WO2007059685A1

WO2007059685A1 - Astragalus calycosin with the function of resisting coxackievirus

Info

Publication number: WO2007059685A1
Application number: PCT/CN2006/002910
Authority: WO
Inventors: Chenggang Huang; Yuanyuan Zhang; Haiyan Zhu; Chunhui Ma; Guan Ye; Mingsong Fan; Yihong Tang; Zhixiong Li; Zhaolin Sun
Original assignee: Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences
Priority date: 2005-11-23
Filing date: 2006-10-30
Publication date: 2007-05-31
Also published as: CN1813711B; CN1813711A

Abstract

The present invention discloses the antiviral activity of calycosin 7-O-β-D-glucoside from Astragalus against Coxackievirus, and the application of calycosin 7-O-β-D-glucoside in developing medicine for treating viral myocarditis caused by Coxackievirus.

Description

Use of an isoflavone compound

The invention relates to extracting active ingredients from traditional Chinese medicines, and is proved to be effective for treating viral myocarditis by pharmacological tests, more specifically, extracting and isolating an isoflavone active ingredient from the radix astragali or other traditional Chinese medicines - verapamil isotope - 7 - oxygen - D - Calycosin 7-0-β-D- glucoside, which can be used in the preparation of a drug against Coxsackie virus and a disease caused by Coxsackie virus. Background technique

Coxsackievirus can cause a variety of diseases, such as viral myocarditis, epidemic chest pain, aseptic encephalitis, meningitis, Keshan disease and viral epilepsy, which seriously endanger human health.

At present, the main method for clinical treatment of Coxsackie virus diseases such as viral myocarditis is to use common antiviral drugs, immunosuppressants, interferons and cardiomyocyte nutrients, but the curative effect is not satisfactory.

In recent years, a large number of studies have shown that traditional Chinese medicine has shown great advantages in the clinical treatment of Coxsackie virus diseases such as viral myocarditis. However, most of the traditional Chinese medicines are prescribed by doctors or compound crude drugs, such as Shengmaiyin. , Dongdong Xinxin oral liquid, etc., lack of effective ingredients, clear mechanism of action, affirmative effect, advanced dosage form, taking convenient Chinese medicine preparations.

Chinese medicine Huangqi qi and solid table, diuretic toxin. The components such as astragaloside extracted from Astragalus membranaceus have various pharmacological activities. Journal of the Second Military Medical University, 1999, 20 (9 ): 666- 668 reported that astragaloside has a good therapeutic effect on mice with Coxsackie B3 viral myocarditis; Chinese Journal of Traditional Chinese Medicine, 2001, 26 (12): 850-853 reported that astragaloside can counteract the increase in brain microvascular permeability caused by histamine; Chinese Journal of Pharmacology, 2003, 19 (8): 892-895 reports that total glycosides of Astragalus membranaceus activates hepatic stellate in vitro. Cell proliferation and collagen production are significantly inhibited, which may be one of the mechanisms of anti-fibrosis; "Natural Product Research and Burst, 1994, 6 (2): 1-5" reported that astragaloside and verbasco Flavonoids can improve the ability of hatching red blood cells to deform, which may be an important mechanism for improving the hemorheology of jaundice. Patent CN01126608. 2 discloses that isoflavones and glycoside have good anti-ischemic effect; Patent CN00119451. 8 discloses the use of astragaloside IV in the treatment of viral myocarditis; patent CN01126609. 0 discloses that astragaloside has anti-myocardial fibrosis. No reports of vermiculone isoflavone-7-oxo-β-D-glucopyranoside against Coxsackie Virus and treatment of viral myocarditis. Summary of the invention

The object of the present invention is to overcome the shortcomings of the conventional multi-flavored decoction, and to chemically separate the isoflavone active ingredient - the isoflavone - 7-oxo-β-D-glucopyranoside, from the astragalus or other traditional Chinese medicine. Pharmacological tests are applied to the preparation of anti-coxsackie virus and drugs for the treatment of viral myocarditis. The medicament of the present invention can be prepared by the following method: After pulverization of astragalus or other Chinese medicinal materials, three times (2 hours, one hour, one hour) of refluxing with 70% ethanol, filtering, combining the filtrates, recovering the solvent under reduced pressure, and concentrating to an alcohol-free taste. , obtained xanthine extract I. The extract I was mixed with water and filtered, and the filtrate was adsorbed with a macroporous resin, and the impurities were eluted with water, and then eluted with 70% ethanol, and the eluate was concentrated under reduced pressure to obtain an extract II. The extract II was separated by silica gel column chromatography, eluting with a gradient of chloroform-methanol, and the same fractions were combined, evaporated to dryness, and the solvent was evaporated to dryness, and the mixture was allowed to stand, and the white powder was precipitated and filtered to obtain white powdery crystals. That is, the compound of the present invention, carophyllin 7-O-β-D-glucoside, is obtained. The structure is as follows:

Its molecular formula is ₂₂ Λ. The molecular weight is 446, and the chemical name is 3'-hydroxy-4'-methoxyisoflavone-7-oxo-β-D-glucopyranoside. In the present invention, the isoflavone-7-oxo-β-D-glucopyranoside obtained by the above method is first tested for efficacy.

The results of the in vitro antiviral test performed on primary cultured cardiomyocytes indicate the TC _{5 of the} drug of the present invention. IC ₅ for 177. 84 ug/ml, X-inch CVB ₃ virus. At 17.78 ug/ml, the therapeutic index (TI) was 10.00, indicating that the drug of the present invention has a significant inhibitory effect on CVB ₃ virus in vitro.

A model of viral myocarditis was established by intraperitoneal vaccination with Coxsackie B3 virus in Balb/c mice. The drug-administered 40 mg/kg and 20 mg/kg were administered intragastrically in the high- and low-dose groups. The model control group and the normal control group were administered. The rats were given the same amount of normal saline for 12 days. On the 15th day, the mice were observed for color, eating, and death. The results showed that the two dose groups were significantly reduced by the Coxsackie B ₃ virus. The mortality of mice caused by viral myocarditis significantly reduced the heart index of mice with viral myocarditis, and effectively decreased the serum levels of LDH and GOT in mice with viral myocarditis caused by Coxsackie B3 virus.

Note: The test method refers to "Modern Pharmacological Experimental Methods" (Volume 2, P ₁₄₂₅ , edited by Zhang Juntian, Beijing Medical University, China Union Medical University, United Press, 1998 edition). ' detailed description

The present invention will be further described below in conjunction with specific embodiments without any limitation.

Example 1. Preparation of the medicament of the present invention Take the Astragalus membranaceus, pulverize, extract three times with 70% ethanol reflux (2 hours, 1 hour, 1 hour), filter, combine the filtrate, recover the solvent under reduced pressure, and concentrate to a non-alcoholic taste to obtain a xanthine extract I. The extract I was mixed with water and filtered, and the filtrate was adsorbed with a macroporous resin (AB-8 type), and the impurities were eluted with water, and then eluted with 70% ethanol, and the eluate was concentrated under reduced pressure to obtain an extract II. Extract II was chromatographed on a 200-300 mesh silica gel column, eluting with chloroform-methanol gradient, TLC detection, 10% sulfuric acid-ethanol color development, combining the same fractions, evaporated to dryness, methanol hot solution, standing, white floc The precipitated material was filtered off with suction to give a white powdery crystal. Example 2: Effect of the drug of the present invention on coxsackievirus (CV)-infected cardiomyocytes (in vitro)

Cytotoxicity test:

Take 15 newborn rats of SD rats for 1 to 3 days, remove the heart, and perform primary culture of cardiomyocytes. The obtained cell suspension is cultured in a 24-well culture plate for 24 hours until it is attached, and the culture solution is discarded after 24 hours. . Grouped as follows:

Normal control group: Add culture medium per well 1. 0ml;

Dosing group: A gradient dilution of the drug solution of the present invention was added. OmL/well, four replicate wells per dilution.

The cell survival was observed under an inverted phase contrast microscope, and the TC ₅ of the primary cultured cardiomyocytes of the drug of the present invention was calculated according to the Reed-Mench method. .

Anti-Coxsack B3 virus test:

15 newborn rats of SD rats were taken 1 to 3 times, the heart was taken out, and the cardiomyocytes were cultured in primary culture. The obtained cell suspension was cultured in a 24-well culture plate for 24 hours until it was attached, and the culture solution was discarded after 24 hours. . Grouped as follows:

Normal control group: adding culture solution per well l.

Virus control group: 0. 2ml CVB ₃ virus solution (TCID ₅ = 10 - ' ^5a , 1 (Γ ⁵ dilution) per well, incubate for 1 hour, discard the supernatant, clear three times with PBS solution, add culture solution 1 Oral culture;

The administration group: 0.2 ml of CVB ₃ virus solution (TCID^IO" ⁴ - ⁵⁶ , 10 ^-5 dilution) was added to each well, and the mixture was incubated for 1 hour, the supernatant was discarded, and washed three times with PBS solution, and the gradient dilution was added to the present invention. Drug solution. l. OraL/well, four replicate wells per dilution.

After 48 hours of culture, observation was carried out under an inverted phase contrast microscope. Cardiomyocytes will slowly die in culture, pseudopods break, cells shrink, round, and the refractive index increases. The cell death was significantly increased after being infected by the virus, and a large area of necrosis was observed. If the drug is effective after administration, the mortality of the virus-infected cells can be reduced. The IC _{5 of the} drug of the present invention against primary cultured cardiomyocytes was calculated according to the Reed- Muench method. . ·

+ Result: TC _{5 of the} medicament of the invention. It is 177. 84 ug/ml, and 1 for CVB ₃ virus. The therapeutic index (TI) was 10.00, which was 17.78 ug/ml, indicating that the drug of the present invention has a significant inhibitory effect on CVB ₃ virus in vitro. Example 3: Effect of the drug of the present invention on mortality of mice with viral myocarditis (in vivo)

The drug of the present invention was divided into two dose groups, and Balb/c mice were randomly divided into 4 groups:

Normal control group: intraperitoneal inoculation 0. 1ml normal saline, daily, equal amount of normal saline; Virus control group: Each intraperitoneal cavity was inoculated with 0. lmlCVB ₃ virus solution (TCID ₅ = 10 - ⁵⁶ , 10 - ⁵ dilution), and then given the same amount of normal saline daily;

The drug group I of the present invention has a high dose: the same method as the above method of inoculation of CVB ₃ virus solution, the same day, the drug solution of the invention 40mg / kg • d, continuous administration for 12 days;

The pharmaceutical group II of the present invention has a low dose: the same as the third group method, the drug solution of the present invention is administered at a dose of 20 mg/kg*d, and is continuously administered for 12 times;

After observing for 15 days, various conditions such as mouse color and eating were observed, and the mortality of each group of mice was counted.

Results: Both doses of the drug of the present invention significantly reduced the mortality of mice with viral myocarditis caused by Coxsackie B ₃ virus, as shown in Table 1.

Table 1 Effect of the drug of the present invention on morbidity and mortality of mice with viral myocarditis Group Animal number (only) Mortality (number of cases)

Normal control group 6 0 ( 0)

Virus control group 15 40% (6) 'The drug group of the invention I 15 26. 7% (4) *

Drug group of the invention

Π 15 26. 7% (4) *

Note: Using the chi-square test, the drug-administered group compared with the virus group, * has statistical significance. Example 4: Effect of the drug of the present invention on heart index of mice with viral myocarditis (in vivo)

Balb/c mice were grouped and dosed as in Example 2. After 12 days of continuous administration, on the 15th day, all the mice were dissected. The body weight of the mouse was weighed before dissection, and then the heart weight was weighed.

Heart index = heart weight / weight

When an animal is infected with a virus, the food intake is reduced, the body weight is reduced, and the heart is enlarged, fibrotic, and heavier. Therefore, the heart index can be used to measure the degree of heart disease. The greater the heart index, the more severe the heart disease becomes.

Results: Both dose groups of the drug of the present invention significantly reduced the cardiac index of mice with viral myocarditis caused by Coxsackie B ₃ virus, as shown in Table 3.

Table 2 Effect of the drug of the present invention on heart index of mice with viral myocarditis

Cardiac index (X10- ³⁾

Normal control group 4. 14±0· 59

Virus control group 5. 60±0. 73

The drug group of the invention I 5. 05±0. 63*

The medicament of the invention II 5. 14±0. 58*

Note: The drug-administered group is compared with the virus group: * p<0. 05, ** p<0. 01

Example 5: Effect of the drug of the present invention on serum LDH and GOT contents in mice with viral myocarditis (in vivo) Balb/c mice were grouped and administered at the same dose as in Example 2. After 12 days of continuous administration, on the 15th day, blood was taken from the eyeball, and serum was centrifuged to measure the contents of lactate dehydrogenase (LDH) and aspartate aminotransferase (GOT) in the serum.

Aspartate aminotransferase and lactate dehydrogenase are biochemical markers of myocardial damage. In the acute phase of viral myocarditis, both release are elevated and can be maintained for a considerable period of time. Therefore, if the drug can effectively reduce the myocardial enzyme spectrum, it means that the drug effectively reduces the degree of heart disease.

Results: Both dose groups of the present invention significantly improved LDH and GOT sputum in the serum of mice with viral myocarditis caused by Coxsackie B ₃ virus. The results are shown in Table 3. Effect of the drug of the invention on the degree of epithelial lesions in mice with viral myocarditis

Group LDH (unit / 100ml) GOT (Carmen) Normal control group 314.08 ± 57.64 21.46 ± 4.49

Virus control group 496.05±55.63 25.93±5.29

The drug group of the present invention I 459.18±51.31* 21.65±5.00* The drug group of the present invention

461.22±52.51* 21.88±3.90*

II

Note: The drug-administered group compared with the virus group: * p<0.05, ** p<0.01

Claims

Rights request

1. A structural formula is as follows

The use of the isolated active ingredient, muskyl isoflavone-7-oxo-β-D-glucopyranoside, from the astragalus or other botanicals is used in the preparation of a medicament for treating diseases caused by Coxsackie virus.

The use of the active ingredient muskyl isoflavone-7-oxo-β-D-glucopyranoside according to claim 1, which is characterized in that it is used as a viral myocarditis caused by Coxsackie virus, epidemic chest pain, A drug for aseptic encephalitis, meningitis, Keshan disease, and viral epilepsy.

The use of the active ingredient muliformis-7-oxo-β-D-glucopyranoside according to claim 1, which is characterized in that it is used as a medicament for treating viral myocarditis.