Leonurine crystal and use thereof in preparation of insulin sensitizer, hypoglycemic drug and lipid-lowering drug

Abstract

This invention belongs to the modern pharmaceutical field of Traditional Chinese Medicine, and relates to an herbal extract of Chinese Motherwort and its application in pharmacy, which specifically relates to the crystal structure of a Chinese Motherwort extract: Leonurine, and its application in the preparation of medicine. The chemical name of the above-mentioned Leonurine is 4-guanidino-1-)butyl 4-hydroxy-3,5-dimethoxybenzoate. The invention by specific methods prepares leonurine as 6 kinds of crystals with different crystal forms. Specifically, there are six different structures of leonurine sulfate crystals, two of them are hydrate, two are anhydrous crystal form, one is methanol solvate, one is ethanol solvate. The leonurine crystal forms of this invention can applicate in preparing medicine such as insulin sensitizer, hypoglycemic and lipid-lowering drugs. The above mentioned insulin sensitizers are particularly useful in treating insulin resistance syndrome, and the above mentioned lipid-lowering drugs are useful in the treatment of disorders of lipid metabolism, hyperlipidemia and their complications.

Claims

1. A crystalline form of leonurine hemisulfate wherein a powder X-ray diffraction pattern having characteristic peaks in degrees 2θ at 9.55°, 10.64°, 16.06°, 21.94°, 22.16°, 24.27°, 25.62° and 26.77°.

2. The crystalline form according to claim 1, wherein a powder X-ray diffraction pattern is shown in FIG. 15.

3. The crystalline form according to claim 1, wherein the crystalline form belongs to triclinic system, space group thereof is P-1, unit cell parameters thereof are a=7.857(5) Å, b=13.913(8) Å, c=17.314(10) Å, α=103.993(12)°, β=99.612(12)°, γ=100.482(15), and unit cell volume thereof is 1761.2(18) Å.sup.3.

4. The crystalline form according to claim 1, wherein the crystalline form is obtained under a condition of, in the 25° C. suspension experiment, in a solvent of methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, nitromethane, ethyl acetate, isopropyl acetate, isopentyl alcohol, methyl tert-butyl ether, toluene, methyl isobutyl ketone, n-hexane, n-heptane, ethyl ether, dichloromethane, trichloromethane, petroleum ether or water; or in the 50° C. suspension experiment, in a solvent if methanol, ethanol, acetone, methyl ethyl ketone, isopropyl acetate, methyl tert-butyl ether, toluene, n-hexane, n-heptane, ethyl ether, trichloromethane, petroleum ether or water; or in the 25° C. and 50° C. mixing solvent suspension and volatilization of some of solvents.

5. The crystalline form according to claim 1, wherein a powder X-ray powder diffraction pattern is shown in the following table, TABLE-US-00012 2θ Relative intensity (%) 5.26 18.6 7.24 13.6 9.55 27.1 10.64 62.7 11.60 11.0 12.90 20.8 13.33 17.7 14.09 11.0 16.06 100.0 16.69 12.6 17.19 18.6 17.90 11.0 19.36 14.2 19.74 19.6 21.94 34.7 22.16 31.9 23.33 12.6 24.27 74.4 25.62 86.4 26.46 10.7 26.77 30.6 29.68 10.4 31.28 8.8 35.45 9.8 36.95 9.1 38.02 8.2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a differential scanning calorimetry (DSC) diagram of crystal form B.

(2) FIG. 2 is a thermogravimetric analysis (TGA) diagram of crystal form B.

(3) FIG. 3 is a Raman spectra (Raman) diagram of crystal form B.

(4) FIG. 4 is an XRPD diagram of leonurine crystal form E.

(5) FIG. 5 is an XRPD diagram of crystal form C.

(6) FIG. 6 is an XRPD diagram of crystal form D.

(7) FIG. 7 is an XRPD diagram of crystal form B.

(8) FIG. 8 is an XRPD diagram of crystal form F.

(9) FIG. 9 is a differential scanning calorimetry (DSC) diagram of crystal form F.

(10) FIG. 10 is a thermogravimetric analysis (TGA) diagram of crystal form F.

(11) FIG. 11 is a Raman spectra (Raman) diagram of crystal form F.

(12) FIG. 12 is a DVS diagram of crystal form A. Experiments show that crystal form A has no or almost no hygroscopicity. In a range of 0-95% RH, the dehydration and hygroscopicity of crystal form A is very weak.

(13) FIG. 13 is a powder polarizing image of crystal form A (200 times).

(14) FIG. 14 is a single crystal polarizing image of crystal form A (100 times).

(15) FIG. 15 is an XRPD diagram of crystal form A.

(16) FIG. 16 is the molecular structure of leonurine crystal form A.

(17) FIG. 17 is the two-dimensional molecular structure of leonurine crystal form A.

(18) FIG. 18 is the three-dimensional molecular structure of leonurine crystal form A.

(19) FIG. 19 shows the effects of three kinds of dosage of one acute administration of leonurine crystal form A on insulin sensitivity curve, where the insulin sensitivity curve of the 60 mg/kg dose group of leonurine crystal form A decreased significantly at the 60 minute time point (P=0.038), indicating that leonurine crystal form A increased the insulin sensitivity of the rat.

(20) FIG. 20 shows the effects of three kinds of dosage for continuous administration of leonurine crystal form A for 7 days on insulin sensitivity curve, where the insulin sensitivity curves of the 30 and 60 mg/kg/d dose group of leonurine crystal form A decreased significantly at the 30 minute time point (compared with the control group, P=0.004 and P=0.018, respectively); the insulin sensitivity curves of the three dose groups, including 30, 60 and 120 mg/kg/d of leonurine crystal form A, decreased significantly at the 60-minute time point (compared with the control group, P=0.011, P=0.018, and P=0.017); the insulin sensitivity curves of the 30 and 60 mg/kg/d dose group of leonurine crystal form A decreased significantly at the 90-minute time point (compared with the control group, P=0.009 and P=0.008, respectively).

(21) FIG. 21 shows the effects of chronic administration of leonurine crystal form A for 3 weeks on weight of rats. At the fourth week (one week after discontinuation), it was observed that leonurine crystal form A significantly decreased the weight of rats when the group dose was 60 mg/kg/d (compared with the solvent control group, P=0.033); however, the 120 mg/kg/d dose group of leonurine crystal form A had a declining trend of weight, but the difference was not statistically significant (compared with the solvent control group, P=0.057).

(22) FIG. 22 shows the effects of chronic administration of leonurine crystal form A for 3 weeks on fasting blood glucose of rats. After 3 weeks of continuous administration, the 120 mg/kg/d dose group of leonurine crystal form A significantly decreased fasting blood glucose of rats (compared with the solvent control group, P=0.009); however, the 60 mg/kg/d dose group of leonurine crystal form A had a declining trend of fasting blood glucose, but the difference was not statistically significant (compared with the solvent control group, P=0.083). At the fourth week, it was observed that both the 60 and 120 mg/kg/d dose groups of leonurine crystal form A decreased fasting blood glucose (compared with the solvent control group, P=0.016 and 0.043, respectively).

(23) FIG. 23 shows the effects of chronic administration of leonurine crystal form A for 3 weeks on glucose tolerance tests of rats, leonurine crystal form A could significantly decreased the glucose tolerance curve at the 15- and 60 minute-time point; at the 15 minutes time point, the glucose tolerance curve moved down, and the 30 mg/kg/d dose group of leonurine crystal form A could decrease the level of blood glucose, improve the body's tolerance to glucose when (compared with the solvent control group, P=0.012); at the 60-minute time point, the glucose tolerance curve also moved down, and the 30 and 120 mg/kg/d dose groups of leonurine crystal form A could decrease the level of blood glucose, improve the body's tolerance to glucose (compared with the solvent control group, P=0.026 and 0.005, respectively).

(24) FIG. 24 shows that chronic continuous administration of leonurine crystal form A could decrease the level of serum total cholesterol (TC) of hyperlipidemia mice. Total cholesterol (TC); Control; Model; Leonurine; ***, compared to the model group, P<0.001.

(25) FIG. 25 shows that leonurine crystal form A (medium dose group) could decrease the level of serum triglyceride of ApoE knockout mice. Total triacylglycerol (TG); Control; Model; Leonurine; *, compared to the model group, P<0.05; **, compared to the model group, P<0.01; ***, compared to the model group, P<0.001.

(26) FIG. 26 shows that leonurine crystal form A (medium dose group) could decrease the level of serum low density lipoprotein of ApoE knockout mice. LDL-cholesterol (LDL); Control; Model; Leonurine; **, compared to the model group, P<0.01; ***, compared to the model group, P<0.001.

(27) FIG. 27 shows that chronic continuous administration of leonurine crystal form A for 12 weeks could decrease the level of serum total cholesterol (TC) in the New Zealand white rabbit hyperlipidemia model. Total cholesterol (TC); Control; Model; Leonurine; **, compared to the model group, P<0.01; ***, compared to the model group, P<0.001.

(28) FIG. 28 shows that chronic continuous administration of leonurine crystal form A for 12 weeks could decrease the level of total triacylglycerol (TG) in the hyperlipidemia model of New Zealand white rabbit. Total triacylglycerol (TG); Control; Model; Leonurine; ***, compared to the model group, P<0.001.

(29) FIG. 29 is a Carotid atheromatous plaque micro-ultrasonographic which showed that atherosclerotic plaque formation at the bifurcation of the common carotid artery in each group was observed from the long axial section of the artery after 8 weeks of high-fat diet and administration, and the arrow points to the plaque in the thickening of the blood vessels. In the model group, the blood vessels were significantly thickened, and the plaque thickness of the drug group was significantly decreased and showed a dose-dependent effect.

(30) NC, normal diet group; MD, atherosclerotic model group; ST, Atorvastatin group; AS, aspirin group; SCM198-L, low dose group of leonurine crystal form A; SCM198-M, medium dose group of leonurine crystal form A; SCM198-H, high dose group of leonurine crystal form A; the white ruler represents 1 mm.

(31) FIG. 30 is a statistical comparison diagram of intimal-medial thickness (IMT) data at the largest plaque of carotid atheromatous plaque in the micro-ultrasonic end-diastole. The plaque thickness of model group was significantly thickened, and medium and high dose group of leonurine crystal form A could decrease plaque thickness;

(32) NC, normal diet group; MD, atherosclerotic model group; ST, Atorvastatin group; AS, aspirin group; SCM198 4 mg/kg/d, low dose group of leonurine crystal form A; SCM198 8 mg/kg/d, medium dose group of leonurine crystal form A; SCM198 16 mg/kg/d, high dose group of leonurine crystal form A; #P<0.01, compared with the normal group; *P<0.05, compared with the model group.

DETAILED DESCRIPTION OF THE INVENTION

(33) The following examples illustrate more specifically the preparation, method for the identification of Leonurine Crystal, drug-delivery way, forms of preparation, operating conditions and the order, as shown in this invention and examples can be changed appropriately within the limits of the spirit of the invention.

EXAMPLE 1

Preparation of Leonurine Crystal by Suspension Crystallization

(34) Take about 20 g leonurine sulfate raw material medicine, mix with 1 mL solvent respectively under the condition of 25° C. and 50° C. for at least 24 h, then filter solution separately, the solid part is dried in air for 10 min, and subsequently taking an X-ray powder diffraction (XRPD) detection. If it is observed that the measured XRPD spectrogram is different from the raw material spectrogram, further measurements are conducted (such as DSC, TGA, IR, DVS, etc.). The liquid part is volatilized in a vacuum to determination of approximate solubility of raw material medicine in solvents by gravimetric analysis. XRPD detection was conducted for the solid precipitated after solvent volatilization. If it is observed that the measured XRPD spectrogram is different from the raw material spectrogram, further measurements are conducted (such as DSC, TGA, IR, DVS, etc.).

(35) Gravimetric analysis method: Accurately take the filtrate of a certain volume (usually 0.5 mL) and put it into a dry and weighed container, recorded as M0 mg, weigh the total weight accurately after volatilizing the solvent in a vacuum, recorded as M1 mg. Then the mass of the precipitated solid is M1-M1, the volume of the solvent is V mL, according to this, the approximate solubility of the raw material in the solvent is X=(M1-M0)/V mg/mL.

(36) Table 2 shows the results of suspension crystallization in solvent under the condition of 50° C.

(37) TABLE-US-00002 TABLE 2 the results of suspension crystallization in solvent under the condition of 50° C. Solvent XRPD DSC TGA Raman Methanol — — — — Ethanol — — — — Isopropanol Crystal FIG. 1 FIG. 2 FIG. 3 Form B (FIG. 7) Acetone — — — — Methyl ethyl ketone — — — — Acetonitrile Crystal FIG. 1 FIG. 2 FIG. 3 Form B (FIG. 7) Tetrahydrofuran Crystal FIG. 1 FIG. 2 FIG. 3 Form B (FIG. 7) Nitromethane Crystal FIG. 1 FIG. 2 FIG. 3 Form B (FIG. 7) Ethyl acetate Crystal FIG. 1 FIG. 2 FIG. 3 Form B (FIG. 7) Isopropyl acetate — — — — Isoamyl alcohol Crystal FIG. 1 FIG. 2 FIG. 3 Form B (FIG. 7) Methyl t-butyl ether — — — — Toluene — — — — Methyl isobutyl Crystal FIG. 1 FIG. 2 FIG. 3 ketone Form B (FIG. 7) Hexane — — — — Heptane — — — — Diethyl ether — — — — Dichloromethane Crystal FIG. 1 FIG. 2 FIG. 3 Form B (FIG. 7) Chloroform — — — — Petroleum ether — — — — Water — — — —

(38) where, (1) Using “-” to indicate that no change has been detected compared to the raw material. (2) If it detects any changes compared to raw materials, using the DSC mark the Onset temperature, the TGA mark the weightlessness temperature and its percentage, and mark the corresponding spectrogram number respectively.

(39) The leonurine crystal form B with XRPD data characteristics as shown in Table 1 was obtained by the above method especially the solvent combination, the DSC, TGA and Raman spectral data characteristics are shown in FIG. 1, FIG. 2 and FIG. 3 respectively; The above data provides the basis for the identification of the leonurine crystal form B.

EXAMPLE 2

Preparation of Leonurine Crystal by Evaporative Crystallization

(40) Prepare 2 groups of leonurine sulfate samples, each group weighed 96 parts, about 3 mg per part, add solvent according to the design shown in Table 3, mix, and dissolve. Slowly volatilize to dry under the condition of 25° C. and 50° C. respectively (this process will take 24 h to several days), then collect solids, test and analyze XRPD, DSC, TGA and Raman spectroscopy. The aforementioned suspension experiments are used for the insoluble ones under this condition.

(41) TABLE-US-00003 TABLE 3 Slow volatile solvent table (showing the solvent combination scheme for the preparation of leonurine crystal by evaporative crystallization) 1 2 3 4 5 6 7 8 9 10 11 12 A 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul MeOH EtOH i-PrOH IAA Acetone MEK ACN THF NM EA MTBE Toluene B 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 200 ul 400 ul MeOH EtOH i-PrOH IAA Acetone MEK ACN THF NM EA MTBE Toluene 200 ul 200 ul 200 ul 200 ul 200 ul 200 ul 200 ul 200 ul 200 ul 200 ul 200 ul 200 ul H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O 200 ul 200 ul 200 ul MeOH MeOH MeOH C 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul MeOH EtOH i-PrOH IAA Acetone MEK ACN THF NM EA MTBE Toluene 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O H.sub.2O 200 ul 200 ul 200 ul MeOH MeOH MeOH D 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul MeOH EtOH i-PrOH IAA Acetone MEK ACN THF NM EA MTBE Toluene 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul Hex Hex Hex Hex Hex Hex Hex Hex Hex Hex Hex Hex E 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul MeOH EtOH i-PrOH IAA Acetone MEK ACN THF NM EA MTBE Toluene 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul Hep Hep Hep Hep Hep Hep Hep Hep Hep Hep Hep Hep F 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul MeOH EtOH i-PrOH IAA Acetone MEK ACN THF NM EA MTBE Toluene 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 200 ul 200 ul MTBE MTBE MTBE MTBE MTBE MTBE MTBE MTBE MTBE MTBE MeOH MeOH G 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul MeOH EtOH i-PrOH IAA Acetone MEK ACN THF NM EA MTBE Toluene 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 200 ul 200 ul Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene EtOH EtOH H 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul MeOH EtOH i-PrOH IAA Acetone MEK ACN THF NM EA MTBE Toluene 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 400 ul 200 ul MIBK MIBK MIBK MIBK MIBK MIBK MIBK MIBK MIBK MIBK MIBK MIBK

(42) Where, MeOH=Methanol; EtOH=Ethanol; i-PrOH=Isopropanol; IAA=Isoamyl alcohol; MEK=Methyl ethyl ketone; ACN=Acetonitrile; MTBE=Methyl tert-butyl ether; MIBK=Methyl isobutyl ketone; NM=Nitromethane; THF=Tetrahydrofuran; EA=Ethyl acetate; Hep=N-heptane; Hex=Hexane

(43) Experimental result: According to the solvent combination scheme shown in Table 3, after the solvent is volatilized, several kinds of leonurine crystals as shown in Table 4 below can be obtained.

(44) TABLE-US-00004 TABLE 4 Experiment results of slow solvent evaporation crystallization at 25° C. obtained according to the solvent combination scheme shown in Table 3. 1 2 3 4 5 6 7 8 9 10 11 12 A — — — — — — — — — — — — B — — Crystal — — — Crystal — — Form E Form E (FIG. 4) (FIG. 4) C — — Crystal Crystal — — — — — — Form E Form E (FIG. 4) (FIG. 4) D — E F G H Powder volatilization disappeared and could not be determined

(45) Where, (1) Using“-”to indicate that no crystal form changes have been detected compared to leonurine sulfate raw material. (2) If it's detected that there are any changes in the crystal form compared with leonurine sulfate raw material, the number of corresponding XRPD spectrogram is shown in parentheses. (3) The blank indicates that suspension crystallization are made because the sample cannot be dissolved.

(46) As shown in Table 4, according to the solvent combination scheme listed in Table 3, the solvents slowly volatile under the condition of 25° C., and the crystal form E of the leonurine can be obtained in four solvent combination schemes. This crystal has the XRPD spectrogram features shown in Table 1 and FIG. 4.

(47) According to the solvent combination scheme shown in table 3, suspension crystallization is used for samples that cannot be dissolved, and the results are shown in Table 5, crystal form C and crystal form D can be obtained by suspension crystallization test; The XRPD data for identifying crystal form C is shown in Table 1, and the spectrogram is shown in FIG. 5; The XRPD data for identifying crystal form D is shown in Table 1, and the spectrogram is shown in FIG. 6.

(48) TABLE-US-00005 TABLE 5 Experiment results of suspension at 25° C. obtained according to the solvent combination scheme shown in Table 3. 1 2 3 4 5 6 7 8 9 10 11 12 A — — — — — — — — — — — — B — — — C — — D Crystal Crystal — — — — — — — — — Form C Form D (FIG. 5) (FIG. 6) E Crystal Crystal — — — — — — — — — — Form C Form D (FIG. 5) (FIG. 6) F Crystal — — — — — — — — — Crystal Crystal Form C Form C Form C (FIG. 5) (FIG. 5) (FIG. 5) G Crystal — — — — — — — — — — — Form C (FIG. 5) H Crystal — — — — — — — — — — Form C (FIG. 5)

(49) where, (1) Using “-” to indicate that there is no change have been detected compared to leonurine sulfate raw materials. (2) If it detects any changes compared to raw materials, identify the crystal form and mark its XRPD spectrogram number in parentheses. (3) The blank indicates that evaporative crystallization are made because the sample can be dissolved.

(50) In this example, by designing different solvent combination schemes, after eliminating the solvent combination scheme that does not produce new crystal form (as compared to the synthesized leonurine sulfate raw material medicine), the leonurine crystal are prepared by evaporative crystallization in a soluble solvent combination scheme to obtain leonurine crystal form E. In the samples that could not be dissolved, the suspension crystallization was used for obtaining the leonurine crystal form C and D. In this example, the identification of different leonurine crystal forms is based on XRPD data and spectrogram. The solvent slow evaporation crystallization experiment was repeated according to the solvent combination scheme shown in Table 3 under the condition of 50° C., and the sample in which the leonurine could not be dissolved was used as a suspension crystallization test. The experiment results show that according to the solvent combination scheme shown in Table 3, volatilize the solvent can obtain several kinds of leonurine crystals as shown in Table 6 below.

(51) TABLE-US-00006 TABLE 6 Experiment results of slow solvent evaporation crystallization at 50° C. obtained according to the solvent combination scheme shown in Table 3. 1 2 3 4 5 6 7 8 9 10 11 12 A — — Crystal Crystal — — Crystal Crystal Crystal Crystal — — Form B Form B Form B Form B Form B Form B (FIG. 4) (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 7) B — Crystal Crystal — — — — Crystal — — Crystal Form E Form E Form E Form E (FIG. 4) (FIG. 4) (FIG. 4) (FIG. 4) C Crystal — Crystal Crystal — — — — — — — — Form E Form E Form E (FIG. 4) (FIG. 4) (FIG. 4) D — — — — E F G H

(52) Where, (1) Using “-” to indicate that there is no change have been detected compared to to the raw material. (2) If any change is detected compared to the raw material, mark the identified crystal form and mark its XRPD spectrogram number in parentheses. (3) The blank indicates that suspension crystallization are made because the leonurine sample cannot be dissolved.

(53) In the experiment shown in Table 6, in which the leonurine sample could not be dissolved was continuously subjected to a suspension crystallization test under the condition of 50° C., and the results are shown in Table 7:

(54) TABLE-US-00007 TABLE 7 Experiment results of suspension crystallization at 50° C. obtained according to the solvent combination scheme shown in Table 3. 1 2 3 4 5 6 7 8 9 10 11 12 A B Crystal Form E (FIG. 4) C D — — Crystal — Crystal — — — Form B Form B (FIG. 7) (FIG. 7) E — — Crystal Crystal — — Crystal — Crystal — — — Form B Form B Form B Form B (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 7) F — — Crystal Crystal — — Crystal Crystal — — Crystal Crystal Form B Form B Form B Form B Form C Form C (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 5) (FIG. 5) G — Crystal Crystal Crystal — — Crystal Crystal — — — Crystal Form D Form B Form B Form B Form B Form D (FIG. 6) (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 6) H Crystal — Crystal Crystal — — Crystal Crystal — Crystal — — Form C Form B Form B Form B Form B Form B (FIG. 5) (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 7) (FIG. 7)

(55) Where, (1) Using “-” to indicate that no change has been detected compared to leonurine sulfate raw materials; (2) If any change is detected compared to leonurine sulfate raw materials, identify the crystal form and mark its XRPD spectrogram number in parentheses; (3) The blank indicates that evaporative crystallization is made because the sample can be dissolved.

(56) The above experiment results show that under the condition of 50° C., according to the solvent combination scheme of Table 3, the combination of the crystal form of the leonurine is significantly different from that is obtained at 25° C. Specifically, the leonurine crystal form B and E were obtained in the volatile crystallization experiment (Table 6). In the suspension crystallization experiment, four crystal forms of leonurine were obtained, specifically crystal forms B, C, D and E (Table 7). Identification of all of the above-mentioned leonurine crystal form is based on the corresponding XRPD data and spectrogram shown.

EXAMPLE 3

Cooling Crystallization from Hot Concentrated Solution

(57) About 6 mg leonurine sulfate raw material was taken and dissolved in solvent, heated to 60° C. until completely dissolved, then cooled to room temperature, filtered, measure the solid, or put it into the refrigerator at 4° C. until the crystals are precipitated.

(58) TABLE-US-00008 TABLE 8 Cooling crystallization results in hot concentrated solution solvent XRPD DSC TGA Raman MeOH Crystal Crystal Crystal Crystal Form F Form F Form F Form F (FIG. 8) (FIG. 9) (FIG. 10) (FIG. 11) EtOH — — — — CHCl.sub.3 — — — — MTBE — — — —

(59) Where, (1) Using “-” to indicate that no change has been detected compared to to the raw material of the leonurine sulfate; (2) If any changes is detected compared to leonurine sulfate raw materials, using XRPD, DSC, TGA and Raman to identify and mark the corresponding spectrogram number. The DSC marks the T.sub.onset temperature, and the TGA marks the starting weightlessness temperature;

(60) Through the above experiments, the leonurine crystal form F was obtained by cooling crystallization in a hot solution of MeOH, and was determined by XRPD, DSC, TGA and Raman, confirmed as Form F. Identification of crystal form is based on the corresponding XRPD data and spectrogram.

EXAMPLE 4

Single Crystal Analysis of Leonurine Crystal Form A

(61) The results of the DVS experiment on the synthetic leonurine sulfate raw material medicine (as shown in FIG. 12) show that the raw material medicine itself is monohydrate crystal form, when the relative humidity is from 0 to 95%, the raw material medicine moisture absorption is about 0.15%, with hygroscopicity. In this invention, it is named as leonurine crystal form A, which is monohydrate crystal form, square block crystal (as shown in FIG. 13), no crystal transformation before melting, dehydrates at 120˜150° C. and transform into anhydrous crystal form B, and decomposes at about 250° C. In the range of 0˜95% relative humidity, the crystal form does not change, and the hygroscopicity changed slightly, showing no or almost no hygroscopicity. Under the condition of, in the suspension experiment at 25° C., in methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, nitromethane, ethyl acetate, isopropyl acetate, isoamyl alcohol, methyl tert-butyl ether, toluene, methyl isobutyl ketone, n-hexane, n-heptane, ether, dichloromethane, chloroform, petroleum ether, water and other solvents; and under the condition of, in the suspension experiment at 50° C., in methanol, ethanol, acetone, methyl ethyl ketone, isopropyl acetate, methyl tert-butyl ether, toluene, n-hexane, n-heptane, diethyl ether, chloroform, petroleum ether, water and other solvents; and under most solvent conditions in suspension and volatilization of mixed solvents at 25° C. and 50° C., form A can be obtained. FIG. 12 shows the DVS pattern of form A, and the hygroscopicity test results show that Form A has no or almost no hygroscopicity. In a range of 0˜95% RH, the dehydration and water absorption behavior of the crystal form is very weak. FIG. 14 shows single crystal polarized photo of crystal form A, single crystal of form A can be obtained by dissolved in acetonitrile/water or methyl ethyl ketone/water or acetone/water=2/1 (V/V) solution, or ethanol/water or acetone/water or methyl ethyl ketone/water or acetonitrile/water or tetrahydrofuran/water=1/1 (V/V) solution, or methyl tert-butyl ether/water/methanol=2/2/1 (V/V/V) solution and then slowly volatilize at room temperature; The separated single crystal of leonurine crystal form A was analyzed by X-ray diffraction, and the data thereof is shown in Table 1, the XRPD diagram is shown in FIG. 15.

(62) The structure of the leonurine crystal form A single crystal was determined by Bruker Smart Apex II single crystal X-ray diffractometer, instrument parameters: light source: Mo target; X-ray: Mo—K (=0.71073 Å); detector: CCD surface detector; resolution: 0.77 Å; current voltage: 50 kV, 30 mA; exposure time: 10 s; surface detector to sample distance: 50 mm; test temperature: 296 (2) K. The structure of leonurine crystal form A single crystal was analyzed by analyzing its X-ray diffraction data.

(63) Structural analysis and refine process: after the integration by substitution of the diffraction data by the SAINT program, the data is subjected to empirical absorption correction using the SADABS program. The single crystal structure was analyzed by direct method using SHEXLT2014, and the structure was refined by least square method; the hydrogen atom refinement process was obtained by isotropic calculation; the hydrogen atoms on O and N are obtained by residual electron density; the hydrogen atoms on C—H are obtained by calculation of hydrogenation, and refine it by adopting the riding model.

(64) The above measurement and data analysis gave the single crystal data of the leonurine crystal form A as shown in Table 9; the spatial positional parameters of all atoms in the molecule are shown in Table 10, based on the above data, the molecular structure of the leonurine crystal form A is depicted (FIG. 16). The leonurine crystal form A is hemisulfate monohydrate, is colorless lamellar crystal (FIG. 14). It belongs to monoclinic system, space group is P-1. The asymmetric unit contains two leonurine molecules, one sulfate ion and two water molecules. The guanidyls in two leonurine molecules and the sulfate ion form an ion bond trimer, a two-dimensional structure is formed between two trimers by hydrogen bondings of the guanidyls and the sulfate ions, to form a multimeric molecular plane layer (FIG. 17). The multi-layered multimeric molecular plane are superimposed in a parallel form, the two-dimensional structure molecular plane forms a three-dimensional laminated network structure of the leonurine crystal form A by hydrogen bondings of water molecules (FIG. 18). Three-dimensional structure shows that the hydrophilic layers formed by guanidyls and sulfate ions and the hydrophobic groups of leonurine are alternately arranged.

(65) TABLE-US-00009 TABLE 9 Data of leonurine crystal form A single crystal Molecular Formula C.sub.28H.sub.48N.sub.6O.sub.16S Crystal form A Collecting Temperature 296 K X-ray Generator Mo Kα Crystal Color Colorless and transparent Crystal Shape lamellar Crystal System triclinic Space Group P-1 a = 7.857(5) Å b = 13.913(8) Å c = 17.314(10) Å Unit Cell Parameters α = 103.993(12)° β = 99.612(12)° γ = 100.482(15)° Unit Cell Volume 1761.2(18) Å.sup.3 Calculate Density 1.427 mg/cm.sup.3

(66) TABLE-US-00010 TABLE 10 Spatial positional parameters of all atoms in leonurine single crystal form A (Functional atomic coordinates and isotropic or equivalent isometric displacement parameters) x y z U.sub.iso*/U.sub.eq O1 0.2706 (3) 0.97028 (15) 0.55640 (12) 0.0300 (5) O2 0.3705 (3) 1.10861 (17) 0.51794 (13) 0.0447 (6) O3 0.0092 (3) 1.01092 (15) 0.22144 (12) 0.0338 (5) O4 −0.1928 (3) 0.82326 (16) 0.20137 (12) 0.0341 (5) H4 −0.1505 0.8377 0.1630 0.051* O5 −0.1885 (3) 0.73474 (15) 0.31752 (12) 0.0346 (5) N1 0.5878 (3) 0.85074 (18) 0.95391 (15) 0.0323 (6) H1A 0.4982 0.8173 0.9127 0.039* H1B 0.6269 0.8205 0.9901 0.039* N2 0.7988 (3) 0.99643 (18) 1.02438 (14) 0.0319 (6) H2A 0.8498 1.0603 1.0301 0.038* H2B 0.8369 0.9655 1.0603 0.038* N3 0.6076 (3) 0.99373 (18) 0.90795 (14) 0.0284 (6) H3 0.6512 1.0596 0.9188 0.034* C1 0.6638 (4) 0.9466 (2) 0.96149 (17) 0.0243 (6) C2 0.4778 (4) 0.9423 (2) 0.83226 (17) 0.0289 (7) H2C 0.3569 0.9326 0.8431 0.035* H2D 0.4989 0.8745 0.8086 0.035* C3 0.4922 (4) 1.0054 (2) 0.77270 (17) 0.0293 (7) H3A 0.6173 1.0225 0.7680 0.035* H3B 0.4577 1.0700 0.7942 0.035* C4 0.3752 (4) 0.9502 (2) 0.68841 (17) 0.0290 (7) H4A 0.4108 0.8863 0.6657 0.035* H4B 0.2498 0.9324 0.6925 0.035* C5 0.3941 (4) 1.0176 (2) 0.63333 (16) 0.0280 (7) H5A 0.3707 1.0843 0.6589 0.034* H5B 0.5167 1.0295 0.6247 0.034* C6 0.2735 (4) 1.0259 (2) 0.50316 (17) 0.0277 (6) C7 0.1451 (4) 0.9732 (2) 0.42395 (17) 0.0262 (6) C8 0.0364 (4) 0.8775 (2) 0.41243 (17) 0.0262 (6) H8 0.0396 0.8461 0.4555 0.031* C9 −0.0761 (4) 0.8290 (2) 0.33707 (17) 0.0260 (6) C10 −0.0784 (4) 0.8749 (2) 0.27377 (16) 0.0252 (6) C11 0.0285 (4) 0.9717 (2) 0.28693 (17) 0.0265 (6) C12 0.1422 (4) 1.0215 (2) 0.36255 (16) 0.0253 (6) H12 0.2163 1.0871 0.3719 0.030* C13 0.0999 (5) 1.1133 (2) 0.23333 (19) 0.0384 (8) H13A 0.0766 1.1313 0.1819 0.058* H13B 0.2277 1.1205 0.2515 0.058* H13C 0.0579 1.1588 0.2748 0.058* C14 −0.1957 (5) 0.6871 (2) 0.3817 (2) 0.0407 (8) H14A −0.2797 0.6205 0.3607 0.061* H14B −0.2347 0.7300 0.4259 0.061* H14C −0.0775 0.6780 0.4026 0.061* O6 0.5617 (3) 0.41591 (16) 0.38037 (12) 0.0338 (5) O7 0.4343 (3) 0.28020 (18) 0.41548 (14) 0.0529 (7) O8 0.7916 (3) 0.34316 (17) 0.70748 (13) 0.0415 (6) O9 1.0084 (3) 0.52479 (17) 0.74073 (13) 0.0388 (6) O10 1.0101 (3) 0.63530 (16) 0.63798 (13) 0.0425 (6) N4 0.5054 (3) 0.53333 (18) 0.10860 (14) 0.0296 (6) H4C 0.6177 0.5643 0.1294 0.036* N5 0.2264 (3) 0.54760 (18) 0.05537 (15) 0.0344 (6) H5C 0.1882 0.4810 0.0419 0.041* H5D 0.1529 0.5857 0.0445 0.041* N6 0.4516 (3) 0.69053 (18) 0.11043 (16) 0.0352 (6) H6A 0.3785 0.7284 0.0983 0.042* H6B 0.5632 0.7192 0.1348 0.042* C15 0.3934 (4) 0.5902 (2) 0.09185 (17) 0.0273 (6) C16 0.4560 (4) 0.4229 (2) 0.09497 (18) 0.0305 (7) H16A 0.5634 0.3956 0.0923 0.037* H16B 0.3715 0.3919 0.0416 0.037* C17 0.3724 (4) 0.3918 (2) 0.16078 (18) 0.0332 (7) H17A 0.3434 0.3166 0.1479 0.040* H17B 0.2600 0.4144 0.1606 0.040* C18 0.4931 (4) 0.4366 (2) 0.24612 (17) 0.0314 (7) H18A 0.6187 0.4432 0.2421 0.038* H18B 0.4765 0.5054 0.2706 0.038* C19 0.4504 (4) 0.3691 (2) 0.29975 (17) 0.0345 (7) H19A 0.3238 0.3603 0.3024 0.041* H19B 0.4722 0.3011 0.2769 0.041* C20 0.5404 (4) 0.3608 (2) 0.43333 (18) 0.0323 (7) C21 0.6630 (4) 0.4073 (2) 0.51512 (17) 0.0283 (7) C22 0.7778 (4) 0.5035 (2) 0.53459 (18) 0.0316 (7) H22 0.7771 0.5420 0.4961 0.038* C23 0.8931 (4) 0.5422 (2) 0.61116 (18) 0.0310 (7) C24 0.8936 (4) 0.4849 (2) 0.66691 (17) 0.0287 (7) C25 0.7767 (4) 0.3908 (2) 0.64644 (18) 0.0287 (7) C26 0.6610 (4) 0.3505 (2) 0.57091 (18) 0.0297 (7) H26 0.5821 0.2855 0.5575 0.036* C27 0.6701 (4) 0.2497 (2) 0.69559 (19) 0.0374 (8) H27A 0.7000 0.2226 0.7420 0.056* H27B 0.6765 0.2011 0.6453 0.056* H27C 0.5496 0.2608 0.6912 0.056* C28 1.0149 (5) 0.6960 (2) 0.5826 (2) 0.0446 (9) H28A 1.1095 0.7580 0.6069 0.067* H28B 0.9006 0.7145 0.5713 0.067* H28C 1.0378 0.6573 0.5316 0.067* S1 0.01981 (10) 0.76948 (5) 0.03277 (4) 0.0261 (2) O13 0.1906 (3) 0.81191 (16) 0.09135 (14) 0.0414 (6) O14 −0.1179 (3) 0.81781 (16) 0.06340 (12) 0.0334 (5) O15 0.0315 (3) 0.78906 (16) −0.04547 (13) 0.0369 (5) O16 −0.0327 (3) 0.65947 (15) 0.02365 (13) 0.0342 (5) O11 0.7557 (3) 0.27715 (18) 0.15072 (15) 0.0474 (6) H11A 0.7592 0.2361 0.1799 0.071* H11B 0.7949 0.2554 0.1085 0.071* O12 0.8776 (3) 0.60262 (16) 0.16410 (13) 0.0380 (5) H12A 0.9110 0.6250 0.1262 0.057* H12B 0.8846 0.6524 0.2051 0.057* H9 1.012 (5) 0.474 (2) 0.767 (2) 0.054 (11)*

EXAMPLE 5

Accelerated Stability Tests of Various Leonurine Crystal Forms and Selection of Dominant Crystal Form

(67) After being placed in a stability chamber at 40° C. and 70% relative humidity for 10 days, the crystal forms A and E remain unchanged, and the crystal form B, crystal form C, crystal form D, and form F are all transformed into crystal form A;

(68) At 40° C. and relative humidity of 60%-80%, the 10-day stability test (Table 11) showed that the leonurine crystal form A was most stable, with no crystal transformation behavior, and the hygroscopicity was not obvious. The crystal form B, C, D and F all appeared crystal transformation; although the crystal form E did not appear crystal transformation, the hygroscopicity is strong, and the hygroscopicity is about ten times that of the crystal form A. Therefore, leonurine crystal form A is the preferred medicinal crystal form, it is a monohydrate with stable performance and can be obtained repeatedly by various conditions.

(69) TABLE-US-00011 TABLE 11 Comparison of various new leonurine crystal forms Crys- Melt- tal ing Crystal Form point hygroscopicity stability A — 60% RH, water absorption 0.07% stable 80% RH, water absorption 0.12% B 190.8° 60% RH, water absorption 0.14% 40° C., 70% RH, C. 80% RH, water absorption 0.30% 10 days, trans- form into A C — 60% RH, water absorption 0.56% 40° C., 70% RH, 80% RH, water absorption 1.05% 10 days, trans- form into A D — 60% RH, water absorption 1.63% 40° C., 70% RH, 80% RH, water absorption 0.98% 10 days, trans- form into A E — 60% RH, water absorption 0.07% stable 80% RH, water absorption 0.12% F 192.2° 60% RH, water absorption 0.14% 40° C., 70% RH, C. 80% RH, water absorption 0.30% 10 days, trans- form into A
Where: RH: Relative Humidity

EXAMPLE 6

Experiment of Single Dosage Acute Administration of Leonurine Crystal Form A to Improve Insulin Sensitivity in Rats

(70) Thirty-two Sprague-Dawley male rats (body weight 180-220 g) were randomly divided into 4 groups, gave control solvent and three different doses of leonurine crystal form A respectively. The control solvent group was given an equal volume of DMSO, and the treatment groups were administered with three doses of 30 mg/kg, 60 mg/kg and 120 mg/kg respectively, 8 rats per group, where the leonurine crystal form A was dissolved in DMSO at concentrations of 30 mg/ml, 60 mg/ml and 120 mg/ml respectively. The volume of solvent obtained per rat is one millilitre per kilogram of body weight. The route of administration is gavage, weighing before gavage, and taking blood from tail vein to measure blood glucose. Each rat was tested for insulin sensitivity 1 hour after administration of drugs or control solvent, the dose of insulin selected was 0.5 units per kilogram of body weight, intraperitoneal injection of insulin, blood glucose was measured before injection and at 15, 30, 60, 90, and 120 minutes after injection. The data were analyzed by SPSS 11.5 statistical software, and the results were expressed as mean ±standard error of the mean (mean±SEM), pairwise comparison between groups was examined by one-way analysis of variance (ANOVA), P<0.05 was considered significant difference between the groups. The experiment results (FIG. 19) showed that the downward shift of the insulin sensitivity curve of the rat was observed when leonurine crystal form A was administered at a dose of 60 mg/kg, and downward shift of the curve reached statistical significance (P=0.038) at the 60 minutes time point, indicating that the leonurine crystal form A increased the sensitivity of the rat to insulin. Compared with the DMSO control group, the leonurine crystal form A had a tendency to move down the insulin sensitivity curve at the 60-minute time point with the dose of 120 mg/kg, but no statistically significant difference was observed (P=0.079). In the 30 mg/kg dose group of leonurine crystal form A, the insulin sensitivity curve was basically coincident with the control group, and there was no statistically significant difference.

EXAMPLE 7

Experiment of Chronic Continuous Administration of Leonurine Crystal Form A to Improve Insulin Sensitivity in Rats

(71) Twenty Sprague-Dawley male rats (body weight 180-220 g) were randomly divided into 4 groups, give control solvent and three different doses of leonurine crystal form A respectively. The control solvent group was given an equal volume of DMSO, and the treatment groups were administered at three doses of 30 mg/kg, 60 mg/kg and 120 mg/kg respectively, 5 rats per group, where the leonurine crystal form A was dissolved in DMSO at concentrations of 30 mg/ml, 60 mg/ml and 120 mg/ml respectively. The volume of solvent used per rat is one millilitre per kilogram of body weight. The rats were administered intragastrically with the drugs or control solvent once a day for 7 consecutive days, and performing insulin sensitivity test on the 7th day. The rats were treated with the drugs or control solvent on the day of the test, and weighed before administration, then taking blood from tail vein to measure blood glucose. Each rat was tested for insulin sensitivity 1 hour after administration of the drugs or control solvent, and the rats were not fed food for 4 hours before the insulin sensitivity test to make them fasted but still given water. The insulin dose in the insulin sensitivity test is 0.75 units per kilogram of body weight, and insulin was injected through intraperitonealion. Blood glucose was measured before injection and at 15, 30, 60, 90, and 120 minutes after injection. The data were analyzed by SPSS statistical software (version 11.5), and the results were expressed as mean ±standard error of the mean (mean±SEM). Multiple group comparisons were tested by one-way analysis of variance (ANOVA). P<0.05 was considered as statistically significant. The experiment results (FIG. 20) showed that the downward shift of the insulin sensitivity curve of the rat was observed when leonurine crystal form A was administered at a dose of 30, 60 and 120 mg/kg/d. The insulin sensitivity curve was moved down significantly by different doses of the leonurine crystal form A at three time points (30 min, 60 min and 90 min). At 30 minutes point, leonurine crystal form A increased insulin sensitivity in both 30 and 60 mg/kg/d dose groups (P=0.004 and P=0.018, respectively) compared with the control group. At 60 minutes point, leonurine crystal form A increased insulin sensitivity in both 30, 60 and 120 mg/kg/d dose groups (P=0.011, P=0.018 and P=0.017) compared with the control group. At 90 minutes point, leonurine crystal form A increased insulin sensitivity in both 30 and 60 mg/kg/d dose groups(P=0.009 and P=0.008, respectively) compared with the control group. The above experiment results show that chronic continuous administration of leonurine crystal form A can increase insulin sensitivity.

EXAMPLE 8

Experiment of Chronic Continuous Administration of Leonurine Crystal Form A Reduced Body Weight, Fasting Blood Glucose and Increased Glucose Tolerance in Rats

(72) Forty male Sprague-Dawley rats weighed 180 to 220 g were randomly divided into 4 groups (10 rats each group): control group are treated with equal volume of DMSO, and the treatment groups were administered at 30 mg/kg per day, 60 mg/kg per day, and 120 mg/kg per day of leonurine crystal form A respectively, where leonurine crystal form A was dissolved in DMSO at concentrates of 30 mg/ml, 60 mg/ml and 120 mg/ml, and the volume of vehicle administered intragastrically in each rat was 1 ml per kg of body weight. The rats were treated with leonurine crystal form A or DMSO once a day continuously for 3 weeks, and the body weight and fasting blood-glucose were detected at third week. The rats were fasted (allowed water ad libitum) for 12 h before experiment. Fasting blood-glucose and body weight were tested after 1 h of administered drugs. The rats were not given drugs on the next days for one week, and the body weight and fasting blood-glucose were detected at forth week. The data were analyzed by SPSS statistical software (version 11.5), and results were expressed as the mean±standard error of the mean (mean±SEM). Multiple group comparisons were tested by one-way analysis of variance (ANOVA). P<0.05 was considered as statistically significant.

(73) The results suggested that the body weight of rats was significantly reduced at fourth week (the drug discontinued for one week) after administration of leonurine crystal form A at the dose of 60 mg/kg/d continuously for 3 weeks (p=0.033 vs vehicle group). The body weight of rats administrated of leonurine crystal form A at the dose of 120 mg/kg/d had the trend to reduce, but had not statistically difference (p=0.057 vs vehicle group) (FIG. 21).

(74) Fasting blood-glucose results showed significant reduce after administration of leonurine crystal form A at the dose of 120 mg/kg/d (p=0.009 vs vehicle group), but had not statistically difference at the dose of 60 mg/kg/d (p=0.083 vs vehicle group), after continuously administrated for 3 weeks (FIG. 22). However, fasting blood-glucose of rats were both significantly reduced after administration of leonurine crystal form A at the dose of 60 mg/kg/d and 120 mg/kg/d at forth week (p=0.016 and p=0.043 vs vehicle group, respectively) (FIG. 22).

(75) Eighteen male Sprague-Dawley rats weighed 180 to 220 g were randomly divided into 4 groups, and with vehicle or three different dose of leonurine crystal form A treatment respectively: control group are treated with equal volume of DMSO, and treatment groups are treated with 30 mg/kg per day, 60 mg/kg per day, or 120 mg/kg per day of leonurine crystal form A. Leonurine crystal form A was dissolved in DMSO at concentrates of 30 mg/ml, 60 mg/ml and 120 mg/ml, and the volume of vehicle administered intragastrically in each rat was 1 ml per kg of body weight. The rats were treated with leonurine crystal form A or DMSO once a day for 3 weeks. Oral glucose tolerance test (OGTT) was detected at third week, and the rats were fasted 12 h before OGTT experiment. OGTT experiment was performed 1 h after administered of drugs. Blood glucose and body weight were tested before OGTT. OGTT: Rats were given 25% glucose solution (2 g/kg of body weight, 8 ml/kg) by intraperitoneal injection, and blood glucose concentration was determined from the tail vein at 15, 30, 60 and 120 min. The drug was discontinued for one week after OGTT, and the body weight and fasting blood-glucose were detected at forth week.

(76) The data were analyzed by SPSS statistical software (version 11.5), and results were expressed as the mean±standard error of the mean (mean±SEM). Multiple group comparisons were tested by one-way analysis of variance (ANOVA). P<0.05 was considered as statistically significant. The glucose tolerance test showed that the glucose tolerance curve was significantly decreased at both 15 and 60 min time points. At 15 min time point, the glucose tolerance curve was shifted down. And the leonurine crystal form A at the dose of 30 mg/kg/d could reduce the degree of elevation of blood glucose and increase the tolerance of glucose to the body (P=0.012 vs vehicle group) (FIG. 23). Moreover, at 60 min time point, the glucose tolerance curve also was moved down at the dose of 30 mg/kg/d and 120 mg/kg/d. The leonurine crystal form A at the dose of 30 mg/kg/d and 120 mg/kg/d could both reduce the degree of elevation of blood glucose and increase the tolerance of glucose to the body (P=0.026 and P=0.005 vs vehicle group, respectively) (FIG. 23).

(77) The results suggested leonurine crystal form A can reduce body weight, fasting blood glucose and increase glucose tolerance in rats. Based on the previous toxicological study showed that the toxicity of leonurine crystal form A was very low, and far lower than the toxic dose. Therefore, the leonurine crystal form A can be considered with the therapeutic effect of disease instead of toxic effects.

EXAMPLE 9

Experiment of Chronic Continuous Administration of Leonurine Crystal Form A Reduced Lipid in Hyperlipidemia Mice

(78) Fifteen C57BL/6J mice (male, 6-8 weeks) and seventy-five ApoE knockout mice (male, 6-8 weeks) were used for experiments. C57BL/6J mice were fed with normal diet, and ApoE knockout mice were fed for 1 week with a diet of normal/high fat diet ratio of 1/1. All the mice were randomly divided into 6 groups (C57BL/6J mice as a group): 1) Control group (C57B/6J, Normal diet, n=15); Administered intragastrically with ultrapure water; 2) Model group (Vehicle group, ApoE, High fat diet, n=15); Administered intragastrically with ultrapure water; 3) Leonurine crystal form A in low dose group (ApoE, High fat diet, n=15); Administered intragastrically at a dose of 10 mg/kg/day; 4) Leonurine crystal form A in middle dose group (ApoE, High fat diet, n=15); Administered intragastrically at a dose of 20 mg/kg/day; 5) Leonurine crystal form A in high dose group (ApoE, High fat diet, n=15); Administered intragastrically at a dose of 40 mg/kg/day; 6) Atorvastatin group (Positive control group, ApoE, High fat diet, n=15); Administered intragastrically at a dose of 3 mg/kg/day;

(79) Mice were weighed before administered drugs every week, where the dosage of the drug for mice is calculate according to the weight; and housed under 24±1° C. room temperature and 55±5% air humidity condition, and allowed enough food and water ad libitum. After the drug administration continuously for 4 weeks, the mice were anesthetized with pentobarbital sodium according to body weight. Blood (0.5 ml per mouse) was drawn from the inner canthus vein for hematology analysis. Hematology analysis: blood samples were let for standing for 2 h and centrifuged (3000 rpm, 10 min, 4° C.) to obtain serum. The total cholesterol (TC), triacylglycerol (TG) and low-density cholesterol (LDL-C) levels in the serum samples were assessed using automatic biochemical analyzer. Statistical analysis: GraphPad Prism 5 software was used to analyze and draw figure. Results were expressed as the mean±standard (mean±SD), and statistical analysis was performed by one-way analysis of variance (ANOVA) or student's t test. A difference with P<0.05 was considered statistically significant. Statistical significance was set at *P<0.05, **P<0.01, ***P<0.001 versus the model group. The results suggested that the total cholesterol in the serum of ApoE knockout mice fed with high fat diet was higher than wide type fed with normal diet. Atorvastatin group and leonurine crystal form A in middle dose group reduced the total cholesterol in the serum compared with model group (vehicle group).

(80) The results suggested that the triacylglycerol in the serum of ApoE knockout mice fed with high fat diet was higher than wide type (C57BL/J) fed with normal diet. Atorvastatin group and leonurine crystal form A in middle dose group both reduced the triacylglycerol in the serum compared with model group (vehicle group) (FIG. 25).

(81) The results showed that the low-density cholesterol (LDL-C) levels in the serum of ApoE knockout mice fed with high fat diet was higher than wide type (C57BL/J) fed with normal diet. Atorvastatin group and leonurine crystal form A in middle dose group both reduced the LDL-C levels in the serum compared with model group (FIG. 26).

(82) The results suggested that chronic continuous administration of leonurine crystal form A for 4 weeks could reduce the total cholesterol, triacylglycerol and low-density cholesterol levels in the serum of ApoE knockout hyperlipidemia mice. Given that the total cholesterol, triacylglycerol and low-density cholesterol are independent risk factors for cardiovascular diseases, such as atherosclerosis, the effects of leonurine crystal form A can be used to treat disorders of lipid metabolism diseases characterized in high cholesterol, triglycerides and low-density lipids, which in turn reduces the risk of cardiovascular diseases, such as secondary atherosclerosis.

EXAMPLE 10

Experiment of Chronic Continuous Administration of Leonurine Crystal Form A Reduced Total Cholesterol and Triacylglycerol in Hyperlipidemia Rabbits

(83) Forty-eight male New Zealand rabbits weighed 1.8 to 2.2 kg were housed under 12 h dark-12 h light cycles and allowed food and water ad libitum to adapt to the housing environment for one week, and then randomly divided into 6 groups: 1) Control group (Normal diet, n=8); Administered intragastrically with ultrapure water; 2) Model group (High fat diet, n=8); Administered intragastrically with ultrapure water; 3) Leonurine crystal form A in low dose group (High fat diet, n=8); Administered intragastrically at a dose of 4 mg/kg/day; 4) Leonurine crystal form A in middle dose group (High fat diet, n=8); Administered intragastrically at a dose of 8 mg/kg/day; 5) Leonurine crystal form A in high dose group (High fat diet, n=8); Administered intragastrically at a dose of 16 mg/kg/day; 6) Atorvastatin group (Positive control group, ApoE, High fat diet, n=8); Administered intragastrically at a dose of 2.5 mg/kg/day;

(84) Rabbits were weighed before administered drugs every week, where the dosage of the drug for rabbits is calculate according to the weight; and housed in an air-conditioned room under (24±1° C. room temperature and 55±5% air humidity) and allowed enough food and water ad libitum. After 12 weeks of administration, the rabbits were fasted for 12 h, and blood (2 ml) was drawn from the marginal ear vein for hematology analysis. Hematology analysis: blood samples were let for standing for 2 h and centrifuged (3000 rpm, 10 min, 4° C.) to obtain serum. The serum samples were tested according to the protocols recommended by Nanjing Jiancheng Bioengineering Institute. The total cholesterol (TC), triacylglycerol (TG), low-density cholesterol (LDL-C) and how-density cholesterol (HDL-C) levels in the serum samples were assessed using automatic microplate reader and automatic biochemical analyzer. Statistical analysis: GraphPad Prism 5 software was used to analyze and draw figure. Results were expressed as the mean±standard (mean±SD), and statistical analysis was performed by one-way analysis of variance (ANOVA) or student's t test. A difference with P<0.05 was considered statistically significant. Statistical significance was set at *P<0.05, **P<0.01, ***P<0.001 versus the model group.

(85) The results showed that the total cholesterol in the serum of New Zealand white rabbit fed with high fat diet was higher than fed with normal diet. Leonurine crystal form A in high dose group reduced the total cholesterol in the serum compared with model group (vehicle group). However, the total cholesterol in the serum in atorvastatin group had no significant difference compared with model group. It indicated that the effects of leonurine crystal form A in high dose group to reduce total cholesterol were advanced than atorvastatin (FIG. 27).

(86) The results showed that the triacylglycerol in the serum of New Zealand white rabbit fed with high fat diet was higher than fed with normal diet. Atorvastatin group and leonurine crystal form A in middle/high dose group both reduced the triacylglycerol in the serum compared with model group (vehicle group) (FIG. 28).

(87) The results suggested that New Zealand white rabbits are highly susceptible to hyperlipidemia after giving a high-fat diet. However, chronic continuous administration of leonurine crystal form A could ameliorate the symptom of hyperlipidemia, including reduce total cholesterol and triacylglycerol in the serum, and better than the effect of Atorvastatin.

EXAMPLE 11

Experiment of Chronic Continuous Administration of Leonurine Crystal Form A Inhibited the Progression of Atherosclerotic Lesions in the Model of Hyperlipidemia Rabbits

(88) Forty-two male New Zealand rabbits weighed 2.0 to 2.5 kg were fed with normal diet to adapt to the housing environment for one week before experiment, and then the rabbits were fed with high fat diet (1% cholesterol; SF00-221 SpecialtyFeed Australia) for 8 weeks to establish the rabbit model of atherosclerosis. All the animals were randomly divided into 7 groups, and each group contains 6 rabbits: normal diet group (NC group), atherosclerosis model group (MD group), lipid lowering drug Atorvastatin positive control group (ST group, 2.5 mg/kg/day), anti-inflammatory drug aspirin positive group (ASP group, 25 mg/kg/day), leonurine crystal form A in low dose (SCM198-L group, 4 mg/kg/day), leonurine crystal form A in middle dose (SCM198-M group, 8 mg/kg/day) and leonurine crystal form A in high dose (SCM198-H group, 16 mg/kg/day). When the rabbits were started to give high fat diet, various drugs or vehicle (NC and MD groups) were simultaneously administered. The drug was administered once a day continuously for 8 weeks and weighed once a week to adjust the dose of drugs. Rabbits were caged in an air-conditioned room (24±1° C. room temperature and 55±5% air humidity) with 12 h light per day and had free access to water and were fed ad libitum. High-frequency ultrasound detection of carotid plaque: the high-frequency microscopy imaging system (Vevo770) was used to detect carotid atherosclerotic plaque at the end of the 8.sup.th week of the experiment. The rabbits were anesthetized with 3% pentobarbital sodium in the dose of 30 mg/kg. Using the RMV707B probe (resolution 30 MHz) to observe the carotid bifurcation plaque and measure the blood flow velocity on the carotid artery long axis section by two-dimensional ultrasound. Then adjust direction of the probe so that the angle between the probe and the carotid artery is less than 60° to obtain the long-axis view. The intima-media thickness (IMT) is measured at the maximum end-diastolic plaque. The measurement to detected IMT is taking the vertical distance between the lumen-intimal echo to the medial-outer membrane echo, and detecting the atherosclerotic plaque on the right common carotid artery bifurcation proximal. The carotid ultrasound images and the maximum IMT at the plaque were analyzed, and the morphology of atherosclerotic plaques was evaluated. The data were analyzed by Vevo770 microscopy imaging system software. All measurements were repeated twice in the same area and averaged. The results showed that the mean IMT in MD group is higher than control group. Leonurine crystal form A significantly inhibited IMT growth induced by high fat and showed a dose-dependent effect. It indicates that leonurine crystal form A inhibited the formation of atherosclerotic plaques in a dose-dependent way.

EXAMPLE 12

(89) According to a conventional method, the effective amount of leonurine crystal form A were added an appropriate amount of microcrystalline cellulose, starch or other excipients cosolvents were added as well to produce capsules, tablets, powders, granules, pills or other oral dosage forms.