Use of aescin in the preparation of a medicament for preventing spontaneous abortion

Abstract

The present application relates to the technical field of biological medicine, and more particularly to the application of isodon japonicus in the preparation of a drug for preventing and treating spontaneous abortion. The present application analyzes the in-vivo migration components of Jianwei Shoutai Pills, excavates the pharmacodynamic material basis for preventing and treating spontaneous abortion, and uses a CBA / J x DBA / 2 peri-implantation period abortion mouse model to conduct pharmacodynamic experiments. The results show that isodon japonicus can significantly reduce the abortion rate of the CBA / J x DBA / 2 peri-implantation period abortion model mice, and can effectively increase the embryo and placenta wet weight of the CBA / J x DBA / 2 peri-implantation period abortion model mice. That is, isodon japonicus can effectively inhibit spontaneous abortion, has a significant curative effect, has considerable treatment potential, provides a potential new drug selection for clinical drugs for preventing and treating spontaneous abortion, and has a broad application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of halofuginone in the preparation of a drug for preventing and treating spontaneous abortion. BACKGROUND

[0002] Spontaneous abortion refers to the termination of pregnancy before the fetus has the ability to survive, which is caused by natural factors (non iatrogenic) and is a common complication in early pregnancy. 50%-60% of spontaneous abortions are related to chromosomal abnormalities of the embryo. The main clinical manifestations are vaginal bleeding and abdominal pain after menstruation. The spontaneous abortion rate accounts for 10%-15% of all pregnancies, of which more than 80% are early abortion (i.e. before 12 weeks of pregnancy). The causes of spontaneous abortion include embryonic factors, maternal factors, paternal factors and environmental factors. Older pregnant women, previous history of spontaneous abortion, and underweight or overweight before pregnancy are all high-risk factors for spontaneous abortion. With the continuous decline of the birth rate, spontaneous abortion, as a common complication of pregnancy that seriously affects women's reproductive health, has also attracted increasing attention.

[0003] Under the guidance of traditional Chinese medicine theory, Jianwei Shuitai Pill, which has the effect of tonifying kidney and preventing miscarriage, is the base formula of most kidney-tonifying and miscarriage-preventing compounds in clinical practice. It is composed of Cuscuta chinensis Lam., Taxillus delavayi (Franch.) Danser and Dipsacus asperoides. Cuscuta chinensis Lam. can warm yang and benefit yin, and can make the kidney strong to make the embryo grow. Taxillus delavayi (Franch.) Danser can nourish blood and prevent miscarriage, and Dipsacus asperoides can nourish liver and kidney, and can also consolidate the uterus to maintain the embryo. Although Jianwei Shuitai Pill has a significant effect on preventing and treating spontaneous abortion, its complex composition and unknown effective substances and mechanism hinder its clinical application and development. Therefore, clarifying the effective substance basis of Jianwei Shuitai Pill is an important prerequisite for clarifying the kidney-tonifying and miscarriage-preventing targets and mechanism of Jianwei Shuitai Pill. In the previous study, based on the chemical components of traditional Chinese medicine, the effective components of Jianwei Shuitai Pill were screened, and it was found that total flavonoids of Cuscuta chinensis Lam., total polysaccharides of Taxillus delavayi (Franch.) Danser and total saponins of Dipsacus asperoides were effective components, and hyperoside, dipsacus saponin VI and the like were effective monomers. However, this screening method has the disadvantages of low throughput and not considering the actual metabolic process of traditional Chinese medicine compounds in vivo, which may cause deviation in the subsequent study of targets and mechanism of traditional Chinese medicine.

[0004] LC-MS technology (Liquid Chromatograph Mass Spectrometer) has been widely used in the analysis of chemical components of traditional Chinese medicine, and is also one of the commonly used technologies for studying metabolomics. After oral administration of traditional Chinese medicine compounds, they are digested in the body through organs such as the intestine and the liver, and exist in the form of original or exogenous metabolites. These types of effective substances are absorbed into the blood, and directly or indirectly act on the targets to exert the efficacy; or they may produce endogenous metabolites by disturbing the body metabolism to exert the efficacy. SUMMARY

[0005] In order to solve the above problems, on the basis of the existing drugs for preventing and treating spontaneous abortion, the application provides an application of oridonin in preparation of a drug for preventing and treating spontaneous abortion.

[0006] In one aspect, the application provides an application of oridonin in preparation of a drug for preventing and treating spontaneous abortion.

[0007] Further, the drug for preventing and treating spontaneous abortion prepared from oridonin can improve the wet weight of embryos.

[0008] Further, the drug for preventing and treating spontaneous abortion prepared from oridonin can improve the wet weight of placentas.

[0009] Further, oridonin is obtained by extraction and separation from Chinese herbal medicine or obtained by chemical synthesis.

[0010] Further, the drug for preventing and treating spontaneous abortion is an oral preparation, a sublingual tablet, a pill or a needle injection.

[0011] Further, the oral preparation is a tablet, a capsule, a granule, a pill, a drop, a fruit juice or a syrup.

[0012] Further, the needle injection is an injection, a powder injection or a freeze-dried powder injection.

[0013] In another aspect, the application also provides a drug for preventing and treating spontaneous abortion, which comprises oridonin and a pharmaceutically acceptable excipient.

[0014] Further, the pharmaceutically acceptable excipient comprises any one or a combination of several of disintegrants, wetting agents, binding agents, filling agents, absorption promoters, solvents, lubricants, surfactants, flavoring agents, sweetening agents, antioxidants, preservatives or pigments.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] 1. The application proves that oridonin can significantly reduce the abortion rate of abortion model mice and effectively improve the wet weight of embryos and placentas of the abortion model mice. That is, oridonin can effectively inhibit spontaneous abortion, has significant curative effect and has considerable treatment potential, thereby providing a potential new drug selection for clinical drugs for preventing and treating spontaneous abortion and having a broad application prospect.

[0017] 2. The application proves that oridonin monomer, which plays a key therapeutic role in the compound formula of Jianwei Shuitai Pill, is the key therapeutic compound for preventing and treating spontaneous abortion by analyzing the in-vivo migration components of Jianwei Shuitai Pill, thereby making the treatment for preventing and treating spontaneous abortion more accurate and reducing the occurrence of side effects to a certain extent. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0019] Figure 1 The multiple statistical analysis of the plasma metabolites of the mice in Example 2 of the present application is shown in (A) PCA score plot, (B) OPLS-DA score plot between the control group and the model group, (C) 200x permutation test of the OPLS-DA model between the control group and the model group, (D) OPLS-DA score plot between the model group and the Jianwei Shuitai Pill group, and (E) 200x permutation test of the OPLS-DA model between the model group and the Jianwei Shuitai Pill group.

[0020] Figure 2 The PCA analysis and OPLS-DA analysis of the placental metabolites of the mice in Example 2 of the present application are shown in (A) PCA score plot, (B) OPLS-DA score plot between the control group and the model group, (C) 200x permutation test of the OPLS-DA model between the control group and the model group, (D) OPLS-DA score plot between the model group and the Jianwei Shuitai Pill group, and (E) 200x permutation test of the OPLS-DA model between the model group and the Jianwei Shuitai Pill group.

[0021] Figure 3 The differential metabolite analysis of the mice in Example 2 of the present application is shown in (A) differential metabolite volcano plot between the control group and the model group, (B) differential metabolite volcano plot between the model group and the Jianwei Shuitai Pill group, and (C) significant differential metabolite heat map between the model group and the Jianwei Shuitai Pill group.

[0022] Figure 4 The differential metabolite analysis of the mice in Example 2 of the present application is shown in (A) differential metabolite volcano plot between the control group and the model group, (B) differential metabolite volcano plot between the model group and the Jianwei Shuitai Pill group, and (C) significant differential metabolite heat map between the model group and the Jianwei Shuitai Pill group.

[0023] Figure 5The influence of dunnianoside on the CBA / J x DBA / 2 abortion model mice of Example 2 of the present application, wherein: (A) mouse embryo, placenta stereoscopic graph (model group placenta shows absorption embryo); (B) the influence of dunnianoside on the abortion rate of CBA / J x DBA / 2 abortion model mice; (C) the influence of dunnianoside on the embryo wet weight of CBA / J x DBA / 2 abortion model mice; (D) the influence of dunnianoside on the placenta wet weight of CBA / J x DBA / 2 abortion model mice; Note: * represents P<0.05 compared with the control group; # represents P<0.05 compared with the model group. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be described clearly and completely in combination with specific examples. Obviously, the described examples are only some of the examples of the present application, but not all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0025] Sample processing of traditional Chinese medicine in Example 1

[0026] The composition of Shoutai Pill is Cuscuta, Taxilli Herba, Radix Dipsaci and Colla Corii Asini. Cuscuta is the monarch drug, and Taxilli Herba and Radix Dipsaci are the ministerial drugs, which have the functions of tonifying kidney and stabilizing fetus. They are used for treating fetus movement instability and pregnancy abortion. In the formula, Cuscuta tonifies kidney and essence, and the kidney can protect fetus when it is strong; Taxilli Herba and Radix Dipsaci tonify liver and kidney, and strengthen the fetus; and Colla Corii Asini nourishes yin blood, so that the fetus is stable. The four drugs together have the function of tonifying kidney and stabilizing fetus. Reduced Shoutai Pill is Colla Corii Asini reduced Shoutai Pill, which retains Cuscuta, Taxilli Herba and Radix Dipsaci, and is used for treating fetus movement instability and pregnancy abortion.

[0027] Cuscuta, Taxilli Herba and Radix Dipsaci are put in according to the ratio of 2:1:1, and the first time is 10 times the volume of distilled water, which is boiled for 60 min. After the traditional Chinese medicine is poured out, 8 times the volume of distilled water is added and boiled for 45 min. The two traditional Chinese medicine liquids are mixed, filtered, concentrated in a rotary evaporator, and dried into powder. 200 g of dry powder is obtained from 1 kg of raw material, and stored at 4°C.

[0028] The sample preparation for liquid chromatography-mass spectrometry analysis is as follows:

[0029] (1) 3 g of reduced Shoutai Pill powder is weighed, dissolved in a 100 mL volumetric flask, 75 mL of 53% methanol is added, and the mass is weighed after being tightly capped to obtain the initial mass M1.

[0030] (2) The conical flask is placed in a 50°C ultrasonic water bath for 30 min (power 500 W, frequency 40 KHz).

[0031] (3) 85℃ water bath pot heating reflux 1h, after cooling, weighing, adding 50% methanol to M1.

[0032] (4) The traditional Chinese medicine solution was transferred to a 50 mL centrifuge tube and centrifuged at 2500 rpm for 10 min.

[0033] (5) The filtrate was filtered with filter paper, and 50 mL of the middle filtrate was taken to a new centrifuge tube.

[0034] (6) 5 mL of the filtrate was taken and made up to 50 mL with 53% methanol.

[0035] (7) 1 mL was taken with a 2 mL syringe, filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a sample bottle. The sample name, concentration and date were labeled, and the sample was sent for liquid chromatography-mass spectrometry analysis.

[0036] Example 2 CBA / J x DBA / 2 mouse model verification

[0037] 1. Mouse grouping, modeling and administration

[0038] Randomly selected 15 SPF CBA / J female mice, 5 SPF BALB / c male mice and 8 DBA / 2 male mice, mating with CBA / J x DBA / 2 to model, and CBA / J x BALB / c mating as control group. Vaginal smears were taken before mating, and female and male mice in estrus were selected for mating at 1:1, and the cage was combined at 17:00-18:00 in the afternoon. The next day, the tampons were checked at 8:00, and the day when the tampons were seen was considered as the 0.5th day of pregnancy. They were divided into control group, model group, Jia Wei Shu Tai pill group and halofuginone group (N=5).

[0039] Preparation of Jia Wei Shu Tai pill solution: 8 g of Jia Wei Shu Tai pill powder was dissolved in 40 mL of distilled water, with a concentration of 0.2 g / mL, i.e. 1 g (crude drug) / mL. The conversion factor between humans and mice is 12.33, and according to the conversion of clinical equivalent dose, the required low dose for mice is = 40 g x 12.33 ÷ 60 kg = 8.22 g (crude drug) / kg (mouse weight).

[0040] Preparation of halofuginone solution: 30 mg of halofuginone was dissolved in 1.2 mL of DMSO, 4.8 mL of PEG300, 0.6 mL of Tween-80 and 5.4 mL of saline, respectively, with a concentration of 2.5 mg / mL. The dose for mice was 10 mg / kg.

[0041] On the 13.5th day of pregnancy, heart blood was taken, and the plasma was separated after standing at 4℃ for 4 h and stored at -80℃; the embryo and placental tissues were separated and weighed.

[0042] 2. Mouse plasma sample processing

[0043] Take 100 μL of each group of mouse plasma samples in 1.5 mL EP tubes, add 400 μL of 80% methanol to precipitate the protein, vortex after shaking, and stand in ice water bath for 5 min, centrifuge at 15000 x g in 4°C centrifuge for 20 min. Take 100 μL of supernatant in a new 1.5 mL EP tube, add 51 μL of distilled water to dilute the methanol concentration to 53%, centrifuge at 15000 x g in 4°C centrifuge for 20 min, take the supernatant and load into the sample bottle, and wait for sample analysis.

[0044] Take an equal volume of each experimental sample and mix as a quality control (QC) sample, and sample every 5 needle cycles during the sampling process.

[0045] 3. Mouse placental tissue sample processing

[0046] Weigh 100 mg of mouse placenta, grind thoroughly in liquid nitrogen, and transfer to a 1.5 mL EP tube, add 500 μL of 80% methanol to precipitate the protein, vortex after shaking, and stand in ice water bath for 5 min, centrifuge at 15000 x g in 4°C centrifuge for 20 min. Take 100 μL of supernatant in a new 1.5 mL EP tube, add 51 μL of distilled water to dilute the methanol concentration to 53%, centrifuge at 15000 x g in 4°C centrifuge for 20 min, take the supernatant and load into the sample bottle, and wait for sample analysis. Take an equal volume of each experimental sample and mix as a quality control (QC) sample, and sample every 5 needle cycles during the sampling process.

[0047] Take an equal volume of each experimental sample and mix as a quality control (QC) sample, and sample every 5 needle cycles during the sampling process.

[0048] 4. Chromatographic conditions

[0049] Chromatographic column: Hypesil Gold column (C18)

[0050] Column temperature: 40°C

[0051] Flow rate: 0.2 mL / min

[0052] Positive ion mode mobile phase: A (0.1% formic acid) and B (methanol)

[0053] Negative ion mode mobile phase: A (5 mM ammonium acetate, PH = 9.0) and B (methanol)

[0054] The chromatographic gradient elution program is as follows Table 1:

[0055] Table 1 Chromatographic gradient elution program

[0056]

[0057] 5. Mass spectrometry conditions

[0058] Mass spectrometry analysis was performed on a quadrupole orbiting ion trap mass spectrometer equipped with a thermospray ion source. The positive and negative ion source spray voltage was 3.5 kV, the sheath gas flow rate was 35 psi, the auxiliary gas flow rate was 10 L / min, the ion transfer tube temperature was 320 °C, the auxiliary gas heating temperature was 350 °C, and the mass-to-charge ratio scan range was 100-1500.

[0059] 6. Data processing

[0060] The raw data was imported into CD3.1 search software for processing. Each substance was subjected to simple screening of retention time, mass-to-charge ratio, etc. Different samples with a retention time deviation of 0.2 min and a mass deviation of 5 ppm were subjected to deconvolution, peak alignment; then, peak extraction and peak area quantification were performed on samples with a mass deviation of 5 ppm, a signal intensity deviation of 30%, a signal-to-noise ratio of 3, combined with minimum signal intensity and adduct ions, etc. The target ions were integrated, the molecular formula was predicted through the molecular ion peak and the fragment ion, and compared with mzCloud, mzVault and Masslist. The background ions were removed using the 53% methanol sample. The sample raw peak area / QC sample metabolite quantification sum was obtained, the relative peak area was obtained, and the compounds with a relative peak area greater than 30% in the QC sample were deleted to obtain the metabolite identification and relative quantification results.

[0061] 7. Multivariate statistical analysis

[0062] In the multivariate statistical analysis section, the data was converted using the metabolomics data processing software metaX for principal component analysis (PCA). Further, a supervised multivariate statistical method, i.e., partial least squares discriminant analysis (OPLS-DA), was used for statistical analysis of the two groups of samples. The VIP (Variable Importance in the Projection) value of the OPLS-DA model (threshold > 1) was used in combination with the P value of the t-test (P < 0.05) to find differentially expressed metabolites. The qualitative method for differentially expressed metabolites was to search online databases (HMDB) (compare the mass-to-charge ratio m / z or the accurate molecular mass mass of the mass spectrum, with an error limit of 0.01 Da).

[0063] 8. Results

[0064] After peak alignment, peak correction, deconvolution, etc., a total of 773 metabolites were identified in the mouse plasma. PCA analysis can reflect the differences between the metabolites of each group of samples as a whole, and the results show that the overall distribution of the plasma metabolites of the control group, the model group and the Jianshi Shoutaiwan group has a small degree of differentiation (e.g.,Figure 1 A) To maximize the differences between groups, further supervised OPLS-DA analysis was performed. The results showed that there were significant differences in the distribution of plasma metabolites between the control group and the model group, and between the model group and the Shuitai Pill group (e.g. Fig. 2A). Figure 1 B, D). Seven cycles of cross-validation were performed on the OPLS-DA model. The R2Y and Q2Y of the OPLS-DA model between the control group and the model group were 0.873 and 0.21, respectively, and the R2Y and Q2Y of the OPLS-DA model between the model group and the Shuitai Pill group were 0.92 and 0.717, respectively. Both R2Y and Q2Y were close to 1, indicating that the model was good. The 200x permutation test was used to determine whether the OPLS-DA model was over-fitted. The test results showed that R2>Q2 and -1 Figure 1 C, E).

[0065] A total of 774 metabolites were identified in the mouse placental tissue. PCA analysis showed that the overall distribution of placental metabolites in the control group, the model group, and the Shuitai Pill group had little distinction, and the overall distribution of placental metabolites in the Shuitai Pill group was more similar to that in the control group (e.g. Fig. 3A). Figure 2 A) Further supervised OPLS-DA analysis showed that there were significant differences in the distribution of placental metabolites between the control group and the model group, and between the model group and the Shuitai Pill group (e.g. Fig. 3A). Figure 1 B, D). Seven cycles of cross-validation were performed on the OPLS-DA model. The R2Y and Q2Y of the OPLS-DA model between the control group and the model group were 0.975 and 0.708, respectively, and the R2Y and Q2Y of the OPLS-DA model between the model group and the Shuitai Pill group were 0.966 and 0.474, respectively. Both R2Y and Q2Y were close to 1, indicating that the model was good. The 200x permutation test was used to determine whether the OPLS-DA model was over-fitted. The test results showed that R2>Q2 and -1 Figure 2 C, E).

[0066] Significant difference metabolites were screened with the conditions of VIP>1, FC>1.2 or <0.833, and P<0.05. The results showed that there were no significant difference metabolites in the plasma between the control group and the model group (e.g. Fig. 2A). Figure 3 A), and there were 3 significant difference metabolites in the plasma between the Shuitai Pill group and the model group: Dangguiglycoside, 6-(3-hydroxy-2-butyl)-5-hydroxymethyl-4-methoxy-2H-pyran-2-one, and 3,4-dihydroxyphenylpropionone (e.g. Fig. 2A). Figure 3B-C). By comparative analysis with the ingredients of traditional Chinese medicine, it was found that among them, sarracenin was the prototype compound, and 6-(3-hydroxy-2-butyl)-5-hydroxymethyl-4-methoxyl-2H-pyran-2-one was a drug-derived metabolite. This result showed that sarracenin and 6-(3-hydroxy-2-butyl)-5-hydroxymethyl-4-methoxyl-2H-pyran-2-one were the migrating substances of Jianwei Shuitai Pills in the natural abortion model mice, and were the important pharmacodynamic material basis for preventing and treating spontaneous abortion.

[0067] The results showed that there were 14 significantly different placental metabolites between the model group and the control group (such as Figure 4 .A), and there were 12 significantly different placental metabolites between the Jianwei Shuitai Pills group and the model group (such as Figure 4 .B). Among them, the placental metabolites significantly adjusted by Jianwei Shuitai Pills included: N1-[1-(3-isopropenylphenyl)-1-methylethyl]-3-oxobutanamide (down-regulated), N-[1-(aminocarbonyl)-3-methylbutyl] tert-butyl carbamate (down-regulated), and 2E,4E-N-(2-methylpropyl)dodecane-2,4-diamide (up-regulated) (such as Figure 4 .C). In addition, the unique differential metabolites in the placenta of the Jianwei Shuitai Pills group and the model group included: sarracenin, 2-(2-amino-3-methylbutanamide)-3-phenylpropanoic acid, 3-[3-(β-D-glucopyranosyloxy)-2-methoxyphenyl]propanoic acid, L-aspartic acid, alanine-isoleucine, norbupleurophol, LPC 14:0, LPE 22:6, PC (14:1e / 2:0) (such as Figure 4 .D). By comparative analysis with the ingredients of traditional Chinese medicine, it was found that among them, sarracenin was the prototype compound, and 3-[3-(β-D-glucopyranosyloxy)-2-methoxyphenyl]propanoic acid was a drug-derived metabolite, and the remaining differential metabolites were endogenous or food-derived metabolites. Tracing the drug-derived metabolite 3-[3-(β-D-glucopyranosyloxy)-2-methoxyphenyl]propanoic acid, it was found that 3-[3-(β-D-glucopyranosyloxy)-2-methoxyphenyl]propanoic acid was a drug-derived metabolite produced by the metabolism of quercetin and chlorogenic acid in the intestinal tract and placental tissue.

[0068] Comprehensive analysis found that sarracenin was a common migrating component of Jianwei Shuitai Pills in the plasma and placental tissue of mice, indicating that sarracenin was an important pharmacodynamic material for Jianwei Shuitai Pills to prevent and treat abortion.

[0069] And further in vivo verification of the efficacy of sarracenin showed that sarracenin intervention could significantly reduce the abortion rate of abortion model mice, and there was no significant difference in the curative effect of Jianwei Shuitai Pills compound ( Figure 5A-B). And can effectively improve the abortion model mouse embryo and placental wet weight Figure 5 C-D). Therefore, when the drug can effectively inhibit spontaneous abortion, its curative effect is remarkable, has considerable treatment potential, provides a potential new drug selection for clinical prevention and treatment of spontaneous abortion, and has broad application prospect.

[0070] The above further describes the present application with specific examples, but it should be understood that the specific description herein should not be understood as limiting the essence and scope of the present application, and various modifications made by those skilled in the art after reading the above description are within the scope of the present application.

Claims

1. The use of swertisin as the only active ingredient in the preparation of a drug for preventing and treating spontaneous abortion.

2. Use according to claim 1, characterized in that, The drug can increase the wet weight of the embryo.

3. Use according to claim 1, characterized in that, The drug can increase the wet weight of the placenta.

4. Use according to claim 1, characterized in that, The swertisin is obtained by extraction and separation from Chinese herbal medicine or by chemical synthesis.

5. The use according to claim 1, characterized in that, The drug is an oral preparation or a needle injection.

6. Use according to claim 5, characterized in that, The oral preparation is a tablet, capsule, granule, pill, drop, fruit juice or syrup.

7. Use according to claim 5, characterized in that, The needle injection is an injection solution or a powder injection.

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