CN101683387B

CN101683387B - Medication and preparation method and application thereof

Info

Publication number: CN101683387B
Application number: CN2008102234408A
Authority: CN
Inventors: 罗国安; 李连达; 王义明
Original assignee: GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
Current assignee: GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
Priority date: 2008-09-27
Filing date: 2008-09-27
Publication date: 2012-07-11
Anticipated expiration: 2028-09-27
Also published as: CN101683387A

Abstract

The present invention discloses a medication and a preparation method and application thereof Active components of the medication are ginseng extract and salvia miltiorrhiza extract; a weight ratio between the ginseng extract and the salvia miltiorrhiza extract is 1:(0.1-20); a weight percentage content of total ginsenoside in the ginseng extract is larger than 50%; and a weight percentage content of salvianolic acid in the salvia miltiorrhiza extract is larger than 50%. As shown in the result of Wistar rat myocardial infarction experiment and myocardial ischemia experiment, the reconstitution of total ginsenoside and salvianolic acid has an optimal therapeutic effect, can effectively control Wistar rat myocardial infarction area and improve myocardial ischemia situation, furthermore, has a same therapeutic effect with an original medicinal material reconstitution prescription. Pharmaceutical ingredient of the medication of the invention is clear and the quality of the medication can be controlled easily.

Description

A kind of medicine and preparation method thereof and application

Technical field

The present invention relates to a kind of medicine and preparation method thereof and application, particularly relating to a kind of is the medicine and preparation method thereof and application of raw material with Radix Ginseng and Radix Salviae Miltiorrhizae.

Background technology

Cardiovascular and cerebrovascular disease is one type of disease of serious harm human health, and along with the increase of social pressure and the aggravation of China's aged tendency of population, cardiovascular disease is high at the sickness rate of China.Therefore, the medicine of exploitation treatment cardiovascular disease has great social significance.

The Chinese crude drug Radix Ginseng is the root of araliaceae ginseng plant's Radix Ginseng (Panax.ginsengC.A.Mey).The root sweet in the mouth of Radix Ginseng, little hardship, warm in nature have regulating QI nourish blood, the calm the nerves effect of Fructus Alpiniae Oxyphyllae, the cough-relieving of promoting the production of body fluid, nourishing and fit keeping function.Mainly contain compositions such as saponin, volatile oil (Panaxynol, Panacene, sterol, fatty acid), sugar, aminoacid, peptide and vitamins in the Radix Ginseng; Up to the present; From Radix Ginseng Rubra, Radix Ginseng and Radix Ginseng, be divided into from obtaining 40 kinds of ginsenosides, promptly ginsenoside R0, Ra1, Ra2, Ra3, Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, 20-glc-Rf, Rg1, Rg2,20 (R)-ginsenoside-Rg2, Rg3,20 (S)-ginsenoside-Rg3, Rg5, Rh1,20 (R)-ginsenoside-Rh1, Rh2,20 (S)-ginsenoside-Rh2, Rh4, Ri, Rs1, Rs2, malonyl ginsenoside Rb1, Rb2, Rc, Rd, Radix Notoginseng ginsenoside R1, Radix Panacis Quinquefolii saponin R1,20 (R)-ginsenoside-La, F4,25-hydroxy-ginsenoside-Rg2,25-hydroxy-ginsenoside-Rg2, Ia, Ib, koryoginsenoside-Rg1 and-Rg2.The stem of Radix Ginseng, leaf and flower also contain ginsenoside's constituents, also can replace Radix Ginseng to be used as medicine.In addition, the processing of root process, the process of preparing Chinese medicine with Radix Ginseng are prepared into Radix Ginseng, Radix Ginseng Rubra, also can be used as medicine.

The Chinese crude drug Radix Salviae Miltiorrhizae is the exsiccant root and rhizome of labiate Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bunge.).Radix Salviae Miltiorrhizae has pharmacological action widely aspect cardiovascular disease; Wherein salvianolic acid is one type of organic acid chemical compound that contains phenolic hydroxyl group; It is ingredient important in the red sage root water soluble ingredient; Can alleviate hypoxic-ischemic blood vessel endothelium injury caused, promote the blood vessel endothelium hypertrophy, have the myocardial cell injury, atherosclerosis, anticoagulant and the antithrombotic effect that improve due to the hypoxic-ischemic.The effect that DA Sal-A, B, C, D, E, F, G, H, I and different salvianolic acid C etc. have very strong anti peroxidation of lipid and remove free radical; Two the composition DA Sal-As (SalA) that wherein content is the highest and the activity of salvianolic acid B (SalB) are the strongest, and the cell membrane damage that lipid peroxidation is caused has effects such as significant protection and atherosclerosis.When the oxygen-derived free radicals that produces in the organism metabolism process can not be eliminated, can produce harmful damage to body, wherein myocardial cell is injured particularly remarkable.Oxygen-derived free radicals has the active effect of obvious suppression potassium-channel, and SalA then has the effect that promotes that potassium-channel is open, and can reverse the activity of the potassium-channel that is suppressed by oxygen-derived free radicals, thereby plays a protective role.

At present, the method that the reuse n-butanol extraction is handled behind the general many employing ethanol extractions of the method for distilling of saponin in the Radix Ginseng, the shortcoming of this method is that extracting operation is loaded down with trivial details, is easy to generate emulsion, makes troubles to operation.In addition, the boiling point of n-butyl alcohol higher (117.7 ℃) is difficult to volatilization and removes, thereby it is easy to residual in extract.The method for distilling that adopts water extract-alcohol precipitation is also arranged, but technology is loaded down with trivial details, energy consumption is big.More the water-soluble phenolic acids component of Radix Salviae Miltiorrhizae adopts water extract-alcohol precipitation, water to carry the back and crosses that resin is crossed macroporous resin (Chinese patent CN1384090A) or polyamide column (Chinese patent CN1242364A) behind precipitate with ethanol (Chinese patent CN1129572A), the water extract-alcohol precipitation again, water is carried the back successively with polyamide column and macroporous resin chromatography (Chinese patent CN1459448A), reduces pressure then or methods such as lyophilization obtain.But there is following shortcoming in the method for above extraction Radix Salviae Miltiorrhizae total phenolic acids: 1, in the prepared Radix Salviae Miltiorrhizae extract, the content of Radix Salviae Miltiorrhizae total phenolic acids is not high, and productive rate is lower, generally 2～4%; 2, adopt the high concentration ethanol precipitate with ethanol, the industrialization production security is reduced greatly, the ethanol consumption is big, and production cost is high; 3, high concentration ethanol precipitate with ethanol step influences the productive rate of Radix Salviae Miltiorrhizae total phenolic acids in the Radix Salviae Miltiorrhizae extract; 4, need a large amount of condensed water, be not easy to industrialized great production, operation is comparatively complicated; 5, generally choose danshensu and protocatechualdehyde index components, but the protocatechualdehyde toxic side effect represents the activity of Radix Salviae Miltiorrhizae very unreasonable with danshensu as control red sage formulation quality.

Summary of the invention

The purpose of this invention is to provide a kind of medicine and preparation method thereof and application, this curable substance and/or prevention angiopathy, the differentiation of promotion mesenchymal stem cells MSCs become cardiac-like muscle cell with the inducing bone mesenchymal differentiation of stem cells.

Said angiopathy can be cardiovascular disease, cerebrovascular disease or peripheral vascular disease; Said cardiovascular disease can be coronary heart disease, angina pectoris, myocardial ischemia or anoxia or myocardial infarction, and said cerebrovascular disease can be ischemic cerebrovascular or vascular dementia, and said peripheral vascular disease can be rhomboembolia type cerebral vasculitis or phlebothrombosis.

Medicine provided by the present invention, the raw material that its active component is made up of Radix Ginseng and Radix Salviae Miltiorrhizae is processed; Said Radix Ginseng be root, stem, the leaf of plant Radix Ginseng and spend at least a; Said Radix Salviae Miltiorrhizae is the root of plant Radix Salviae Miltiorrhizae; The ratio of weight and number of said Radix Ginseng and Radix Salviae Miltiorrhizae is 1: (0.08-20).Wherein, Radix Ginseng and Radix Salviae Miltiorrhizae can also can be the bright medical material of Radix Ginseng and Radix Salviae Miltiorrhizae for concocting back Radix Ginseng and Radix Salviae Miltiorrhizae.

The active component of this medicine specifically can be Radix Ginseng extract and Radix Salviae Miltiorrhizae extract, and the ratio of weight and number of said Radix Ginseng extract and Radix Salviae Miltiorrhizae extract is 1: (0.1-20); In the said Radix Ginseng extract, the quality percentage composition of Radix Ginseng total saponins is greater than 50%, is preferably more than to equal 80%, as 80-83%; Wherein, Radix Ginseng total saponins comprises ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rf; The quality percentage composition of said ginsenoside Rg1 in Radix Ginseng total saponins is more than or equal to 2%; The quality percentage composition of ginsenoside Re in Radix Ginseng total saponins is more than or equal to 2.5%, and the quality percentage composition of ginsenoside Rb1 in Radix Ginseng total saponins is more than or equal to 2%; In the said Radix Salviae Miltiorrhizae extract, the quality percentage composition of salvianolic acid is greater than 50%, is preferably more than to equal 80%, as 89-95%.

Medicine of the present invention when needed, can add pharmaceutically acceptable carrier, processes tablet, granule, capsule, drop pill, oral cavity disintegration tablet, injection, infusion solution, slow-release tablet agent, slow controlled release micro pill, aerosol or inhalant; Said carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier or the lubricant etc. that pharmaceutical field is conventional, can also add flavouring agent or sweeting agent etc.

Radix Ginseng extract in the medicine of the present invention and Radix Salviae Miltiorrhizae extract can prepare according to following method:

1) with ethanol water or water Radix Ginseng is carried out reflux, extract,, the extracting solution that obtains is centrifugal, get supernatant, obtain ginsenoside's extracting solution; With ethanol water or water Radix Salviae Miltiorrhizae is carried out reflux, extract,, the extracting solution that obtains is centrifugal, get supernatant, obtain the salvianolic acid extracting solution;

2) adopting macroporous adsorptive resins to above-mentioned steps 1) ginsenoside's extracting solution of obtaining adsorbs, first water or ethanol water eluting water solubility impurity, and then with ethanol aqueous wash deresination post, the collection eluent obtains the Radix Ginseng total saponins eluent; Adopting macroporous adsorptive resins to above-mentioned steps 1) the salvianolic acid extracting solution that obtains adsorbs, first water eluting water solubility impurity, and then with ethanol aqueous wash deresination post, the collection eluent obtains the salvianolic acid eluent;

3) to above-mentioned steps 2) the Radix Ginseng total saponins eluent that obtains carries out drying, obtains Radix Ginseng extract; To above-mentioned steps 2) the salvianolic acid eluent that obtains carries out drying, obtains Radix Salviae Miltiorrhizae extract.

When preparing ginsenoside's extracting solution above-mentioned steps 1), in the said ethanol water, alcoholic acid volumn concentration is 50-95%, is preferably 90%; The ratio of weight and number of said Radix Ginseng and ethanol water is 1: (6-15), be preferably 1:10; Said return time is 30min-120min, is preferably 60min; When preparing the salvianolic acid extracting solution in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 10-60%, is preferably 50%; The ratio of weight and number of said Radix Salviae Miltiorrhizae and ethanol water is 1: (5-15), be preferably 1:10; Said return time is 30min-120min, is preferably 30min;

When preparing ginsenoside's eluent said step 2), during the eluting water solubility impurity, alcoholic acid volumn concentration is less than 10% in the said ethanol water; During the eluting resin column, alcoholic acid volumn concentration is 50-90% in the said ethanol water, is preferably 60-70%; Resin extender in the said macroporous adsorptive resins is a following A) or B):

A) any one in following three kinds of resins: HPD100 type resin, HPD700 type resin and S-8 type resin;

B) by in these four kinds of resins of HPD100 type, HPD700 type, S-8 type and AB-8 type any one with SephadexLH-20 gel, C18 bonding mutually with the C8 bonding at least a composite resin of forming in these three kinds mutually;

When preparing the salvianolic acid eluent said step 2), during the eluting resin column, alcoholic acid volumn concentration is 30-90% in the said ethanol water, is preferably 40-60%; Resin extender in the said macroporous adsorptive resins is a following A) or B):

In the said step 3), it is 50-60 ℃ that Radix Ginseng total saponins eluent and salvianolic acid eluent are carried out exsiccant temperature.

Radix Ginseng extract in the medicine of the present invention and Radix Salviae Miltiorrhizae extract can also prepare according to following method:

1) with Radix Ginseng and Radix Salviae Miltiorrhizae according to 1: ratio of weight and number (0.08-20) mixes; With ethanol water or water the mixture of Radix Ginseng and Radix Salviae Miltiorrhizae is carried out reflux, extract; The extracting solution that obtains is centrifugal, get supernatant, obtain containing the mixed extract of ginsenoside and salvianolic acid;

Said Radix Ginseng be root, stem, the leaf of plant Radix Ginseng and spend at least a;

Said Radix Salviae Miltiorrhizae is the root of plant Radix Salviae Miltiorrhizae;

2) adopting macroporous adsorptive resins to above-mentioned steps 1) mixed extract that obtains adsorbs; Elder generation's water eluting water solubility impurity; And then, collect eluent with ethanol aqueous wash deresination post, obtain containing the mixing eluent of Radix Ginseng total saponins and salvianolic acid;

3) to above-mentioned steps 2) the mixing eluent that obtains carries out drying, obtains the mixed extract of Radix Ginseng extract and Radix Salviae Miltiorrhizae extract.

When preparing mixed extract in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 30-90%%, is preferably 60%; The ratio of weight and number of said Radix Ginseng and Radix Salviae Miltiorrhizae mixture and ethanol water is 1: (6-15), be preferably 1:10; Said return time is 30-120min, is preferably 45min;

When preparation mixed eluent said step 2), during the eluting resin column, alcoholic acid volumn concentration was 30-90% in the said ethanol water, is preferably 50-70%; Resin extender in the said macroporous adsorptive resins is a following A) or B):

In the said step 3), it is 50-60 ℃ that the mixing eluent is carried out exsiccant temperature.

Another object of the present invention provides manufacturing method for above mentioned medicine.

Medicine provided by the present invention can adopt any one method in following two kinds of methods to prepare.

Wherein a kind of method for preparing may further comprise the steps:

3) to above-mentioned steps 2) the Radix Ginseng total saponins eluent that obtains carries out drying, obtains the Radix Ginseng extract powder; To above-mentioned steps 2) the salvianolic acid eluent that obtains carries out drying, obtains the Radix Salviae Miltiorrhizae extract powder;

4) with above-mentioned steps 3) the Radix Ginseng extract powder and the Radix Salviae Miltiorrhizae extract powder mixes that obtain, obtain medicine of the present invention.

When preparing ginsenoside's extracting solution in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 50-95%, is preferably 90%; The ratio of weight and number of said Radix Ginseng and ethanol water is 1: (6-15), be preferably 1:10; Said return time is 30min-120min, is preferably 60min; When preparing the salvianolic acid extracting solution in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 10-60%, is preferably 50%; The ratio of weight and number of said Radix Salviae Miltiorrhizae and ethanol water is 1: (5-15), be preferably 1:10; Said return time is 30min-120min, is preferably 30min;

Other a kind of method for preparing of medicine provided by the present invention may further comprise the steps:

3) to above-mentioned steps 2) the mixing eluent that obtains concentrates, drying, obtains the mixed extract powder of Radix Ginseng and Radix Salviae Miltiorrhizae, obtains medicine of the present invention.

When preparing mixed extract in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 30-90%, is preferably 60%; The ratio of weight and number of said Radix Ginseng and Radix Salviae Miltiorrhizae mixture and ethanol water is 1: (6-15), be preferably 1:10; Said return time is 30-120min, is preferably 45min;

Medicine of the present invention and preparation method thereof has the following advantages:

1, medicine effect material base of the present invention is clear and definite, comprises Radix Ginseng total saponins and Radix Salviae Miltiorrhizae total phenolic acids that proportion compatibility is clear and definite; Through experiment of rat acute myocardial infarction and myocardial ischemia experiment; The result shows, adopts the compatible drug action with the best of Radix Ginseng total saponins and Radix Salviae Miltiorrhizae total phenolic acids, can effectively suppress the rat myocardium block area; Improve the myocardial ischemia situation; And kept the curative effect identical with crude drug compatibility side, and drug component of the present invention is clear, and quality is controlled easily.

2, in the step of extracting Radix Salviae Miltiorrhizae total phenolic acids, take the method for direct alcohol extraction, overcome the defective that needs a large amount of condensed water in the prior art; Simplified production stage; Optimized working condition, free from environmental pollution, have the advantage that step is simple, easy to operate, be easy to industrialization;

3, the inventive method has been avoided the problem that causes the salvianolic acid active component to lose because of precipitate with ethanol in the prior art, has the advantage that active component loses less, finished product yield is high;

4, the inventive method has avoided in the prior art percolation to extract the long shortcoming that expends time in, and has the high advantage of extraction efficiency;

5, adopt the Radix Salviae Miltiorrhizae total phenolic acids content in the Radix Salviae Miltiorrhizae extract that the inventive method obtains high, improved the utilization rate of red rooted salvia greatly;

When the medicine that 6, adopts the inventive method preparation to treat cardiovascular disease carried out industrialization production, other method that adopts at present can make production cost reduce greatly;

7, the inventive method process stabilizing, mass discrepancy is little between prepared each batch of medicine that goes out, and can pass through chromatographic fingerprinting simultaneously, and the component of Radix Ginseng total saponins in the medicine and Radix Salviae Miltiorrhizae total phenolic acids is carried out assay, can effectively control the quality of medicine of the present invention.

Description of drawings

Fig. 1 for medicinal granule of the present invention to the Electrocardiographic influence curve figure of acute myocardial ischemia rat due to the isoproterenol

Fig. 2 intervenes the idiographic flow of Chinese Small-sized Medulla Sus domestica mescenchymal stem cell vitro differentiation process experiment for medicinal granule of the present invention

When Fig. 3 induces 30d for pastille serum, intervene the situation that the MSCs cellular morphology changes

When Fig. 4 induced 30d for pastille serum, each organized the 2-DE electrophoresis detection result of cell

When Fig. 5 induces 30d for pastille serum, the situation of change of Partial Protein

Fig. 6 is the expression of albumen Lamin A/C in the MSCs atomization

Fig. 7 intervenes proteic 2-DE collection of illustrative plates in the MSCs directed differentiation process for pastille serum

Fig. 8 intervenes proteic difference variation tendency in the MSCs directed differentiation process for pastille serum

The specific embodiment

Below in conjunction with specific embodiment and accompanying drawing, the present invention is further specified.

The preparation of the medicine of embodiment 1, treatment cardiovascular disease

The medicine of the treatment cardiovascular disease of present embodiment is made up of Radix Ginseng extract and Radix Salviae Miltiorrhizae extract, and wherein, the method for preparing of Radix Ginseng extract and Radix Salviae Miltiorrhizae extract is following:

One, the preparation of Radix Ginseng extract

Obtain 10 purpose Radix Ginseng coarse powder after getting the root crushing screening of Radix Ginseng (Panax.ginsengC.A.Mey).Accurately take by weighing Radix Ginseng coarse powder 10kg, add the 80L volumn concentration and be 90% ethanol and carry out reflux, extract,, extraction time is 45min; Collect extracting solution, and then in residue, to add the 80L volumn concentration be that 90% ethanol carries out reflux, extract,, extraction time is 45min; Repeat the said extracted process more once.Three times extracting solution are mixed, be cooled to room temperature, filter, get filtrate decompression and be concentrated into nothing alcohol flavor, obtain 2.3kg extract extractum; Get 2.3kg extract extractum add 14L water when being diluted to 20 ℃ the density of solution be 1.06g/ml, obtain 15L ginsenoside extracting solution.

Adopt the good low pole macroporous adsorptive resins of pretreatment (in resin column, to fill with polystyrene or to gather divinylbenzene be that the model of skeleton is the resin extender of HPD100; This low pole macroporous adsorptive resins and HPD100 resin extender are available from Cangzhou, Hebei precious grace chemical industry company limited); 15L ginsenoside extracting solution to above-mentioned acquisition carries out separation and purification, and bed volume is 21L behind the filler.Ginsenoside's extracting solution washes resin column to the water of resin column bed volume with the flow velocity of 2bv/h (1bv/h is promptly per hour by the flow of 1 times of bed volume) with 3 times after chromatographic column absorption, remove water-solubility impurities such as polysaccharide, discards eluent; 4 times of volumn concentrations to the resin column bed volume of reuse are 70% the ethanol flow velocity eluting resin column with 1bv/h, collect eluent, obtain the Radix Ginseng total saponins eluent; With Radix Ginseng total saponins eluent concentrating under reduced pressure is thick clear paste, and thick clear paste again through 60 ℃ of constant temperature constant pressure and dries, is obtained 500g Radix Ginseng extract powder.

Adopt the HPLC method to detect the content of Radix Ginseng total saponins in the Radix Ginseng extract powder, concrete chromatographic condition is: with the octadecylsilane chemically bonded silica is filler, is mobile phase A with the acetonitrile, is Mobile phase B with water, gradient elution.Flow velocity: 1ml/min, the detection wavelength is 203nm.Theoretical cam curve is calculated by the ginsenoside Rg1 peak, is not less than 6000.Concrete condition of gradient elution is following:

The result shows; In the above-mentioned Radix Ginseng extract powder that obtains; The quality percentage composition of Radix Ginseng total saponins is 80%; The quality percentage composition of ginsenoside Rg1 in Radix Ginseng total saponins is 3.4%, and the quality percentage composition of ginsenoside Re in Radix Ginseng total saponins is 3.7%, and the quality percentage composition of ginsenoside Rb1 in Radix Ginseng total saponins is 4.1%; The quality percentage composition of ginsenoside Rf in Radix Ginseng total saponins is 0.12%.

Two, the preparation of Radix Salviae Miltiorrhizae extract

Get the root 10kg of Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bunge.), be cut into decoction pieces, carry out reflux, extract, after adding the 100L volumn concentration and be 50% soak with ethanol 4h, extraction time is 30min; Collect extracting solution, and then in residue, to add the 100L volumn concentration be that 50% ethanol carries out reflux, extract,, extraction time is 30min.Twice extracting solution is mixed, obtain the 150L Radix Salviae Miltiorrhizae extract, again this 150L Radix Salviae Miltiorrhizae extract is carried out sucking filtration, is concentrated into 50L.

Adopt macroporous adsorptive resins (the styrene type resin that with the model is HPD700 is a filler, available from Cangzhou, Hebei precious grace chemical industry company limited), the 50L Radix Salviae Miltiorrhizae extract of above-mentioned acquisition is carried out separation and purification, bed volume is 65L behind the filler.Radix Salviae Miltiorrhizae extract is after chromatographic column absorption; With 3 times to the water of resin column bed volume flow velocity flushing resin column with 2bv/h (1bv/h is promptly per hour by the flow of 1 times of bed volume); Discard eluent; And then to use 3 times of volumn concentrations to the resin column bed volume be 60% the ethanol flow velocity eluting resin column with 1bv/h, collects eluent, obtains the salvianolic acid eluent; The salvianolic acid eluent is carried out concentrating under reduced pressure, and obtaining 775g is that proportion is the thick clear paste of 1.2 (60 ℃); Thick clear paste again through 60 ℃ of constant temperature constant pressure and dries, is obtained 620g Radix Salviae Miltiorrhizae extract powder.

Adopt the HPLC method to detect the content of Radix Salviae Miltiorrhizae total phenolic acids in the Radix Salviae Miltiorrhizae extract powder; Concrete chromatographic condition is: be filler with the octadecylsilane chemically bonded silica, with by the blended solution of following volume ratio as mobile phase: methanol: acetonitrile: formic acid: water=30:10:1:59; Detect wavelength 286nm.Theoretical cam curve is pressed the salvianolic acid B peak and is calculated, and is not less than 2000.

Accurately take by weighing the Radix Salviae Miltiorrhizae extract powder of the above-mentioned acquisition of 2.5g, add 40ml water, use supersound process 0.5h behind the immersion 1h, get supernatant; In residual residue, add 40ml water again, supersound process 0.5h gets supernatant; Repetitive process once again.The supernatant mixing and the water of three collections are settled to 250ml, as supplying test agent.To supply 5 times of test agent dilutions, get the measuring bottle that 2ml places 50ml, add dehydrated alcohol to 10ml; Add 4ml quality percentage composition again and be 0.3% dodecyl sodium sulfate and 2ml (wherein according to the blended mixed liquor of forming by liquor ferri trichloridi and potassium ferricyanide solution of the volume ratio of 1:0.9; The quality percentage composition of liquor ferri trichloridi is 0.6%, and the quality percentage composition of potassium ferricyanide solution is 0.9%), mixed solution is shaken up; 0.1molL is used after placing 5min in the dark place ^-1The hydrochloric acid solution standardize solution shakes up, and after 20min was placed in the dark place, spectrophotometer 720nm detected at the place.The salvianolic acid B standard solution of getting the 1mg/mL for preparing dilutes 2,5,10,20,50 and 100 times respectively, detects the record result with said method.With concentration mgmL ^-1Being abscissa, is vertical coordinate with absorbance A bs value, obtains regression equation through the excel software processes.Go out the content and the productive rate of total phenolic acid according to the regression equation calculation of salvianolic acid B standard solution.

The result shows that in the above-mentioned Radix Salviae Miltiorrhizae extract powder that obtains, the quality percentage composition of Radix Salviae Miltiorrhizae total phenolic acids is 89.8%, and the productive rate of Radix Salviae Miltiorrhizae total phenolic acids is 6.6%.

Three, the preparation of the medicine of treatment cardiovascular disease

With the Radix Ginseng extract powder of 210g above-mentioned steps one preparation and the Radix Salviae Miltiorrhizae extract powder mixes of 90g above-mentioned steps two preparations, obtain medicine of the present invention.

The preparation of the medicine of embodiment 2, treatment cardiovascular disease

The method for preparing of Radix Ginseng extract and Radix Salviae Miltiorrhizae extract is with embodiment 1.With 200g according to the Radix Ginseng extract powder of the method for the foregoing description 1 step 1 preparation and 20g according to the Radix Salviae Miltiorrhizae extract powder mixes that the method for the foregoing description 1 step 2 prepares, obtain medicine of the present invention.

The preparation of the medicine of embodiment 3, treatment cardiovascular disease

The method for preparing of Radix Ginseng extract and Radix Salviae Miltiorrhizae extract is with embodiment 1.With 20g according to the Radix Ginseng extract powder of the method for the foregoing description 1 step 1 preparation and 400g according to the Radix Salviae Miltiorrhizae extract powder mixes that the method for the foregoing description 1 step 2 prepares, obtain medicine of the present invention.

The effect experiment of embodiment 4, treatment cardiovascular disease medicine

1, Acute Myocardial Ischemia in Rats and myocardial infarction effect experiment

Get the medicine of the foregoing

description

1,2 and 3 preparations respectively, the medicinal liquid that uses normal saline to join as 1g/ml carries out effect experiment.

Get 75 of SD rats (available from this laboratory animal company of Beijing agate), be divided into 5 groups at random, be respectively normal group, model group, positive controls, high dose medicament group and low-dose drugs group, 15 every group.Wherein model group, positive controls, high dose medicament group and the equal abdominal cavity of low-dose drugs group rat multi-point injection isoproterenol (ISO), every day 1 time, each 10mgkg ^-1Body weight is injected 2d continuously, makes up the acute myocardial ischemia rat model, and normal group gives the normal saline of same dose.After the modeling success, high dose medicament group rat is according to 320mgkg ^-1The dosage of body weight is irritated the drug extract that stomach gives the treatment cardiovascular disease of the foregoing description 1,2 and 3 preparations respectively; Low-dose drugs group rat is according to 64mgkg ^-1The dosage of body weight is irritated the drug extract that stomach gives the treatment cardiovascular disease of the foregoing description 1,2 and 3 preparations respectively; The positive controls rat is according to 200mgkg ^-1The dosage of body weight is irritated stomach and is given FUFANG DANSHEN DIWAN (available from sky, Tianjin Shi Li company); Model group rat and normal rats are according to 7.5mlkg ^-1The dosage of body weight is irritated stomach and is given the normal saline with volume.Below respectively organize and be administered once common administration 7 days every day.

Behind last 1 administration 1h, with urethane according to 1.3gkg ^-1The dosage of body weight is respectively organized rat and is carried out intraperitoneal injection of anesthesia above-mentioned.Each group rat dorsal position is fixed, connect MPA2000 multiple tracks bio signal analytical system (II leads), the normal ECG in the rat 5min respectively organized in record, and the unusual rat of generation is discarded need not.After waiting to stablize, given the normal saline except that normal rats, all the other respectively organize the equal abdominal cavity of rat multi-point injection isoproterenol (ISO), and injection measurement is 10mgkg ^-1Body weight.Behind the record injection ISO 0.5,1,2,5,10,15 and respectively organize the electrocardiogram of rat during 20min, observe the variation of J point (terminal point of QRS wave group and T ripple junction) respectively.With the mV number of its reduction as index, electrocardiogram J point value (mV) before each time point electrocardiogram J point value (mV)-lumbar injection ISO behind Δ J (electrocardiogram J point changing value)=lumbar injection ISO.

Possess one of following condition person and be judged as rat heart muscle ischemia positive criteria:

1) J point skew >=0.1mV downward or upward;

2) the T wave height is alarmmed, and surpasses with leading 1/2 of R ripple;

3) the T wave height is alarmmed the displacement of companion J point.

Line with QRS complex wave starting point is made isoelectric line, and the degrees of offset of ordering with J is a diagnosis basis, and selects the index of the millivolt average of heart beating calculating J point change in 2 seconds as degree of myocardial ischemia.

Electrocardiographic to influence the result as shown in Figure 1 to the acute myocardial ischemia rat due to the isoproterenol (ISO) for the drug extract of the treatment cardiovascular disease of embodiment 1.As can be seen from the figure, after each organized rats by intraperitoneal injection ISO, significant change all took place in electrocardiogram J point, after the of short duration rising, along with the prolongation of time, descended gradually; The inductive rat electrocardiogram J point of antagonism ISO raises and does in order to the administration group optimumly, and each time point △ J of administration group rat compares with the model group rat, difference remarkable (P < 0.05).

Surveyed after the electrocardiogram, give embodiment 1 medicine respectively organize SD rat 1h behind ip ISO, open the abdominal cavity and get blood from postcava, the wherein content of LDH, CK, SOD and MDA is measured in the freezing preservation of separation of serum.The result is as shown in table 1.

LDH, CK, SOD and MDA value in the table 1 acute myocardial ischemia rat blood serum (x ± s, n=9)

Compare with model group, * P 0.05, * * P 0.01, * * * P < 0.001.

The result shows, with compared with normal, model group rat LDH and CK value significantly raise (P < 0.01), significantly reduction of SOD value (P < 0.05), MDA value extremely significantly raise (P < 0.001).Compare with model group, all significantly reductions of the LDH of administration group, CK and MDA value (P 0.05).For LDH, high dose group and low dose group all are better than positive controls, and for CK, the high dose group effect is best, and low dose group and positive controls are close, and for SOD and MDA, three's effect is close.

After getting the blood agglomeration bundle, the heart of SD rat is respectively organized in taking-up rapidly, after cleaning up with normal saline; Reject non-cardiac muscular tissue, blot, weigh with filter paper; In refrigerator freezing 30 minutes, after the taking-up ventricle is cut into the thick myocardium sheet of 0.1cm, place nitro blue tetrazolium (NBT) solution; Temperature is incubated dyeing 15min in 37 ℃ of water-baths; The non-orchid of cardiac muscle is dyed district's (infarct) downcut, claim weight in wet base, the ratio that the non-orchid of calculating myocardium is dyed district's weight and total cardiac weight is myocardial infarction area (myocardial infarction area=(infarct heart heavy/total cardiac weight) * 100%).The result is as shown in table 2.

Table 2 acute myocardial ischemia rat heart infarct size (x ± s, n=9)

Compare with model group, < 0.05***P < 0.001 for * P.

The result shows, with compared with normal, and the cardiac infarction area of model group rat significantly raise (P < 0.001); Compare with model group, high dose medicament group rat myocardium block area obviously reduces, and has significant difference (P < 0.05).

2, the drug extract of treatment cardiovascular disease promotes Chinese Small-sized Medulla Sus domestica mescenchymal stem cell (MSCs) vitro differentiation process experiment

Concrete experiment flow is as shown in Figure 2.

Respectively with the drug solution of the foregoing

description

1,2 and 3 with 10mlkg ^-1The dosage of body weight carries out gastric infusion to the SD rat, irritates stomach every day 2 times, and successive administration 3d behind the last administration 1h, gets the serum of rat, adds in the culture medium, processes 4% pastille serum.

The mesenchymal stem cells MSCs of Chinese Small-sized pig (MSCs) (available from animal housing of BJ Union Hospital) is incubated in the DMEM/F12 culture medium behind extraction, purification, makes cell density reach 100/100 μ L.Chinese Small-sized pig MSCs is divided into three groups of A, B, C, and the A group is MSCs propagation group; The B group is induced group for 5-aza, promptly in incubation, adds U-18496 (5-aza) (available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), and making its final concentration in culture medium is 10 μ mol/L; The C group promptly adds 5-aza for the pastille serum group in incubation, making its final concentration in culture medium is 10 μ mol/L, adds 4% pastille serum of above-mentioned preparation simultaneously.

Give embodiment 1 medicine respectively organize cell culture 30d the time cellular morphology as shown in Figure 3.Wherein, Fig. 3 A, 3B and 3C represent above-mentioned A, B, three experimental grouies of C respectively.The result shows; The pastille serum that in culture medium, adds above-mentioned preparation; Cellular morphology is changed; In conjunction with single situation that cellular morphology changes when inducing the MSCs directed differentiation with 5-aza, show tentatively that pastille serum is induced at 5-aza to play the effect that suppresses cell proliferation, promotes cell differentiation in the MSCs directed differentiation process.Induce at 5-aza with the pastille serum of the medication preparation of

embodiment

2 or 3 and also can play the effect that suppresses cell proliferation, promotes cell differentiation in the MSCs directed differentiation process.

When medicine above-mentioned that gives embodiment 1 respectively organized cell culture 30d, extract albumen respectively, carry out the 2-DE electrophoresis, it is following to carry out the electrophoretic actual conditions of proteome analysis 2-DE: albumen applied sample amount: 150 μ g; The linear adhesive tape (17cm) of 2-DE deposition condition and parameter: pH5-8IPG; 10kV focuses on 60kVh down; 12%PAGE is used for protein isolate, the about 8h of constant current electrophoresis (30mA/gel) operation.Silver dyes and detects the gel protein point, and the result is as shown in Figure 4.Wherein, Fig. 4 A, 4B and 4C represent above-mentioned A, B, three experimental grouies of C respectively.The result shows that pastille serum can be intervened proteic expression in the MSCs atomization.

Above-mentioned when respectively organizing cell culture 30d, the situation of change of Partial Protein is as shown in Figure 5.Twice repetition established in experiment.Among Fig. 5, abscissa is represented different albumen, just lists out significance in the table 3 and changes albumen; Vertical coordinate is represented each proteic expression.

Table 3 pastille serum is intervened differential protein evaluation situation in the MSCs atomization

As can be seen from Figure 5; Similar Lamin A/C is not expressed before the MSCs differentiation, can detect this proteic expression after the differentiation, under the effect of pastille serum; This protein expression presents about 5.7 times rise trend, shows that pastille serum can promote the atomization of MSCs; Antioxidase 2 appears in the MSCs atomization and increases progressively trend, and after pastille serum acts on the MSCs atomization, has suppressed the expression of antioxidase 2.

The expression of Lamin A/C in the MSCs atomization is as shown in Figure 6.From Fig. 6, also can find out; Similar Lamin A/C is not expressed before the MSCs differentiation, can detect this proteic expression after the differentiation, under the effect of pastille serum; This protein expression demonstrates rise trend, shows that pastille serum can promote the atomization of MSCs.

The albumen 2-DE collection of illustrative plates of intervening different time points (6d and 30d) in the MSCs directed differentiation process with the pastille serum of the medication preparation of embodiment 1 is as shown in Figure 7.Wherein, proteic 2-DE collection of illustrative plates when proteic 2-DE collection of illustrative plates when Fig. 7 A intervenes MSCs directed differentiation 6d for pastille serum, Fig. 7 B are intervened MSCs directed differentiation 30d for pastille serum.Through comparison, can analyze at pastille serum and intervene proteic variation tendency in the atomization the 2-DE collection of illustrative plates of these two time points.Listed among Fig. 8 in twice repeated experiments, some significances changed proteic expression before and after the MSCs differentiation was induced in the intervention of pastille serum.Among Fig. 8, abscissa is represented different albumen, and each proteic numbering and title are as shown in table 4; Vertical coordinate is represented each proteic expression.6C1 representes experiment for the first time, and the expression during pastille serum group 6d, 6C2 are represented experiment for the second time; Expression during pastille serum group 6d, 30C1 are represented experiment for the first time, the expression during pastille serum group 30d; 30C2 representes experiment for the second time, the expression during pastille serum group 30d.

Table 4 directed differentiation process Identification of Fusion Protein result

Above experimental result shows; Intervene in the MSCs directed differentiation process at pastille serum; Partial Protein is participated in cell proliferation or atomization according to its molecular structure characteristics and in intracellular distribution situation through the various signals approach, and causes thus presenting difference in the above-mentioned multiple protein expression.Induce down at 5-aza, MSCs is in-vitro directed to be divided in the myocardial cell process, expresses the atomization that the albumen that presents difference has been participated in MSCs, and the differentiation that pastille serum and 5-aza synergism can be induced MSCs embodies in the expression of GAP-associated protein GAP to some extent.With the pastille serum of the medication preparation of

embodiment

2 or 3 and the differentiation that the 5-aza synergism also can be induced MSCs.

3, the inducing bone mesenchymal differentiation of stem cells becomes the gene expression of cardiac-like muscle cell to detect

Existing many bibliographical information 5-aza can induce BMMSCs to break up to myocardial cell, and the mechanism of its differentiation maybe some cytosine demethylation causes in the cell DNA owing to 5-aza can make.

Get MSCs cell after 5-aza induces in the above-mentioned steps 2 (be B group cell) and the MSCs cell (being that C organizes cell) after the pastille serum of the medication preparation of 5-aza and embodiment 1 is induced 30 days; Extract its total RNA respectively; And reverse transcription is cDNA; With this cDNA is template, designs primer PCR increase following 10 gene: Il1a, Pla2g2a, Ccl20, Ccl2, Mmp9, IL6, Sncg, 2 ' 5 '-AS, Pem, 3 α HSD respectively.Concrete primer is following:

According to rise, the downward modulation of gene expression, and the physiological function of time point, acting duration and the gene of pastille serum inducing action is divided into five kinds of binding modes with pastille serum inducing action mode: component is induced mode, the omnidistance rise of component to induce mode for a long time, is promoted model of action, model of action that suppresses the MSCs canceration that the MSCs metabolism is grown and the model of action that strengthens the basic indication of cell life.

1) component is induced mode for a long time: gene Il1a, Ccl20, Ccl2, Pla2g2a and Mmp9 are similar to the responsive mode of pastille serum, are active phase with 10 days, 20 days time points.Variable quantity with 10 days time point gene Il1a, Ccl20, Ccl2, Pla2g2a and Mmp9 compares, and overall trend is that the 5-aza group is similar to or is better than the pastille serum group; But compare with continuous action time length, the pastille serum group is in the time of 10 days, 20 days, and gene Il1a, Ccl20, Ccl2, Pla2g2a and Mmp9 have the expression ascendant trend, obviously are better than 5-aza and induce group.The medicine that above-mentioned steps three preparation is described has more slowly, more stable effect.

2) component is omnidistance raises that to induce mode: Mmp9 (matrix metal peptidase 9) be a kind of proteolytic enzyme; Experiment confirm is arranged; MMP9 overexpression in the atherosis unstability speckle of human artery, increased activity, and can decompose the collagen fragment in the atheromatous plaque.Pastille serum is induced under the stimulation; The Mmp9 gene is all presented mileometer adjustment and is reached in 10 days to 40 days time; When the medicine that shows above-mentioned steps three preparation was used for the acute myocardial infarction treatment, stimulation Mmp9 gene expression that can persistence was raised, and acts on the atheromatous plaque place that myocardial infarction produces; Make the substrate degradation of speckle, thereby be that newborn myocardial cell substitutes the downright bad myocardial cell creation advantageous conditions in speckle place.

3) promote the model of action that the MSCs metabolism is grown: Il 6 (interleukin-6) gene quantification testing result shows that Ill 6 gene expressions obviously raise when having only 10 days time points of pastille serum group.Explain that pastille serum has tangible intervention effect to Il 6 genes, and the Best Times that plays a role is 10 days.When 20 days, 30 days and 40 days, Il 6 gene expressions all present downward modulation in all samples, in the time of 30 days downward modulation the most obvious, consider that this moment, apoptosis began.

4) suppress the model of action of MSCs canceration: Sncg (homotype nucleoprotein γ) gene quantification testing result shows, 5-aza induces that the Sncg gene all had obvious expression decline in group and the pastille serum group in the time of 30 days.Sncg genovariation is that multiple cancer worsens key factor, and is relevant with growth, infiltration, transfer and the prognosis of cancerous cell.

5) model of action of enhancing cell life basis indication: Pem (polymorphic epithelium mucoitin) gene is the intracellular signal transduction molecule, in becoming somatic cell, does not express, but expression is arranged in sexual cell, breaks up relevant with gonad development with embryonic cell; 2 ' 5 '-AS (2 ' 5 ' oligo-adenylate synthetase) gene is the media that interferon plays a role, and has antiviral, suppresses the synthetic and biological functions such as cell growth, adjusting immunoreation of DNA; 3 α HSD (3 α-hydroxyl-steroid dehydrogenase) are one of enzymes initial in the steroid metabolism approach; TOTAL BILE ACID TBA in the serum (TBA) is one type and has 3 alpha-hydroxy steroid derivatives; Under 3 α HSD catalysis, dehydrogenation generates corresponding 3-ketosteroid, in this process with coenzyme NAD+be reduced to NADH+H+; Further regulate the activity of glucose-6-phosphate dehydrogenase, the metabolic cycles of involved in sugar.

Analyze the function of above each gene and find, 10 big types of basi gene functions keeping with organism life all have and relate to.Interpretation of result shows; Il1a, Pla2g2a, Ccl20, Ccl2, these 5 genes of Mmp9 possibly bring into play major function inducing differentiation MSCs to become on the mechanism of action of cardiac-like muscle cell, and other 5 genes are promoting the MSCs metabolism to grow, suppress the canceration process of MSCs, strengthen and bringing into play complementary function aspect the indication of cell life basis respectively.Therefore think that the process that the drug-induced MSCs of above-mentioned steps three preparations is divided into cardiac-like muscle cell is the result of multi-functional, many networks, polygenic coordinative role.

Immunofluorescence result shows; The conversion ratio of inducing that 5-aza induces group to induce MSCs to be divided into cardiac-like muscle cell is higher than the pastille serum group; But the gene quantification testing result shows that the expression change of the key gene of pastille serum group lasts longer than 5-aza and induces group, considers that 5-aza inducing action produce effects is fast; Can bring into play its effect at short notice; And the pastille serum group is on the contrary, shows the inducing action that the medicine of the foregoing

description

1,2 or 3 preparations maybe be bigger to the material base of genetic expression-gene performance scope, double, and to reach the required time of produce effects longer by measuring matter in this effect.So, think that the drug-induced MSCs of the foregoing

description

1,2 or 3 preparations is divided into the effect of cardiac-like muscle cell, The apparent phenomenon is obvious not as 5-aza, but the efficient of improving of the nature basis of effect is better than 5-aza.

The preparation of the medicinal granule of embodiment 5, treatment cardiovascular disease

One, the preparation of the medicinal granule of treatment cardiovascular disease

Get the Radix Ginseng extract powder 100g of the foregoing description 1 step 1 preparation and the Radix Salviae Miltiorrhizae extract powder 10g of the foregoing description 1 step 2 preparation; With the two mix homogeneously; Add 360g dextrin (weight that adds dextrin is Radix Ginseng extract powder and Radix Salviae Miltiorrhizae extract powder weight sum 3 times) and 240g cane sugar powder (weight that adds cane sugar powder is Radix Ginseng extract powder and Radix Salviae Miltiorrhizae extract powder weight sum 2 times) again; Mix homogeneously; Being made for granularity is 16 purpose drug particles, under 60 ℃ of conditions drug particles is carried out drying, the medicinal granule of the cardiovascular disease that obtains medical treatment.

Two, zoopery

Concrete experimental technique is with embodiment 4.The result shows that the medicinal granule of present embodiment is identical with the effect of the medicine of

embodiment

1,2 and 3 to the therapeutic effect of rat acute myocardial infarction and myocardial ischemia.

The preparation of the drug injection of embodiment 6, treatment cardiovascular disease

One, the preparation of Radix Ginseng extract

(pollen of Panax.ginsengC.A.Mey is broken to obtain 10 purpose Flos Ginseng coarse powder after sieving to get Radix Ginseng.Accurately take by weighing Flos Ginseng's coarse powder 150g, add the 1500ml volumn concentration and be 90% ethanol and carry out reflux, extract,, extraction time is 45min; Collect extracting solution, and then in residue, to add the 1500ml volumn concentration be that 90% ethanol carries out reflux, extract,, extraction time is 45min.Extracted twice liquid is mixed, be cooled to room temperature, filter, get filtrate decompression and be concentrated into nothing alcohol flavor, obtain 32g extract extractum; Get 32g extract extractum add 90ml water when being diluted to 20 ℃ the density of solution be 1.06g/ml, obtain 96ml ginsenoside extracting solution.

Adopt the good low pole macroporous adsorptive resins of pretreatment (in resin column, to fill with polystyrene or to gather divinylbenzene be that the skeleton model is the resin extender of HPD700; This low pole macroporous adsorptive resins and HPD700 resin extender are available from Cangzhou, Hebei precious grace chemical industry company limited); 96ml ginsenoside extracting solution to above-mentioned acquisition carries out separation and purification, and bed volume is 117mi behind the filler.Ginsenoside's extracting solution washes resin column to the water of resin column bed volume with the flow velocity of 2bv/h (1bv/h is promptly per hour by the flow of 1 times of bed volume) with 3 times after chromatographic column absorption, remove water-solubility impurities such as polysaccharide, discards eluent; 4 times of volumn concentrations to the resin column bed volume of reuse are 60% the ethanol flow velocity eluting resin column with 1bv/h, collect eluent, obtain the Radix Ginseng total saponins eluent; With Radix Ginseng total saponins eluent concentrating under reduced pressure is thick clear paste; With thick clear paste again through 60 ℃ of constant temperature constant pressure and dries; Obtain 9g Flos Ginseng extract powder (wherein the content of Radix Ginseng total saponins reaches 83% (assay method is with embodiment 1)); The quality percentage composition of ginsenoside Rg1 in Radix Ginseng total saponins is 2.3%, and the quality percentage composition of ginsenoside Re in Radix Ginseng total saponins is 2.6%, and the quality percentage composition of ginsenoside Rb1 in Radix Ginseng total saponins is 3.1%; The quality percentage composition of ginsenoside Rf in Radix Ginseng total saponins is 0.015%.

Two, the preparation of Radix Salviae Miltiorrhizae extract

Get the root 10kg of Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bunge.), be cut into decoction pieces, decoction pieces was pulverized 10 mesh sieves process the Radix Salviae Miltiorrhizae coarse powder, carry out reflux, extract, after adding the 100L volumn concentration and be 50% soak with ethanol 4h, extraction time is 30min; Collect extracting solution, and then in residue, to add the 100L volumn concentration be that 50% ethanol carries out reflux, extract,, extraction time is 30min.Twice extracting solution is mixed, obtain the 150L Radix Salviae Miltiorrhizae extract, again this 150L Radix Salviae Miltiorrhizae extract is carried out sucking filtration, is concentrated into 50L.

Adopt macroporous adsorptive resins (the styrene type resin that with the model is HPD300 is a filler, available from Cangzhou, Hebei precious grace chemical industry company limited), the 50L Radix Salviae Miltiorrhizae extract of above-mentioned acquisition is carried out separation and purification, bed volume is 65L behind the filler.Radix Salviae Miltiorrhizae extract is after chromatographic column absorption; With 3 times to the water of resin column bed volume flow velocity flushing resin column with 2bv/h (1bv/h is promptly per hour by the flow of 1 times of bed volume); Discard eluent; And then to use 3 times of volumn concentrations to the resin column bed volume be 40% the ethanol flow velocity eluting resin column with 1bv/h, collects eluent, obtains the salvianolic acid eluent; The salvianolic acid eluent is carried out concentrating under reduced pressure, and obtaining 820g is that proportion is the thick clear paste of 1.2 (60 ℃); Thick clear paste again through 60 ℃ of constant temperature constant pressure and dries, is obtained 660g Radix Salviae Miltiorrhizae extract powder (wherein the productive rate of Radix Salviae Miltiorrhizae total phenolic acids is 6.6%).Through detecting, in the Radix Salviae Miltiorrhizae extract powder, the content of Radix Salviae Miltiorrhizae total phenolic acids is 94.7% (assay method is with embodiment 1).

Three, the preparation of the injection of treatment cardiovascular disease

Get the Radix Ginseng extract powder 10g of above-mentioned steps one preparation and the Radix Salviae Miltiorrhizae extract powder 200g of above-mentioned steps two preparations, add 630ml water for injection again, stir, under 4 ℃ of conditions, leave standstill 24h.Draw supernatant, in supernatant, add injection in the 0.05g injection with the ratio of active carbon/100ml supernatant and use active carbon, be incubated 30min under 40 ℃ of conditions, with 0.22 μ m filtering with microporous membrane; Get filtrating, adding water for injection again, to make the gross weight of mixed liquor be 840g, stir, and the ampoule embedding, pressure sterilizing is 30 minutes under 115 ℃ of conditions, packing, the drug injection of the cardiovascular disease that obtains medical treatment.

Four, zoopery

Concrete experimental technique is with embodiment 4.The result shows that the drug injection of present embodiment is identical with the effect of the medicine of

embodiment

The preparation of the medicament sustained-release tablets of embodiment 7, treatment cardiovascular disease

One, the preparation of Radix Ginseng extract

Accurately take by weighing the blade 8kg that gets Radix Ginseng (Panax.ginsengC.A.Mey), add the 64L volumn concentration and be 90% ethanol and carry out reflux, extract,, extraction time is 45min; Collect extracting solution, and then in residue, to add the 64Lml volumn concentration be that 90% ethanol carries out reflux, extract,, extraction time is 45min; Repeat the said extracted process more once.Three times extracting solution are mixed, be cooled to room temperature, filter, get filtrate decompression and be concentrated into nothing alcohol flavor, obtain 1.15kg extract extractum; Get 1.15kg extract extractum add 4.1L water when being diluted to 20 ℃ the density of solution be 1.06g/ml, obtain 4.4L ginsenoside extracting solution.

Adopt the good low pole macroporous adsorptive resins of pretreatment (with polystyrene or to gather divinylbenzene be skeleton; In resin column, filling model is the resin extender of S-8; Available from Cangzhou, Hebei precious grace chemical industry company limited); 4.4L ginsenoside extracting solution to above-mentioned acquisition carries out separation and purification, and bed volume is 5.7L behind the filler.Ginsenoside's extracting solution is after chromatographic column absorption; To use 3 times of volumn concentrations to the resin column bed volume be 5% ethanol with the flow velocity flushing resin column of 2bv/h (1bv/h is promptly per hour by the flow of 1 times of bed volume); Remove water-solubility impurities such as polysaccharide, discard eluent; 4 times of volumn concentrations to the resin column bed volume of reuse are 70% the ethanol flow velocity eluting resin column with 1bv/h, collect eluent, obtain the Radix Ginseng total saponins eluent; With Radix Ginseng total saponins eluent concentrating under reduced pressure is thick clear paste; With the thick clear paste of the 450g that obtains again through 60 ℃ of constant temperature constant pressure and dries; Obtain 320g Radix Ginseng extract powder (wherein the content of Radix Ginseng total saponins reaches 81% (assay method is with embodiment 1)); The quality percentage composition of ginsenoside Rg1 in Radix Ginseng total saponins is 2.1%, and the quality percentage composition of ginsenoside Re in Radix Ginseng total saponins is 2.6%, and the quality percentage composition of ginsenoside Rb1 in Radix Ginseng total saponins is 2.5%; The quality percentage composition of ginsenoside Rf in Radix Ginseng total saponins is 0.03%.

Two, the preparation of Radix Salviae Miltiorrhizae extract

Adopt macroporous adsorptive resins (the styrene type resin that with the model is HPD300 is a filler, available from Cangzhou, Hebei precious grace chemical industry company limited), the 50L Radix Salviae Miltiorrhizae extract of above-mentioned acquisition is carried out separation and purification, bed volume is 63L behind the filler.Radix Salviae Miltiorrhizae extract is after chromatographic column absorption; With 3 times to the water of resin column bed volume flow velocity flushing resin column with 2bv/h (1bv/h is promptly per hour by the flow of 1 times of bed volume); Discard eluent; And then to use 3 times of volumn concentrations to the resin column bed volume be 50% the ethanol flow velocity eluting resin column with 1bv/h, collects eluent, obtains the salvianolic acid eluent; The salvianolic acid eluent is carried out concentrating under reduced pressure, and obtaining 812g is that proportion is the thick clear paste of 1.2 (60 ℃); Thick clear paste again through 60 ℃ of constant temperature constant pressure and dries, is obtained 640g Radix Salviae Miltiorrhizae extract powder (wherein the productive rate of Radix Salviae Miltiorrhizae total phenolic acids is 6.4%).Through detecting, in the Radix Salviae Miltiorrhizae extract powder, the content of Radix Salviae Miltiorrhizae total phenolic acids is 91.6% (assay method is with embodiment 1).

Three, the preparation of the medicament sustained-release tablets of treatment cardiovascular disease

Get the Radix Ginseng extract powder 50g of above-mentioned steps one preparation and the Radix Salviae Miltiorrhizae extract powder 50g of above-mentioned steps two preparations; With the two mix homogeneously; Add 100g hydroxypropyl emthylcellulose (specification is SH4000) (weight that adds hydroxypropyl emthylcellulose is the weight sum of Radix Ginseng extract powder and Radix Salviae Miltiorrhizae extract powder) and 100g lactose (weight that adds lactose is the weight sum of Radix Ginseng extract powder and Radix Salviae Miltiorrhizae extract powder) again, mix homogeneously, being made for granularity is 20 purpose granules; Under 60 ℃ of conditions granule is carried out drying; Add the 1.5g magnesium stearate again, make magnesium stearate be overlying on Manufactured particle surface (weight of magnesium stearate account for slow releasing tablet gross weight 0.5%), carry out tabletting at last again; The medicament sustained-release tablets of the cardiovascular disease that obtains medical treatment, every heavy 1g.

Four, zoopery

Concrete experimental technique is with embodiment 4.The result shows that the medicament sustained-release tablets of present embodiment is identical with the effect of the medicine of

embodiment

Embodiment 8, a kind of preparation of treating the medicinal tablet of cardiovascular disease

One, the preparation of Radix Ginseng and Radix Salviae Miltiorrhizae mixed extract

250g Radix Ginseng Rubra decoction pieces is mixed with the 250g salvia piece; Add the 4000ml volumn concentration and be 50% ethanol and carry out reflux, extract; Extraction time is 45min; Collect extracting solution, and then in residue, to add the 3500ml volumn concentration be that 50% ethanol carries out reflux, extract,, extraction time is 45min; Repeat the said extracted process more once.Three times extracting solution are mixed, be cooled to room temperature, filter, get filtrate decompression and be concentrated into nothing alcohol flavor, obtain 127g extract extractum; Get 127g extract extractum adding 600ml water and dilute, obtain the 640ml mixed extract.

Adopt the good low pole macroporous adsorptive resins of pretreatment (in resin column, to fill with polystyrene or to gather divinylbenzene be that the model of skeleton is the resin extender of HPD100; Available from Cangzhou, Hebei precious grace chemical industry company limited); 640ml mixed extract to above-mentioned acquisition carries out separation and purification, and bed volume is 790ml behind the filler.Mixed extract washes resin column to the water of resin column bed volume with the flow velocity of 2bv/h (1bv/h is promptly per hour by the flow of 1 times of bed volume) with 7 times after chromatographic column absorption, remove water-solubility impurity, discards eluent; 3 times of volumn concentrations to the resin column bed volume of reuse are 40% ethanol with the flow velocity eluting resin column of 2bv/h once; To use 3 times of volumn concentrations to the resin column bed volume be 65% ethanol with the flow velocity eluting resin column of 2bv/h once; Collect twice eluent and, obtain the mixing eluent of Radix Ginseng total saponins and salvianolic acid its merging; The mixing eluent of Radix Ginseng total saponins and Radix Salviae Miltiorrhizae total phenolic acids is carried out drying, obtain the mixed extract powder of 22g Radix Ginseng and Radix Salviae Miltiorrhizae.Wherein, The content of Radix Ginseng total saponins is 31.3%; The quality percentage composition of ginsenoside Rg1 in Radix Ginseng total saponins is 4.1%; The quality percentage composition of ginsenoside Re in Radix Ginseng total saponins is 4.3%, and the quality percentage composition of ginsenoside Rb1 in Radix Ginseng total saponins is 3.9%, and the quality percentage composition of ginsenoside Rf in Radix Ginseng total saponins is 0.4%; The content of Radix Salviae Miltiorrhizae total phenolic acids is 33.7% (assay method is with embodiment 1).

Two, the preparation of the medicinal tablet of treatment cardiovascular disease

Take by weighing the Radix Ginseng of 22g above-mentioned steps one preparation and the mixed extract powder of Radix Salviae Miltiorrhizae; Add 22g starch (adding the weight of starch and being equal in weight of mixed extract powder) and 11g lactose (weight that adds lactose is the half the of mixed extract powder weight); Mix homogeneously, processing granularity is 16 purpose granules, through dry; Tabletting, the medicinal tablet of the cardiovascular disease that obtains medical treatment.

Four, zoopery

Concrete experimental technique is with embodiment 4.The result shows that the medicinal tablet of present embodiment is identical with the effect of the medicine of

embodiment

Embodiment 9, a kind of preparation and effect experiment thereof of treating the medicine decoction of cardiovascular disease

One, the preparation of the medicine decoction of treatment cardiovascular disease

Get the Radix Ginseng decoction pieces and mix by the weight ratio of 7:3, in the 100g mixture that obtains, add the 800ml decocting and boiled 1 hour, filter with salvia piece; Get decoction liquor and filtering residue respectively; In filtering residue, add the 600ml decocting again and boiled 1 hour, twice decoction liquor is merged, filter, concentrate; Obtain 1100ml filtrating, this filtrating is the medicine decoction of treatment cardiovascular disease.In this decoction; The content of Radix Ginseng total saponins is 3.4mg/ml; Ginsenoside Rg1's content is the content 0.18mg/ml of Rg1, and the content of ginsenoside Re in Radix Ginseng total saponins is 0.23mg/ml, and the content of ginsenoside Rb1 in Radix Ginseng total saponins is 0.14mg/ml; The content of Radix Salviae Miltiorrhizae total phenolic acids is 1.1mg/ml (assay method is with embodiment 1).

Two, the effect experiment of the medicine decoction of treatment cardiovascular disease

Method according to the foregoing description 4 makes up the acute myocardial ischemia rat model; And be divided into model group and administration group; Administration group rat is irritated the medicine decoction that stomach gives the treatment cardiovascular disease of above-mentioned steps one preparation according to the dosage of 5.0ml/kg body weight; Model group gives the normal saline of same dose, investigate this medicine to the influence of acute myocardial ischemia rat heart infarct size due to the isoproterenol (ISO) (x ± s, n=9).The result is as shown in table 5.

The medicine decoction of table 5 treatment cardiovascular disease is to the influence of acute myocardial ischemia rat heart infarct size

Compare with model group, < 0.05**P < 0.01 for * P

The result shows that the administration group can effectively suppress the heart infarct size to a certain extent.

Claims

1. medicine that treats and/or prevents angiopathy, its active component is Radix Ginseng extract and Radix Salviae Miltiorrhizae extract; The ratio of weight and number of said Radix Ginseng extract and Radix Salviae Miltiorrhizae extract is 1: (0.1-20);

In the said Radix Ginseng extract, the quality percentage composition of Radix Ginseng total saponins is greater than 50%; In the said Radix Salviae Miltiorrhizae extract, the quality percentage composition of salvianolic acid is greater than 50%;

Said Radix Ginseng extract and Radix Salviae Miltiorrhizae extract prepare according to following method:

1) with ethanol water or water Radix Ginseng is carried out reflux, extract,, the extracting solution that obtains is centrifugal, get supernatant, obtain ginsenoside's extracting solution; With ethanol water Radix Salviae Miltiorrhizae is carried out reflux, extract,, the extracting solution that obtains is centrifugal, get supernatant, obtain the salvianolic acid extracting solution;

3) to above-mentioned steps 2) the Radix Ginseng total saponins eluent that obtains carries out drying, obtains Radix Ginseng extract; To above-mentioned steps 2) the salvianolic acid eluent that obtains carries out drying, obtains Radix Salviae Miltiorrhizae extract;

When preparing ginsenoside's extracting solution in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 50-95%; Said return time is 30min-120min;

When preparing the salvianolic acid extracting solution in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 10-60%; Said return time is 30min-120min;

When preparing ginsenoside's eluent said step 2), during the eluting water solubility impurity, alcoholic acid volumn concentration is less than 10% in the said ethanol water; During the eluting resin column, alcoholic acid volumn concentration is 50-90% in the said ethanol water; Resin extender in the said macroporous adsorptive resins is a following A) or B):

When preparing the salvianolic acid eluent said step 2), during the eluting resin column, alcoholic acid volumn concentration is 30-90% in the said ethanol water; Resin extender in the said macroporous adsorptive resins is a following A) or B):

2. medicine according to claim 1; It is characterized in that: in the said Radix Ginseng total saponins; The quality percentage composition of ginsenoside Rg1 in Radix Ginseng total saponins is more than or equal to 2%; The quality percentage composition of ginsenoside Re in Radix Ginseng total saponins is more than or equal to 2.5%, and the quality percentage composition of ginsenoside Rb1 in Radix Ginseng total saponins is more than or equal to 2%.

3. medicine according to claim 1 and 2 is characterized in that: said angiopathy is a cardiovascular disease; Said cardiovascular disease is coronary heart disease, angina pectoris, myocardial ischemia or anoxia or myocardial infarction.

4. medicine according to claim 3 is characterized in that: the dosage form of said medicine is tablet, granule, capsule, drop pill, suppository, injection, infusion solution, aerosol or inhalant.

5. medicine that treats and/or prevents angiopathy, the raw material that its active component is made up of Radix Ginseng and Radix Salviae Miltiorrhizae is processed, according to following method preparation:

Said Radix Ginseng be root, stem, the leaf of Radix Ginseng plant and spend at least a;

Said Radix Salviae Miltiorrhizae is the root of Radix Salviae Miltiorrhizae plant;

3) to above-mentioned steps 2) the mixing eluent that obtains carries out drying, obtains the mixed extract of Radix Ginseng extract and Radix Salviae Miltiorrhizae extract;

The ratio of weight and number of said Radix Ginseng extract and Radix Salviae Miltiorrhizae extract is 1: (0.1-20);

When preparing mixed extract in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 30-90%; Said return time is 30-120min;

When preparation mixed eluent said step 2), during the eluting resin column, alcoholic acid volumn concentration was 30-90% in the said ethanol water; Resin extender in the said macroporous adsorptive resins is a following A) or B):

6. medicine according to claim 5; It is characterized in that: in the said Radix Ginseng total saponins; The quality percentage composition of ginsenoside Rg1 in Radix Ginseng total saponins is more than or equal to 2%; The quality percentage composition of ginsenoside Re in Radix Ginseng total saponins is more than or equal to 2.5%, and the quality percentage composition of ginsenoside Rb1 in Radix Ginseng total saponins is more than or equal to 2%.

7. according to claim 5 or 6 described medicines, it is characterized in that: said angiopathy is a cardiovascular disease; Said cardiovascular disease is coronary heart disease, angina pectoris, myocardial ischemia or anoxia or myocardial infarction.

8. medicine according to claim 7 is characterized in that: the dosage form of said medicine is tablet, granule, capsule, drop pill, suppository, injection, infusion solution, aerosol or inhalant.

9. method for preparing arbitrary said medicine among the claim 1-4 may further comprise the steps:

4) with above-mentioned steps 3) the Radix Ginseng extract powder and the Radix Salviae Miltiorrhizae extract powder mixes that obtain, obtain arbitrary described medicine among the claim 1-4;

When preparing ginsenoside's extracting solution in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 50-90%; Said return time is 30min-120min; When preparing the salvianolic acid extracting solution in the said step 1), in the said ethanol water, alcoholic acid volumn concentration is 10-60%; Said return time is 30min-120min;

10. method for preparing arbitrary said medicine among the claim 5-8 may further comprise the steps:

3) to above-mentioned steps 2) the mixing eluent that obtains carries out drying, obtains the mixed extract powder of Radix Ginseng and Radix Salviae Miltiorrhizae, is arbitrary described medicine among the claim 5-8;