WO2003075938A1 - Coumarins extractions and their uses for treating hypertension disease - Google Patents

Abstract

The invention relates to a kind of Coumarin Extraction coming from Chinese herbs and containing Imperatorin and Isoimperatorin. The invention also relates to a use of the extraction for treating hypertension disease. Especially, the invention provides a kind of Coumarin extraction coming from Angelica dahurica. Furthermore the invention also provides an extract method adopted by the invention, which is Super-critical CO2 Extraction. The invention also provides a pharmaceutical composition containing the extraction. The extract method of the invention is simple and practical, the extraction has a good purity, and the formula of the invention is unique, but also the pharmaceutical composition containing the extraction has a new use for treating hypertension disease.

Description

 Coumarin extract and its application in the treatment of hypertension

 The present invention relates to a coumarin extract from a Chinese medicinal raw material medicine, in particular to a coumarin extract from a paeony drug raw material. The invention also relates to the use of such extracts in the treatment of hypertension. The invention also provides a pharmaceutical composition using the extract. technical background

 Hypertension is a common cardiovascular and cerebrovascular disease in many countries around the world, especially in the elderly population. Many complications of hypertension are common, such as stroke, myocardial infarction, heart failure, coronary heart disease, and diabetes. The survey results show that the population of patients with hypertension is increasing and showing a younger development trend. Simple diastolic hypertension is more common in middle-aged people, mostly males; simple systolic hypertension is more common in older people, and most females (Li Kuibao et al. "Prospective study of the prognosis of simple diastolic hypertension", Journal of Cardiovascular Diseases ", Vol. 21, No. 1, February 2002). At present, although the pathogenic factors and pathogenesis of hypertension are not very clear, many systems in the body are known to be closely related to the maintenance of blood pressure, such as the vascular endothelial relaxation factor-contracting factor system. The function of the system is affected by the internal and external environment. Changes in their effects, thereby participating in the regulation of blood pressure in the body.

 '' The human system includes a variety of receptors and receptors, which are abundant in the heart and blood vessels. The functions of these receptors are closely related to a variety of related transmission shields. Anyone that can affect the production, release and metabolism of the transmitter, or affect its binding to the receptor, can cause changes in blood pressure. It has been reported that the level of nitric oxide (NO) synthase gene expression in spontaneously hypertensive rats is significantly lower than that in control rats, and that endothelial cell dysfunction may be an important cause of hypertension.

Because the body involved in a wide range of blood pressure regulation system or organization, thus acting on the system, many different types of antihypertensive drugs (Kwok Shing was like "anti-hypertensive drug research new progress,""Chinese Pharmaceutical Journal", ⁱⁿ September 2001 the first Volume 36 Number 9).

There are currently non-drug and drug treatments for the treatment of this type of disease. Among the common therapeutic drugs are diuretics, beta blockers, calcium channel antagonists such as nifedipine, o receptors Blockers, angiotensin-converting enzyme inhibitors (ACEI), etc. However, these drugs can cause side effects. Diuretics can cause hypokalemia and orthostatic 4 氐 blood pressure, β blockers can cause conduction blocks, and calcium channel antagonists such as nifedipine may increase myocardial ischemia. , Alpha receptor blockers can cause orthostatic hypotension, and angiotensin-converting enzyme inhibitors (ACEI) can cause electrolyte disorders (Huang Zhenhua, "Simple systolic hypertension and its treatment", Comprehensive Clinical Medicine, Volume 14, Number 2, 1998).

 Statistical results show that the current antihypertensive drugs have a better effect on reducing diastolic blood pressure, and have less effect on reducing systolic blood pressure.

 Paeonia lactiflora is a plant of the umbelliferae family, paeonia Angelica clahurica (Fisch ex Hoffm.) Ben th. E ^ o. /. According to the 2000 edition of the Chinese Pharmacopoeia, the temperature of the white peony is mild, the taste is spicy, and it is attributed to the stomach, large intestine, and lung meridian. It has the functions of scattered wind and dehumidification, Tongqiao analgesic, swelling and pus. It is used for cold headache, brow rib pain, nasal congestion, nasal abyss, toothache, leucorrhea, sore and sore.

It has been reported that the white pupa mainly contains volatile oil components, coumarin compounds and trace elements. The 1977 edition of the Dictionary of Traditional Chinese Medicine reported that the active components of coumarins contained in white peony include Byak-angelicin, Byak-angelicol, Bakbumin, Dehydrated Bakbumin , Bethustrin, Oxpeucedanine, Imperatorin, Isoimperator in, Phellopterin, Xanthotoxin, Angelicotoxin "Dongying" (Scopoletin), Isobyakangelicol (Anhydrobya-kangelicin) and Neobyakangel icol (Neobyakangel icol), Alloi-soimperatorin (5-isopropyl)- 8-hydroxy psoralen (5-Methyl- 8- hydro- xypsoralen), 7-demethylsuberosin (7-demethylsuberosin), 5- (2-f group-3-methoxy-3-methyl Butoxy) psoralen [5- (2- Hydroxy- 3- methoxy- 3- methyl- butoxy) psoralen], tert-o-methyl b. Yakangelicin, secondary -o-acetic acid white angelica (Sec-o-acetylbyakangeliein), 8-hydroxy-7-isopent-2-enyl coumarin (8-Hydroxy-7-i sopent-2-ene- coumarins), nidlin> Cedrelopsin, Sen -. Byakangelicol evening etc. This also Bu Carotene, bergamot lactone, and oxidized precursin; in addition, the white and white peony contain inorganic elements Ca, Cu, Fe, Zn, Mn, Ni, Mg, Co, Cr, Mo, Na, P, etc., among which The content of Na, Mg, Fe, Ca, and P is higher.

 In the study of coumarins in Paeonia lactiflora, the inventors have unexpectedly found that the obtained extract containing isoimperatorin and imperatorin is effective in the treatment of hypertension diseases, so The present invention has been completed.

 The coumarin extracts of the Chinese medicine provided by the present invention, especially the coumarin extracts extracted from the Chinese medicine Paeonia lactiflora, have no toxic and side effects compared with the existing medicines, and have a stronger effect on reducing systolic blood pressure than diastolic blood pressure. In the past, antihypertensive drugs have a poor effect on reducing systolic blood pressure. Summary of invention

 The present invention provides a coumarin extract from a traditional Chinese medicine raw material medicine, which contains at least imperatorin and isoimperatorin. The extract can be used for the treatment of hypertension.

 In particular, the present invention provides the above-mentioned coumarin extracts from Chinese medicine Paeonia lactiflora, and further proposes new medicinal uses of the effective ingredients of Paeonia lactiflora.

 The present invention also provides a supercritical carbon dioxide fluid extraction method for the above-mentioned extracts from the raw materials of Paeonia lactiflora to obtain an extract with a high purity of effective ingredients.

 In the study of the above-mentioned coumarin extracts, the present invention also provides a pharmaceutical composition using the extract as an active ingredient, which can be used for the treatment of hypertension, and the composition may further include other raw materials. Extracts, such as chuanxiong extract and kudzu root extract.

 The coumarin extract of Chinese medicine provided by the present invention contains at least imperatorin and isoimperatorin, and the total coumarin component contained in each gram of extract is not less than 300 milligrams of imperatorin . In particular, imperatorin contains not less than 80 mg per gram of extract.

 The extract of the present invention may be derived from any drug substance containing the coumarin component, preferably from a coumarin-based active ingredient in the drug substance of Paeonia lactiflora.

Through experiments on the composition and the effects of drugs, the present invention particularly provides a white peony extract obtained by applying a supercritical carbon dioxide fluid extraction method, and has relatively high coumarin active ingredients. Content, and maintain the ingredients of natural raw materials.

 Through pharmacodynamic research, it was proved that the above-mentioned extract (or coumarin effective part) of the present invention can be used for the treatment of hypertension diseases. Therefore, the present invention also provides a pharmaceutical composition for treating hypertension, which includes a therapeutically effective amount of the above-mentioned Chinese herbal coumarin extract and an acceptable pharmaceutical adjuvant in pharmacy.

 The pharmaceutical composition of the present invention may further include other traditional Chinese medicine extracts, for example, one or more of traditional Chinese medicine extracts such as Chuanxiong, Puerariae, Angelica, Panax notoginseng and Salvia miltiorrhiza. Detailed description of the invention

 The present invention first provides a coumarin extract from a traditional Chinese medicine, which contains at least imperatorin and isoimperatorin. The term "coumar in" as used in the present invention refers to compounds of different substituents known in the art that use cis-o-hydroxycinnyllactone as the mother core.

 According to the foregoing description, the extract of the present invention is derived from traditional Chinese medicine. In addition to containing imperatorin and isoimperatorin, it may also include any effective part of coumarins. One, or several kinds of, bacicralin, dehydrated bacillin, alloprollin, etc. The extract may be derived from any traditional Chinese medicine raw material containing these ingredients. According to a preferred embodiment of the present invention, the extract is derived from the active ingredients of coumarins in the raw medicine of Paeonia lactiflora. The extract also preferably has a fingerprint as shown in FIG.

 The extract of the present invention can be obtained by any feasible method, and preferably can be obtained by supercritical carbon dioxide fluid extraction, so that the product has a relatively high purity and yield.

Therefore, another aspect of the present invention is to prepare the coumarin extract by a method of supercritical carbon dioxide extraction. According to the method of the present invention, supercritical carbon dioxide fluid is used for extraction. In the extraction process, C ₂ -C ₃ lower alcohol can be used as a carrier.

Preferably, in the extraction method of the present invention, a supercritical fluid of carbon dioxide is used as a solvent, the extraction pressure is 20-35Mpa, and the extraction temperature is 30-50 ° C. The selected C ₂ -C ₃ lower alcohol is added as a carrier in the solvent to obtain the desired extraction product. The lower alcohol may include ethanol, propanol, isopropanol, etc. The concentration of the lower alcohol is about 1% to 15% (based on the weight of the medicinal material to be extracted). According to a preferred solution of the present invention, ethanol or isopropanol is used as a carrier, and the concentration is about 10%. Extraction The completion of the process is based on the composition of the extracted product meeting the requirements of the present invention, which can generally be controlled at about

More than 3 hours.

In the present invention, raw raw materials can be used, and processed pieces can also be used. Only according to the preferred method of the present invention, the use of Paeonia lactiflora pieces or raw Paeonia lactiflora medicine as raw material, if it is unprocessed Paeonia crumba medicine, the processing such as washing, drying and slicing should be performed as a preliminary treatment. The extraction method is as follows: Crush, sieve, place in the extraction kettle, after the heating device works normally, turn on the compression pump to pressurize to 20 ~ 35MP, temperature 40 ~ 50 ° C, add 1% ~ 15% ethanol as a carrier, and take 40 ~ 50kg after / extraction h 0) flow rate of ₂ cycles of separation vessel pressure of 5 ~ 10MP, the temperature is 30 ~ 50 ° C, the product was extracted to meet the requirements determination, entrainer evaporated to dryness, to give coumarins Angelica supercritical active ingredient Extract. The properties of the extract are yellow to reddish brown oil or solid.

 In addition to the above, other operations involved in implementing the method of the present invention, such as volatilization of the extraction solution, can be heated and volatilized, or can be naturally volatilized directly at normal temperature or other feasible methods. The operation belongs to the basic skills in the field, and the processing, pulverization, sieving, and extraction of the traditional Chinese medicine decoction pieces can all adopt conventional operations in the art or other feasible operations, and the present invention is not particularly limited. The obtained extract can be used to determine whether it meets the requirements by any conventional detection and analysis methods, such as thin-layer chromatography, ultraviolet spectrophotometry, and other analytical methods such as those contained in an appendix to the 2000 edition of the Chinese Pharmacopoeia. It is a routine operation in the art, and the present invention is not particularly limited.

 In order to verify the medicinal effect of the coumarin extract of the present invention, in addition to the component detection, the inventors also performed further cell membrane chromatography analysis on the extract (Cel l Membrane Chromatography, CMC λ cell membrane chromatography is a bionic The biological affinity chromatography method can be used to determine the specific interactions between drugs and compound molecules and cell membranes and membrane receptors. In the experiments of the present invention, aortic cell membrane color language models are used, and the calcium antagonist verapamil is used as a control. The active ingredients in the total effective parts of the raw materials of Paeonia lactiflora were screened.

The test results show that under CMC screening conditions, the total effective site of Paeonia lactiflora in the present invention contains active ingredients similar to verapamil (VP) and imperatorin and isoimperatorin, in which imperatorin and The effect of isoimperatorin on vascular cell membranes and membrane receptors is stronger than that of the positive control. In short, the present inventors unexpectedly discovered in the medicinal research on Paeonia lactiflora that the above-mentioned effective parts of coumarins are effective for the treatment of hypertension, and determined that they contained a similar effect to the calcium channel blocker paravirmi Active ingredient. Further pharmacodynamic tests confirmed that effective parts of coumarins could make the receptor-regulated calcium channels have a strong non-competitive inhibitory effect; a significant inhibition of aortic strip contraction indicates that it has a voltage-regulated calcium channel Inhibition. The effective part of Paeonia lactiflora can reduce the arterial vascular resistance. It has a significant chronic reduction effect on spontaneous hypertension and a significant acute reduction effect on renal hypertension. Therefore, the present invention also proposes a new use of the effective part of coumarins in the treatment of hypertension, and is particularly applicable to the treatment of primary and secondary hypertension. That is, it is used for treating diastolic and systolic blood pressure diseases of primary and secondary hypertension.

 Based on the above research results, the present invention also provides a pharmaceutical composition that can be used to treat hypertension, including a therapeutically effective amount of the traditional Chinese medicine coumarin extract of the present invention and an acceptable pharmaceutical adjuvant in pharmacy.

 According to the pharmaceutical composition of the present invention, the effective portion of the coumarins can also be compounded with other traditional Chinese medicine ingredients to achieve better therapeutic effects. The traditional Chinese medicine ingredients can be, for example, hawthorn, chuanxiong, kudzu root, angelica, sanqi, The active ingredients in APIs such as Danshen can come from any extraction method.

 According to a preferred embodiment of the present invention, the medicinal composition further contains an active ingredient extract of Ligusticum chuanxiong. Based on the consideration of synergistic efficacy, the Ligusticum chuanxiong extract contains not less than n-hexane extract.

40 wt, and not less than 9 ml of chuanxiong volatile oil per 100 grams, can be extracted by any feasible method. The weight ratio of the Chuanxiong active ingredient extract to the Chinese medicinal coumarin extract is 1: 3 to 3: 1, preferably 1: 2 to 2: 1, and more preferably 1: 1. In the pharmaceutical composition of the present invention, the supercritical carbon dioxide extract of Ligusticum chuanxiong is preferably used, and the properties of the product are yellow to brown-red oily substance, and the aroma and taste are bitter. Ethanol as the extraction process using entrainers, i.e., with 1% to 15% ethanol as entrainer, extraction pressure 8-15Mpa, the extraction temperature 40- 60 ° C, (flow rate of 30-50kg / h 0 _2, the separation kettle Pressure and temperature are 3-7 Mpa and 25-35 ° C.

According to another preferred embodiment of the present invention, the pharmaceutical composition may further comprise a fat-soluble extract of puerarin, the extract containing isoflavones of not less than 300 mg in terms of puerarin per gram, and Each 1 gram of extract contains no less than 15 mg of puerarin and daidzein, and the extract can be obtained by ethanol extraction. Brief description of the drawings:

 Figure 1 shows the HPLC chromatograms of iso-imperatorin (a), imperatorin (b), and total effective fraction (c) of Amaranth.

 Figure 2 shows the chromatograms of total effective fractions (a), verapamil (b), isoimperatorin (c), and imperatorin (d) of scutellaria baicalensis on the vascular cell membrane column.

 Fig. 3 is the dose-response curve of the contraction rate of the isolated aortic tissue contraction of rabbits induced by NE with the effective components of Paeonia lactiflora.

 Figure 4 is the blood pressure curve of the chronic hypotensive effect of SHR systolic blood pressure by the active ingredients of Paeonia lactiflora.

 Figure 5 is the blood pressure curve of the active antihypertensive effect of SHR on diastolic blood pressure. Specific implementation plan

 The specific implementation and therapeutic effect of the present invention will be described in detail with reference to the preferred embodiments and the accompanying drawings.

 It should be noted that: the terms "coumarin extract", "coumarin effective part" and "total effective part" used in the present invention have the same meaning.

 White: The crushed pieces of medicinal materials are crushed, sieved through a 20-mesh sieve, 450g of powdered medicine is weighed, and placed in an 1L extraction kettle. After the refrigeration device, extraction kettle, separation kettle and heating device work normally, turn on the compression pump

30MP, temperature 30 ° C, plus about 10% ethanol entrainer to C0 ₂ flow rate of 45kg / h cycle extraction, the separation vessel pressure and temperature were 5MP, 40 ° C, extraction After 3h, the fractionated extract, The carrier is dried, and the supercritical total active site extract of the white peony is obtained, which is stored in the refrigerator for use. Legal analysis of thin-layer chromatography of Paeonia lactiflora extract

'' Take 40 mg of the above-mentioned Paeonia lactiflora extract and place it in a 10 ml volumetric flask, add an appropriate amount of ethyl acetate, shake to dissolve, bring the volume to the mark, and shake the hook as the test solution. Imperatorin and isoimperatorin As a reference solution, a solution of 1 mg / ml was prepared by adding ethyl acetate respectively. According to the thin-layer chromatography (Chinese Pharmacopoeia 2000 edition, Appendix VIB) test, draw 5 μs of each of the above solutions, and point them on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as the binder. Take petroleum ether (60 ° C ~ 90 ° C) -diethyl ether (3: 2) as the developing agent, unfold it below 25Ό, take it out, dry it, and inspect it under UV lamp (365 nm). Fluorescence spots of the same color are displayed at positions corresponding to the chromatogram of the reference substance. The results showed that imperatorin and isoimperatorin were contained in the extract. Analysis of Total Coumarin by UV Spectrophotometry

 Conditions: UV spectrophotometer (Shimadzu UV-260);

 Dilution solvent: methanol-ether (4: 1);

 Reference substance: imperatorin;

 Absorption wavelength: 302mn.

 The specific operation is determined according to the ultraviolet spectrophotometry (Appendix VA of the 2000 edition of the Chinese Pharmacopoeia), and a standard curve is drawn with the absorbance (A) versus the solution concentration (C, μξ / l).

The present results show that the extract of each 'lg containing coumarin compound imperatorin (C ₁₆ H ₁₄ 0 ₄₎ meter, of not less than 300mg _o

HPLC chromatography

Conditions: The color i column is LiChrosorb ODS C ₁₈ (150x4.6ram, 5 μm);

 Mobile phase: methanol-water (60:40);

 Flow rate: 1.0 ml / min; Detection wavelength: 302nm;

 Methanol is used as a dissolving agent.

 The specific operation was determined by external standard method according to high performance liquid chromatography (Chinese Pharmacopoeia, 2000 edition, Appendix VIB).

HPLC fingerprint analysis: As shown in Figure 1, under the condition of chromatographic analysis, the HPLC fingerprint of the extract of the total effective part of Paeonia lactiflora can obtain six common peaks, which are in agreement with the imperatorin and isoimperatorin standards. Method ("Appendix VIB of the Chinese Pharmacopoeia" 2000 edition) comparison of measurement results (peak 1 in Figure 1 is imperatorin and peak 6 is isoimperatorin.), As the total effective part Index peaks for qualitative identification. At the same time, the area normalization method was used to determine imperatorin as the standard, and the effective part content was greater than 80%.

 Experimental results show that the extracts of the effective components of the coumarins mentioned above include imperatorin, isoimperatorin, oxidized precursin, biccate baicalein, dehydrated biccate baicalein, alloprocarcin, etc. Any one or a combination of several. Chromatographic screening

 A rabbit aorta cell membrane stationary phase column was installed (50 x X 2 legs, I. D.);

Mobile phase: 50mmol / L phosphate buffer (ρΗλ ⁴ );

 Flow rate: 0.5ml / min;

 Detection wavelength: 236nm;

 Column temperature: 37 ° C.

 Take appropriate amounts of the above extracts from the total effective parts of Paeonia lactiflora, verapamil (VP), imperatorin and isoimperatorin respectively, add pyridine 100 μΐ to dissolve, centrifuge for 5min (6000r / min), and extract the supernatant 5 μ 1 injection measurement, VP is the positive control drug, record the CMC screening color i chart, as shown in Figure 2, curve a is the chromatogram of the total effective part of Paeonia lactiflora, curve b is the chromatogram of verapamil, curve c Is the color term for isoimperatorin, and curve d is the chromatogram of imperatorin. Peak 2 is imperatorin and isoimperatorin, peak 1 is other coumarins, peak 3 is verapamil, peak 4 is isoimperatorin, and peak 5 is imperatorin. Under CMC screening conditions, the total effective fraction of this white peony contains active ingredients similar to verapamil (VP) and imperatorin and isoimperatorin. Among them, imperatorin and isoimperatorin and The strength of the vascular cell membrane and membrane receptor is greater than that of the positive control.

 In vitro pharmacological study of coumarin extract from peony

 1. Rabbit aortic strip test

Healthy rabbits were sacrificed and blood was sacrificed by common carotid artery. The chest was opened to expose the heart, the aorta was separated, and the inferior vena cava artery near the heart was cut and placed in oxygen-saturated Tyrode's nutrient solution to remove connective tissue on the surface of the blood vessel wall. Use scissors to cut the aorta into a spiral strip with a width of 3mm and a length of 15mm. Both ends are threaded and ligated. The lower end is fixed on the ventilation hook at the bottom of the bath with a volume of 10ml. On the tension transducer, the tension transducer is connected to an automatic benchtop balance recorder. The bath was continuously and evenly fed with 0 ₂ , the arterial strip was loaded with lg, the bath temperature: 37 ° C ± 0.5 ° C, pH: 7.4 ± 0.1. When the specimens were equilibrated, the Tyrode's nutrient solution (37 ° C) was replaced every 30 minutes in the bath; after each dosing, the Tyrode's nutrient solution was rinsed 3 times. The contraction of the aortic strip was recorded on the recorder after being transformed by the tension transducer. Verapamil (VP) was used as a positive control for comparison.

 2.Effects on NE-induced aortic contraction

 After the samples were equilibrated for 1-2 hours, noradrenaline (NE) was added to the bath tube by the cumulative administration method, and the dose-response curve of the samples to NE was continuously recorded; then the samples were washed with nutrient solution, and the blood vessel tension was restored to the baseline level. Different dosage groups of the active ingredient of Paeonia lactiflora were added to the bath tube, and the dose-response curve of NE on vascular smooth muscle contraction was re-tested. The dose-response curve of the positive drug (VP) was measured in the same way. Experiments have shown that the difference: the dose of active ingredients of Amaranth shifts the dose-effect curve of the active pulse to NE contraction to the right, and the maximum inhibitory effect is reduced.

 3. Effect on aortic contraction induced by high K + depolarization

After the specimens were equilibrated for 1-2 hours, 1 mol / L KC1 1 ml was added to make the aorta contraction reach a steady state. Different concentrations of active ingredients or positive drugs (VP) of Paeonia lactiflora were added to the bath tube in a single administration method, and the contraction tension of the aortic strip was recorded. See Table 1-1. Experiments show that the maximum inhibitory rate of active ingredients of different doses of Amaranth on aortic contraction induced by high K ⁺ depolarization is 22.5%, while the maximum inhibitory rate of voltage-regulated calcium channel-specific inhibitor VP is 95.5 %.

Table 1-1 Effect of active ingredients of Amaranth on aortic contraction caused by high K + depolarization Group's concentration (mg / ml) Mean inhibition rate 0

 S white group-active ingredient group of Paeonia 0.15 4.62

 Paeonia lactiflora active ingredient group 0.5 14.0

 Paeonia lactiflora active ingredient group 1.5 22.5

Verapamil Group 1.0 95.3 4.Effects on the contractile components of NE exocalcin and exocalcium

After the specimen is equilibrated for 1 to 2 hours, NE is added to make the vasoconstriction response reach a steady state. Rinse with calcium-free nutrient solution. After equilibration, add the same dose of NE to cause vasoconstriction. When the steady state is reached, add Ca ^{2+ to} restore the normal Ca ²⁺ concentration. The vessel will constrict to a maximum in one step. Then rinse with calcium-free solution, add different concentrations of active ingredients of Paeonia lactiflora or VP after equilibration, and record the effects of each drug on the two contractile components of NE. See Table 1-2. Experiments have shown that the effective components of Paeonia lactiflora have a dose-dependent inhibitory effect on the contraction of aorta by intra- and intra-external calcium. At the same time, it showed that its effect of blocking external calcium inflow was stronger than that of VP. Effects of active ingredients of Paeonia lactiflora on the contraction of aorta by intra- and intra-external calcium

Ca ^{2 + -free} liquid Ca ²⁺ liquid group concentration

 (mg / ml) Maximum contraction average Maximum contraction average

 (%) Inhibition rate (%) () Inhibition rate (%)

• Blank group-40.9 ± 2.5-59.1 ± 2.9-Active ingredients of Amaranth 0.45 35.4 ± 1.1 13.4 54.0 + 2.9 8.63 Active ingredients of Amaranth 2.25 17.6 ± 2.1 56.9 22.7 + 2.0 61.6 Active ingredients of Amaranth 4.50 1.43 ± 0.9 96.5 10.0 ± 2.1 83.1 Vera The pamid group 0.20 5.7 ± 1.8 90.6 41.8 ± 2.6 28.3 The dose-response curve of the active ingredients of Paeonia lactiflora that caused NE to induce isolated aortic contraction shifted non-parallel to the right and the maximum response decreased, indicating that it had a non-receptive effect on receptor-regulated calcium channels. Competitive inhibitory effect; inhibits aortic contraction caused by high K + depolarization, indicating that it has inhibitory effect on voltage-regulated calcium channels. It is suggested that the effect of the active ingredients of Paeonia lactiflora on the aortic strips caused by NE is the result of inhibiting the release of intracellular calcium and the influx of external calcium, thereby reducing the arterial vascular resistance.

 5. Chronic Antihypertensive Effect of Active Ingredients of Paeonia lactiflora on SHR

 Take a certain amount of the extract of the effective ingredient of Paeonia lactiflora, and prepare 0.5, 5, 10 mg / ml of medicinal solution with 0.5% CMC (sodium carboxymethyl cellulose), respectively. Nifedipine tablets: produced by Xi'an Bohua Pharmaceutical Co., Ltd., batch number 0003007, 10mg each, ground, 0.5% CMC (Carboxamidine Cellulose Sodium) was formulated into a drug solution with a concentration of 0.5mg / ml.

Animals: Spontaneously hypertensive rats (SHR), 6 weeks old, weighing 180 ± 30g, purchased from the Animal Experiment Department of Fuwai Hospital, Chinese Academy of Medical Sciences, certificate number: Yidongzi No. 0bu 3080. Feed animals Keep at room temperature 26 ± 3. C, artificial lighting for 12 hours daily, caged by sex, free drinking water, pellets about

Feed feeding.

 Forty male SHRs were selected, both male and male, at a room temperature above 28 ° C, and the blood pressure of the tail artery of rats was measured indirectly using a HEM-80F volume impedance sphygmomanometer. Experiment after 1 week of training. Blood pressure was measured continuously for 5 days before the drug, once a day, and 3-5 data were measured each time, and the average value was taken. The SHR was stratified randomly into 5 groups based on the mean blood pressure 5 days before the drug, with 8 in each group: (1) control group: 0.5% CC; (2) nifedipine group: 5mg / kg; (3)-(5 ) Active ingredient group of Paeonia lactiflora: 100, 50, 25mg / kg group. Each group was administered orally, once a day, with a volume of 10 ml / kg, for 20 consecutive days. The blood pressure was measured the next day before and 2 hours after the drug. After stopping the drug, the blood pressure during the recovery period was measured continuously for 8 days.

 1). Chronic reduction of systolic blood pressure in SHR

 As shown in Figure 4, the systolic blood pressure of each group was basically stable during the 5 days before the drug administration, and maintained at 20.8-21.6 kPa. The control group had small fluctuations in systolic blood pressure during the entire administration period. Active ingredients of Paeonia lactiflora 100, 50 mg / kg group and nifedipine The systolic blood pressure in the group decreased slowly from 1 to 15 days after administration, and the systolic blood pressure was maintained between 15.6 and 16.9 kPa from 15 to 19 days after the administration. 6 days basically returned to pre-dose levels.

The systolic blood pressure in the 25 mg / kg active ingredient of A. lobata also decreased slowly from 1 to 9 days after administration, and remained at 18.2 to 19.5 kPa between 11 and 19 days. 6 days after stopping the drug, it basically returned to the pre-dose level. The specific changes of each group can also refer to the following table 2-1 and table 1-1. Table ² -1 Effect of effective ingredients of Paeonia lactiflora on SHR systolic blood pressure (kPa) (n = 8) systolic blood pressure (kPa)

 White cricket White cricket White cricket

22.2 ± 2.9 21.1 ± 3.2 21.3 ± 2.3 20.9 ± 2.9 20.7 ± 2, 6

2 22.0 ± 2.9 20.3 ± 2.6 20.4 ± 1.4 21.7 ± 2.0 21.2 ± 1.3

21.1 ± 2.5 20.9 ± 1.8 20.8 ± 1.8 22.2 ± 2.6 21.6 ± 1.2 21.1 ± 1.7 20.5 ± 1.7 21.2 ± 1.3 20.7 ± 2.9 20.4 ± 1.4 21.6 ± 2.1 21.3 ± 2.2 20.0 ± 1.8 21.4 + 2.2 21.1 ± 1.6

21.6 ± 1.3 20.8 ± 1.0 20.8 ± 1.2 21.3 ± 1.7 20.9 ± 0.3 1 Before drug 22.8 ± 2.0 22.4 + 2.3 22, 6 ± 1.7 21.8 ± 1.6 20.9 ± 2.3 After drug 22.0 ± 2. 0 18.6 ± 2.3 ^ΔΔ 20.4 ± 2.1 ^ΔΔ 19.1 ± 2.9 ^Δ 20.3 ± 2.0

3 Before drug 21.3 ± 2. 6 20.7 ± 2.3 20.3 ± 2.1 21.8 ± 3.9 20.2 ± 1.4 After drug 21.2 ± 3. 4 18.5 ± 2.5 18.7 ± 3.0 19.1 ± 3.1 18.8 ± 1.7

5 Before drug 22.9 ± 1. 3 20.2 ± 3.0 19.4 ± 1.3 19.4 ± 1.6 19.5 ± 2 · 3 After drug 21.8 ± 3. 0 19.8 ± 2.2 19.0 ± 2.1 17.8 ± 2.1 18.1 ± 1.6

7 Before drug 21.4 ± 1. 0 18.3 ± 2.5 20.3 ± 3.0 20.4 ± 2 · 2 20.4 ± 2.3 After drug 20.5 ± 2. 0 17.6 ± 2.1 17.4 ± 2.6 18.1 ± 2.9 19.0 ± 1.4

9 Before drug 21. SiS- 0 19.4 soil 2.1 19.4 + 2.1 18.6 ± 2.1 19.8 ± 2.2 give

SO.4 ± 1.2 2 17.2 ± 3.1 16.5 ± 1.8 ^Δ 18.1 ± 1.0 17.7 ± 0.9 ^Δ

 11 Before drug 21.2 ± 0.9 9 19.1 ± 3.2 25.1 ± 3.5 19.1 ± 3.1 19.1 ± 1.4 period

22.6 ± 1. 8 17.0 ± 2.5 16.5 ± 2.0 16.1 ± 1.8 ^Δ 18.8 ± 1.6

13 Before drug 20.3 + 1. 0 15.6 ± 1.3 18.1 + 1.7 17.6 + 2.2 19.6 ± 1.6 After drug 20.7 + 1. 3 15.2 ± 1.4 17.0 ± 2.3 15.5 ± 2.2 16.6 ± 1.

15 Before drug 21.1 ± 1. 7 16.9 ± 1.8 17.4 ± 1.8 17.8 ± 2.6 19.5 ± 12 After drug 21.3 ± 2. 5 16.0 + 1.0 16.1 ± 1.3 16.4 ± 2.0 18.8 ± 1.0

17 Before drug 20.8 ± 1. 8 16.5 ± 1.8 16.2 ± 1.4 16.8 ± 1.8 19.1 ± 1.6 After drug 20.7 ± 0.9 9 17.0 ± 2.1 16.2 + 1.6 17.0 ± 0.9 19.2 ± 1.0

19 Before drug 21.4 + 1. 0 16.1 + 1.8 15.9 + 0.9 16.1 ± 2.0 18.3 ± 1.0 After drug 20.5 + 1. 2 16.8 ± 1.8 16.1 ± 1.3 16.1 ± 1.3 19.0 ± 2.0

1 21.2 ± 1. 4 17.3 + 2.5 16.5 ± 1.3 18.1 ± 2.1 19.0 ± 1.0

2 21.2 + 1. 3 17.7 ± 1.3 17.7 ± 2.0 18.5 ± 1.6 19.2 ± 1.3 Stop

 3 21.8 ± 1. 6 17.9 ± 1.6 18.8 ± 3.1 18.1 ± 1.4 20.1 ± 1.8

 4 20.4 + 1. 2 20.0 ± 2.3 19.8 ± 1.7 18.3 ± 1.6 20.0 ± 1.4 periods

 6 21.2 ± 1. 0 20.2 + 1.4 20.5 ± 2.0 20.3 ± 2.5 20.5 ± 2.0

8 21.1 + 1. 8 20.8 ± 1.6 21.1 ± 20 21.8 ± 1.4 21.3 ± 1.6 Note: ί test, compared with the pre-drug blood pressure of the day, ^Δ 0.05, ^ΔΔ 0.01 Table 2-2 Effects of active ingredients in Paeonia lactiflora on SHR systolic blood pressure (kPa) (n = 8)

 Reduced systolic blood pressure (kPa)

 Days of nifedipine

 Control group

 5mg / kg 100mg / kg 50rag / kg 25mg / kg-1.2 ± 2.1 — 1.6 ± 2.9 0.3 ± 3.0-0.4 ± 1.8 0.0 ± 2.2 after drug-0.4 ± 2.3 2.2 ± 3.1 1.6 ± 2.2 2.2 ± 3.3 0.6 ± 2.0 before drug 0.1 ± 2.6 0.1 ± 3.0 0.0 ± 2.6-0.4 ± 4.0 0.9 ± 1.7 0.4 ± 4.0 2.3 ± 3.0 2.1 ± 3.5 2.2 ± 2.9 2.1 ± 1.6 before medication 1.4 + 2.0 0.8 ± 3.4 1.4 ± 2.0 2.0 ± 2.9 1.4 ± 2.3 After drug-0.3 ± 3.1 1.2 ± 2.3 1.7 + 2.0 3.5 ± 2.7+ 2.9 ± 1.6+ before drug 0.1 ± 1.8 2.5 ± 2.2+ 1.2 ± 3.0 1.0 + 2.3 0.5 ± 2.3 after drug 0.8 ± 1.4 3.4 + 2.2 + 3.4 ± 2.1 + 3.2 ± 2.2+ 2.0 ± 1.4 before drug 0.4 ± 3.0 1.4 ± 2.5 1.4 ± 2.5 2.7 + 2.7 1.3 ± 2.5 after drug 1.0 ± 1.3 3.8 ± 3.8 4.3 ± 2.6 * 3.4 + 2.3 + 3.2 ± 1, 0 * before drug 0.6 ± 1.8 1.7 ± 3.0 2.7 ± 3.6 2.3 ± 2.7 2.0 ± 1.4 after drug-0.9 ± 2.9 3.8 ± 2.6 * 4.3 ± 2.2 * 5.2 ± 2.9 * 2.2 ± 3.0+ before drug 1.3 ± 2.0 5.3 ± 1.4 * 2.6 ± 0.9 3.9 ± 2.3+ 1.4 ± 1.6

13

 0.8 ± 2.1 5.6 ± 2.2 * 3.8 ± 2.6 + 5.8 ± 3.4 * 3.0 ± 1.8 + before drug 0.5 ± 1.6 4.0 ± 2.7 * 3.4 + 2.2 * 3.6 ± 1.7 * 1.4 ± 1.2

15

 0.3 ± 1.8 4.8 ± 2.0 * 4.7 ± 1.8 * 4.9 ± 2.0 * 2.2 ± 1.3 + before drug 0.8 ± 1.2 4.4 ± 2.5 * 4.6 ± 1.8 * 4.7 + 1.8 * 2.0 ± 1.6

17

 1.3 ± 1.3 3.8 + 2.7+ 4.6 ± 2.0 * 4.4 ± 2.0 * 1.7 ± 1.3 before drug 0.0 ± 1.7 4.7 ± 1.8 * 4.8 ± 1, 6 * 5.3 ± 2.1 * 2.6 ± 1.2 *

19

 Post-dose 0.9 ± 2.0 4.0 ± 2.1 * 4.7 + 1.6 * 5.2 ± 1.8 * 2.1 ± 1.8

1 0.4 ± 2.1 3.5 ± 2.7 4.3: 1.2 * 2.0 +

 0.4 ± 1.6 3.1 ± 1.8 * 3.1: 2.r 1.7 soil medicine 3 0.3 ± 1.7 3.0 ± 1.8 * 2.0:: 3.4 0.8 soil

 4 1.2 ± 2.3 0.8 ± 3.0 0.6 ± 1.4 (.9 soil period

6 0.4 ± 1.8 0.6 ± 1.7 0.3 ± 2.3 0.4 ± 8 0.4 ± 1.6 0.0 + 1.7 — 0.3 ± 1.7 -0.3 + Note: Compared with the control group, the test is + .05,. *. Oi; from Table 2-1 It can be seen from Table 2-2 that the change of systolic blood pressure in each group was small in the 5 days before the medicine, and maintained at 20.8 ~ 21.6 kPa. The systolic blood pressure of the control group did not fluctuate much during the administration period. The systolic blood pressure of each administration group tended to decrease. The decrease of systolic blood pressure in the 100, 50 and 25 mg / kg groups of Astragalus started from 5 or 7 days after administration. Compared with the control group, the difference was significant (<0.05); the difference between the 100 and 50mg / kg dose groups to 9 or 11 days after the drug was more obvious (<0.01), and thereafter remained at the same level. Comparing the systolic blood pressure before and after the day of administration, there is a tendency to decrease after administration, which is more obvious at 1-9 days after administration, and there is a significant difference between 1 day after administration and before administration. Compared with the control group, the systolic blood pressure of the nifedipine group was significantly different from that of the control group from the 7th day of administration. The low value of P bar was significantly different, and the same hypotensive level was maintained from 11 days to 3 days after the drug was discontinued. The difference at 1 day was significant. After 20 days of administration, the systolic blood pressure of the above four groups decreased by an average of 4.7 ± 1.6, 5.2 ± 1.8, 2.1 ± 1.8, 4.0 ± 2. lkPa. The large and medium doses of Paeonia lactiflora extract group and nifedipine group were still significantly different from the control period 1-3 days after drug withdrawal. The systolic blood pressure of each dose group recovered to the control phase level 4-6 days after discontinuation.

 2). Chronic reduction of diastolic blood pressure in SHR

 As shown in Figure 5, the diastolic blood pressure in each group did not change much in the 5 days before the medicine. The control group did not change significantly after administration of diastolic blood pressure. The diastolic blood pressure of the active ingredients of Baiji 100, 50 mg / kg group and nifedipine group decreased slowly after administration, and the dose of 13 ~ 19 ^^ was maintained between 9.1-10.4 kPa with small fluctuations. The diastolic blood pressure decreased less in the 25 mg / kg active ingredient of Amaranth. Each group recovered within 4 to 6 days.

It can also be seen in Tables 2-3 to 2-4 that the diastolic blood pressure in each group did not change much in the 5 days before the medicine, and maintained at 12.5-13 kPa. The diastolic pressure in the control group changed little throughout the administration period. The diastolic blood pressure of the active ingredients of 100 and 50 mg / kg group started from 7 days or 11 days after administration, and the decrease value was significantly different from that of the control group (<0.05 or <0.01), and remained basically unchanged from 11 days to 20 days after administration. Same level. However, compared with the control group, the decrease in diastolic blood pressure at 2 hours after daily medication was significantly different from that of the control group at the same time (P <0.05 or P <0.01). Compared with the control group, the effective value of the effective ingredient of A. baicalensis 25mg / kg group was significantly lower than that of the control group before d3 and 13d (<0.05). The effect of P in the nifedipine group was basically the same as that of the effective ingredient of Paeonia lactiflora at 100 mg / kg. The diastolic blood pressure of each group was decreased before and after the day of administration. 19 days after the administration of the ^four groups were decreased amplitude of ^{2.9 ± 1.2, λ 2 ± 1.} 6, 3.0 ± 2.1, 1.6 ± 1.2kPa. The diastolic blood pressure in each administration group recovered within 4 to 6 days.

Observation of the effect of the active ingredients of Paeonia lactiflora on SHR blood pressure shows that the effective ingredients of Paeonia lactiflora have a significant chronic reduction effect on SHR blood pressure. Discontinued Drugs Table 2-3. Effect of Active Ingredients of Paeonia lactiflora on SHR Diastolic Pressure (n = 8)

 4 12683 Diastolic blood pressure (kPa)

 Days-Nifedipine White peony White peony control group White: £ 50mg / kg

 5mg / kg. 100mg / kg 25mg / kg

1 12.8 ± 1.0 13.4 + 3.5 13.1 ± 2.3 11.8 ± 1.7. 12.8 ± 1.8

2 13.9 + 2.6 12.1 ± 1.7 13.4 + 2.5 12.5 ± 1.6 13.0 ± 2.0

3 12.6 ± 2.9 12.5 ± 1.7 12.4 ± 2.0 12.4 ± 1.8 13.9 ± 1.8 before 4 13.0 ± 1.3 13.0 ± 1.0 12.0 ± 2.0 12.0 ± 2.0 12.6 + 1.2

 5 13.0 ± 2.0 13.0 ± 1.0 12.0 ± 1.0 13.9 ± 1.4 12.4 ± 1.7

X ± s 13.0 ± 0.9 12.9 ± 1.2 12.5 ± 1.0 12.5 ± 1.2 13.0 ± 0.9 Approx. Fl'J 13.8 ± 1.6 13.0 ± 1.0 14.3 ± 1.8 13.8 ± 1. 3 12. 6 ± 2.3 After drug 12.4 ± 0.9 11.2 ± 2.1 ^Δ 13.3 + 2.2 11.2 ± 1.8 ^ΔΔ 12. 4 ± 2.3 before drug 13.9 ± 2.1 12.0 + 1.7 12.4 + 2.2 13.4 ± 1. 0 12. 0 ± 1.7 after drug 12.5 ± 1.8 11.8 ± 1 · 6 11.0 ± 1.8 11.8 ± 1. 7 ^Δ 12. 5 ± 1.4 before drug 13.8 ± 2.3 12.9 ± 2.3 12 · 5 ± 1 · 6 12.4 ± 1. 3 12. 9 ± 2.0 after drug 13.3 ± 2.1 11.2 ± 1.3 11.6 + 2.0 10.9 ± 1. 7 11. 7 ± 1.4 before drug 12.5 + 1.3 11.0 ± 2 · 3 11.7 ± 2.5 13.0 ± 2. 3 13. 0 ± 1.8 after drug 13.0 ± 2.6 9 · 9 ± 0.5 10.3 ± 1.7 11.0 ± 1. 8 12. 0 ± 1.4 drug Before 12.6 + 1.7 11.6 ± 1.8 12.1 ± 1.8 11.6 ± 1. 8 12. 0 ± 1.7 after drug 12.1 + 2.1 10.3 ± 2.1 9 · 8 ± 1.2 ^ΔΔ 11.0 ± 1. 2 10. 9 ± 1.3 before drug 12.6 ± 2.0 11.3 ± 0.8 11.3 + 2.7 12.0 ± 3. 1 11. 8 ± 1.2 after drug 14.2 ± 1.4 10 · 1 ± 1.2 ^Δ 9.8 ± 1. 2 9.6 ± 1. 2 12. 0 ± 1. · 3 before drug 14.5 + 1.7 9.5 ± 1 · 0 10.7 ± 1.2 10 · 4 ± 1. 8 12. 2 ± 1.2 After treatment 13.4 ± 2.1 9.4 ± 0 · 9 9 · 9 ± 1.6 9.8 ± 2. 1 10. 9 ± 1 · 0 about 'J 12.6 ± 2.2 10.3 ± 0.9 10.5 ± 1 · 6 10.7 ± 1. 4 12. 5 ± 1.2 after drug 12.2 ± 3.0 9.6 ± 1.6 9.8 ± 1.6 10.3 ± 1. 6 11. 0 ± 1.2 before drug 12.6 ± 1.7 10.3 ± 1.6 9.8 ± 0.9 10.1 ± 1. 4 11. 7 ± 0.9 after drug 12.0 ± 1.0 9.8 ± 1.0 10.1 ± 1.3 10.1 ± 1. 4 11. 3 ± 1 · 2 Drug hundred 13.0 ± 1.3 10.1 ± 1.0 9.8 ± 0 · 8 10.7 ± 1. 3 11. 6 ± 2.0 After drug 12.0 ± 0.8 9.8 ± 1.4 9.8 + 0.5 10.3 ± 1. 0 11. 4 + 0.5

12.5 ± 1.8 10.7 ± 1. 2 9.9 ± 0.9 10. 8 ± 1. 0 12.5 + 1.6

12.6 ± 1.8 11.0 ± 1. 3 10.4 ± 1.7 11. 3 ± 1. 0 11.4 ± 1.6

12.6 + 1.0 10.4 ± 0. 8 11.0 ± 1.8 10. 3 ± 1. 2 12.7 ± 1.8

12.1 ± 1.7 11.6 ± 1. 7 11.3 ± 1.6 10. 8 ± 2. 0 12.2 ± 1.4

11 · 7 ± 1 · 3 12.1 ± 1. 0 12.4 ± 1.0 11. 3 ± 1. 3 12.9 ± 1.3

.. 12.2 ± 1.2 13.1 ± 1 6 12.1 ± 1.4 13. 3 ± 1 4 13.4 ± 2.0 Note: test, comparison with the day of blood ^pressure, Δ Ρ <0.05, ΔΔ Ρ prodrug <0.01 Discontinuation medicine

 Table 2-4. Effects of effective components of Paeonia lactiflora on SHR diastolic blood pressure (n = 8)

 Diastolic blood pressure reduction (kPa)

 Days of nifedi

 5mg / kg 100rag / kg 50mg / kg 25mg / kg before drug 0.6 ± 1.2-0.3 ± 1.4-1.8 ± 2.3-1.3 ± 1.6 0.3 ± 2.9 after drug 0.8 ± 1.2 1.6 ± 2.6-0.8 ± 2.7 1.3 ± 2.1 0.6 ± 2.6 Before the drug 0.9 ± 1.8 0.9 ± 1.6 + 0.1.2.5-0.9 ± 1.4 1.0 ± 2.0 + After the drug 0.5 ± 2.0 0.9 ± 2.3 1.4 ± 2.2 0.6 ± 1.7 0.5 ± 2.2 Before the drug -0.8 ± 2.1 0.0 ± 3.0 0.1 ± 2.3 0.1.2.0 0.1 + 2.5 after drug-0.3 + 2.3 1.6 ± 1.4 0.9 ± 2.0 1.6 ± 2.2 1.3 ± 1.8 before drug 0.6 ± 1.0 1.7 ± 2.2 + 0.8 ± 2.2 -0.5 ± 2.0 0.0 ± 1.8 after drug 0.1 ± 2.7 3.0 ± 1.0 * 2.3 ± 1.8 + 1.4 ± 1.3 1.0 ± 2.1 0.5 ± 1.8 before drug 1.2 ± 2.3 0.4 ± 1.7 0.9 ± 2.1 1.0 ± 1 · 8 after drug 1.0 ± 1.4 2.6 + 2.9 2.7 ± 1.7 + 1.4 · 1.7 2.0 ± 1.0 before drug 0.4 ± 2.0 1.6 ± 1.4 1.2 ± 2.6 0.5 ± 3.0 1.0 ± 1.4

11

 After the medication-1.0 ± 1.6 2.6 ± 1.4 * 2.7 ± 1.6 * 2.9 ± 2.1 * 1.0 ± 1.8 + before the medication 0.6 ± 2.3 3.4 ± 1.0 * 2.0 ± 1.3 2.1 ± 1.6 0.6 ± 1.0

13

 After medication — 0.3 ± 2.5 3.5 ± 1.8 * 2.7 ± 1.4 * 2.7 ± 2.6 + 2.1 ± 1.3 + before medication 0.5 ± 2.3 2.6 ± 1.7 + 2.0 ± 2.1 1.8 ± 0.8 0.5 ± 0.6

15

 0.8 ± 2.7 3.1 + 2.2+ 2.7 ± 1.7 2.2 ± 1.3 1 · 8 ± 1 · 6 before drug 0.5 ± 1.6 2.6 ± 2.3 + 2.7 ± 1.4 * 2.3 ± 2.0 + 1.3 ± 1.4

17

 1.2 ± 1.3 3.1 ± 1.3 * 2.3 ± 1.7 2.3 ± 2.1 1.7 ± 1.2 before drug 0.1 ± 1.4 2.7 ± 1.7 * 2.7 ± 1.4 * 1.8 ± 1.7 + 1.4 ± 2.2 before drug

19

 1.0 + 1.0 3.0 + 2.1 + 2.9 ± 1.2 * 2.2 ± 1.6 1.6 ± 1.2

0.6 ± 2.2 2.1 ± 1.7 2.6 ± 1.3 + 1.7 ± 1.7 0.5 ± 1.4 0.5 ± 2.2 1.8 ± 2.2 2.2 ± 2.1 1.2 ± 1.7 1.6 ± 2.0

0.4 ± 1.2 2.5 ± 1.0 1.6 ± 1.8 2.2 ± 1.2 * 0.1 ± 1.3

1.0 ± 2.1 1.3 ± 1.7 1.2 ± 1.8 1.7 ± 2.2 0.6 ± 1.6 1.4 + 2.0 0.8 ± 1.6 0.1 ± 1.2 1.2 + 2.0 0.0 + 1.4 0.9 ± 2.0-0.3 ± 2.1 0.4 ± 1.4-0 · 8 ± 1.3 -0.4 ± 2.0 Note: ί test, compared with the control group, + 0.05, * 0.01 6. Acute antihypertensive effect of extracts of active ingredients of Paeonia lactiflora on renal hypertension rats.

 Take a certain amount of the extract of the active ingredient of Paeonia lactiflora, and formulate them with 2.5, 5, 10 mg / ml of medicinal solution with 0.5% CMC (sodium carboxymethyl cellulose). Captopril: Hunan Zhengtai Pharmaceutical Co., Ltd., a Sino-US joint venture, is produced by Puge Company, batch number 000902, 25mg per tablet, pulverized, and formulated with 0.5% CMC (carboxymethylcellulose sodium) in a concentration of 1.25mg / ml. liquid.

 'Animals: SD rats, provided by the Experimental Animal Center of Xi'an Jiaotong University. The animal quality qualification certificate numbers are Shaanxi Dongdong No. 08-005. Animals were kept at room temperature of 26 ± 3 ° C, artificial lighting for 12 h each, divided into cages according to sex, drinking water freely, and feeding with pellets.

 Preparation of renal hypertensive rats: 70 SD rats, half male and half female, were adapted to the environment for 1 week, and the tail blood pressure was measured by indirect manometry. Anesthetize with ether, fix the abdomen, make a 2cm long incision on one side of the back and waist, expose the renal pedicle, and narrow the renal artery with a silver clip of about 0.3mm in diameter. The other kidney is removed. Suture the incision. After 3 consecutive days, penicillin was ip. After 40 days, 27 animals survived and their blood pressure was measured. Ten patients with systolic blood pressure greater than 145 mmHg were selected for experimental use.

 Animals were randomly divided into 5 groups according to systolic blood pressure, with 2 animals in each group. (1) Control group: 0.5% CMC; (2) Captopril group: 12.5mg / kg; (3), (4), (5) are the active ingredients of Amaranth 100, 50, 25mg / kg group. Designed with 2 5 X 5 Latin squares. Once intragastrically, the volume is 10ml / kg. Blood pressure was measured indirectly through the tail at 1, 1, 4, 6, and 8 hours after treatment. The washing period was 3 days, and the next experiment was carried out for a total of 5 rounds.

 1). Acute reduction of systolic blood pressure in renal hypertension rats

As can be seen from Table 3-1 to Table 3-2, there was no significant change in the control group after the systolic blood pressure prodrug. The systolic blood pressure reduction value of the active ingredients of A. pallidus in the 100, 50 mg / kg group and captopril group was significantly different from that in the control group (<0.05 or cadaver <0.01), and maintained until 4, 2, and 4 hours after drug administration, respectively. The systolic blood pressure reduction value of 25 mg / kg of active ingredient of Paeonia lactiflora at 1h after treatment was significantly different from that of the control group (P <0.05). The systolic blood pressure of each administration group decreased most obviously at 1 h after drug administration, and the reductions were 2.5 ± 2.1, 2.2 ± 1.6, 3.5 ± 2.2, and 1.6 ± 1.3 mmHg. After 2 hours, the systolic blood pressure of each administration group gradually increased, but 8 hours was still lower than the pre-dose blood pressure. Table 3-1. Acute lowering effect of active ingredients of Paeonia lactiflora on renal hypertension rats (n-10) blood pressure (kPa)

 Post-dose time

 (h) Baiji

 Control group captopril white £ 50mg / kg white peony 25mg / kg

 100mg / kg

 0 20.3 ± 1.7 19.8 ± 1.4 19.2 ± 1.0 20.5 ± 1.0 19.6 ± 1.9 1 20.4 ± 2.3 16.2 ± 1.4 16.9 + 2.6 18.3 ± 1.6 18.1 ± 1.6 2 19.9 ± 1.6 16.6 + 1.2 16.8 ± 1.2 18.1 ± 2.2 18.2 ± 2.3 4 19.5 ± 2.2 17.4 ± 1.4 17.4 ± 1.2 18.3 ± 1.2 18.1 ± 2.2 6 19.6 ± 1.4 17.8 ± 1.4 18.1 + 1.4 18.6 ± 1.3 17.7 ± 2.9 8 19.9 ± 1.2 18.3 ± 1.2 18.1 ± 1, 4 18.2 ± 1.6 18.1 ± 2.4 Table 3 -2. Acute reduction effect of active ingredients of Paeonia lactiflora on systolic blood pressure in renal hypertension rats (n = 10) Value of systolic blood pressure reduction after drug administration (kPa)

 Time Control Captopril Amaranth 100mg / kg white; ££ 50mg / kg amaranth 25mg / kg

1-0.1 ± 1.6 3.5 ± 2.2 * 2.5 ± 2.2 + 2.2 ± 1.6 * 1.6 ± 1.3 +

2 0.4 + 1.4 3.1 ± 2.2 * 2.5 ± 1.4 * 2.5 + 2.3+ 1.3 ± 2.5

4 0.8 ± 1.7 2.3 + 1.3+ 1.6 ± 1.6 2.2 ± 1.3 + 1.6 ± 1.7

6 0.6 ± 1.7 2.0 ± 16 1.1 ±± 1.4 2, 0 ± 1.6 2.0 ± 1.6

8 0.3 ± 1.6 1.4 ± 2.3 1.3 ± 1.0 2.3 ± 1.6 * 1.4 ± 1.4 Note: ί test, compared with the control group, + ρ <0.05, * ρ <0.01

 2). Acute reduction of diastolic blood pressure in renal hypertension rats

It can be seen from ΛΛ ^ 3-3 and Table 3-4 that the diastolic blood pressure of each administration group has a tendency to decrease, which is not significant compared with the control group. Compared with the pre-drug group, the diastolic blood pressure reduction values of the active ingredients of A. pallidum at 100, 50, and 25 mg / kg were significantly different at 1, 2, 4 h, 1, 4, and 1 h after treatment (<0.05 or <0.01). 1, 2, 4h values decrease significantly different drugs captopril chopsticks (<ft or P <0.0l) _o Table 3. Angelica 3- acute reducing effect of the active ingredient of the diastolic blood pressure of renal hypertensive rats ([eta] = 10) Diastolic blood pressure (kPa)

 Post-dose time.

 (h) Baiji

 Control group Captopril Amaranth 50rag / kg Amaranth 25mg / kg

 100mg / kg

0 12.7 ± 2.6 12.0 ± 1.6 11.7 ± 1.3 12.5 ± 2.0 12.3 ± 2.0 1 12.4 ± 1.6 10.0 ± 1.0 + 10.1 + 1.6 + 11.2 ± 1.3 + 11.2 ± 1.2 + 2 11.8 + 2.3 9.6 ± 1.2 + 9.9 ± 0.9+ 11.0 ± ± 1.4 11.0 ± 1.6 4 12.0 + 1.6 10.4 + 1.3+ 10.4 ± 1.2 + 11.4 + ± 1.4 + 11.7 ± 1.7 6 12.4 ± 1.7 10.7 ± 0.9 10.5 ± 0.6 11.2 ± 1.6 11.2 + 1.7 8 11.8 ± 16 10.9 ± 1.0 11.0 ± 0.6 10.9 ± 1.7 11.2 ± 0.8 Note: Test, compared with pre-drug blood pressure, + ^ 0.05, * P <Q.01 Table 3- 4. Acute reduction of diastolic blood pressure by active ingredients of Paeonia lactiflora in renal hypertensive rats (n = 10) Diastolic blood pressure reduction (kPa)

 (h) Control group Captopril Amaranth 100mg / kg Amaranth 50rag / kg Amaranth 25mg / kg

1 0.3 ± 2.3 2.0 + 2.3 1.6 ± 1.6 1.3 ± 1.7 1.1 ± 1.4

2 0.9 ± 2.3 2.4 ± 1.6 1.6 ± 1.4 1.6 + 2.5 1.2 ± 2.5

4 0.7 ± 2.3 1.6 ± 0.9 1.3 ± 1.4 1.1 ± 1.3 0.6 + 2.1

6 0.3 ± 2.5 1.3 ± 1.6 1.2 ± 1.6 1.4 ± 2.3 1.1 ± 2.2

8 0.9 ± 2.5 1.1 + 2.3 0.7 ± 1.3 1.6 ± 2.9 1.1 ± 2.0 test, compared with each group> 0.05

 Observation of the effect of the effective components of Paeonia lactiflora on SHR blood pressure showed that the effective components of Paeonia lactiflora had a significant acute reduction in blood pressure in renal hypertensive rats, and a stronger effect on reducing systolic blood pressure than diastolic blood pressure. Pharmaceutical composition example 1

 The extracted Chinese herbal medicine scutellaria coumarins can be used alone or in combination with other traditional Chinese medicine extracts or active ingredients to prepare medicines for treating hypertension and its complications.

 Preparation of chuanxiong extract

Take the Chuanxiong decoction pieces, crush them, pass through a 20-mesh sieve, weigh 2kg of powder, and place them in a 5L extraction kettle. After the refrigeration device, the extraction kettle and the separation kettle heating device work normally, turn on the compression pump to maintain the pressure at 10.0 MP, and the temperature is 50. ° C, add ethanol entrainer to about 10%, 40kg / C0 ₂ flow rate of circulation h, extraction, separation vessel pressure of 5.0 MP, temperature 30Ό, circulation extraction after 4h, fractionated extract, evaporated to dryness entrainer, to give Ligusticum chuanxiong supercritical extract is a yellow to reddish brown oily substance, with a delicate aroma and slightly bitter taste.

Character determination: (1) Identification by thin layer chromatography, according to thin layer chromatography (Chinese Pharmacopoeia 2000 edition, Appendix VIB), dissolved with ethyl acetate, on a silica gel G plate with n-hexane-ethyl acetate ( 17: 3) As a developing agent, inspect under an ultraviolet lamp at 365 nm. In the chromatogram of the test product, a fluorescent spot of the same color is displayed at a corresponding position with the reference medicinal material. (2) Take 2.5g of Chuanxiong extract, add 25ml of n-hexane, sonicate for 20min, filter out the supernatant, wash the filter paper with a small amount of n-hexane, combine the supernatants, and dry the n-hexane on 70 ° C ~ 8 (TC water bath) Alkane, weighed as the extract, this product contains not less than 40% by weight of n-hexane extract. (3) according to the volatile oil determination method ("Chinese Pharmacopoeia" The appendix XD of the 2000 edition determined that the volatile oil was extracted and read with a volatile oil extractor, and the content of the volatile oil in the test product was calculated. This product contains not less than 9ml of chuanxiong volatile oil per 100g.

 Pharmacological study: The total effective component extracts of supercritical coumarins obtained from Paeonia lactiflora and the supercritical extract from Ligusticum chuanxiong were mixed in different proportions, and noradrenaline was provided as a control, and healthy rabbits were used as experimental animals to perform the composition Experiment on inhibition of aortic contraction induced by NE.

Preparation for the test solution: distilled water as a solvent, were prepared having a concentration of 10. Omg cm- Angelica sample ¹ and 30.0 mg cm "Chuanxiong sample ^1, was placed in the spare tank...

Preparation and determination of animal specimens: Take healthy rabbits, sacrifice, open the chest to expose the heart, 'disconnect the aorta, remove the connective tissue on the surface of the blood vessel wall, cut the aorta into a spiral artery specimen with a width of 4mm and a length of 3 ~ 4cm , Placed in the bath tube of Tyrode's nutrient solution, the two ends were threaded and ligated, the lower end was fixed on the hook with oxygen, the upper end was suspended on the tension signal through the sensor, and 0 _{2 was} continuously passed to maintain the bath temperature: 37 ° C ± 0.5 ° C. The specimens were equilibrated for 1 hour, and the nutrient solution was changed every 20 minutes.

 Optimal ratio test: Rinse the specimen several times until the specimen shrinkage value returns to near the baseline. After recording a period of normal contraction value, add norepinephrine (NE) 60μ to the bath tube. After the contraction curve reaches the maximum and maintains for 2 minutes, the ratio of the effective part of the peony coumarins to the supercritical extract of Chuanxiong is 1 : 1 solution, record the shrinkage value. Repeat the operation to obtain the drug inhibition ratios of the effective part of Paeonia coumarins and the supercritical extract of Ligusticum chuanxiong respectively as 2: 1, 3: 1, 1: 2, 1: 3. The results are shown in the attached table.

Attached table. Inhibition rates obtained by different proportions of drugs (η = 4)

The results showed that the different ratios of the total effective component extracts of supercritical coumarins of Paeonia lactiflora and Chuanxiong had a significant inhibitory effect on the aortic contraction induced by NE, and the dose-effect relationship was obvious. The compatibility ratio of leguminoid extracts with respect to the supercritical extract of Ligusticum chuanxiong is 1: 3 to 3: 1, preferably 1: 2 to 2: 1, and more preferably 1: 1. Drug group ^ ^ Example 2

Ge Ming take

 Take the Kudzu root pieces, crush them, and pass through a 20 mesh sieve. Weigh the drug-bearing powder with 8 times the amount of 85% B twice. Each time for 3 hours, filter, combine the extracts, and dry the solvent. The extract is divided into two parts, a supernatant and a precipitate, through a water sedimentation process, and then passed through a macroporous adsorption resin and an ethanol gradient elution to obtain Kudzu root extracts I and II, which are combined into Kudzu azalea.

Character determination: (1) Identification by thin-layer chromatography, according to thin-layer chromatography (Chinese Pharmacopoeia 2000 edition, Appendix VIB), taking puerarin as a reference substance, dissolving in methanol, and using carboxymethylcellulose sodium as ^! 交 G

Methanol-water (7: 2.5: 0.25) was used as a developing agent, and viewed under an ultraviolet lamp at 365 nm. In the chromatogram of the test product, a fluorescent spot of the same color was displayed at a position corresponding to the control medicinal material; daidzein, methanol Dissolve it as a reference solution, and use chloroform-methanol (8.3: 1.7) as a developing agent on a silica gel G plate using carboxymethylcellulose sodium as a reagent. View under UV light at 365nm. Fluorescent spots of the same color are displayed at the positions corresponding to the reference. (2) Take Pueraria is 25mg, add methanol to make up the volume, Puerarin is the reference solution, and under the color Fusi, (color column: ODS C ₁₈ (150ramx4.6ram, 5μαι) methanol: water (42: 58 ) as the mobile phase; 1. Oml / min; detection wavelength: 2 ⁵ 0nm) under the assay, the test is calculated for each phrase FIG relative retention time for the major color Pueraria plain language peaks at relative retention time 0.128, 0.161, 0.22 The five chromatographic peaks of 0.349 and 1.00 are common peaks, which are used as index peaks for the general qualitative analysis of fingerprints on the Ge effect part.

Determination of content: (1) Determined according to ultraviolet spectrophotometry (Chinese Pharmacopoeia 2000 edition, Appendix VA). Alcohol is dissolved, puerarin is used as a reference substance, and its absorption is measured at 250nm.!, A standard curve is drawn by the absorbance (A) versus the solution concentration (C, μ g / ral). This product contains isoflavones per lg. The element shall not be less than 350m _g . (2) Measured in accordance with (Appendix VID of the 2000 edition of the Chinese Pharmacopoeia), methanol: water (42:58) is the mobile phase, the wavelength is 250nm, the volume is dissolved by methanol, and puerarin and daidzein are used as reference substances. It is determined that the content of puerarin per lg of this product shall not be less than 15mg, and that: 苷 aglycone shall not be less than 15mg.

 Pharmacological study Jie Siming, the compatibility of the total active ingredient extracts of Paeonia lactiflora and Pueraria lobata extract also had significant inhibitory effects on aortic contraction in NE i hair. The compatibility ratio is 1: 3 to 3: 1, preferably 1: 2 to 2: 1, and more preferably 1: 1.

Claims

Rights request

1. Coumarin extracts, which contain at least imperatorin and isoimperatorin, and the total coumarin content per gram of the extract is not less than 300 mg in terms of imperatorin.

 2. The coumarin extract according to claim 1, wherein each gram of the extract contains no less than 80 g of imperatorin.

 3. The coumarin extract according to claim 1 or 2, which is derived from an active ingredient of coumarins in a drug substance of Paeonia lactiflora.

 4. The coumarin extract according to any one of claims 1 to 3, which has a fingerprint as shown in FIG.

 5. The coumarin extract according to any one of claims 1-4, which is derived from a supercritical dioxide secondary extract of a paeony drug.

 6. The method for preparing a coumarin extract according to any one of claims 1 to 5, characterized in that supercritical carbon dioxide fluid is used for extraction.

7. The preparation method according to claim 6, characterized in that the supercritical carbon dioxide fluid is used as the carrier in the extraction process with C _2- (: ₃ lower alcohol).

 8. The preparation method according to claim 7, wherein the concentration of the carrier is 1% to 15% of the drug substance to be extracted.

 9. The preparation method according to claim 7 or 8, wherein the carrier is ethanol or isopropanol.

 10. The preparation method according to any one of claims 6-9, wherein the extraction pressure during supercritical extraction is 20-35Mpa, and the extraction temperature is 30-50. C.

 11. The coumarin extract according to any one of claims 1 to 5 is used for treating hypertension.

.

12. The coumarin extract according to any one of claims 1 to 5 for use in the treatment of primary and secondary hypertension.

13. The coumarin extract according to any one of claims 1 to 5 for use in the preparation and treatment of hypertension Drugs for the disease.

 14. The coumarin extract according to any one of claims 1-5, for preparing a medicament for treating primary and secondary hypertension.

 15. A pharmaceutical composition for treating hypertension, comprising a therapeutically effective amount of a coumarin extract according to any one of claims 1-5 and a pharmaceutical adjuvant acceptable in pharmacy.

 16. The pharmaceutical composition according to claim 15, further comprising a supercritical carbon dioxide extract of Ligusticum chuanxiong, and a weight ratio of the coumarin extract to 1: 3 to 3: 1.

 17. The pharmaceutical composition according to claim 15 or 16, further comprising a fat-soluble active ingredient of pueraria root in a weight ratio of 1: 3 to 3: 1 with respect to the coumarin extract.