CN1552416A

CN1552416A - Medicine composition application for preparing hypertension medicine

Info

Publication number: CN1552416A
Application number: CNA031408443A
Authority: CN
Inventors: 阎希军; 吴廼峰; 张伯礼; 高秀梅
Original assignee: Tianjin Tasly Pharmaceutical Co Ltd
Current assignee: Tasly Pharmaceutical Group Co Ltd
Priority date: 2003-06-03
Filing date: 2003-06-03
Publication date: 2004-12-08
Anticipated expiration: 2023-06-03
Also published as: CN100502908C

Abstract

An application of an invented medicinal composition in preparing the medicines for treating primary hypertension and protecting heart and kidney is disclosed.

Description

The application of a kind of pharmaceutical composition in preparation treatment hypertension disease medicament

Technical field

The invention belongs to field of medicaments, be specifically related to the application of a kind of pharmaceutical composition in preparation treatment hypertension disease medicament.

Background technology

Hypertension is global great public health problem.Studies have shown that both at home and abroad hypertension not only causes abnormal hemodynamics, sugar, insulin, fatty obstacle, and cause the grievous injury of the heart, brain, kidney target organ, be coronary heart disease, the main hazard factor of apoplexy, thereby become emphasis in the cardiovascular disease preventing and controlling.Studies show that untreated hypertension can obviously increase the infringement of target organs such as the heart, brain, kidney, blood vessel and the incidence rate of nonfunction.The practice guidelines that World Health Organization (WHO) in 1994 and international hypertension alliance (WHO/ISH) formulate is pointed out: through the hyperpietic of treatment, the general mortality rate of coronary heart disease, apoplexy obviously descends but does not reach normal population level of the same age yet.This is directly relevant with target organ damage.

Therefore, hypertensive therapeutic goal not instrument brings high blood pressure down to desirable level, and will reduce or reverse the pathological change of target organ, reduces the cardiovascular and cerebrovascular vessel events incidence, improves life quality, is only the hypertensive key of treatment.Differentiation of tcm is treated primary disease, does not lie in merely to bring high blood pressure down, and its emphasis point is to adjust the balance of the bright sun of body, thereby obviously improves symptom, promotes many-sided comprehensive functions such as recovery that the heart, brain, kidney, vascular pathological change.Therefore, Chinese medicine is had whole conditioning in mind and blood pressure lowering, and correcting functional disorder simultaneously improves the body symptom, improves the quality of living, and has potential advantages in this respect.

Heart is one of target organ of involving of hypertension, and main infringement is left ventricular hypertrophy (LVH).Research in recent years also shows during hypertension LVH, the hypertrophy of myocardial cell is arranged not only, and normal with the hypertrophy of myocardium Interstitial cell and the abnormal stacking of collagen fiber, causes the generation of myocardial fibrosis (MF).MF shows as mainly that the original collagen of matter increases slightly between cardiac muscle, and new collagen is deposited between the cardiac muscle of original shortage collagen in the matter, and the collagen volume ratio increases.Collagen fiber in the cardiac muscle interconnect, and form a huge rack, are wrapped in various cells, make heart become as a whole, finish the function of heart jointly.Left ventricular hypertrophy and development of myocardial fibrosis go down left ventricle diastole and contractile function, finally cause heart failure; Can make the reserve capabillity of coronary artery circulation reduce the coronary heart disease that increases the weight of and deposit; Also can cause the generation of various arrhythmias and sudden death.

Aspect kidney, just point out kidney and hypertension correlate as far back as Bright in 1826.Perera followed the tracks of and followed up a case by regular visits to 500 routine untreated primary hypertension patients the fifties, found that from diagnosis the extremely dead mean survival time is 20 years, and wherein only having hypertension not have the complication time is 15 years, occurs the target organ complication later on.The incidence rate of renal complication is 42%, is only second to cardiac complication, and about 10% dies from renal failure, further specifies the harm of human hypertension to kidney.

At present, treat hypertension clinically based on chemicals, its main feature is a symptomatic treatment, and produce effects is fast, and effect is strong, and controlling blood pressure water half preferably, as angiotensin converting enzyme inhibitor, and captopril etc.But, the effect of chemicals mainly is the some links at pathological change in the hypertension pathogenic process, action pathway, action target spot are single relatively, can not take into account abnormalities of sugar/lipid metabolism in the hypertension incidence process, carbohydrate tolerance and reduce and effectively protect vascular endothelial cell and function thereof and influencing each other and complicated link such as adjusting between them, and these links all play an important role for the protection target organ; The side effect of chemicals has simultaneously limited some special populations and has used.

The compound of Chinese medicine hypertension is not only the simple addition of each flavor Chinese medicine hypotensive effect, and it is to regulate by the integration of multipath, many target spots, performance hypotensive effect, blood pressure lowering characteristics are that effect relaxes, and compare with chemicals, side effect is little, is fit to take for a long time; And because the complexity and the multi-component characteristics thereof of Chinese medicine compound itself, in its performance hypotensive effect, effectively adjusting other system that the higher blood pressure level caused unusually, bring into play its organ protection effect.

Kidney aspect, motherland's medical science think that hypertensive basic pathology is hyperactivity of YANG due to deficiency of YIN, and then cause the generation and the QI and blood lifting imbalance of pathological factor such as wind-fire expectorant silt, and sick position is mainly at Liver and kidney.The kidney being the origin of congenital constitution, hides Kidney-Yinly, and residence Yuanyang should Gu Zang and should not reveal.The early stage renal damage person of hypertension system suffers from a deficiency of the kidney can not astringency inducing precise and tiny, and expectorant is turbid in addition, and blood stasis resistance kidney network is stopped up and rushed down outward.The visible TG of clinical examination (triglyceride), TC (cholesterol), LDL (low density lipoprotein, LDL) etc. and urine β ₂-MG (β ₂Microglobulin), IgG (immunoglobulin G), Alb (albumin) increase and kidney damage sign such as polyuria, further develop as seen to feel sick vomiting, abdominal distention, renal failure symptoms such as anasarca.[tight east such as Yan Dong, Jiang Weimin, Tang Shuhua. the early stage renal damage 35 routine observation of curative effect of blood pressure lowering kidney tonifying granule therapy hypertension. Chinese medicine research [J] .1999, L5 (I): 6-7.] observation is light, moderate essential hypertension 35 routine oral blood pressure lowering kidney tonifying granule (Herba Bidentis Bipinnatae, Radix Polygoni Multiflori Preparata, Fructus Corni, Radix Scrophulariae, Radix Stephaniae Tetrandrae) 3 months, find that blood pressure reduces, and its blood metabolism and hematuria LgG, β ₂The effect of this side's hypertension and hyperlipemia and protection kidney is pointed out in-MG, the Alb effect that all has clear improvement.Tang Shude etc. [Tang Shude etc. the Radix Polygoni Multiflori is treated early stage kidney damage blood stasis type hyperpietic 28 examples. Chinese combination of Chinese and Western medicine magazine [J] .1994,5; 302-303.] studies show that Radix Polygoni Multiflori hypotensive effect protection kidney, alleviate injury of kidney, reach and prevent and treat the early stage renal damage purpose of hypertension.

The inventor has applied for the pharmaceutical composition patent, the patent No.: 93100050.5, form by Radix Angelicae Sinensis, Rhizoma Chuanxiong etc.It is under the guidance of motherland's theory of medicine, and sums up through clinical practice for many years and to form, and has the effect of the headache that the treatment internal injury causes, is applicable to symptoms such as dizziness that angioneurotic headache, migraine and hypertension causes, headache clinically.In this prescription ratio, can make any dosage form on the pharmaceutics.Granule dosage form is wherein produced by Tianjin Tasly Pharmaceutical Co., Ltd, and name is called " blood-nourishing and brain-refreshing granules "." function with cure mainly " got permission is the suppressing the hyperactive liver that nourishes blood, promoting blood circulation to remove obstruction in the collateral.Be used for various headaches, traumatic cranial nerve syndrome due to the blood deficiency and excessive liver-YANG, stagger, susceptible to lose temper due to restlessness, insomnia and dreamful sleep etc.Be mainly used in the headache due to treatment blood deficiency, blood stasis, the hyperactivity of YANG due to deficiency of YIN clinically.Because its definite curative effect since listing, has won the trust of extensive patients.

Through clinical experimental research in recent years, the range of application of blood-nourishing and brain-refreshing granules has obtained further expansion.Zhu Yuhuan [blood-nourishing and brain-refreshing granules treatment chronic tension headache 45 examples, Shandong journal of Chinese medicine is 21 the 1st phases 22 of volume of year January J.2002] has reported with the ZHENNAONING to be the contrast medicine, the treatment chronic tension headache, and the result has obtained satisfied effect.Chen Yingxian [blood-nourishing and brain-refreshing granules and the clinical observation of flunarizine hydrochloride capsule for treating migraine, the Acta Pharmaceutica Sinica J. of PLA the 17th the 2nd phase 100 of volume] observed blood-nourishing and brain-refreshing granules and the migrainous curative effect of flunarizine hydrochloride capsule for treating, the result shows that blood-nourishing and brain-refreshing granules and flunarizine hydrochloride capsule unite use and can obviously improve curative effect.So far find report blood-nourishing and brain-refreshing granules treatment essential hypertension and to the report of target-organ protection aspects such as the heart, kidney.

Summary of the invention

The present invention is on the basis of existing patent [93100050.5], the new purposes invention of finishing, and the composition of prescription is as follows by weight ratio:

Radix Angelicae Sinensis 4～9%, Rhizoma Chuanxiong 4～9%, the Radix Paeoniae Alba 2～8%, Radix Rehmanniae Preparata 2～8%, Ramulus Uncariae Cum Uncis 10～15%, Caulis Spatholobi 10～15%, Spica Prunellae 10～15%, Semen Cassiae 10～15%, Concha Margaritifera 10～15%, Rhizoma Corydalis 4～9%, Herba Asari 0.5～2%;

Best proportioning is:

Radix Angelicae Sinensis 6.75%, Rhizoma Chuanxiong 6.75%, the Radix Paeoniae Alba 5.4%, Radix Rehmanniae Preparata 5.4%, Ramulus Uncariae Cum Uncis 13.5%, Caulis Spatholobi 13.5%, Spica Prunellae 13.5%, Semen Cassiae 13.5%, Concha Margaritifera 13.5%, Rhizoma Corydalis 6.75%, Herba Asari 1.34%.

This patent drug compositions is implemented by following scheme:

The material of getting it filled according to the above ratio, extracting in water 3 times, each 1 hour, merge extractive liquid, suitably concentrated, the ethanol that adds 2 times of amounts, leave standstill 24 hours precipitations, get supernatant concentration and become extractum, relative density is 1.3～1.4, paste-forming rate is 10%, and qinghuo reagent, sucrose and dextrin are made granule in 1: 3: 1 ratio.Wherein, preparation part can also be made any dosage form on other pharmaceuticss according to the process of routine.

Headache has close getting in touch with hypertension, but at aspects such as pathogenesis and Therapeutic Method tangible difference is arranged again.At present; treatment headache and hypertensive medicine two classes are arranged respectively clinically; the medicine of treatment headache not necessarily has antihypertensive effect, even also can cause other untoward reaction, says nothing of to have the protective effect that causes the target organ of damage for hypertension.

Said blood-nourishing and brain-refreshing granules confirms can obviously reduce spontaneous hypertensive rat (spontaneously hypertensive rat, arterial pressure SHR) at certain dosage through zoopery.Result of study shows, the former medicine blood-nourishing and brain-refreshing granules that belongs to the treatment headache has the hypertensive effect of anti-constitutional, has simultaneously for hypertension to cause that the target organs such as the heart, kidney of damage have protective effect.

In order to understand content of the present invention better, below by granule dosage form---the blood-nourishing and brain-refreshing granules of this patent drug, the result of the test in hypertension animal model illustrates its new purposes in pharmaceutical field.

Spontaneous hypertensive rat claims that again (Spontaneous Hypertension Rat, SHR), its pathogeny and development and change approach the mankind to original hypertensive rat, and blood pressure level is higher more stable, are a kind of essential hypertension models.The influence of this scale-model investigation blood-nourishing and brain-refreshing granules to systolic blood pressure, clear and definite its hypotensive effect and intensity are adopted in this experiment.And used high-throughout gene chip detecting technique, blood-nourishing and brain-refreshing treatment back SHR rat kidney difference expression gene has been screened, observed the variation of left ventricle gene expression profile simultaneously.Attempt from above observation, to study the hypotensive effect of this medicine, and inquire into the mechanism of Chinese medicine the protective effect of hypertension target organ damage from molecular level.

First: blood-nourishing and brain-refreshing granules is to the influence of spontaneous hypertensive rat blood pressure

1. laboratory animal and instrument

60 of SHR rats; male and female half and half; 14 ages in week; body weight 210.89 ± 39.34; 10 of this especially big Mus (WKY) of Tokyo prestige; male and female half and half, as normal control, body weight 220.17 ± 37.67g (Shanghai Research Institute of Hypertension; the animal quality certification number: the moving word of doctor 0237-1 number); RBP-1B type rat blood pressure cardiotachometer (Beijing Chinese-Japanese Friendship Clinic Medical Inst), blood-nourishing and brain-refreshing granules (Tianjin Tasly Pharmaceutical Co., Ltd, lot number 20020404); Bezoar pill for lowering blood pressure (Tianjin Zhongxin Pharmaceutical Group Co; lot number 0484035), captopril (Tianjin Hua Xin pharmaceutical factory, lot number 20010901)

2. experimental technique

(1) pressure testing method

Adopt arteria caudalis volume noinvasive manometry.Before the pressure measurement, rat is put into 37 ± 1 ℃ of electric heating warming casees, suitable mouse cage is selected in about 12～13 minutes of preheating then, is fixed in pressure measurement on the pressure measurement platform.Under the rat rest state, pressure measurement is 3-5 time continuously, averages as the pressure measurement result.In the pressure measurement process, it is soft that action is wanted, and avoids causing the rat uneasiness.Before the formal experiment, about 1 week of adaptability pressure measurement.Treat that rat conforms, behind the blood pressure stabilization, beginning is experiment formally.Institute's measuring blood pressure is systolic pressure in this research.

(2) grouping and administration

After the adaptability pressure measurement finishes, calculate the basic blood pressure of every rat, press the blood pressure layering, be divided into 6 groups by table of random number again, 10 every group is contrast with WKY:

The WKY group

The model group distilled water

Bezoar pill for lowering blood pressure group 0.168g/kg/d

Captopril group 7.785g/kg/d

Blood-nourishing and brain-refreshing granules group 31.5g crude drug/kg/d

All medicines all are configured to solution by the 1ml/100g body weight, gastric infusion before experiment.

Survey rat blood pressure morning every day.The 1st week is measured rat blood pressure every day, the 2nd, 3,4,6,8 weeks weekly measuring blood pressure once, each survey continuously to average in three days be this blood pressure in week.Survey body weight on every Mondays one time, adjust dosage at any time.After the first administration, every 1 hour measuring blood pressure 1 time, continuous 4 hours.Rat feeding in constant temperature (22 ± 2 ℃) room ventilation cabinet, 5～6 in every cage, standard feed is freely drunk water.

(3) result's statistics: carry out statistical procedures with the Spss10.0 software kit, data are with (x ± s) expression, paired t-test is relatively used in front and back, relatively checks with t between group.

3. result

One, single-dose is to the influence of SHR systolic pressure

Single gavages blood-nourishing and brain-refreshing granules, and the spontaneous hypertensive rat systolic pressure is descended.Began to show hypotensive effect after administration in about 1 hour, the peak is behind the medicine between 2～4 hours.The after-contraction drops was promptly variant in 2 hours, reached the about 9mmHg in blood pressure lowering peak in 3 hours.After 24 hours.See Table 1, accompanying drawing 1.

Table 1 blood-nourishing and brain-refreshing granules is to the influence (mmHg) of SHR systolic pressure (x ± s)

2h behind the 1h medicine behind the group dosage n medicine prodrug

(g/kg/d)

Model group 10 183.9 ± 8.7 188.9 ± 13.2 188.7 ± 14.0

Bezoar pill for lowering blood pressure group 0.17g 10 185.6 ± 16.3 184.1 ± 15.9 178.8 ± 11.75

Captopril group 7.79mg 10 184.8 ± 13.9 174.4 ± 11.2 ^c168.9 ± 10.4 ^b

Blood-nourishing and brain-refreshing granules group 31.5 10 181.9 ± 9.4 177.5 ± 6.7 173.9 ± 7.7 ^a

Annotate: with comparison before the administration ^aP＜0.05, t=3.11; ^bP＜0.001, t=5.65; ^cP＜0.05, t=2.80

Continuous table 1 blood-nourishing and brain-refreshing granules is to the influence (mmHg) of SHR systolic pressure (x ± s)

Dosage

24h behind the 4h medicine behind the 3h medicine behind the group n medicine

(g/kg/d)

Model group 10 189.7 ± 11.9 189.5 ± 12.1 187.1 ± 9.5

Bezoar pill for lowering blood pressure group 0.17g 10 181.2 ± 27.1 179.3 ± 10.6 180.0 ± 12.8

Captopril group 7.79mg 10 173.5 ± 12.3 ^b177.5 ± 14.2 181.1 ± 12.5

Blood-nourishing and brain-refreshing granules group 31.5 10 172.7 ± 7.7a 175.4 ± 9.8 179.9 ± 8.5

Annotate: with comparison before the administration ^aP＜0.05, t=3.01; ^bP＜0.01, t=3.50.

Two, 8 weeks of blood-nourishing and brain-refreshing granules successive administration are to the influence of SHR systolic pressure

Gavage blood-nourishing and brain-refreshing granules continuously, can suppress the trend that the spontaneous hypertensive rat blood pressure increases with the increase at age, after 2 weeks of administration, blood-nourishing and brain-refreshing granules group and model more promptly have statistical significance (p＜0.05); After 8 weeks of administration, systolic pressure sees Table 2, accompanying drawing 2 than the low 26.1mmHg of model group.

Table 2 blood-nourishing and brain-refreshing granules is to the influence (mmHg) of SHR systolic pressure (x ± s)

Dosage

Group example base plinth blood pressure 1W 2W 3W

(g/kg/d)

WKY organizes 10 129.4 ± 4.2 ^*130.6 ± 7.5 ^*124.0 ± 6.4 ^*124.6 ± 5.9 ^*

Model group 10 181.6 ± 13.9 187.1 ± 9.3 188.8 ± 9.4 193.1 ± 11.9

Bezoar pill for lowering blood pressure 0.1 10 182.7 ± 12.2 185.1 ± 19.3 181.6 ± 10.7 181.2 ± 12.9 ^*

Group 68g

Captopril group 7.785mg 10 181.3 ± 14.3 174.8 ± 12.1 ^*170.7 ± 10.98 ^*169.7 ± 7.9 ^*

Blood-nourishing and brain-refreshing 31.5 10 181.9 ± 9.4 179.1 ± 10.3 178.3 ± 7.3 ^*174.1 ± 8.3 ^*

The grain group

Annotate: ^*Compare with model group ^*P＜0.05, ^*P＜0.01

Continuous table 2 blood-nourishing and brain-refreshing granules is to the influence (mmHg) of SHR systolic pressure (x ± s)

Dosage

Group N 4W 6W 8W

(g/kg/d)

WKY organizes 10 127.5 ± 11.2 ^*121.6 ± 6.8 ^*117.5 ± 5.6 ^*

Model group 10 192.1 ± 105 199.9 ± 13.1 199.3 ± 11.5

Bezoar pill for lowering blood pressure group 0.168g 10 178.8 ± 8.4 ^*177.9 ± 13.8 ^*181.9 ± 10.9 ^*

Captopril group 7.785g 10 169.7 ± 8.6 ^*169.7 ± 12.9 ^*171.1 ± 6.9 ^*

Blood-nourishing and brain-refreshing granules group 31.5 10 175.2 ± 6.7 ^*173.9 ± 5.5 ^*173.2 ± 5.68 ^*

Annotate: ^*Compare with model group ^*P＜0.05, ^*P＜0.01

Second portion: blood-nourishing and brain-refreshing granules is to the influence of spontaneous hypertensive rat left ventricular hypertrophy index of correlation

Left ventricular hypertrophy (LVH) is one of hypertension most common complication.Long-term higher blood pressure level causes afterload to increase, heart is in compensatory state for a long time, myocardial cell hypertrophy, hypertrophy, and activate heart part and systemic factor, as renin angiotensin aldosterone system (RAAS), ET-NO system etc., promote the further plumpness of ventricle, the cardiac muscle interstitial proliferation causes vicious cycle, develops into myocardial fibrosis.Myocardial fibrosis is one of common cause that causes clinically heart failure.The SHR rat model in 14 ages in week is adopted in this experiment, in 8 weeks of successive administration, observes blood-nourishing and brain-refreshing granules to the reverse of SHR left ventricular hypertrophy and to the protective effect of early stage myocardial fibrosis.

1. laboratory animal and key instrument

60 of SHR rats, age in male and female half and half, 14 week; body weight 210.89 ± 39.34,10 of this especially big Mus (WKY) of Tokyo prestige, male and female half and half; body weight 220.17 ± 37.67 (Shanghai Research Institute of Hypertension; the animal quality certification number: the moving word of doctor 0237-1 number), blood-nourishing and brain-refreshing granules (day Shi Li group, lot number 20020404); Bezoar pill for lowering blood pressure (Tianjin Zhongxin Pharmaceutical Group Co; lot number 0484035), captopril (Tianjin Hua Xin pharmaceutical factory, lot number 20010901).Polyacrylamide, methene bisacrylamind, pepsin etc. (U.S., Sigma).Hydroxyproline standard substance (special reagent center, North China, lot number 20020215).Hypervelocity refrigerated centrifuge (Germany, Beckman), DYY-IIIB electrophresis apparatus (Liuyi Instruments Plant, Beijing), CS-9300PC thin layer chromatogram scanner (Japan, Tianjin, island), 722RS type spectrophotometer (the Shanghai second analytical tool factory), PHS-2C acidometer (Shanghai thunder magnetic instrument plant), DY8f-I type electric driven glass refiner (Ningbo Xin Mangke device institute).

2. experimental technique

(1) grouping and administration

60 of SHR rats are divided into 6 groups by table of random number, and 10 every group is contrast with WKY10:

WKY

The model group distilled water

Bezoar pill for lowering blood pressure group 0.168g/kg/d

Captopril group 7.785g/kg/d

Blood-nourishing and brain-refreshing granules group 35.5g crude drug/kg/d

(2) draw materials

After 8 weeks of administration, weigh (BW), carotid artery sacrificed by exsanguination rat opens breast and takes out heart rapidly, the residual blood of normal saline flushing, blot, cut off auricle, atrium, trunk etc., take by weighing heavy whole-heartedly along the atrioventricular junction place, carefully wipe out right ventricular free wall, accurately take by weighing left ventricle+interventricular septum weight (LVW), and calculate the ratio of LVW and BW, as the index of left ventricular hypertrophy.Get stage casing cross section cardiac muscular tissue, drop into 10% formalin fixed.Get the about 1m of left ventricular free wall cardiac muscular tissue ³, insert in ice-cold 2.5% glutaraldehyde (Ph7.2) fixing immediately.All the other left ventriclies are wrapped up with tinfoil, preserve standby in the input liquid nitrogen.

(3) tectology is observed: get the cardiac muscular tissue of 10% formalin fixed, and the routine paraffin wax embedding, dehydration, section, row VG dyeing, myocardial cell is yellow under the light microscopic, and collagen fiber take on a red color.Row HE dyeing, the morphosis of observation myocardial cell.Get the cardiac muscular tissue of 2.5% glutaraldehyde, osmic acid is fixed, dehydration, and embedding, the row ultrathin section is with transmission electron microscope observation cardiac muscle and blood vessel structure.

(4) mensuration of the total collagen content of cardiac muscle

Get an amount of cardiac muscular tissue, weigh, be placed on the 6h that dewaters in 95% ethanol, used the acetone defat then 48 hours.The defat sample is copied in the case in 110 ℃ and is dried, and pulverization after the title dry weight is preserved.Accurately take by weighing a certain amount of dry powder of organizing, in ampoule, add 5ml 6mol/L hydrochloric acid (6mol/L), seal, copy in the case hydrolysis 5 hours in 125 ℃, then hydrolyzed solution is moved in the 25ml volumetric flask, add 2～2.5ml NaOH (6ml/L), make pH value near 6, be diluted to scale, placement is spent the night.Draw diluent 2ml and be placed in the test tube, add 1ml chloramine-T solution (0.05ml/L), shake up, placed 20 minutes under the room temperature.Add 1ml and cross solution chlorate (3.15ml/L), shake up.After 5 minutes, add 1ml paradime thylaminobenzaldehyde solution (10%), shake up, insulation is 20 minutes in 60 ℃ of water-baths, colour developing, cooling, colorimetric under the 550nm wavelength.The making of standard curve: accurately take by weighing standard 100mg and be dissolved in the 0.01ml/L hydrochloric acid, be settled to 100ml, be titer, its concentration is 1000 μ g/ml.Get titer 2ml, be diluted to 100ml, its concentration is 20 μ g/ml, dispose successively 2,5,8,10,16 μ g/m variable concentrations titer, press the aforesaid operations colorimetric, according to concentration and optical density drawing standard curve, calculate regression coefficient.

What myocardial collagen concentration (mg/mg cardiac muscle dry powder)=[(sample absorbance/titer absorbance) * concentration of standard solution * extension rate * 7.46] ÷ weighed organizes dry powder heavy

Annotate: the 7.46th, the coefficient when hydroxyproline is scaled collagen.

(5) mensuration of myocardium I, III Collagen Type VI ratio

The extraction of cardiac muscle I, III collagen mixture: accurately take by weighing a certain amount of cardiac muscular tissue, shred, add the normal saline of 2ml pre-cooling, homogenate in the ice bath.Add the 0.05mol/L Tris-HCl buffer (pre-cooling) that 8ml contains 5mol/ml NaCl in the homogenate, mixing was put into ice bath 2-4 hour.4 ℃, 3000-4000r/min, centrifugal 15min gets precipitation, with the cold deionized water wash of capacity, desalts, and adds the acetic acid of the 0.5mol/L of 20 times of volumes, piping and druming, mixing.Add the pepsin of capacity, make final concentration be not less than 2mol/L, mixing, (4 ℃) carry out restricted degraded 48 hours under low temperature.The whole operation process must be below 4 ℃.Add 0.2mol/L EDTA-Na ₂, final concentration is 20mmol/L, stops degraded.4 ℃, 3000r/min, centrifugal 10min gets supernatant, adds the NaCl grind to superfine end, slowly adds, the limit edged stirs, to final concentration be 2mol/L, 4 ℃ left standstill 24 hours.4,35000 * g, centrifugal 45 minutes, the collecting precipitation thing was used cold deionized water wash, removed to desalt.Gains are I, III type mixing collagen.Freezing preservation.

The preparation of SDS-polyacrylamide gel and electrophoresis: separation gel preparation: contain 30% acrylamide and 0.8%N, the storing solution 6mol of N '-methylene bisacrylamide, 1.5mol/L Tris-HCl buffer 8ml, 10%SDS0.18ml, deionized water 3.8ml, TEMED 15 μ l, 10% Ammonium persulfate. 0.16ml.Concentrate the glue preparation, contain 30% acrylamide and 0.8%N, the storing solution 0.68ml of N '-methylene bisacrylamide, 0.5mol/L Tris-HCl buffer 1.8ml, 10%SDS0.04ml, deionized water 1.5ml, TEMED 5 μ l, 10% Ammonium persulfate. 0.05ml.Get about 5-8mg I, III type mixing collagen, add 60～80 μ l deionized waters, with sample treatment liquid 60～80 μ l sample dissolution that contain 5% beta-mercaptoethanol, 100 ℃ were boiled 5 minutes, get the every hole 20 μ l of supernatant point sample, put voltage 100V, to the separation gel forward position, transfer voltage to 200V, electrophoresis to tracer dye stops when arriving apart from separation gel lower curtate 1～2cm.Take out gel, dyeing is 2 hours in dyeing liquor, vibration simultaneously, and dyeing is finished, and it is transparent to the gel background to decolour with destaining solution.

Gel after the decolouring is put CS-9300PC type thin layer chromatogram scanner, do spectral scan, determine that maximum absorption wavelength is 560 nanometers; At 560 nano wave length places electrophoretic band is scanned then, calculate the ratio of I type and III Collagen Type VI peak area, be the ratio of I, III Collagen Type VI.

3. result

One, blood-nourishing and brain-refreshing granules is to the influence of SHR Left ventricular mass index

Each dosage group of blood-nourishing and brain-refreshing granules all has the effect that reduces left ventricular mass, and blood-nourishing and brain-refreshing granules group and model group relatively have statistical significance (p＜0.01).The blood-nourishing and brain-refreshing granules group has the effect of the Left ventricular mass index of reduction, compares with model group, and the blood-nourishing and brain-refreshing granules group has statistical significance (p＜0.01), is better than Captopril group, sees Table 3.

Table 3 blood-nourishing and brain-refreshing granules is to the influence of SHR left side room index (x ± s)

Dosage

Group n LVW (g) BW (g) LVW/BW (mg/g)

(g/kg/d)

WKY organizes 10 0.600 ± 0.09 ^a257.90 ± 52.5 2.35 ± 0.19 ^b

Model group 10 0.907 ± 0.08 229.40 ± 49.9 4.08 ± 0.68

Bezoar pill for lowering blood pressure group 0.168g 10 0.858 ± 0.12 239.44 ± 34.7 3.62 ± 0.54

Captopril group 7.785mg 10 0.767 ± 0.17 ^c223.30 ± 42.5 3.45 ± 0.50 ^d

Blood-nourishing and brain-refreshing granules group 31.5 10 0.724 ± 0.09 ^e217.00 ± 28.3 3.35 ± 0.38 ^f

Annotate: compare with model group ^aP＜0.01, t=7.72; ^bP＜0.01, t=7.73; ^cP＜0.05, t=2.39; ^dP＜0.05, t=2.34;

^ep＜0.01，t＝4.65； ^fp＜0.0?1，t＝2.92； ^gp＜0.05，t＝2.54。

Two, blood-nourishing and brain-refreshing granules is to the influence of chamber, SHR left side collagen

Each medicine group all can reduce left ventricle collagen content and I, III Collagen Type VI ratio.Wherein the blood-nourishing and brain-refreshing granules group is having statistical significance aspect the left ventricle collagen content reducing, and each group all has statistical significance (p＜0.01) aspect I, the III Collagen Type VI ratio improving.Histological observation finds around the model group rat aorta large-area collagen hypertrophy is arranged, and the large tracts of land collagen distribution that is dispersed in is also arranged in the cardiac muscle, and obviously more than WKY group rat.Collagen distribution is also arranged around the blood-nourishing and brain-refreshing granules group blood vessel, but obviously be less than model group, also have in the cardiac muscular tissue to be dispersed in to be point-like or thread collagen distribution, also fewer, see Table 4,5; Accompanying drawing 3 is to shown in Figure 8.

Table 4 blood-nourishing and brain-refreshing granules is to the influence of chamber, SHR left side collagen content (x ± s)

Dosage collagen

Group n

(g/kg/d) (μ g/mg cardiac muscle dry powder)

WKY organizes 10 28.19 ± 5.29 ^b

Model group 10 36.39 ± 5.92

Bezoar pill for lowering blood pressure group 0.168g 9 32.63 ± 4.82

Captopril group 7.785mg 10 30.74 ± 7.89

Blood-nourishing and brain-refreshing granules group 31.5 10 30.11 ± 7.32 ^a

Annotate: compare with model group ^aP＜0.05, t=2.10; ^bP＜0.01 t=3.27.

Table 5 blood-nourishing and brain-refreshing granules is to the influence of SHR left side chamber I, III collagen ratio (x ± s)

Dosage

Group n I, III collagen ratio

(g/kg/d)

WKY organizes 10 2.44 ± 0.85 ^a

Model group 9 6.03 ± 2.01

Bezoar pill for lowering blood pressure group 0.168g 9 2.81. ± 1.23 ^b

Captopril group 7.785mg 7 3.00 ± 1.21 ^c

Blood-nourishing and brain-refreshing granules group 31.5 9 2.69 ± 1.12 ^d

Annotate: compare with model group ^aP＜0.01, t=5.16; ^bP＜0.01, t=4.09; ^cP＜0.01, t=3.5 1; ^dP＜0.01, t=4.36; ^eP＜0.01, t=4.33; ^fP＜0.01, t=4.07.

Three, blood-nourishing and brain-refreshing granules is to the influence of chamber, SHR left side tectology

Morphological observation shows that model group rat heart muscle fibre thickening, spacing are dwindled; Or the increase of cardiac muscle fiber atrophy spacing, the myocardium swelling of part gap is unclear, some cardiac muscle fiber interruptions arrangements disorder, and comparatively tangible large tracts of land cardiac muscle hydropic degeneration, cellular content becomes graininess, and fracture is merged, even necrosis is arranged, vessel wall thickening; The distribution of blood-nourishing and brain-refreshing granules group cardiac muscle fiber approaches normally, and above-mentioned lesion degree is also lighter, and myocardial cell hydropic degeneration, fracture and the necrosis of kitchen range are arranged.As accompanying drawing 9 to shown in Figure 16.Transmission electron microscope results shows that distance increases between the model group myocardial cell, and blood capillary is also visible narrow or closed, the visible platelet adhesion of narrow blood vessel intracavity, and endotheliocyte drink vacuole increases, and the basement membrane material thickens on every side; The outer visible a large amount of courageous and upright material of blood vessel oozes out, organelles such as visible therebetween monokaryon phagocyte, fibroblast, erythrocyte and mitochondrion, lysosome, and collagen fiber obviously increase and heart cell secretion collagen phenomenon, and as seen bigger blood vessel thickens.Blood-nourishing and brain-refreshing granules group capillary structure is better, do not have and obviously ooze out, tube chamber is open, vascular endothelial cell is near parenchyma, tube chamber is obviously open, does not have the blood plasma material on every side and oozes out or only have on a small quantity and ooze out, not organelles such as show cell and mitochondrion, lysosome, a small amount of collagen material calmness, cell tissue is better.Figure 17 is to shown in Figure 22.

Below as can be seen, blood-nourishing and brain-refreshing granules can be improved the LVH that long-term hypertension causes, and reduces myocardium interstitial collagen protein content, reduces the ratio of myocardium I, III Collagen Type VI, thereby improve the framework of myocardial collagen net, prevent or alleviated the process of LVH and left ventricular remodeling.The performance of tectology is consistent with The above results.

Third part: blood-nourishing and brain-refreshing granules is to the influence of spontaneous hypertensive rat left ventricle gene expression profile

This experiment is in clear and definite blood-nourishing and brain-refreshing granules blood pressure lowering and alleviate on the basis of left ventricular hypertrophy, further adopts the influence of biochip technology research blood-nourishing and brain-refreshing granules to SHR left ventricle related gene expression, inquires into this medicine to alleviating the mechanism of ventricular hypertrophy.

1. laboratory animal: first.

2. reagent: TRIzol, isopropyl alcohol, chloroform, 75% ethanol (RNase-free), Milli-Q water (RNase-free) dehydrated alcohol, dNTPs, Cy5-dCTP and Cy3-dCTP, hybridizing reagent 1, labelled reagent I, hybridizing reagent 2, labelled reagent II, reverse transcription, labelled reagent III, reverse transcription primer, the reagent 1 of developing a film, reverse transcription, buffer, the reagent 2 of developing a film, DTT, the reagent 3 of developing a film, 4000 cDNA chips of rat (R40s).Mentioned reagent provides by Boxing Gene Chip Co., Ltd., Shanghai

3. instrument and equipment: electric driven glass refiner, electronic balance, low-temperature and high-speed centrifuge, low-temperature and high-speed desk centrifuge, superclean bench, ice machine, electric heating constant temperature tank, electrophoresis tank, electrophresis apparatus, microwave oven, gel imaging instrument, desk centrifuge, nucleic acid quantification analyser, liquid-transfering gun, adjustable electric furnace, vortex mixer, hybridization case, hybridization cabin, S-200 purification column, vacuum concentration instrument, coverslip, ScanArry4000 chip scanner

4. experimental technique

(1) grouping and administration: same first, but only observe SHR model group and blood-nourishing and brain-refreshing high dose group in this experiment.Choose and gavage each 10 of the SHR that raise blood-nourishing and brain-refreshing granules 8 all SHR and the same period, weigh, put to death rat, open breast and take out heart rapidly, with the normal saline flushing that DEPC handled, clean residual blood, in the about 100mg of left ventricle apex of the heart position clip cardiac muscular tissue, put into frozen pipe rapidly, (the tissue clip carries out on ice in preservation in the input liquid nitrogen; The used apparatus of whole process, tinfoil, beaker etc. are all used the 0.1%DEPC solution-treated, high-temperature sterilization or oven for baking sterilization).Per 5 apexes of the heart organize mixed in equal amounts as 1 sample in each group, 2 samples of medicine group, and 2 samples of model group carry out total RNA respectively and extract.

(2) total RNA extracts

Behind the samples weighing that ultralow temperature is preserved, be transferred in the stone roller alms bowl with the liquid nitrogen pre-cooling,, constantly add liquid nitrogen therebetween with the pestle tissue of milling, Powdered until being milled into, be transferred in the homogenate pipe that adds an amount of TRIzol reagent, place ice bath, carry out homogenate.Homogenate is transferred in the 15mL centrifuge tube, in 4 ℃, 12000g, centrifugal 10min.Draw supernatant and change new 15mL centrifuge tube over to, place 5min at 15-30 ℃.In homogenate, add chloroform, cover tight centrifuge tube lid, firmly shake centrifuge tube, place 3min at 15-30 ℃.In 4 ℃, 12000g, centrifugal 15min.From centrifuge, take out centrifuge tube carefully, draw supernatant to another 15mL centrifuge tube.Upwards reset and add into isopropyl alcohol, put upside down the abundant mixing liquid of centrifuge tube gently, place 10min at 15-30 ℃.In 4 ℃, 12000g, centrifugal 10min.Supernatant discarded adds 75% ethanol 5mL along tube wall lentamente, puts upside down washing centrifuge tube tube wall gently, carefully discards ethanol.Add 75% ethanol 10mL again, of short duration vortex on vortice; In 4 ℃, the centrifugal 10min of 8000g.Carefully abandon supernatant, of short duration centrifugal, remove all supernatants, drying precipitated 5min in superclean bench with the liquid-transfering gun suction.The Milli-Q water that adds RNase-ffee dissolves the RNA post precipitation ,-80 ℃ of preservations fully.

(3) probe mark and hybridization

Preparation prehybridization solution: in the Eppenderf pipe that hybridizing reagent 1 joins, behind the vibration mixing, add hybridizing reagent 2 mixings.The prehybridization solution for preparing is put into 95 ℃ of water-bath degeneration 2min, the slide for the treatment of prehybridization is put into 95 ℃ of water-bath degeneration 30sec, slide is promptly put into dehydrated alcohol 30sec after taking out, and dries.The prehybridization solution of degeneration is added in the point sample zone of slide, and covered is put into 42 ℃ of prehybridization 5～6hr of hybridization case.

Label probe: Cy5-dCTP labelling blood-nourishing and brain-refreshing granules medicine group, Cy3-dCTP labelling SHR model group.(following carry out in ice bath) adds following reagent (reacting final volume is 50 μ L, and following reagent is RNase-free) successively in a sterilized 1.5mL Eppendorf pipe: ddH2O, 23 μ L; Reverse transcriptase primer, 5 μ L; Total RNA, 50～100 μ g

The vibration mixing places 70 ℃ of water-bath 10min.After the taking-up, place on ice rapidly.Add following reagent respectively: reverse transcriptase buffer, 10 μ L; DTT, 5 μ L; DNTPs, 4 μ L.Then in the darkroom, add following reagent: reverse transcriptase, 2 μ L; Cy5-dCTP or Cy3-dCTP, 3 μ L.

Beat tube wall with the mixing sample with the finger bullet, maniluvium 2min.The Eppendorf pipe is placed 42 ℃ of water-bath 2h.In the Eppendorf pipe, add labelled reagent I 4 μ L successively, add labelled reagent II 4 μ L behind 65 ℃ of water-bath 10min.Mixing merges matched group, experimental group.Lucifuge, vacuum are drained to the 50 μ L.Use DNA purification column (or ethanol precipitation) purify DNA.Cylinder thermal agitation on vortex mixer is shaken up molten resin in suspending.The vertical small cap of post is unscrewed 1/4th circles, break the seal head of disconnected post lower end off with the fingers and thumb.Post is placed the Eppendorf pipe of a 1.5mL, post is placed another new 1.5mL Eppendorf pipe, remove vertical medicated cap, sample slowly is added to the centre of resin upper surface, note not stirring cylinder with the centrifugal 1min of 3000rpm.With the centrifugal 2min of 3000rpm, purified sample flows out, and is collected in the Eppendorf pipe of supporting usefulness.Add labelled reagent III 8 μ L, vacuum is drained.

Hybridization: add 6.5 μ L hybridizing reagent I in the probe tube of draining, fully mixing makes the probe dissolving.Add 6.5 μ L hybridizing reagent II again, mixing is standby.The slide of prehybridization is taken out, use ddH ₂O washes away coverslip.Probe is placed 95 ℃ of water-bath degeneration 2min; Slide places 95 ℃ of water-bath degeneration 30sec, and slide takes out and soaks dehydrated alcohol 30sec, and probe places on ice rapidly after taking out.Probe is placed on the chip, cover, place the hybridization cabin,, put into the hybridization of 42 ℃ of hybridization casees and spend the night (16～18h) with the Parafilm sealing with coverslip.

Develop a film: the cleaning mixture 1 flushing slide with 0.5%, remove coverslip.Prepare two color jars, the reagent 2 of developing a film of 0.5% the reagent 1+2% that develops a film, 5% the reagent 3 of developing a film are housed respectively, put into 60 ℃ of water-baths.Slide immersed in above two color jars successively wash 10min.Cleaning mixture 1 flushing slide with 0.5% dries back scanning.

(4) detect and analyze: gene chip carries out the laser scanning of two wavelength, reuse ImaGene3.0 software analysis cy3, two kinds of fluorescence signal intensities of cy5 and ratio by the ScanArray4000 scanner that General Scanning company produces.The cy3 mark intensity of all data item is multiplied by the Normalize coefficient, draws adjusted cy3 ^*Calculate the adjusted Ratio value of all genes (cy5/cy3 ^*).Filter out Ratio greater than 2 or less than 0.5 data, Ratio＞2 these genes of explanation are high expressed in the blood-nourishing and brain-refreshing group, and Ratio＜0.5 this gene of explanation is expressed for low in the blood-nourishing and brain-refreshing group.

5. result

1. the gene expression profile of differential expression: the gene expression profile of scanning spectra, X-axis, Y-axis are coordinate with cy3 fluorescence intensity level and cy5 fluorescence intensity level respectively, each data point is represented the hybridization signal of a gene point on the chip, more shows that near X-axis or Y-axis this point gene differential expression significantly.If data point is red, the ratio of then representing Y value and X value belongs to non-differential expression substantially between 0.5 to 2.0; If data point is yellow, then represent the ratio (this point belongs to differential expression probably) outside 0.5 to 2.0 scope of Y value and X value.See accompanying drawing 23,24.

2. the gene number of differential expression: two chip positive genes are overlapping 20, and wherein the gene of high expressed has 4, low express have 16.Unknown gene has 14, and known has 6 to see the following form.

Gene title Ratio1 Ratio2

Glycine?methyltransferase(Gnmt) 0.353 0.132

LTBP-2?like?protein(Ltbp2) 0.451 0.212

beta-galactoside-alpha?2，6-sialyltransferase 0.485 0.437

CD36?antigen 2.251 2.743

heart?fatty?acid?binding?protein(Fabp3) 2.002 3.610

Propionyl?Coenzyme?A?carboxylase，beta?polypeptide(Pccb) 2.010 2.174

Blood-nourishing and brain-refreshing granules is by collaborative the playing a role of last mediation downward modulation several genes.Its improvement may be relevant with the LTBP-2 down regulation of gene expression with the mechanism that alleviates LVH and MF.Gnmt is intravital a kind of enzyme, is the key protein matter of methyl metabolism in the control agent, and its down-regulated expression is for guaranteeing that the supply based on methyl group in the body of S-adenosylmethionine has important function.CD36, Fabp3, Pccb are important active substances in the body, and in the metabolism of regulating materials such as heart fat acid, cholesterol, protecting myocardial cell prevents that from all there is positive effect the atherosclerosis aspect.

The 4th part: blood-nourishing and brain-refreshing granules is to the influence of SHR renal function and form

This experiment is inquired into the kidney protective effect of blood-nourishing and brain-refreshing granules by observing the change of SHR kidney of rats function and pathology.

1 material and method

1.1 laboratory animal: SHR rat and WKY rat, 13 ages in week, body weight 210.89 ± 39.34g, this especially big Mus (WKY) of Tokyo prestige, male and female half and half, body weight 220.17 ± 37.67g, available from Shanghai hypertension institute, the quality certification number: No. the 02631st, the moving word of doctor, raise in conventional animal room laminar flow cabinet, 26 ± 2 ℃ of room temperatures, humidity constant humidity (55 ± 5%) is divided cage by male and female, 5 in every cage, freely drink water, standard feed is fed, the environment peace and quiet.

1.2 main agents and instrument: blood urea nitrogen (BUN) (lot number 020701), serum creatinine (Scr) (lot number 320041), uric acid (UA) (lot number 380031) test kit, available from middle Bei Bei control biotech inc, instrument: 95 editions semi-automatic biochemical analyzers of 200 types (originating from Dutch prestige figure)

1.3 medicine: blood-nourishing and brain-refreshing granules is provided by Tianjin Tasly Pharmaceutical Co., Ltd, lot number: 20020407.Captopril: produce lot number: 20010901 by Tianjin Hua Xin pharmaceutical factory.Bezoar pill for lowering blood pressure: be Darentang Traditional Chinese Medicine Factory, Tianjin's product, lot number: C484035.

1.4 grouping and administration: measuring blood pressure training every day is 1 time before the formal experiment, continuously 7d.Treat that rat conforms, behind the blood pressure stabilization, SHR be divided into 6 groups at random, i.e. blood-nourishing and brain-refreshing granules high dose group 31.56g/kg/d; Nine groups of 0.168g/kg/d of Calculus Bovis blood pressure lowering, captopril 7.78mg/kg/d, model group and WKY matched group give the distilled water of equal volume, and route of administration is for irritating stomach, and administration time is regularly administration of every morning, freely ingests and drinks water, and feeds for 8 weeks altogether.

1.5 renal function index is measured: after eight weeks of perfusion, 3% pentobarbital sodium anesthesia, the carotid artery intubate is got the about 5ml of blood, low-temperature centrifugation 3500r, 15 minutes.Get serum,, detect above index at 200 type semi-automatic biochemical analyzers according to Scr, BUN, the strict operation of UA test kit description.

1.6 collection of specimens: get rat one side kidney, remove peplos, get the upper pole piece branch put people's 10% neutral formalin liquid soak fixing, conventional dehydration, paraffin embedding.Other gets 1mm ³Renal cortex, 2.5% glutaraldehyde is 2h fixedly, is used to make electron microscope specimen.The thick about 4 μ m of paraffin section, VG and HE dyeing.The conventional embedding of Electronic Speculum, the ultrathin section location, ultrathin section is through the two dyeing of a plumbous uranium, and JEM-1200EX type transmission electron microscope observing is also taken pictures.

1.7 statistical procedures: all indexs all use mean ± standard deviation to represent, adopt T check between SPSS10.0 work group.

2. experimental result

2.1 blood-nourishing and brain-refreshing granules is to the influence of SHR renal function

Cr, Ua content in 22 all SHR rat blood serums all are significantly higher than the normal control group, P＜0.01.Each administration group all obviously reduces, and is the most obvious with the blood-nourishing and brain-refreshing granules group, compares with model group, and Scr has significant difference, t=2.690, P＜0.001; Ua all has downward trend in each administration group, and the blood-nourishing and brain-refreshing granules group has statistical significance, high dose t=2.580, P＜0.01, low dosage t=2.452, P＜0.05; BUN Bezoar pill for lowering blood pressure group has significant difference, P＜0.05.See Table 3 and Fig. 3,4,5.

The 5th part. blood-nourishing and brain-refreshing granules influences spontaneous hypertensive rat (SHR) Re-A-A

The purpose of this experiment is to study blood-nourishing and brain-refreshing granules treatment spontaneous hypertensive rat (SHR) and inquires into it to circulation and nephridial tissue local renin-angiotensin system (renin-angiotensin system, RAS) active influence.

1 material and method

1.1 laboratory animal: the same.

1.2 main agents and instrument: Angiotensin II (Ang II), aldosterone (Ald) test kit, available from the clean auspicious company in Tianjin.Instrument SN-695B type is put and is exempted from the γ measuring instrument.Ang II variation within batch difference＜5%, batch variation are all＜10%; Ald variation within batch difference＜10%, batch variation are all＜15%.

1.3 medicine: the same

1.4 grouping and administration: the same

1.5 nervous plain I, II in the blood plasma, aldosterone are measured: experiment 8 week back drug withdrawals, in next day weigh (BW), with pentobarbital sodium 3% intraperitoneal anesthesia, the carotid artery intubate is got blood 5ml, that puts into pre-cooling respectively contains aprotinin, EDTA-Na ₂In the anticoagulant tube, shake up, put back in the ice-water bath immediately and cool off, 4 ℃ then, 1000 rev/mins, centrifugal 10 minutes, to get blood plasma and put-20 ℃ of preservations, radioimmunoassay method is measured.

1.6 the nervous plain II in the renal tissue, the mensuration of aldosterone: after anesthesia at 8 weekends is put to death, win the laboratory animal kidney rapidly, be stored in-80 ℃ of refrigerators behind the liquid nitrogen freezing.Get the 400mg renal tissue and shred back adding 5mL0.5mol/L acetic acid solution, again test tube is put into 100 ℃ of water-baths and boiled 15min, cooling back homogenate.Then at 4 ° of low-temperature centrifugations 20 minutes (1400r/min).Get supernatant, deposit for-20 ℃, radioimmunoassay method is measured.

2. experimental result,

2.1 blood-nourishing and brain-refreshing granules is to the influence of SHR plasma angiotensinogen-aldosterone system

There is not notable difference between each group of the nervous plain I of each administration group blood plasma medium vessels.ALD blood-nourishing and brain-refreshing granules group, Captopril group and Bezoar pill for lowering blood pressure group all are lower than model group, and significant difference is arranged.Blood-nourishing and brain-refreshing granules group t=4.101, P＜0.001.See Table 6.

Table 6 is respectively organized the nervous plain and aldosterone measurement result of blood plasma medium vessels (x ± s)

Group N Ang I AngII (pg/ml) Ald (ng/ml)

WKY organizes 10 8.9 ± 1.3 332.8 ± 88.2 0.24 ± 0.04 ^▲

Model group 10 9.0 ± 1.0 349.7 ± 93.6 0.31 ± 0.07

Captopril group 10 9.1 ± 0.5 216.8 ± 137.1 0.25 ± 0.07 ^▲

Calculus Bovis blood pressure lowering group 10 9.3 ± 0.6 294.9 ± 131.8 0.25 ± 0.07 ^▲

Blood-nourishing and brain-refreshing granules group 10 9.3 ± 0.6 287.8 ± 229.3 0.21 ± 0.02 ^▲

With model group comparison ▲ P＜0.05 ▲ ▲ P＜0.01 ▲ ▲ ▲ P＜0.001

2.2 blood-nourishing and brain-refreshing granules is to the influence of nervous element-aldosterone system in the SHR renal tissue

Blood-nourishing and brain-refreshing granules group Ang II content is lower than model group, significant difference is arranged, t=7.070, P＜0.001.Captopril group and Bezoar pill for lowering blood pressure group all are lower than model group, and significant difference is also arranged, P＜0.05; Blood-nourishing and brain-refreshing granules makes the Ald content in the SHR nephridial tissue descend t=2.956, P＜0.01.Captopril also has significant difference, t=2.706, P＜0.05.See Table 7.

The nervous plain II of each administration group nephridial tissue medium vessels of table 7, aldosterone content (x ± s)

Group N Ang II (pg/mg) Ald (pg/mg)

WKY organizes 10 6.91 ± 3.38 ^{▲ ▲ ▲}15.00 ± 4.50 ^▲

Model group 10 11.9 ± 1.96 20.25 ± 4.10

Captopril group 10 9.35 ± 3.25 ^▲15.00 ± 4.50 ^▲

Bezoar pill for lowering blood pressure group 10 7.59 ± 1.53 ^▲16.50 ± 6.38

Blood-nourishing and brain-refreshing granules group 10 6.25 ± 1.31 ^{▲ ▲ ▲}14.63 ± 5.63 ^▲

With model group comparison ▲ P＜0.05 ▲ ▲ P＜0.01 ▲ ▲ ▲ P＜0.001

SHR treatment blood-nourishing and brain-refreshing granules can make the Ald content in circulation blood, the nephridial tissue obviously descend after 8 weeks, and nephridial tissue Ang II content is also descended, and is excellent with middle high dose especially.Ald, Ang II generate the performance hypotensive effect to this explanation blood-nourishing and brain-refreshing granules in the kidney by reducing, and stop the kidney pathological lesion, produce the kidney protective effect.See Table 8.

Table 3 is respectively organized renal function index result (x ± s)

Group N Scr (μ mol/L) UA (μ mol/L) BUN (mg/dl)

WKY organizes 10 48.3 ± 6.8 ^{△ △}127.5 ± 34.7 ^{△ △ △}26.2 ± 5.8

Model control group 10 55.2 ± 7.2 194.4 ± 56.2 26.1 ± 5.1

Captopril group 10 50.3 ± 9.9 148.4 ± 23.8 ^△25.5 ± 4.3

Bezoar pill for lowering blood pressure group 10 49.5 ± 4.9 149.4 ± 41.1 ^△20.4 ± 3.3 ^△

Blood-nourishing and brain-refreshing granules group 10 46.2 ± 7.9 ^{△ △ △}147.4 ± 20.1 ^{△ △}22.8 ± 3.8

Compare △ P＜0.05 △ △ P＜0.01 with model control group

2.2 nephridial tissue pathological manifestations

VG and HE dyeing observed result: the visible glomerule silght enlargement of model group, kidney small artery tube wall thickens, interstitial cell hyperplasia around the tremulous pulse.Inflammatory exudation and inflammatory cell infiltration are arranged in the renal cortex.Can see collagen fiber around the tremulous pulse and increase, the flesh layer plumpness of tremulous pulse, inner membrance proliferation of fibrous tissue, the narrow and small even locking of tube chamber, indivedual slight atrophys of glomerule.Focal interstitial cell hyperplasia is many with glomerule on every side.Glomerule shows as hypertrophy in various degree, the corresponding hypertrophy of renal tubules, visible protein cast; Between the visible lymphocyte of matter, mononuclear cell be kitchen range infiltration and fibrosis.Blood-nourishing and brain-refreshing granules group and model group comparing difference are obvious, except that the slight hypertrophy of part Interstitial cell, most of visible glomerule recovers normally not see obvious interstitial cell hyperplasia on every side substantially, inflammatory cell and inflammatory exudation and collagen hypertrophy are not obvious, do not see that glomerule increases and atrophy.(seeing accompanying drawing 25 to 28)

The electron microscopic observation result: the narrow or complete closure of capillary lumen between the visible renal tubules of model group, endotheliocyte drink vacuole obviously increases, rare normal capillary structure; Blood vessel has a large amount of blood plasma materials to ooze out outward, wherein visible RBC, mitochondrion, lysosome and collagen fiber calmness.The damage of part renal cells, organelle overflows.Narrow in the blood-nourishing and brain-refreshing granules group nephridial tissue, the closure except that the minority blood capillary, most of capillary structure is better, do not have and obviously ooze out, tube chamber is open, vascular endothelial cell is obviously open near, excess of the kidney cell plastid tube chamber, does not have the blood plasma material on every side and oozes out or only have on a small quantity and ooze out, do not see mitochondrion, lysosome, RBC and collagen material calmness, do not see disorganization.(seeing accompanying drawing 29,30)

By this experiment as can be known, after blood-nourishing and brain-refreshing granules treated for 8 weeks, BUN, Scr, the Ua of treatment group SHR obviously reduced; Tissue slice is not seen glomerular sclerosis, and kidney small artery tube wall, tube chamber are near normal.Illustrate that blood-nourishing and brain-refreshing granules when reducing the SHR blood pressure, has good minimizing hamartoplasia effect, can delay the infringement of renal function, thereby play the effect of protection kidney.

The 6th part: blood-nourishing and brain-refreshing granules is to the influence of spontaneous hypertensive rat kidney gene expression profile

This experiment is on the basis of clear and definite blood-nourishing and brain-refreshing granules blood pressure lowering and alleviating hypertension renal damage, further adopt of the influence of biochip technology research blood-nourishing and brain-refreshing granules, inquire into the mechanism of this medicine blood pressure lowering and alleviating hypertension renal damage to SHR kidney related gene expression.

1. material

1.1 laboratory animal: the same.

1.2 reagent: TRIzol, isopropyl alcohol, chloroform, 75% ethanol (RNase-free), Milli-Q water (RNase-free) dehydrated alcohol, dNTPs, Cy5-dCTP and Cy3-dCTP, hybridizing reagent 1, labelled reagent I, hybridizing reagent 2, labelled reagent II, reverse transcription, labelled reagent III, reverse transcription primer, the reagent 1 of developing a film, reverse transcription, buffer, the reagent 2 of developing a film, DTT, the reagent 3 of developing a film, 4000 cDNA chips of rat (R40s).Mentioned reagent provides by Boxing Gene Chip Co., Ltd., Shanghai

1.3. instrument and equipment: electric driven glass refiner, electronic balance, low-temperature and high-speed centrifuge, low-temperature and high-speed desk centrifuge, superclean bench, ice machine, electric heating constant temperature tank, electrophoresis tank, electrophresis apparatus, microwave oven, gel imaging instrument, desk centrifuge, nucleic acid quantification analyser, liquid-transfering gun, adjustable electric furnace, vortex mixer, hybridization case, hybridization cabin, S-200 purification column, vacuum concentration instrument, coverslip, ScanArry4000 chip scanner

2. method

2.1 grouping and administration: the same, observe SHR model group and blood-nourishing and brain-refreshing group.

Choose and gavage each 10 of the SHR that raise blood-nourishing and brain-refreshing granules 8 all SHR and the same period, weigh, put to death rat, open abdomen and take out kidney rapidly, with the normal saline flushing that DEPC handled, clean residual blood, in the about 100mg nephridial tissue of kidney middle part clip, put into frozen pipe rapidly, (the tissue clip carries out on ice in preservation in the input liquid nitrogen; The used apparatus of whole process, tinfoil, beaker etc. are all used the 0.1%DEPC solution-treated, high-temperature sterilization or oven for baking sterilization).Per 5 renal tissue mixed in equal amounts are as 1 sample in each group, 2 samples of medicine group, and 2 samples of model group carry out total RNA respectively and extract.

2.2 total RNA extracts

Behind the samples weighing that ultralow temperature is preserved, be transferred in the stone roller alms bowl with the liquid nitrogen pre-cooling,, constantly add liquid nitrogen therebetween with the pestle tissue of milling, Powdered until being milled into, be transferred in the homogenate pipe that adds an amount of TRIzol reagent, place ice bath, carry out homogenate.Homogenate is transferred in the 15mL centrifuge tube, in 4 ℃, 12000g, centrifugal 10min.Draw supernatant and change new 15mL centrifuge tube over to, place 5min at 15-30 ℃.In homogenate, add chloroform, cover tight centrifuge tube lid, firmly shake centrifuge tube, place 3min at 15-30 ℃.In 4 ℃, 12000g, centrifugal 15min.From centrifuge, take out centrifuge tube carefully, draw supernatant to another 15mL centrifuge tube.Upwards reset and add into isopropyl alcohol, put upside down the abundant mixing liquid of centrifuge tube gently, place 10min at 15-30 ℃.In 4 ℃, 12000g, centrifugal 10min.Supernatant discarded adds 75% ethanol 5mL along tube wall lentamente, puts upside down washing centrifuge tube tube wall gently, carefully discards ethanol.Add 75% ethanol 10mL again, of short duration vortex on vortice; In 4 ℃, the centrifugal 10min of 8000g.Carefully abandon supernatant, of short duration centrifugal, remove all supernatants, drying precipitated 5min in superclean bench with the liquid-transfering gun suction.The Milli-Q water that adds RNase-free dissolves the RNA post precipitation ,-80 ℃ of preservations fully.

2.3 probe mark and hybridization

Label probe: Cy5-dCTP labelling blood-nourishing and brain-refreshing granules medicine group, Cy3-dCTP labelling SHR model group.(following carry out in ice bath) adds following reagent (reacting final volume is 50 μ L, and following reagent is RNase-free): ddH successively in a sterilized 1.5mL Eppendorf pipe ₂O, 23 μ L; Reverse transcriptase primer, 5 μ L; Total RNA, 50～100 μ g

3. detect and analyze: gene chip carries out the laser scanning of two wavelength, reuse ImaGene3.0 software analysis cy3, two kinds of fluorescence signal intensities of cy5 and ratio by the ScanArray4000 scanner that General Scanning company produces.The cy3 mark intensity of all data item is multiplied by the Normalize coefficient, draws adjusted cy3 ^*Calculate the adjusted Ratio value of all genes (cy5/cy3 ^*).Filter out Ratio greater than 2 or less than 0.5 data, Ratio＞2 these genes of explanation are high expressed in the blood-nourishing and brain-refreshing group, and Ratio＜0.5 this gene of explanation is expressed for low in the blood-nourishing and brain-refreshing group.

4. result

The nephridial tissue difference expression gene compares: select spontaneous hypertensive rat SHR model group to make the matched group of SHR Chinese drug-treated group, the ratio of first two kinds of fluorescence signal is less than 0.5 or have 389 greater than 2.0 gene in two chips, wherein the gene of expressing down has 273, and 209 energy land on Genbank; 135 of the genes of last expression, 116 genes can land on Genbank; The ratio of second two kinds of fluorescence signal is less than 0.5 or have 170 greater than 2.0 gene, and wherein the gene of expressing down has 120, and 87 energy land on Genbank; 50 of the genes of last expression, 42 genes can land on Genbank.Renal tissue sample (matched group/experimental group) Two Colour Fluorescence labelling stacking chart explanation: for certain any two kinds of stack fluorescence signals, if the cy3 signal is stronger, this point shows green (down regulation trend); If the cy5 signal is stronger, this some redness (rise trend) that show more; If intensity is similar, i.e. displaing yellow.

In renal tissue sample (matched group/experimental group) the hybridization signal intensity scatterplot, X-axis, Y-axis are coordinate with cy3 fluorescence intensity level (=prospect value-background value) and cy5 fluorescence intensity level respectively, and each data point is represented the hybridization signal of a gene point on the chip; If data point is red, the ratio of then representing Y value and X value belongs to non-differential expression substantially between 0.5 to 2.0; If data point is yellow, then represent the ratio (this point belongs to differential expression probably) outside 0.5 to 2.0 scope of Y value and X value.This shows that blood-nourishing and brain-refreshing granules is by collaborative the playing a role of last mediation downward modulation several genes, has changed SHR rat kidney gene expression profile, the gene of these changes may be relevant with renal damage mechanism with improvement and alleviating hypertension.

Description of drawings

Fig. 1, single-dose are to the influence of SHR rat systolic pressure, and wherein A is a model group, and B is a Calculus Bovis blood pressure lowering group, and C is the captopril matched group, and D is the blood-nourishing and brain-refreshing granules group;

Fig. 2, blood-nourishing and brain-refreshing granules are to the influence of spontaneous hypertensive rat systolic pressure, and wherein A is a model group, and B is a Calculus Bovis blood pressure lowering group, and C is the captopril matched group, and D is the blood-nourishing and brain-refreshing granules group;

Fig. 3, WKY matched group (around the blood vessel) VG * 100;

Fig. 4, model control group (around the blood vessel) VG * 100;

Fig. 5, blood-nourishing and brain-refreshing granules group (around the blood vessel) VG * 100

Fig. 6, WKY matched group (cardiac muscular tissue) VG * 100

Fig. 7, model control group (cardiac muscular tissue) VG * 100

Fig. 8, blood-nourishing and brain-refreshing granules group (cardiac muscular tissue) VG * 100

Fig. 9, WKY matched group cardiac muscle (longitudinal section) HE * 200

Figure 10, WKY matched group cardiac muscle (cross section) HE * 400

Figure 11, model group cardiac muscle (longitudinal section) HE * 200

Figure 12, model group cardiac muscle (longitudinal section) HE * 200

Figure 13, model group cardiac muscle (cross section) HE * 200

Figure 14, blood-nourishing and brain-refreshing granules group cardiac muscle (longitudinal section) HE * 200

Figure 15, blood-nourishing and brain-refreshing granules group cardiac muscle (cross section) HE * 200

Figure 16, blood-nourishing and brain-refreshing granules group cardiac muscle (cross section) HE * 200

Figure 17, WKY matched group (blood capillary) * 7500

Figure 18, WKY matched group (blood capillary) * 13500

Figure 19, model group (blood capillary) * 6000

Figure 20, model group (blood capillary) * 13500

Figure 21, blood-nourishing and brain-refreshing granules group (blood capillary) * 13500

Figure 22, blood-nourishing and brain-refreshing granules group (blood capillary) * 13500

Figure 23, Two Colour Fluorescence labelling stacking chart

Figure 24, hybridization signal intensity scatterplot

Figure 25, model group glomerule and renal tubules HE * 400

Albumen HE in Figure 26, the model group renal tubules * 400

Figure 27, blood-nourishing and brain-refreshing granules group renal tubules HE * 400

Figure 28, blood-nourishing and brain-refreshing granules group glomerule HE * 400

Figure 29, model group kidney blood vessel Electronic Speculum picture * 3750

Figure 30, blood-nourishing and brain-refreshing granules group kidney blood vessel Electronic Speculum picture * 3750

Figure 31, Two Colour Fluorescence labelling stacking chart

Figure 32, hybridization signal intensity scatterplot

The specific embodiment

The present invention is further illustrated below in conjunction with embodiment, and following this embodiment only is used to the present invention is described and to the present invention without limits.

Embodiment 1

(1) gets Radix Angelicae Sinensis 80g, Rhizoma Chuanxiong 80g, Radix Paeoniae Alba 80g, Radix Rehmanniae Preparata 80g, Ramulus Uncariae Cum Uncis 120g, Caulis Spatholobi 120g, Spica Prunellae 120g, Semen Cassiae 120g, Concha Margaritifera 120g, Rhizoma Corydalis 70g, Herba Asari 10g;

(2) with above-mentioned medical material extracting in water 3 times, each 1 hour, merge extractive liquid, suitably concentrated, the ethanol that adds 2 times of amounts leaves standstill 24 hours precipitations, gets supernatant concentration and becomes extractum, relative density is 1.3, and paste-forming rate is 10%, and qinghuo reagent, sucrose and dextrin are made 200 in tablet in 1: 3: 1 ratio.

Embodiment 2

(1) gets Radix Angelicae Sinensis 67.5g, Rhizoma Chuanxiong 67.5g, Radix Paeoniae Alba 54g, Radix Rehmanniae Preparata 54g, Ramulus Uncariae Cum Uncis 135g, Caulis Spatholobi 135g, Spica Prunellae 135g, Semen Cassiae 135g, Concha Margaritifera 135g, Rhizoma Corydalis 67.5g, Herba Asari 14.5g;

(2) with above-mentioned medical material extracting in water 3 times, each 1 hour, merge extractive liquid,, suitably concentrate, add the ethanol of 2 times of amounts, leave standstill 24 hours precipitations, get supernatant concentration and become extractum, relative density is 1.3, and paste-forming rate is 10%, and qinghuo reagent, sucrose and dextrin are made 125 bags of granules in 1: 3: 1 ratio.

Claims

1. the application of pharmaceutical composition in preparation treatment hypertension disease medicament, wherein the weight proportion of each flavour of a drug is as follows: Radix Angelicae Sinensis 4～9%, Rhizoma Chuanxiong 4～9%, the Radix Paeoniae Alba 2～8%, Radix Rehmanniae Preparata 2～8%, Ramulus Uncariae Cum Uncis 10～15%, Caulis Spatholobi 10～15%, Spica Prunellae 10～15%, Semen Cassiae 10～15%, Concha Margaritifera 10～15%, Rhizoma Corydalis 4～9%, Herba Asari 0.5～2%.

2. the application of a kind of compositions according to claim 1 in preparation treatment hypertension disease medicament, wherein the weight proportion of each flavour of a drug is as follows:

3. the application of a kind of pharmaceutical composition according to claim 1 and 2 in preparation treatment hypertension disease medicament is characterized in that described hypertension is an essential hypertension.

4. the application of pharmaceutical composition in preparation reduction systolic pressure medicine, wherein the weight proportion of each flavour of a drug is as follows:

Radix Angelicae Sinensis 4～9%, Rhizoma Chuanxiong 4～9%, the Radix Paeoniae Alba 2～8%, Radix Rehmanniae Preparata 2～8%, Ramulus Uncariae Cum Uncis 10～15%, Caulis Spatholobi 10～15%, Spica Prunellae 10～15%, Semen Cassiae 10～15%, Concha Margaritifera 10～15%, Rhizoma Corydalis 4～9%, Herba Asari 0.5～2%.

5. the application of pharmaceutical composition in the left ventricular hypertrophy medicine of preparation reverse caused by hypertension, wherein the weight proportion of each flavour of a drug is as follows:

6. the application of pharmaceutical composition in the early stage myocardial fibrosis medicine of preparation reverse, wherein the weight proportion of each flavour of a drug is as follows:

7. a pharmaceutical composition delays application in the renal function injury medicine that hypertension causes in preparation, and wherein the weight proportion of each flavour of a drug is as follows: