WO2006089478A1

WO2006089478A1 - The use of isoscutellarin for the manufacture of medicine

Info

Publication number: WO2006089478A1
Application number: PCT/CN2006/000254
Authority: WO
Inventors: Guanzheng Shi
Original assignee: Guanzheng Shi
Priority date: 2005-02-22
Filing date: 2006-02-22
Publication date: 2006-08-31
Also published as: CN1823798A

Abstract

A new use of isoscutellarin(5, 7, 4’-tri-hydroxyl flavone-6-O-glucuronide) for the manufacture of medicine is disclosed. The present invention provides the use of isoscutellarin for the manufacture of medicine for anti-myocardial ischemia, especially of medicine for cardiovascular disease, coronary heart disease or angina cordis. The present invention also provides the use of isoscutellarin in the manufacture of medicine for anticoagulated blood, thrombolysis, especially of medicine for the treatment and prophylaxis in cerebral accident diseases such as cerebral thrombosis, cerebral embolism, cerebral hemorrhage etc. The present invention discloses that the medicine containing isoscutellarin has the effect of vessel extension, increasing in artery flowing, decreasing in blood viscosity and peripheral resistance, platelet reducing and platelet agglutination, etc.

Description

Application of isoflavone glycoside in pharmaceutical industry

The present invention relates to the use of isoflavone glycosides (ISOSCUTELLARIN 5, 7, 4,-trihydroxyflavone-6-0-glucuronide), in particular to its use in the pharmaceutical field.

Background technique

Astragaloside (SCUTELLARIN 5, 6, 4'-trihydroxyflavone - 7-0-glucuronide) is also known as scutellarin. It has the functions of dilating blood vessels, increasing cerebral blood flow and coronary flow, reducing blood viscosity, and improving microcirculation. It is mainly used for the treatment of cerebral thrombosis, cerebral infarction, post-stroke spasm, coronary heart disease, angina pectoris and other diseases. The existing preparations are ordinary tablets, injections, and powder injection needles. O. 19%。 The oral bioavailability of the ordinary tablets is very low, only 0.4 ± 0.19%. Ordinary injections and powder injections have a short half-life and rapid elimination in the body. At present, researches at home and abroad have focused on improving the bioavailability and long-acting preparations of the compound containing 5, 6, 4'-trihydroxyflavone-7-0 glucuronide. No application of isoflavone glycoside in the preparation of drugs for prevention and treatment of cardiovascular and cerebrovascular diseases, and pharmacokinetic studies of two isomers in vivo. Its isoflavone glycoside structure is:

5, 7, 4'-trihydroxyflavone-isoxanthin

(isoxanthinin ISOSCUTELLARIN) The compound has the formula C21 H20 012, a yellow powder, a molecular weight of 464, and is easily soluble in low concentrations of ethanol. According to the literature, it can be extracted from the leaves of the sycamore plant Sterculia foetida or Sterculia colorata.

Summary of the invention

It is an object of the present invention to provide a new use of isoflavone glycosides, i.e. new applications in pharmaceuticals. A further object of the present invention is to provide an isoflavone glycoside having an anti-myocardial ischemic effect. The use of drugs, especially in the preparation of medicaments for the treatment or prevention of cardiovascular diseases, coronary heart disease or angina pectoris.

Another object of the present invention is to provide an application of isoflavone glycoside in the preparation of a medicament having anti-blood and anti-thrombotic effects, in particular to prepare and treat cerebrovascular diseases such as cerebral vascular diseases such as cerebral thrombosis, cerebral embolism and cerebral hemorrhage. The application of drugs for accidental diseases.

The invention establishes a method for determining the blood concentration of the compound in the plasma of the human body (LC/MS/MS); and the pharmacokinetic study of the isoflavone glycoside, the invention adopts the isoflavone glycoside and the jaundice The human kinetic study of the glucoside found that the plasma concentration after oral administration of baicalein was < 5 ng/ml, and the elimination was fast and irregular. The isoflavone glycoside maintains a higher blood concentration in the human plasma, and the curative effect is superior to baicalein; meanwhile, no clinical adverse reactions are observed in clinical trials.

In order to better understand the essence of the present invention, the pharmacological test and results of isoflavone glycoside are used to illustrate its new application in the pharmaceutical field.

100 kg of leaves of Sterculia foetida or Sterculia colorata of the genus Indus, extracted with 60% ethanol equivalent to 15 times the weight of the leaves, refluxed for 40 minutes, and extracted twice by reflux; combined extracts, concentrated under reduced pressure After ethanol, it is extracted with petroleum ether, chloroform, ethyl acetate and n-butanol in sequence. The n-butanol fraction is chromatographed with macroporous adsorption resin, and then eluted with water, 20%, 60%, 80% ethanol to obtain 5 sites. The 20% ethanol layer was chromatographed on silica gel column eluting with chloroform: methanol: water (1: 2: 2); and then eluted with Sephadex LH220, methanol: water (1: 2) to give the compound isoflavin Glycoside 30g.

The isolated isoflavone glycoside is extracted as above, and polyethylene glycol 6000 : polyethylene glycol

4000 (1: 2-6) mixture as the substrate, the coolant selected dimethyl silicone oil: liquid paraffin (3: 2), the raw material isorubicin 10g and the substrate 50g ratio, the liquid temperature is 60-90 ° C The test protocol is the best test plan; the cooling temperature is 0~5 °C, the inner diameter of the drip is 4. 5/7. 0mm dripper, the drip is 2-6cm from the cooling liquid surface, and the drip speed is 20- Under the dropping conditions of 45 drops/min, pellets containing 10 mg of isoflavone glycoside per pellet were prepared.

Take 200mg of the extracted and separated, add water for injection to 1000ml, mix well, and then fill it into the bottle with lmg/5ml/concentration injection solution, and disinfect it to make the product for use.

1. Acute toxicity test in mice

A. Determination of LD50 in intravenously administered mice:

50 healthy mice were taken, weighing 18g~22g, and randomly divided into 5 groups according to body weight. 10 rats in each group, half male and half female, fasted 12 hours before the test. Prepare the test drug according to the low ratio dilution method. The highest dose is 2000mg/kg, the lowest dose is 1000mg/kg, the ratio between groups is r=0.84, the dose is 2000, 1680, 1411. 2, 1185. 4, 995. 7mg/kg; i. 0. 2ml/10g重量重量。

After intravenous administration, the animals showed symptoms of dose-dependent poisoning, and the mice showed symptoms such as decreased spontaneous activity, flank, convulsions, and struggling. 1185. The dead animals were found in the 4 mg/kg or higher dose group. The LD50 value calculated by the Bliss method was 1509. 9 + 142. 7 mg'/kg, and the 95% confidence limit was 1399. 9~ 1685. 3mg/kg ₀ dead animals and observation At the end of the animal necropsy, no significant abnormal changes were observed. Changes in body weight before and after administration. .

Intravenous mice LD50

Dosage number of animals deaths mortality unit 13⁄4 and 95% confidence limit

(mglig) (xi) (only) (only) (%) CO (ιτ 3⁄4)

2000 3.301 10 10 100 6.78

1680 3.225 10 7 70 5.68

1509.9 ± 142.7

1411.2 3.150 10 3 30 4.57

( 1399.9 - 1685.3 )

1185.4 3.074 10 1 10 3.46

995.7 2.998 10 0 0 2.36

Pre- and post-dose weight change table

Dosage number of animals (only) weight change ( g )

(π 3⁄4) before the drug, before the drug, 7 days after the drug, 14 days after the drug

1680 10 3 20.49 ± 0. 92 21.40 ± 0. 43 29.13 土 0. 58

1411.2 10 7 20.45 ± 0. 90 21.57 ± 1. 14 29.27 ± 1. 09

1185.4 10 9 20.53 Earth 0. 85 22.37 ± 1. 14 29.90 ± 0. 71

995.7 10 10 20.52 ± 0. 60 23.00 ± 0· 78 30.58 ± 0. 89

Determination of LD50 in sputum and oral administration mice:

The results of the preliminary test showed that the dead animals were not observed for 3 consecutive days after the administration of the test drugs 2500 and 5000 mg/kg. This indicates that the product is less toxic and its LD50 is difficult to measure, so the maximum tolerated dose test is performed.

After the administration, the mice in the experimental group had no significant symptoms of spontaneous poisoning except for spontaneous activity, and there was no obvious poisoning symptoms. No animal death was observed after 7 days of continuous observation. Changes in body weight before and after administration. Number of animals Weight change ( g )

side

(only) 7 days after the administration of the drug, 14 days after the drug

? 10 19.99 ± 0. 89 22.82 ± 1· 54 30.35 ± 1. 18

10 20.19 ± 0. 64 23.32 ± 1. 15 30.55 + 0. 92 C. Maximum Tolerance Test: Twenty healthy mice were taken, weighing 18w22g, female and male, and fasted for 12 hours before the test. Drinking water freely. 5毫升/10公斤重量。 The test set 5000mg / kg test group, the concentration of 12.5%, the dosage volume of 0. 4ml/10g body weight.

The results showed that after intravenous administration of the experimental drug to the mice, the symptoms of spontaneous activity, flank, convulsions, and struggling were observed in the mice. The death of the animals was dose-dependent. The LD50 value was calculated by the Miss method. The LD50 value was 1509. 9± 142 7mg/kg, 95% confidence limit is 1399. 9~: 1685. 3mg/kg. After the oral administration of the experimental drug, when the dose reached 5000 rag/kg, the animals were generally in good condition except for the reduction of the abdomen and spontaneous activity, and no dead animals were observed. This indicates that the oral administration of LD50 >5000mg/kg _o in mice has a therapeutic effect on focal cerebral ischemia-reperfusion in rats.

In the case of anesthesia, the rats were separated and exposed to the common carotid arteries. The bilateral common carotid arteries were closed with arterial clips to produce bilateral cerebral ischemia. After a certain period of ischemia, the arterial clip can be re-opened for reperfusion of the brain according to the test requirements. Take healthy SD rats, half male and half female, weighing 260-290 grams. They were randomly divided into 3 groups, 15 rats in each group, respectively, 5% sodium carboxymethylcellulose (vehicle group), baicalein 20 mg/kg (control group) and isoflavone 20 mg/kg (test group). Once a day, for 10 consecutive days, 10 days later, anesthesia was performed with 2% sodium pentobarbital (40 mg/kg) ip. A midline incision was made in the neck. The bilateral common carotid arteries were clamped with arterial clips for 20 min. The blood vessels were recanalized, the skin incisions were sutured, and the original conditions were returned for feeding. The mortality of the animals in each group was measured 24 h after cerebral ischemia. Results In the solvent group, 8 died, the mortality rate was 53.3%; 3 in the baicalin group died, the mortality rate was 20%; in the isoflavone group, 1 died, the mortality rate was 6.6%. Therefore, baicalin and isoflavone have a strong protective effect on focal cerebral ischemia-reperfusion injury in rats, but isoflavone has better protective effect than baicalin.

The aim is to block the blood vessels that innervate the brain tissue and simulate a pathological model similar to human stroke to evaluate the effectiveness of the drug.

3. Effect on the coagulation system

(1) Effect on clotting time in mice

105 Kunming mice, male and female, 20 soil and 2 g were randomly divided into 4 groups, respectively, and normal saline and baicalein (control group) and isoflavone glycoside (test group) were respectively given at 20 mg/kg. The doses of 40 mg/kg and 60 mg/kg were intragastrically administered for one week. At the end of the last administration, 1 hour after the last administration, blood was taken from the veins of the mouse in the eye of the eyeballs. The time from the blood inflow into the tube is started. After the blood is filled, the capillary is taken out and placed on the table. The capillary is broken at intervals of about 0.5 cm every 10 s until the appearance of the blood coagulation. The elapsed time is the blood coagulation time, and the average of the data at both ends of the capillary. The value is the blood coagulation time of the mouse. Effect on clotting time in mice (capillary method) (X-soil SD)

Note: Compared with the blank group, **P<0.01

The coagulation test in mice showed that both the baicalein group and the isoflavone group had anticoagulant effects, and the group was particularly effective in the isoflavone group.

(2) Effect on prothrombin time in rabbits

The venous blood of healthy rabbits was taken, mixed with (38m _g /ml) sodium citrate solution 1: 9 for anticoagulation, centrifuged at 3000r/min for 10 minutes, and the supernatant was taken for testing. In the measurement, the mixture was added with rabbit cerebral powder extract solution with different concentrations of baicalein and isoflavone glucoside solution, and the physiological saline solution was 0.1 ml, and then rabbit plasma was added to 0.1 ml, and the mixture was placed in a 37 ° C water bath. Incubate for 30 s, add 0. 025raol/L CaC12 solution to 0.1 ml and start timing. Incline the tube once every 5 s to record the time required for fibrin to solidify (liquid level does not move).

Note: Compared with the blank group, *Ρ<0. 05, **Ρ<0. 01

Xanthosine and isoflavone can significantly prolong clotting time and prothrombin time, which is beneficial to prevent thrombosis.

4. Effect of isoproterenol (ISO) on acute myocardial ischemia induced by gerbils

Forty-eight healthy gerbils, weighing (54±14) g, were used for both male and female. They were randomly divided into six groups, 8 in each group, respectively: (1) saline control group (NS); (2) isoflavone glycoside Group (50mg/kg), the above groups of animals were injected intraperitoneally, for 2 consecutive days, on the third day, the gerbils were anesthetized; the intravenous administration channel was established, and the drugs and doses of the above groups were intravenously given to each group. The drug was given IS02. 5ug/kg 5 minutes after the last administration, and intravenously injected at a constant rate within 5 minutes. The ST-segment changes of the II lead electrocardiogram at different time points within 15 min were observed and recorded; (3) Isoflavone intravenous administration group '(50mg/kg); After the screening was qualified for 30 minutes, IS02. 5ug was injected intravenously at 5 minutes after administration. The ST-segment changes of the II lead electrocardiogram at different time points within 15 min were observed and recorded. The results are shown in Table 1. Table 1 Isoflavone glycosides against ISO induced sand Effect of ST segment on acute myocardial ischemia in rats (X 士s, mV)

Dose to ISO before ST constant speed to ISO after ST segment down shift

(mg kg) Segment (mV) lmin 2min 4min 15min Normal saline 0.016 ±0.031 0.078+0.073 0.154 ±0.050 0.163 ± 0.080 0.031 ±0.053 Isoflavone 50 0.019±0.037 0.026±0.046 0.085 ±0.069* 0.135 ±0.079 0.024 ±0.043 Isoflavone glucoside 50 0.013 ±0.023 0.029±0.039 0.101 ±0.065* 0.119±0.090 0.021 ±0.036 pulse administration

Model control group 0.023 ± 0.032 0.084 + 0.065 0.159 ± 0.046 0.158 ± 0.078 0.025 ± 0.049 The test results showed that the first dose of isoflavone injection was administered intraperitoneally for 2 days, the last intravenous administration, and intravenous injection, isoflavone Both have a shorter, milder effect of alleviating ISO-induced acute myocardial ischemia. Its mechanism of anti-myocardial ischemia may be related to its rapid and long-lasting blood pressure reduction, which reduces the burden on the heart and thus reduces the myocardial oxygen consumption. Isoflavone can increase myocardial S0D activity, reduce MDA, reduce the accumulation of free radicals in the myocardium, and reduce the damage of ISO to the myocardium, suggesting that its anti-cardiac ischemia may be related to its anti-lipid peroxidation.

5. Human dynamics study of isoflavone glycosides:

Baicalein has the functions of dilating blood vessels, increasing arterial flow, lowering blood viscosity, reducing peripheral resistance, and reducing platelet aggregation. It is mainly used for the treatment of coronary heart disease, angina pectoris, myocardial ischemic injury and cerebral thrombosis. The existing preparations include ordinary tablets, injections, and powder injection needles. Ordinary tablet oral bioavailability is extremely low, only 0.4 ± 0. 19 ° /. . Ordinary injections and powder injections have a short half-life and are rapidly eliminated in the body. The human pharmacokinetic study has not been reported.

According to the regulations of the Second Pharmacopoeia of the People's Republic of China (2000 edition), the concentrations of baicalein and isoflavone in the plasma of 20 healthy subjects were determined, and the plasma concentration-time curve was plotted to calculate the drug power. Learning parameters; and according to the amount of isoflavone glycoside in the preparation, respectively, provide the necessary experimental data for the study of human bioavailability of isoflavone glycoside pills, see Table 2: 20 subjects orally breviscapin Pill isobutyl isobutyl plasma concentration of breviscapin (n _g / ml) after 80m _g t-:. AUC _0,

No. 0 α ' (tig ml) (〗〖ι:' (ng' .ml) (ng'h'ml)

1 2.61 0.265 123 8.0 1161 1172

3.~3 0.1 S(5 101 6.0 67S 699

4.96 0.140 6? S.O 600 64~

4 2.42 0.:S6 60 541 545

2.57 0.233 15: 5.0 1343 1357

6 0.91 .75S 3 4.5 ::5 130

- 3.46 0,200 144 lC'.C' 1002 1025 '

S 3.60 0.193 10.0 411 423

10 2.67 0.2 «50 - 520 8.0 45 S3 4640

11 i.95 0.175 16~ 7.0 Si£ SS4

1 2.21 27S 6.0 14 4 1447

15 2.26 ΰ.307 119 S.O 65S 661

14 3.32 0.2 OS 209 S.O 9S4 1001

15 2.$o I'l "•rt"? 2i52 6.0 1090 Let

16 322 0.075 24~ S.O 37U 4726

1 2.34 n.295 59 6.0 54: 370

IS 5.73 0.12: 146 S.O 646 70;

19 i.s: 0.;S1 126 10.0 33S S40

21 2.52 |:|.265 55;" 7.0 2437

: 1 0 1G.0 64} 645

3.26 n.2 f 17S 77 1:?5 1263

!■,;;';:·;: l. S 0.14: 142 1.7 1:56 1271

CV'( _C .) 54.7 ?3.0 70.7 22.6 ?5.1 1.00.7

0.91 O.OS 4.5 128 130

<5 τ'» .Ί6 555 10.0 45SS i~26

Result analysis:

Twenty subjects were given oral drops containing isoflavone 80 mg, and the following results were obtained: The Tmax of isoflavone glycoside in plasma was 7.7 ± 1.7 h _; Cmax was 178 ± 142 ng/ml, and tl/2 was 3.26 ± 1.78h; Calculated by the trapezoidal method, AUCO-t was 1195±1136ngh/ml, and AUC0-∞ was 1263±127 lng.h/ml.

In the human pharmacokinetic study of baicalein, the plasma concentration of baicalein, the main component of the subject after oral administration of baicalein, was <5ng/ml, and the elimination was fast and irregular. It is difficult to perform pharmacokinetic evaluation. And the literature [2] reported that after high-dose baicalin in beagle dogs, the peak plasma concentration was reached between 1 and 3 h, and the absolute bioavailability was between 0.2% and 0.75%, suggesting that baicalein was almost not absorbed orally; The results of the static injection elimination half-life were consistent. ' According to the general pharmacokinetics, isoflavone glycoside has better efficacy than baicalein; at the same time, no clinical adverse reactions have been observed in clinical trials.

Based on the above results, it can be concluded that the advantages of the present invention are:

1. The present invention has developed a new application field for the known compound isoflavone glycoside, and has opened up a new application field.

2. The isoflavone glycoside of the invention is safe, non-toxic, has strong pharmacological effects, and has good pharmacological effects, indicating that there is a good medicinal prospect.

3. The raw materials of the product of the invention are rich in source and low in cost, and the leaf as the extraction site can be fully resourced, the preparation process is simple, and no toxic side effects are observed. It can be formulated into a solid preparation such as an oral preparation such as a tablet, a dropping pill, or a capsule; or a liquid preparation such as an intravenous preparation such as a powder injection or an intravenous solution.

4. The drug made by the product of the invention has the ability to reduce brain water content and MDA content, increase SOD, CAT and GSH-Px activity, and protect Na+, K+-ATPase activity. It is suggested that isoflavone can protect the brain tissue from antioxidant enzymes, inhibit lipid peroxidation, and alleviate the damage of free radicals on brain tissue. It can reduce the water content of brain tissue and inhibit Na+, K+-ATPase and Ca2+ in brain tissue. - A decrease in ATPase activity. This drug suggests that the drug can protect against ischemia-reperfusion brain tissue by protecting ATPase activity, which is superior to baicalein.

5. The drug made by the product of the invention can significantly improve the change of TXB2 of platelet aggregation after urinary thrombolysis, and increase the concentration of tissue plasminogen activator and anticoagulant in blood, and reduce plasmin. The concentration of pro-activator inhibitor (PAI) increases the recanalization rate of urinary hormone thrombin therapy in acute coronary thrombosis. It can reduce the number of platelets, reduce platelet aggregation, promote fibrinolytic activity, inhibit thrombin activity, etc., and has anticoagulant and fibrinolysis effects. Its anticoagulant effect is superior to baicalein.

6. The drug produced by the product of the present invention has an anti-ischemic effect. It can improve myocardial SOD activity, reduce MDA, reduce the accumulation of free radicals in the myocardium, and alleviate the damage of ISO to the myocardium, suggesting that its anti-ischemic effect may be related to its anti-lipid peroxidation.

7. The product of the present invention was subjected to a human body dynamics test, and the plasma concentration of plasma in different subjects after oral administration of isoflavone glycoside was determined by liquid chromatography-mass spectrometry-mass spectrometry. - time curve. According to the plasma concentration-time data, the AUC value was calculated by the trapezoidal method in a semi-logarithmic plot method, and tl/2 was calculated from the concentration point at the end of the elimination phase, and the main pharmacokinetic parameters were determined. Compared with baicalin, it has better pharmacokinetic parameters. It laid a theoretical foundation for the clinical efficacy of the drug. At the same time, no clinical adverse reactions were observed in clinical trials.

DRAWINGS Figure 1 shows the product ion full-scan mass spectrum of baicalein, isoflavone glycoside, baicalin [M+H]+: A, baicalin (B), isoflavinose (ISOSCUTELLARIN) C, baicalin (baicalin)

detailed description

Several embodiments of the present invention will be described below, but the content of the present invention is not limited at all. Example 1:

100 kg of leaves of Sterculia foetida or Sterculia colorata of the paulownia plant were extracted with 60% ethanol equivalent to 15 times the weight of the leaves, refluxed for 40 minutes, and extracted twice by reflux. The extracts were combined, and the ethanol was concentrated under reduced pressure, and then extracted with petroleum ether, chloroform, ethyl acetate and n-butanol. The n-butanol fraction was chromatographed with macroporous adsorption resin, followed by elution with water, 20%, 60%, 80% ethanol to obtain 5 sites, and the 20% ethanol layer was chromatographed on silica gel with chloroform: methanol: water (1) : 2: 2) Elution, followed by Seph _a d _eX LH220 chromatography, methanol: water (1: 2) elution to obtain the compound isoflavone glycoside. The mixture of polyethylene glycol 6000: polyethylene glycol 4000 (1: 2-4) is used as the substrate, and the coolant is selected from the group consisting of dimethicone: liquid paraffin (3: 2), and the starting isoflavone and the substrate are 1:4. Proportion, the temperature of the liquid is 60-80 ° C, the cooling temperature is 0~5 Ό, the drip of the inner diameter of the drip is 4.5/7.0mm, the drip is 3-6cm from the cooling surface, and the drip speed is 25- Under the dropping conditions of 35 drops/min, pellets containing 10 mg of isoflavone glycoside per pellet were prepared.

Example 2:

Take 1000 g of isoflavone glycoside prepared according to the method of Example 1, add an appropriate amount of water for injection, adjust the pH value to 7 with sodium carbonate, stir and dissolve, and add 10-15% of the amount of isoflavone glycoside for injection. Alcohol, sterilized and filtered, determined content, dispensed to 10mg / support, sealed, that is.

Example 3:

The isoflavone glycoside prepared by the method of Example 1 is prepared by using an infusion preparation method well known in the art to prepare an isoflavin sodium chloride injection having a specification of 250 ml: isoflavone 20 mg and sodium chloride. 2.25 g; Prepared as an isoflavone glycoside injection, the specification is 250 mh. Isoflavone 20 mg and glucose 12.5 g.

Example 4:

The isoflavone glycoside prepared by the method of Example 1 260 g, lactose 460 g, starch 420 g, talcum powder

148g. Using a tablet preparation method well known in the art, all components are mixed in a suitable container to form granules, then 2 g of magnesium stearate is added, and mixed, using a 9/32 inch standard concave punching sheet, 10 pieces of weight are

1.3g, its specifications are: 20mg / piece.

Example 5: 100 g of isoflavone glycoside prepared by the method of Example 1 and 32 ml of 10% hydroxypropylmethylcellulose were used. A capsule preparation method known in the art is used to prepare 1000 capsules having a specification of 20 mg/granule.

Example 6: A review of related literatures at home and abroad revealed no reports of human kinetic tests of isoflavone glycosides. The invention conducts human pharmacokinetics research, and uses liquid chromatography-tandem mass spectrometry (LC/MS/MS) to determine the plasma concentration of isoflavone glycoside in human body, and uses the pharmacokinetic behavior of isoflavone glycoside The preparation was evaluated for efficacy.

Analytical method for plasma samples

Plasma sample treatment: Add 100 μM methanol, 100 μ ΐ internal standard solution ( baicalin 400 ng/ml) and 200 μl water to 500 μM ΐ plasma, mix well; force n3 ml extraction solvent ethyl acetate, vortex mixing lmin Reciprocating oscillation lOmin (240 times / min), centrifugation for 5 min (3500 rpm), separate the upper organic phase in another tube, blow dry under a nitrogen flow at 40 ° C, the residue was added to 150 μ ΐ mobile phase to dissolve, vortex mixing, Take 20 μl for LC/MS/MS analysis.

Chromatographic conditions: The mobile phase was methanol: water: formic acid (70: 30: 0.5, ν/ν/ν), flow rate 0.50 ml/min, column temperature: 25 ° C.

Mass spectrometry conditions: ion source is ESI source; source spray voltage is 4kV; heating capillary temperature is 320° C; sheath gas (N2) pressure is 25ΑΛ; auxiliary gas (N2) flow rate is 3Arb; collision gas (Ar) pressure is 1.OmTorr; collision The induced dissociation (CID) voltage is 25 eV; positive ion mode detection; the scanning mode is selective reaction monitoring (SRM), and the ion reactions for quantitative analysis are m/z 463→m/z 287 (SCUTELLARIN and ISOSCUTELLARIN) and m/, respectively. Z447→m/z271 (xanthine); scan time is 0.3s. The corresponding secondary full scan mass spectrum is shown in Figure 1.

As shown in Fig. 1, isoflavone glycoside, baicalein and baicalin mainly produced [M+H]+ excimer ion peaks in the ESI ionization mode, which were m/z 463 and m/z 447, respectively. Selective alignment of the molecular ion peak [M+H]+ for product ion scanning, the main fragment ions generated were m/z 287 and m/z 271, respectively, and these main fragment ions were used as product ions for monitoring during quantitative analysis. According to the Pharmacopoeia of the People's Republic of China (Part 2, 2000 edition) Appendix XIXB pharmaceutical preparations human bioavailability and bioequivalence test guidelines, can be used for pharmacokinetic tests of isoflavone glycoside and baicalein.

Result analysis:

Twenty subjects took the test preparation containing the isoflavone glycoside 80 mg and the reference preparation (bauroside), and obtained the following results: The Tmax of isoflavone glycoside in plasma was 7.7 ± 1.7 h; Cmax score Not 178 ± 142 ng / ml, tl / 2 was 3.26 ± 1.78h; calculated by the trapezoidal method, AUCO-t was 1195 ± 1136 ng 'h / ml, AUC0 - ∞ were 1263 ± 1271 ng. h / ml. The plasma concentration of baicalein was <5 ng/ml, and the elimination was fast and irregular.

Claims

Rights request

1. Use of isoflavone glycoside in the preparation of a medicament having anti-myocardial ischemia effect.

The use of isoflavone glycoside according to claim 1 for the preparation of a medicament having an anti-myocardial ischemic effect, wherein the medicament is used for the preparation of a medicament for treating or preventing cardiovascular diseases.

The use of isoflavone glycoside according to claim 2 for the preparation of a medicament having anti-myocardial ischemic effect, wherein said pharmaceutical use is for the preparation of a medicament for the treatment or prevention of coronary heart disease or angina pectoris.

4. The use of isoflavone glycoside in the preparation of a medicament having anticoagulant effect.

5. The use of isoflavone glycoside in the preparation of a medicament having an anti-lysis effect.

6. Use according to claim 4 or 5, wherein the pharmaceutical use is for the preparation of a medicament for the treatment of sequelae caused by cerebral thrombosis, cerebral infarction and cerebral hemorrhage.