the repeated freeze-thaw stability between -80°C and room temperature, the maximum accuracy RE of the quality control samples of three concentrations under each condition are no more than 15%, which meets the requirements.

[0122] Table 8 Pharmacokinetic Methodology Verification - Solution Stability and Repeated Freeze-Thaw Stability (n = 6)

[0123]

[0124]

[0125] 3.1.7 Dilution Reliability

[0126] After diluting each analyte 10 times and 50 times, 6 replicates were measured, and the accuracy RE and precision RSD were both less than 15%, indicating that the dilution was reliable. See Table 9 for specific data.

[0127] Table 9 Pharmacokinetic Methodology Validation - Dilution Reliability Results (n = 6)

[0128]

[0129]

[0130] 3.2 Pharmacokinetic study results

[0131] Mean blood concentration Plotting the time points, we get Figure 3 Drug-time curve. The various pharmacokinetic parameters of Danggui Shaoyao SEM and Danggui Shaoyao Powder were investigated in Table 10. The pharmacokinetic parameters of various analytes in rats showed that after Danggui Shaoyao Powder was prepared into a self-microemulsion, the pharmacokinetic parameters of Yangchuanxiong lactone A and Baizhu lactone ⅢC max , AUC 0-24 , AUC 0-∞ , peak time in vivo T max , half-life t 1 / 2 There was no significant improvement in the pharmacokinetic parameters (P>0.05), and the relative bioavailability F was 99% and 117%, respectively, with no significant improvement; the bioavailability of paeoniflorin, paeoniflorin, n-butylphenyl peptide, ligustilide, and Tokinolide B in T max ,t 1 / 2 There was no significant change (P>0.05). max , AUC 0-24 , AUC 0-∞ There was a significant improvement in pharmacokinetic parameters.

[0132] Table 10 Pharmacokinetic parameters of each analyte in rats (n=6)

[0133]

[0134]

[0135] Note: P Danggui Shaoyao Powder group, S Self-microemulsion group. Compared with Danggui Shaoyao Powder group, *P<0.05,

[0136] **P<0.01, ***P<0.001.

[0137] 1) Angelica and Paeonia lactone C in self-microemulsion max (440.3±154.5 ng / ml), AUC 0-24 (541.3±100.2 ng / ml·h), AUC 0-∞ (749±150.7 ng / ml·h) was significantly improved, which was better than that of Danggui Shaoyao San group C max (60.3±11.9 ng / ml, P<0.01), AUC 0-24(322.9±47.6 ng / ml·h, P<0.01), AUC 0-∞ (507±134.2 ng / ml·h, P<0.05), and the relative bioavailability F was 168%.

[0138] 2) Peony glycoside C in Angelica and Peony Self-Microemulsion max (7383.6±2252.4 ng / ml), AUC 0-24 (7452.2±2195.6 ng / ml·h), AUC 0-∞ (7836.7±2431.3 ng / ml·h) was significantly improved, which was better than Danggui Shaoyao San group C max (1202.5±377.3 ng / ml, P<0.001), AUC 0-24 (4770.6±1190.9 ng / ml·h, P<0.05), AUC 0-∞ (5187±1396.7ng / ml·h, P<0.05), and the relative bioavailability F was 156%.

[0139] 3) Angelica and Peony Self-Microemulsion Group n-Butyl Peptide C max (19.0±8.3 ng / ml), AUC 0-24 (69.6±36.6ng / ml·h), AUC 0-∞ (107.1±73.5 ng / ml·h) was significantly improved, which was better than Danggui Shaoyao San C max (9.5±1.4 ng / ml, P<0.05), AUC 0-24 (20.5±3.7 ng / ml·h, P<0.05), AUC 0-∞ (30.4±4.5 ng / ml·h, P<0.05), and the relative bioavailability F was 339%.

[0140] 4) Angelica and Paeonia lactiflora self-microemulsion group Ligustilide C max (484.3±204.2ng / ml), AUC 0-24 (749.1±379.7ng / ml·h), AUC 0-∞ (1096.1±684.2ng / ml·h) was significantly improved, better than Danggui Shaoyao San C max (180±89.4ng / ml, P<0.05), AUC 0-24 (163.9±65.9ng / ml·h, P<0.05), AUC 0-∞ (235±115.4ng / ml·h, P<0.05), and the relative bioavailability F was 457%.

[0141] 5) Angelica and Paeonia lactiflora self-microemulsion group Tokinolide BC max (174.2±58.5ng / ml) was significantly improved, better than Danggui Shaoyao San C max (101.1±28.9ng / ml, P<0.05), but AUC 0-24 , AUC 0-∞ There was no difference between the two groups (P>0.05), and the relative bioavailability F was 150%.

[0142] In summary, the Angelica and Paeonia lactiflora self-microemulsion can further increase the blood concentration or oral bioavailability of blood-entering components such as paeoniflorin, paeoniflorin, n-butylphenyl peptide, ligustilide, and Tokinolide B.

[0143] The Angelica and Peony Self-Microemulsion has a small particle size and low Zeta potential after self-emulsification, which can promote the permeability of biological membranes; on the basis that the in vitro dissolution of five chemical components, including ligusticum lactone A, n-butylphenyl peptide, ligustilide, atractylodes lactone III, and Tokinolide B, has been improved, a triple quadrupole liquid chromatography-mass spectrometry technology is used to carry out an oral in vivo pharmacokinetic study of the Angelica and Peony Self-Microemulsion in rats. The blood drug concentration is determined by the standard curve method using clarithromycin as the internal standard, and a series of methodological verifications such as specificity, linearity, precision, accuracy, matrix effect, extraction recovery, and stability have been conducted, proving that the method has good accuracy and repeatability.

[0144] SD rats were divided into two groups, 6 in each group, and were administered with single doses of Danggui Shaoyao powder (prepared into suspension) and the Danggui Shaoyao self-microemulsion described in Example 1 in parallel. Blood was collected from the eye sockets, and the blood drug concentration of each rat at different time points after administration was detected. The pharmacokinetic parameters were calculated using DAS software, and a t-test was performed. The results showed that the analytes T, such as paeoniflorin, paeoniflorin, n-butylphenyl peptide, ligustilide, and Tokinolide B in the Danggui Shaoyao self-microemulsion were significantly higher than those in the control group. max ,t 1 / 2 There was no significant difference (P>0.05), C max , AUC 0-24 , AUC 0-∞ All or part of the parameters were significantly improved, with statistical significance (P<0.05), and the oral relative bioavailability F was between 150% and 457%, indicating that the blood concentration or bioavailability of the above five chemical components was significantly improved.

[0145] Compared with Danggui Shaoyao powder, the in vivo T max ,t 1 / 2 , C max , AUC 0-24 , AUC 0-∞There was no significant change in pharmacokinetic parameters (P>0.05), and the relative bioavailability F was 99% and 117%, respectively, with no significant difference in in vivo absorption. Some researchers compared oral administration of chuanxiong lactone A with intravenous administration. Due to the first-pass effect of the liver and other reasons, the oral bioavailability was only 8%. Therefore, the angelica peony self-microemulsion was still affected by the first-pass effect of the liver and limited its in vivo absorption. Atractylodes lactone III has high permeability, no specific absorption site in the gastrointestinal tract, rapid absorption, slow metabolism, and the improvement of solubility has little effect on its absorption and bioavailability. Therefore, although the in vitro dissolution of angelica peony self-microemulsion is improved compared with angelica peony powder, the relative bioavailability of chuanxiong lactone A and atractylodes lactone III has no significant change, which is also reasonable.

[0146] Comprehensive analysis showed that peony lactone glycosides, paeoniflorin, n-butylphenyl peptide, ligustilide, and Tokinolide B not only have pharmacological activity, but are also the blood-entering components of Danggui Shaoyao Powder. The relative bioavailability of Danggui Shaoyao Self-microemulsion in vivo was greatly improved under the premise of improving the in vitro dissolution or biomembrane permeability, which laid a material foundation for the subsequent research on the pharmacodynamics and quality control of Danggui Shaoyao oral self-microemulsion.

[0147] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field shall fall within the protection scope of the claims of the present invention.

Claims

1. A self-microemulsion of angelica and peony root, characterized in that: The invention is composed of 15.90% Danggui Shaoyao Powder extract, a mixed emulsifier, an auxiliary emulsifier and an oil phase. The Danggui Shaoyao Powder extract is prepared by the following method: mixing and grinding angelica, peony, chuanxiong, atractylodes, orientalis and tuckahoe, extracting by heating and refluxing with a methanol solution, filtering, concentrating and freeze-drying to obtain the extract; wherein, The mixed emulsifier is 18.92%-34.69% Tween 80 and 6.31%-11.56% Kolliphor RH40; the auxiliary emulsifier is 11.21%-36.44% propylene glycol and 5.61%-18.22% glycerol; the oil phase is 4.21%-21.03% caprylic acid capric acid triglyceride; or The mixed emulsifier is 16.82%-33.64% Tween 80 and 8.41%-16.82% Kolliphor HS15; the auxiliary emulsifier is 8.41%-36.44% propylene glycol and 4.21%-18.22% glycerol; and the oil phase is 4.21%-21.03% caprylic capric triglyceride.

2. The angelica and peony root self-microemulsion according to claim 1, characterized in that: It is composed of 15.90% Danggui Shaoyao San extract, a mixed emulsifier, an emulsifier and an oil phase, wherein the mixed emulsifier is 18.92%-33.64% Tween 80 and 8.41%-11.56% Kolliphor RH40 or Kolliphor HS15; the emulsifier is 11.21%-36.44% propylene glycol and 5.61%-18.22% glycerol; and the oil phase is 4.21%-21.03% caprylic capric triglyceride.

3. The angelica and peony root self-microemulsion according to claim 1, characterized in that: It is composed of 15.9% Danggui Shaoyao Powder extract, 31.54% Tween 80, 10.51% Kolliphor RH40, 19.62% propylene glycol, 9.81% glycerol, and 12.62% caprylic / capric triglyceride.

4. The angelica and peony root self-microemulsion according to claim 1, characterized in that: The weight ratio of the angelica sinensis, peony root, ligusticum chuanxiong, atractylodes macrocephala, oriental water plantain and poria is 3:10:5:4:5:

4.

5. The angelica and peony root self-microemulsion according to claim 1, characterized in that: The Danggui Shaoyao Powder extract is prepared by the following method: angelica, peony, chuanxiong, atractylodes, oriental water chestnuts, and poria are mixed and crushed according to a weight ratio, and 50% methanol solution is used to heat and reflux extract twice at a solid-liquid ratio of 1:10, each time for 2 hours, and filtered, concentrated, and then freeze-dried to obtain the Danggui Shaoyao Powder extract freeze-dried powder.

6. The method for preparing the Angelica and Paeonia lactiflora self-microemulsion according to any one of claims 1 to 5, characterized in that: The specific steps are as follows: (1) Weigh the Danggui Shaoyao Powder extract according to the prescribed amount, grind it into powder, and add propylene glycol and glycerin; (2) After constant temperature oscillation in a water bath, ultrasonic heating is performed until the drug solution turns into a transparent reddish brown color; (3) After cooling, add Tween 80, Kolliphor RH40 or Kolliphor HS15, and caprylic / capric triglyceride, stir evenly, and centrifuge to obtain the supernatant, which is the self-microemulsion.

7. The method for preparing the angelica and peony root self-microemulsion according to claim 6, characterized in that: In the step (2), after oscillating in a 37°C water bath constant temperature oscillator for 24 hours, ultrasonic heating at 50°C was performed until the drug solution turned into a transparent reddish brown color.

8. The method for preparing the angelica and peony root self-microemulsion according to claim 6, characterized in that: In step (3), the centrifugal speed is 10,000 rpm and the centrifugation is performed for 30 min.

9. Use of the Angelica sinensis and Paeonia lactiflora self-microemulsion according to any one of claims 1 to 5 in the preparation of hypoglycemic and / or lipid-lowering drugs.

Images