CN111588712A - Use of leonurine and its crystals in the preparation of anti-hyperhomocysteinemia drugs - Google Patents

Abstract

The invention belongs to the field of modern pharmacy of traditional Chinese medicines, relates to a Chinese herbal medicine motherwort extract and application thereof in pharmacy, and particularly relates to application of a medicinal crystal form of leonurine of the Chinese herbal medicine motherwort extract in preparation of a medicine, wherein the leonurine has the chemical name: 3, 5-dimethoxy-4-hydroxy-benzoic acid (4-guanidino) -1-butyl ester; in the invention, leonurine is prepared into 1 hydrate crystal form, and experiments prove that leonurine has the function of reducing Homocysteine (Hcy); the leonurine and the crystal form thereof can be used for preparing medicines for resisting various diseases caused by high Hcy, including cardiovascular and cerebrovascular diseases, senile dementia, cerebral apoplexy, venous embolism, neonatal defect and habitual abortion, are particularly suitable for early intervention of the diseases, have the function of reducing the incidence of early cardiovascular risk factors and other related diseases, and have the disease states that specific other risk factors such as triglyceride, cholesterol, low-density lipoprotein and the like are not increased but the cardiovascular and cerebrovascular diseases are easy to occur.

Description

Use of leonurine and its crystals in the preparation of anti-hyperhomocysteinemia drugs use

technical field

The invention belongs to the field of modernized traditional Chinese medicine pharmacy, and relates to a new use of a crystal of a Chinese herbal medicine Leonurus extract Leonurine in pharmacy, especially the use in the preparation of anti-Homocysteine (Hcy) hyperemia drugs. The anti-Hcy hyperemia drug of the present invention is suitable for preventing and treating diseases caused by Hcy, including cardiovascular and cerebrovascular diseases, senile dementia, stroke, venous embolism, neonatal defects and habitual abortion.

Background technique

The prior art discloses that Homocysteine (Hcy) is a sulfur-containing amino acid, an important intermediate product produced in the process of methionine metabolism, and is generated by demethylation of methionine. Under normal circumstances, the concentration of blood Hcy in the body is maintained at a low level, but genetic factors and secondary reasons will affect the metabolism of blood Hcy, resulting in an abnormal increase in blood Hcy concentration, resulting in hyper-Hcy hyperemia, which greatly increases coronary heart disease and peripheral blood. Risk of vascular disease and cerebrovascular disease. Therefore, the industry believes that excessive blood Hcy is an important disease risk factor; in addition, methionine load can induce hyperHcy; studies have found that both fasting hyperHcy and methionine load hyperHcy can induce cardiovascular disease risk factors.

It is evident that early intervention in major diseases such as cardiovascular disease is a more sensible treatment strategy in clinical practice. In view of the fact that risk factors such as hyper-Hcyemia require a long period of continuous action to lead to the occurrence of related diseases, there is still a sufficient time window for intervention after the diagnosis of hyper-Hcyemia; however, there is no specific A specific drug for this indication of hyperhomocysteinemia has come out; the existing intervention and treatment methods are limited to supplementing food components such as folic acid, vitamin _B6 , B12, etc., and the curative effect _on hyperhomocyemia patients who do not lack the above vitamins in their diets Therefore, the development of new anti-hyperHcy drugs for disease states has important clinical value.

The Chinese herbal medicine Motherwort (Lamiaceae, Herba Leonuri, Chinese Motherwort) was first recorded in ancient books such as "Shen Nong's Materia Medica", "Compendium of Materia Medica", and has the effects of promoting blood circulation, regulating menstruation, diuresis and swelling; Pharmacopoeia of the People's Republic of China (2000 edition) For the treatment of irregular menstruation, dysmenorrhea, amenorrhea, lochia, edema, oliguria, acute nephritis and edema. The previous research of this research team found that motherwort has a series of uses in addition to the above-mentioned known functions, including cardiovascular protection, etc., and further isolated and synthesized its monomeric active ingredient motherwort. use.

Studies have shown that hyperhomocysteinemia has become a risk factor for a variety of other diseases, including diabetes, Alzheimer's disease, pregnancy-induced hypertension, neonatal defects and habitual abortion; there is currently no specific treatment for hyperhomocysteinemia. medicine.

Based on the current state of the prior art, the inventors of the present application intend to provide new uses of leonurine and its crystals in the preparation of anti-hypercysemia drugs, which are especially suitable for early intervention before serious lesions occur. The high level of Hcy in the blood, a risk factor for the disease, reduces Hcy to normal levels, so as to intervene early in the disease caused by high Hcy.

SUMMARY OF THE INVENTION

The object of the present invention is to provide new medicinal uses of Leonurine, an active ingredient of traditional Chinese medicine Leonurus and its medicinal crystal form, in the preparation and treatment of hyperhypercy And drugs for early intervention before important organ damage, to prevent the occurrence or reduce the degree of various diseases caused by high Hcy hyperemia. In view of the fact that hyperhomocysteinemia is an independent major disease risk factor recognized by the medical community at present, it is an important early disease that needs timely treatment; secondary disease.

Studies have shown that hyperhomocysteinemia is an independent and important cardiovascular risk factor, which can directly damage human cells, especially vascular endothelial cells, leading to various cardiovascular and cerebrovascular diseases and peripheral vascular diseases; coronary atherosclerosis Sclerosis can lead to myocardial ischemia, angina pectoris and myocardial infarction; and cerebrovascular disease can lead to serious diseases such as stroke; the anti-hypercysemia drug provided by the present invention is suitable for preventing and treating diseases caused by Hcy, including cardiovascular and cerebrovascular diseases, peripheral Vascular disease, stroke, diabetes, Alzheimer's disease and other neurodegenerative diseases, neonatal defects, habitual abortion, pregnancy-induced hypertension, etc.; it is especially suitable for early intervention in the occurrence of the above-mentioned diseases, which can reduce early cardiovascular risk factors and other related The role of disease incidence.

A further object of the present invention is to use the above-mentioned active ingredients as raw materials of pharmaceutical preparations to prepare anti-Hcy hyperemia drugs.

The leonurine of the present invention is an active ingredient separated from motherwort, and is prepared in large quantities through artificial synthesis, and its chemical structural formula is:

The English name of Leonurine is: Leonurine; chemical name: 3,5-dimethoxy-4-hydroxy-benzoic acid (4-guanidino)-1-butyl ester.

The present application further analyzes the crystal structure of leonurine, obtains the best medicinal crystal form, and finds that the leonurine and its crystal form have the effect of anti-hypercysemia.

The present invention prepares leonurine into crystals of 6 different crystal forms through a specific method, specifically 6 kinds of crystals with different structures of leonurine sulfate, of which 2 are hydrates, 2 are anhydrous crystals, and one is anhydrous crystals. For methanolate, one for ethanolate.

For the convenience of description, the 6 kinds of Leonurine crystals described in the present invention are named as Form A, Form B, Form C, Form D, Form E and Form F, respectively.

The preferred crystal form A of the Leonurine crystal with the above-mentioned anti-Hcy hyperemia of the present invention has the following characteristics:

Described crystal form A Leonurine hemisulfate monohydrate crystal, its molecular structure is as shown in Figure 1;

The Leonurine hemisulfate monohydrate crystal represented by crystal form A is a colorless flaky crystal, belonging to the triclinic crystal system, its space group is P-1, and the asymmetric unit contains two Leonurine molecules, one Sulfate ion and 2 water molecules; the guanidine group of two Leonurine molecules and the negative ion of sulfate group form an ionic bond trimer, and the trimer and the trimer pass through the hydrogen bond of guanidine group and sulfate group A two-dimensional structure is formed to form the following multimer molecular plane layer (as shown in Figure 2);

In the present application, the multi-layer polymer molecular planes of the Leonurine hemisulfate monohydrate crystal represented by the crystal form A are superimposed and arranged in a parallel manner, and the molecular planes of its two-dimensional structure form Leonurine through the hydrogen bonding of water molecules. The following three-dimensional layered network structure of the hemisulfate monohydrate crystal, the hydrophilic layer formed by the guanidine group and the sulfate group is alternately arranged with the hydrophobic group of Leonurine (as shown in Figure 3).

In the present application, the spatial positions of all atoms in the single crystal molecule of the Leonurine hemisulfate monohydrate crystal represented by crystal form A conform to the functional atom coordinates and isotropic or equivalent isotropic displacement parameters shown in Table 1 below.

Table 1. Coordinates and isotropic or equivalent isotropic displacement parameters of all atoms in the single crystal molecule of Leonurine hemisulfate monohydrate crystal

In order to further identify the characteristics of the above-mentioned 6 kinds of Leonurine crystal forms, the present invention carries out X-ray powder diffraction (XRPD) analysis respectively, and obtains the XRPD data shown in the following table 2:

Table 2. XRPD data of six kinds of Leonurine crystals:

More specifically, the six crystal forms of Leonurine in the present invention refer to crystals with the above-mentioned XRPD characteristics.

Further, the leonurine and its crystals of the present invention can be used for preparing medicines for treating hyperHcy hyperemia.

The invention provides a new anti-Hcy hyperemia drug for preventing and treating various diseases caused by Hcy, including cardiovascular and cerebrovascular diseases, peripheral vascular diseases, cerebral apoplexy, diabetes, senile dementia and other neurodegenerative diseases, neonatal Infant defects, habitual abortion, gestational hypertension, etc. It is especially suitable for early intervention in the occurrence of the above-mentioned diseases, and has the effect of reducing the incidence of early cardiovascular risk factors and other related diseases.

The leonurine and its crystals of the present invention are also suitable for specific types of patients with amino acid metabolism disorders in precision medicine practice, that is, patients with high Hcy hyperemia; in view of this, the present invention provides a kind of treatment before clinical lesions occur. Preventive drugs for early blocking; this drug can prevent the damage of the following important organs of the human body caused by high Hcy due to the treatment of diseases secondary to high Hcy, including but not limited to: heart, coronary arteries, cerebral arteries, Damage and lesions of important human blood vessels such as carotid artery, femoral artery, kidney, brain, liver, etc., especially the damage of vascular endothelium in various important organs.

The leonurine crystals of the present invention can be used as pharmaceutical raw materials, directly or mixed with solid or liquid auxiliary materials commonly used in pharmaceutical preparations to prepare suitable preparations, including capsules, tablets, powders, suspensions and compound preparations.

A large number of pharmacological studies have been carried out in the present invention, and the results show that the leonurine and its A-type crystals have the effect of reducing the Hcy level of animals on a high-fat diet.

In the experiment of the present invention, ApoE gene knockout (ApoE ^-/- ) mice are used as high-fat animal models, and are fed with high-fat diets; on the basis of their genetic defects, they are placed in a metabolic disorder state caused by high-fat diets , leading to the obstacles of lipid metabolism and amino acid metabolism, used to verify the regulation of Leonurine and its A-type crystals on blood Hcy levels;

The experimental results showed that the leonurine and its A-type crystals reduced the level of Hcy in the blood of high-fat fed ApoE ^-/- mice.

The present application provides the use of leonurine crystals in the preparation of anti-hypercysemia drugs, the anti-hypercysemia drugs are especially suitable for early intervention in the occurrence of the above-mentioned diseases, and have the advantages of reducing early cardiovascular risk factors and other related The role of disease incidence. In view of the fact that there is no specific drug for treating hyperhomocyemia at present, the present invention provides a new method and a new drug for treating hyperhomocyemia.

Description of drawings

Figure 1 is the molecular structure of Leonurine crystal form A.

Figure 2 is the two-dimensional structure of Leonurine crystal form A molecule.

Figure 3 is the three-dimensional structure of Leonurine crystal form A molecule.

Fig. 4 is a statistical graph of experimental data of Leonurine A-type crystals reducing the Hcy level of lipid metabolism disorder model mice, Leonurine A-type crystals can effectively reduce the Hcy level of model mice; wherein,

From left to right, they are divided into 5 groups: control group (ApoE ^-/- mice fed with normal chow, daily control solvent gavage); ApoE ^-/- mice fed with high-fat diet, daily control solvent gavage Stomach; ApoE ^-/- mice were fed with high-fat diet and gavage with orofatine A crystal solution every day (10 mg/kg/day); ApoE ^-/- mice were fed with high-fat diet and every day of oracleine A crystals The solution was intragastrically administered (20 mg/kg/day); ApoE ^-/- mice were fed with high-fat diet, and the orofatine A crystal solution was administered by intragastric administration (40 mg/kg/day) every day. The sample numbers of the above 5 groups are: 14, 16, 8, 8, and 8, respectively. Data are mean ± standard error; P<0.05 represents a statistically significant difference.

Detailed ways

Example 1 Validation of the effects of leonurine and its A crystals on blood Hcy levels in ApoE ^-/- mice fed with high-fat diet in an animal model of metabolic disorder

The experimental methods included: ApoE ^-/- mice were fed with normal chow in the SPF animal room until 6 weeks of age, and then randomly divided into 5 groups according to the following grouping scheme for feeding and administration:

1) Control group (ApoE ^-/- mice, fed with normal chow, n=14), three times a day for gavage with distilled water (1 ml/kg body weight);

2) Model solvent control group (ApoE ^-/- mice, high-fat diet, n=16), three distilled water gavage (1 ml/kg body weight) once a day;

3) The low-dose group of Leonurine A crystals (ApoE ^-/- mice, high-fat diet, n=8), the Leonurine A crystal solution was administered by gavage once a day (drug dose was 10 mg/kg/day, drug concentration 10 mg/ml);

4) Leonurine type A crystal medium dose group (ApoE ^-/- mice, high-fat diet, n=8), once a day the leonurine type A crystal solution was administered by gavage (drug dose was 20 mg/kg/day, drug concentration 20 mg/ml);

5) The high-dose group of Leonurine A crystals (ApoE ^-/- mice, high-fat diet, n=8), the Leonurine A crystal solution was administered by gavage once a day (drug dose was 40 mg/kg/day, drug concentration 40 mg/ml);

The above-mentioned groups 2-5 to the 6th week of age did not start to switch to high-fat diet feeding, and began to be given orurine A crystal solution or solvent gavage every day at the same time according to the above grouping plan; group 1 continued to be fed with ordinary diets And the solvent was given by gavage every day; after 5 weeks, blood was collected in each group to measure the Hcy level at the same time;

After the 5-week dosing period, mice were anesthetized with sodium pentobarbital according to their body weight, and blood was collected from the eyeballs according to the following steps:

The mice were fasted without food and water the night before (in order to avoid mixing food debris in the bedding, new cages were changed during fasting) for 12-14 hours. Take a clean 1.5mL EDTA anticoagulant EP tube, numbered. Grab the mouse with the left hand, extrude one side of the eyeball, and hold the prepared capillary glass tube with a length of about 3-5cm and a diameter of 0.3mm in the right hand (be careful to use a flat end, do not use a sharp end to prevent the capillary tube from breaking and causing damage in the eyes of the mouse. (continuous injury), insert the inner eyelid of the mouse along a 45° angle (feel a feeling of falling), then twist the capillary, the blood flows into the EP tube along the wall of the tube, about 200-300 μL, carefully pull out the capillary quickly and smoothly, and press with a cotton ball to stop the bleeding . The EP tube after blood collection was placed on ice, and after all the blood collection of all mice was completed, centrifuge at 4° C., 3000 rpm for 15 minutes. Carefully pipet the upper pale yellow liquid (plasma) into a new EP tube (do not pipette the pellet) for subsequent experiments.

Example 2 Detection of mouse plasma Hcy level by enzyme-linked immunosorbent assay

Experimental principle: The kit uses a competitive method to detect the content of Hcy in the sample; add the sample to the enzyme-labeled wells pre-coated with antibodies, and then add the biotin-labeled recognition antigen, incubate at 37 °C for 30 minutes, and the two are combined with the solid phase. The antibody competed for binding to form an immune complex, and the unbound biotin antigen was removed by washing with PBST, then avidin-HRP was added, and incubated at 37 °C for 30 min, avidin-HRP was combined with biotin antigen, and the bound HRP was washed It catalyzes TMB (tetramethylbenzidine) into blue, and then converts into yellow under the action of acid, and has an absorption peak at 450nm wavelength, and the absorption value is negatively correlated with the concentration of antigen in the sample;

Operation steps: Please equilibrate the kit at room temperature for half an hour before use;

Blank well: do not add sample, only add color developer A, B and stop solution for zero adjustment;

Standard well: add 50μl of diluted standard to each well, zero well: add 50μl of standard/sample diluent, and then add 50μl of biotin antigen working solution;

Sample well: add 50μl of sample, then add 50μl of biotin antigen working solution;

Shake gently, cover with sealing film, and incubate in a 37°C incubator for 30 min;

The 25-fold concentrated washing solution is diluted 25-fold with distilled water for subsequent use;

The first washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let it stand for 30 seconds, then discard, repeat this 5 times, and pat dry;

Add 50 μl of avidin-HRP to the standard wells and sample wells, shake gently, cover with sealing film, and incubate in a 37°C incubator for 30 minutes;

The second washing: carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let it stand for 30 seconds, then discard, repeat this 5 times, and pat;

Color development: first add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and develop color at 37°C for 10 minutes in the dark;

Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue turns to yellow immediately);

Determination: Zero the blank well, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The determination should be carried out within 10 minutes after adding the stop solution;

Calculation: Calculate the regression equation of the standard curve according to the concentration and OD value, it is recommended to use special calculation software for calculation, it is recommended to use ELISAcalc for calculation, and the fitting model uses logistic curve (four parameters);

Statistical methods: Statistical software SPSS19.0 was used for data analysis. The measurement data were expressed as mean±SEM; the mean comparison between groups was performed by one way-ANOVA, with P<0.05 indicating statistical difference;

Experimental results: As shown in Figure 4, in APOE knockout mice fed with high-fat diet, Leonurine A crystal can significantly reduce the blood Hcy level of mice at a dose of 20 mg/kg/day. Compared with the model group given the control solvent (group 4 compared with group 2), P=0.001, the difference is statistically significant; Leonurine type A crystals at a dose of 40 mg/kg/day, can Compared with the model group given control solvent (group 5 and group 2), P=0.002, the difference is also statistically significant; Leonurine type A crystal at 10 mg Under the dosage condition of /kg/day, although it failed to significantly reduce the blood Hcy level of mice, when the dose was increased to 20 and 40 mg/kg/day, it showed the effect of significantly reducing the blood Hcy level of mice; Therefore, its effect on reducing blood Hcy level is dose-dependent;

The experimental results showed that leonurine and its A-type crystals reduced the Hcy level in high-fat-fed ApoE ^-/- mice, and the leonurine and its crystals could be used to prepare medicines for the treatment of hyper-Hcy hyperemia, and further to prevent hyperglycemia. Preventive drugs for the occurrence of diseases caused by Hcy hyperemia can block the damage and lesions of the body caused by Hcy at an early stage.

Claims (9)

1. The use of Leonurine (British name: Leonurine; chemical name: 3, 5-dimethoxy-4-hydroxy-benzoic acid (4-guanidino) -1-butyl ester) with the following chemical structural formula in the preparation of medicaments for treating hypercy hemaemia,

the drug reduces the Hcy level in vivo.

2. The use according to claim 1, wherein the leonurine further comprises leonurine hemisulfate monohydrate crystals prepared according to the molecular structure below for the preparation of a medicament for the treatment of hypercycy blood disease, said medicament reducing the level of Hcy in the body;

3. the use according to claim 2, wherein the leonurine hemisulfate monohydrate crystals are colorless plate crystals belonging to monoclinic system, and have space group P-1, and comprise 2 leonurine molecules, 1 sulfate ion and 2 water molecules in an asymmetric unit; wherein, the guanidyl of the two leonurine molecules and the negative ions of the sulfate radical form an ionic bond trimer, and a two-dimensional structure is formed between the trimer and the guanidyl through the hydrogen bonding of the guanidyl and the sulfate radical to form a polymer molecule plane layer;

4. use according to claim 2, characterized in that the planes of the molecules of the multilayer polymer of the crystals of leonurine hemisulfate monohydrate are arranged in a superimposed parallel manner, the planes of the molecules of the two-dimensional structure thereof forming, by hydrogen bonding of water molecules, a three-dimensional layered network of crystals of leonurine hemisulfate monohydrate, the hydrophilic layers of guanidino and sulfate groups thereof forming alternating arrangements with the hydrophobic groups of leonurine;

5. the use according to claim 2, characterized in that the leonurine hemisulfate monohydrate crystals have the spatial positions of all atoms in the single crystal molecules in accordance with the following functional atomic coordinates and the parameters of equirectangular or rather equirectangular displacement;

6. use according to claim 2, characterized in that the crystalline leonurine hemisulfate monohydrate has the following X-ray powder diffraction parameters:

7. the use of leonurine and its hemisulfate monohydrate crystals as claimed in claims 1-6, wherein the leonurine and its hemisulfate monohydrate crystals are used for preparing a medicament for preventing secondary diseases caused by high Hcy, wherein the secondary diseases are various diseases caused by high Hcy, including cardiovascular and cerebrovascular diseases, senile dementia, cerebral apoplexy, venous embolism, neonatal deficiency and habitual abortion.

8. The use according to claims 1-6, characterized in that leonurine and its hemisulfate monohydrate crystals are used for the preparation of a medicament for the prevention of secondary diseases caused by high Hcy, such as cardiovascular and cerebrovascular diseases, senile dementia, cerebral stroke, venous embolism, neonatal deficiency or habitual abortion.

9. Use according to claims 1-6, wherein leonurine and its hemisulfate monohydrate crystals are used for the preparation of a medicament for the prevention of secondary diseases caused by high Hcy, i.e. cardiovascular and cerebrovascular diseases caused by high Hcy and disease states which are not elevated but still susceptible to development of cardiovascular and cerebrovascular diseases at specific other risk factors such as triglycerides, cholesterol and low density lipoproteins etc.

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