CN108030797A - Application of the Flos Chrysanthemi extract in the medicine for preparing prevention myocardial hypertrophy - Google Patents
Application of the Flos Chrysanthemi extract in the medicine for preparing prevention myocardial hypertrophy Download PDFInfo
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Abstract
The present invention provides application of the pharmaceutical composition of Flos Chrysanthemi extract, Flos Chrysanthemi extract officinal salt, or Flos Chrysanthemi extract or Flos Chrysanthemi extract officinal salt pharmaceutic adjuvant composition acceptable with human body in the medicine of prevention myocardial hypertrophy or the disease based on myocardial hypertrophy is prepared；The present invention stimulates inducing mouse cardiac muscle using beta receptor agonist isoproterenol, replicates mouse cardiac muscle hypertrophy model；Using abdominal aorta constriction induced rat myocardial hypertrophy, heart hypertrophy in rats model is replicated, using the Chinese medicine Flos Chrysanthemi extract of various dose, is studied through pharmacodynamics Integral animal experiment, achieves the therapeutic effect of affirmative, provide the foundation for new medicament screen.
Description
(1) technical field
The present invention relates to the new application of Flos Chrysanthemi extract, and in particular to Flos Chrysanthemi extract is preparing prevention myocardial hypertrophy
Medicine in application.
(2) background technology
FLOS CHRYSANTHEMI ALBA from Haizhou of China (Chrysanthemum Morifolium Ramat, CM) is an important kind of China's chrysanthemum, quilt
It is classified as one of eight big, Zhejiang Province medicinal material " eight Zhe's ".The main place of production of FLOS CHRYSANTHEMI ALBA from Haizhou of China is Zhejiang Province Tongxiang City, and national health
One of genunie medicinal materials of integration of drinking and medicinal herbs of first approval of portion, FLOS CHRYSANTHEMI ALBA from Haizhou of China is version in 2010《Chinese Pharmacopoeia》The certified products chrysanthemum recorded
Flower, volatile oil, steroidal, triterpene, phenols, polysaccharide and the micro- main component for CM.
The pharmacological action to chrysanthemum has carried out substantial amounts of research to domestic and foreign scholars, it was demonstrated that chrysanthemum have antibacterial, it is antiviral,
Reduce blood pressure, prevent hyperlipidemia, anti-ageing a variety of pharmacological activity of waiting for a long time, part pharmacological activity and the anti-oxidant of it and removing free radical
Related, wherein flavonoids is the active ingredient that content is more in chrysanthemum extract, has good removing free radical, oxidation resistant
(Kuang Chunlin, Lv Dou, Huang Xia, Gao Wei, Shen radiance, the antioxidation activity of the clear FLOS CHRYSANTHEMI ALBA from Haizhou of China different solvents extract of Zhang Zhi are ground function
It is scientific and technological to study carefully food industry, 2015,21:83-87).In angiocardiopathy field, there is researcher's discovery, FLOS CHRYSANTHEMI ALBA from Haizhou of China water extract is to lacking
Blood injury of myocardium has obvious protective function, may be played by suppressing the generation, aggregation and release of inflammatory factor to ischemic myocardium
Protective effect (Huang Hao, Guo Huanyu, protection of the golden red peak FLOS CHRYSANTHEMI ALBA from Haizhou of China water extract to ischemic myocardium after dog acute myocardial infarction AMI
Act on Zhejiang combination of Chinese tradiational and Western medicine magazines 2011,21 (5):307-310).Flos Chrysanthemi extract (Chrysanthemum
Extract, CE) there is obvious protective function to ischemia injury cardiac muscle, including reduce myocardial infarction area, mitigate myocardial ischemia damage
Hinder scope and degree, the release of the reduction significant enzyme of myocardial damage, the reduction ischemic arrhythmia order of severity, mitigate lipid mistake
Oxidation, increase oxidation resistance (Zhou Xinmei, Yang Jun, the Effect study Zhejiang University masters of the anti-ischemic myocardium damages of summer strong .CE
Academic dissertation .2006.5).And renin angiotensin aldosterone system activation, oxidative stress, cardiac muscle cell apoptosis, cell because
Son triggering inflammatory reaction, NF- κ B overexpressions etc. may be with the development of myocardial hypertrophy and lapsing to closely related.Accordingly, we push away
Resisting cardiac hypertrophy may be played the role of by surveying CE.But CE is applied to relevant disease of the treatment based on myocardial hypertrophy both at home and abroad not
See research report.
(3) content of the invention
It is an object of the present invention to provide Flos Chrysanthemi extract (CE) in preparation prevention myocardial hypertrophy or based on myocardial hypertrophy
Relevant disease medicine in application.
Technical scheme is as follows：
The present invention provides Flos Chrysanthemi extract to prepare prevention myocardial hypertrophy or disease based on myocardial hypertrophy
Application in medicine.
The disease based on myocardial hypertrophy is for example：Hypertension, rheumatic heart disease, pulmonary heart disease, diabetes, expansion
Type cardiomyopathy, hypertrophic cardiomyopathy, coronary heart disease, miocardial infarction or anaemia etc..
The Flos Chrysanthemi extract is extracted by raw material FLOS CHRYSANTHEMI ALBA from Haizhou of China through drying, 80% (volume fraction, similarly hereinafter) ethanol water
Obtain.Specifically, the Flos Chrysanthemi extract is made as follows：
Fresh FLOS CHRYSANTHEMI ALBA from Haizhou of China is placed in air dry oven, 45 DEG C of dryings to constant weight, crushed, crossed 100 mesh sieves, obtain FLOS CHRYSANTHEMI ALBA from Haizhou of China
Powder, solid-liquid ratio 1 is pressed by gained FLOS CHRYSANTHEMI ALBA from Haizhou of China powder：15 (w/v, g/mL) are added in 80% ethanol water, ultrasonic extraction
30min, is collected by filtration filtrate, and filter residue repeats extraction 2 times, merges gained filtrate every time, is concentrated under reduced pressure, vacuum freeze-drying obtains described
Flos Chrysanthemi extract；Ultraviolet absorption method (UV methods) measure general flavone content is 33% ± 3%.
The Flos Chrysanthemi extract also can be commercially available on the market, specifically for example：Have purchased from Zhejiang Sheng Shi biotechnologies
The Flos Chrysanthemi extract of limit company, is pale yellow powder, smell delicate fragrance, raw material are than 10：1, general flavone mass fraction 36%.
The conventional amount used for recommending the Flos Chrysanthemi extract is 120~480mg/kg/d, preferably 150~300mg/kg/d,
More preferred 120~200mg/kg/d, most preferably 150mg/kg/d.Route of administration can be oral or injection administration.
The Flos Chrysanthemi extract can be made into the pharmaceutical preparation of following form：Flos Chrysanthemi extract piece, Flos Chrysanthemi extract
Sustained release tablets or Flos Chrysanthemi extract capsule etc., also, the pharmaceutical preparation is calculated as 120~240mg/ units with Flos Chrysanthemi extract
Metering.
Such as：Flos Chrysanthemi extract piece is calculated as 120~240mg/ pieces with Flos Chrysanthemi extract；The Flos Chrysanthemi extract delays
Release piece and 120~240mg/ pieces are calculated as with Flos Chrysanthemi extract；The Flos Chrysanthemi extract capsule is calculated as 120 with Flos Chrysanthemi extract
~240mg/ capsules.
The form of Flos Chrysanthemi extract officinal salt can also be made in the Flos Chrysanthemi extract, and (such as hydrochloric acid FLOS CHRYSANTHEMI ALBA from Haizhou of China carries
Take thing), or the form of pharmaceutic adjuvant acceptable with human body composition pharmaceutical composition, therefore, present invention additionally comprises the Hangzhoupro
Feverfew extract officinal salt or described pharmaceutical composition are preparing prevention myocardial hypertrophy or the disease based on myocardial hypertrophy
Medicine in application.
The beneficial effects are mainly as follows：The present invention stimulates induction using beta receptor agonist isoproterenol
Mouse cardiac muscle, replicates mouse cardiac muscle hypertrophy model；Using abdominal aorta constriction induced rat myocardial hypertrophy, rat heart muscle fertilizer is replicated
Thick model, using the Chinese medicine Flos Chrysanthemi extract of various dose, studies through pharmacodynamics Integral animal experiment, achieves controlling for affirmative
Therapeutic effect, provides the foundation for new medicament screen.
(4) illustrate
Fig. 1 is the change of each group murine myocardium SOD activity, MDA contents and Serum LDH activity in embodiment 1n
=10, compared with the control group,*P<0.01；Compared with Iso model groups,#P<0.05,##P<0.01。
Fig. 2 for 1 each group murine myocardium of embodiment change (HE dyeing, × 200,N=10)
A：Control group；B：Iso model groups；C：Iso+CE 120mg/kg/d treatment groups；D：Iso+CE 480mg/kg/d are controlled
Treatment group.
Fig. 3-I are 1 each group mouse cardiac myocytes pathological section of embodiment (Masson is dyed, × 200)；(N=10)
A：Control group；B：Iso model groups；C：Iso+CE 120mg/kg/d treatment groups；D：Iso+CE 480mg/kg/d are controlled
Treatment group.
Fig. 3-II are 1 each group mouse cardiac muscle interstitial fibrosis quantitative analysis of embodiment；
N=10, compared with the control group,*P<0.01；Compared with Iso model groups,#P<0.05,##P<0.01。
Fig. 4 changes (ERK1, × 200) for 1 each group mouse cardiac myocytes ERK1 protein expressions of embodiment；
A：Control group；B：Iso model groups；C：Iso+CE 120mg/kg/d treatment groups；D：Iso+CE 480mg/kg/d are controlled
Treatment group.
Fig. 5 changes (ERK2, × 200) for 1 each group mouse cardiac myocytes ERK2 protein expressions of embodiment；
A：Control group；B：Iso model groups；C：Iso+CE 120mg/kg/d treatment groups；D：Iso+CE 480mg/kg/d are controlled
Treatment group.
Fig. 6 quantitatively changes for 1 each group mouse cardiac myocytes ERK1/2 protein expressions of embodiment；
N=10, compared with the control group,*P<0.01；Compared with Iso model groups,#P<0.05,##P<0.01。
Fig. 7 is 2 each group rat heart muscle Histological change of embodiment (HE is dyed, × 200)；
A：Control group；B：AAC model groups；C：AAC+CE 120mg/kg/d treatment groups；D：AAC+CE 480mg/kg/d are controlled
Treatment group；E：AAC+ Valsartan 10mg/kg/d treatment groups (N=8).
Fig. 8 is the change of 2 each group abdominal aorta constriction hypertrophic myocardium tissue ANP, β-MHC of embodiment expression；
A：Control group；B：AAC model groups；C：AAC+CE 120mg/kg/d treatment groups；D：AAC+CE 480mg/kg/d are controlled
Treatment group；E：AAC+ Valsartan 10mg/kg/d treatment groups (N=8).
(5) embodiment
Below by specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Embodiment 1：At the beginning of Flos Chrysanthemi extract suppresses the plump effect of mouse cardiac muscle and its mechanism of isoprel induction
Step research
1 materials and methods
1.1 experimental animal：
Cleaning grade Kunming mouse, male and female have concurrently, and weight (20 ± 2) g, is provided by Zhejiang Province's Experimental Animal Center, qualified
Card number：SCXK (Zhejiang) 2015-0002, in Zhejiang Province, Experimental Animal Center barrier environment Animal Lab. is fed and is tested.
1.2 medicines and reagent：
Flos Chrysanthemi extract (CE), Zhejiang Shengshi Biological Product Co., Ltd, pale yellow powder, smell delicate fragrance, raw material ratio
10:1, general flavone mass fraction 36%；(the Shanghai grass tree pharmacy of isoprenaline hydrochloride (isoprenaline, Iso) parenteral solution
Co., Ltd, lot number 160408)；Lactic dehydrogenase (LDH), malonaldehyde (MDA) and total superoxide dismutase (T-SOD) test
Box, protein quantification testing cassete are purchased from Nanjing and build up bioengineering Co., Ltd；Rabbit-anti ERK1/2 antibody is purchased from U.S. Santa
Cruz Co., Ltds；Horseradish peroxidase, goat anti-mouse antibody IgG are purchased from cell signaling Co., Ltds of the U.S..
1.3 key instrument：
- 70 DEG C of low temperature refrigerators (U.S.'s Forma Products)；Ultraviolet imagery scanner (U.S.'s Bio-Rad Products)；
Foeodyne companies of the U.S. produce image analyzers；Millipore companies of the U.S. produce ultrapure water system；Refrigerated centrifuge and Low-temperature Ice
Case (German Heraeus products)；PCY instrument 9600 (PerkinElmer companies of the U.S.)；Microplate reader (U.S. Bio-Rad550)；Surely
Press current stabilization timing electrophoresis apparatus (Shanghai precision instrument factory)；722N visible spectrophotometers (the limited public affairs of Shanghai precision scientific instrument
Department)；MP200A types electronic balance (Shanghai Second Balance Factory)；(Chinese University of Science and Technology's Innovations have KDC-2046 refrigerated centrifuges
Zhong Jia branch companies of limit company)；Qwin image analysis softwares (German Leica Co., Ltds).
1.4 animal packets and modeling：
Healthy Kunming mouse 60 is taken, half male and half female, is randomly divided into：Blank (control) group, model group, model+CE are low
4 groups, every group 15 of dosage treatment group, model+CE high-dose therapies group.In addition to blank group, other each group mouse are subcutaneously injected
Iso, once a day, first day dosage are 40mg/kg, and second day is 20mg/kg, and the 3rd day is 10mg/kg, and the 4th day starts to protect
Hold 10mg/kg, continuous 14 days.Ad lib, feedwater, establishes myocardial hypertrophy model, the life of equivalent is subcutaneously injected in blank group daily
Brine is managed as control.
1.5 administration：
Administration and modeling are carried out at the same time, and after daily morning injection Iso 4h, give low, high dose CE 120mg/kg respectively
Treated with 480mg/kg gavages, once a day.Blank group and model group give normal saline gavage, once a day, continuous 4
Week.1.6 cardiac vegetative nerves measure：
Mouse fasting for solids but not liquids 12h after the last administration, claims mouse weight (body weight, BW), and eyeball takes blood to put to death,
Heart is taken out in rapid thoracic cavity of opening, and with the physiology salt washing residual blood to the greatest extent of precooling, filter paper weighs after blotting whole-heartedly weighs (heart
Weight, HW), then left ventricle is isolated, claim left ventricular mass (LVW), calculate heart weight index (HW/BW) and left ventricle weight respectively
Volume index (LVWI=LVW/BW).Another muscular tissue of coring makes tissue pathological slice, along atrioventricular ring cut off big blood vessel, atrium and
Right ventricular free wall, remaining interventricular septum, left ventricular free wall are placed in 10% formalin solution and fix 24h.Take out solid
Determine the left ventricle sample in liquid, rinsed through flowing water, graded ethanol dehydration is transparent, and routine paraffin wax embedding is every along long axis of left ventricle line
The section of 5 μ m-thicks is continuously cut every 1mm cross sections, observed under HE and Masson dyeing light microscopes, takes pictures.Observation cardiac muscle
Plump, fibrosis, analyze degree of myocardial fibrosis, each group myocardial histopathology is compared in analysis with Qwin image analysis softwares
Learn change.
1.7 Serum LDHs activity, cardiac muscular tissue SOD activity and MDA assays：
Eyeball of mouse takes blood after 4 DEG C, 2000r/min, centrifuges 10 minutes, separates serum, is said in strict accordance with LDH kits
Bright operation, measures LDH activity.Murine myocardium is taken, prepares tissue homogenate by hand, in 4 DEG C, 2000r/min, centrifuges 10 points
Clock, takes supernatant to be measured, SOD activity and MDA contents, illustrates to operate in strict accordance with kit.
1.8 immunohistochemistry detect mouse cardiac myocytes ERK1/2 protein expressions
Immunohistochemical staining, ERK1/2 protein expressions use SP methods, its key step：Paraffin section de-waxing aquation, 3% pair
Oxygen water soaks 20min, and sheep blood serum is incubated 10min, adds ERK1/2 antibody primary antibody 60min at room temperature, goat anti-mouse IgG two successively
Anti- room temperature 30min, streptavidin-Peroxidase Solution room temperature 10min, DAB colour developing, haematoxylin are redyed, and are dehydrated mounting, micro-
Microscopic observation.With Qwin image analysis softwares, quantitative analysis is carried out to positive expression in histotomy immunohistochemical staining endochylema,
Calculate OD value under mirror.
1.9 statistical analysis：
All data are analyzed with SPSS17.0 statistical softwares.As a result with mean ± standard deviationRepresent, multigroup
Compare and use one-way analysis of variance, two comparison among groupss are examined with t, are considered statistically significant with P ＜ 0.05.
2 results
Influences of the 2.1CE to Iso induced mice cardiac mass parameters：
Compared with blank group, after continuous subcutaneous injection Iso, model group mouse whole-heartedly has substantially with Left ventricular massindex
Increase (P ＜ 0.01), meet《Pharmacological experimental methodology》Myocardial hypertrophy model criteria in the second edition, illustrates myocardial hypertrophy model
It is successfully prepared.After being treated with CE 120,480mg/kg, HWI and LVWI have reduced compared with model group, and difference is statistically significant
(P ＜ 0.05), and 480mg/kg CE group changes become apparent.Sin has mouse cardiac muscle plumpness certain protective effect.As a result
It is shown in Table 1.
1 each group mouse of table whole-heartedly compare with Left ventricular massindex (mg/g,)
Compared with blank group,*P ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01
Influences of the 2.2CE to Iso induced mice myocardial hypertrophy tissue oxygen damage criterions：
After continuous subcutaneous injection Iso, MDA contents are significantly raised in model group mice serum LDH, cardiac muscular tissue, SOD activity
It is obvious to reduce, property that there were significant differences compared with blank group (P ＜ 0.01)；After being treated with CE 120,480mg/kg, each treatment group
LDH, MDA value contrast model group are decreased obviously, and SOD substantially rises, and have the significance difference opposite sex (P ＜ 0.05)；480mg/kg CE groups
Effect is more obvious, shows that CE has certain antagonism, the result is shown in Figure 1 to the plump damage of mouse cardiac muscle.
2.3 myocardial histopathology sections observations
Cutting into slices and show under each group murine myocardium light microscopic, Normal group cardiac muscle cell is red, marshalling, densification,
Kytoplasm uniform coloring, interstitial cell is without hyperplasia.Iso model groups cell changes in myocardial hypertrophy, cardiac muscle cell's volume increase, in circle
Shape or similar round, nucleus volume increase dyeing are deepened, and visible blue fibrosis increases and inflammatory between blood vessel and cardiac muscle cell
Cellular infiltration.High dose Flos Chrysanthemi extract, after treatment, the situation of cardiac damage mitigates compared with model group, and cell arrangement is more neat,
Thickening unobvious, inflammatory cell infiltration mitigate, and kytoplasm coloring is obvious, and interstitial fibrosis substantially mitigates.See Fig. 2, Fig. 3-I, Fig. 3-
II。
2.4 each group mouse cardiac myocytes ERK1/2 protein expressions change
Fig. 4, Fig. 5, Fig. 6 are shown, compared with blank control group, total ERK1/2 albumen relative expressions in model group cardiac muscular tissue
The obvious increase of amount, with blank control group comparing difference significantly (equal P<0.05).After CE treatments, the egg of total ERK1/2 in cardiac muscular tissue
White expression is obvious to be reduced, with model group comparing difference significantly (equal P<0.05).
Conclusion：
1st, the myocardial hypertrophy model group mouse cardiac myocytes volume increase of Iso inductions, rounded or similar round, cell nucleome
Product increase dyeing is deepened, and visible blue fibrosis increases and inflammatory cell infiltration between blood vessel and cardiac muscle cell；Blood in model group
Clear LDH is horizontal, the rise of cardiac muscle MDA contents and SOD of heart tissue activity decline, while the expression rise of ERK1/2 albumen, with control group
Compared to significant difference；
2nd, treated using CE, the fibrosis that can obviously improve the mouse cardiac muscle plumpness of Iso inductions changes, and reduces Iso inductions
Mouse cardiac muscle hypertrophy model Serum LDH level, cardiac muscle MDA contents, rise SOD of heart tissue activity, suppress the table of its ERK1/2 albumen
Reach, show good myocardium protecting action, wherein the effect of heavy dose CE is stronger；
3rd, CE has protective effect to the mouse cardiac muscle plumpness that Iso is induced, its mechanism may be with suppressing ERK1/2 albumen
Expression, reduce active horizontal related, the CE energy scavenging activated oxygens of Serum LDH level, cardiac muscle MDA contents, rise SOD of heart tissue
And lipoid peroxidization resistant, improve the oxidation resistance of body.
Embodiment 2：The protective effect for the heart hypertrophy in rats that Flos Chrysanthemi extract induces abdominal aorta constriction and its machine
Reason research.
L materials and methods
1.1 material
1.1.1 main agents and instrument
Flos Chrysanthemi extract (CE), Zhejiang Shengshi Biological Product Co., Ltd, pale yellow powder, smell delicate fragrance, raw material ratio
10:1, general flavone mass fraction 36%；Valsartan, China Resources Saike Pharmaceutical Co., Ltd., lot number：20161008；In endochylema
Calcium ion concentration fluorescence detection reagent kit (fura-3/AM), the upper graceful bio tech ltd of Hypon；The atrium profit of mountain sheep anti mouse
Sodium peptide (atrial na triuretic peptide, ANP) and β-myoglobulin heavy chain (β-myosin heavy chain, β-
MHC) polyclonal antibody, horseradish enzyme mark rabbit-anti sheep IgG, Wuhan doctor's moral Bioisystech Co., Ltd；Rat blood pressure meter, Shanghai
Yu Yan scientific instrument Co., Ltd；Fluorescence microscope, PTll048, Japan；Ultraviolet specrophotometer, Ultmspec-2000 types,
Pharmacia；Electrophoresis data process＆analysis system, Kodak Company；HP Sonos5500 color ultrasonic devices, U.S.'s favour
General company.
1.1.2 experimental animal
Healthy SD rat 50, male and female have concurrently, and weight (243 ± 21) g, is provided by Zhejiang Province's Experimental Animal Center, qualified
Card number：SCXK (Zhejiang) 2015-0002, in Zhejiang Province, Experimental Animal Center barrier environment Animal Lab. is fed and is tested.
1.2 experimental method
1.2.1 the preparation of myocardial hypertrophy model and experiment packet
50 rats underwent constriction of abdominal aorta (abdominal aorta coarctation, AAC), pre-operative anxiety
12h, free water.After pressing 30mg/kg intraperitoneal injection of anesthesia with 3% yellow Jackets, dorsal position four limbs are fixed.Under aseptic condition
Under left costal margin 0.5cm, by abdomen median line at 0.5cm, longitudinal incision 2.5cm, successively chorista, into abdominal cavity.Blunt separation
Abdominal aorta sheath, peels off abdominal aorta.No. 1.0 sutures are worn below the renal artery branch of left and right, are put along blood vessel direction of travel
Put No. 7 syringe needles and (wherein 10 only thread do not ligature as Sham-operated control group) are ligatured together with abdominal aorta, then extract pin out
Head.Intraperitoneal instillation penicillin (400,000 u/mL) in right amount, then closes abdomen.Close cage raising, normal diet.Abdominal aorta constriction is performed the operation
Survival of rats 35, Sham-operated control group survival of rats 8；The cause of the death mainly causes bleeding and anesthesia with damaging inferior caval vein in art
It is related.Postoperative 4 weeks measure abdominal aorta constriction operated rats arteria caudalis blood pressures, 32 rat blood pressure (systolic blood
Pressure, SBP) >=140mm Hg, as being successfully, reproduced hypertension model, and 32 are divided at random：Myocardial hypertrophy group is (raw
Manage brine l ml/d, ig), CE groups (120mg/kg/d/, ig), CE groups (480mg/kg/d, ig) and Valsartan group (10mg/kg/
D, ig), every group 8.Sham-operated control group rat 8 (physiological saline 1ml/d, ip).
1.2.2 the processing of animal model
After drug therapy 8 weeks, weight, tail arterial blood pressure are measured again；Color Sonography measure diastasis left ventricular posterior wall and
Chamber interval thickness；Then rat is put to death, claims the wet quality of left ventricle (containing interventricular septum), detection left ventricular mass index (LVMI).It is left
Ventricular organization's part puts and 12~24h is fixed in 10% buffered formaldehyde liquid of people, row section after conventional dehydration, transparent, waxdip, embedding
Handle, rat heart muscle fibre diameter (MFD) is measured under 10 × 40 times of light microscope, the Pathomorphology for analyzing cardiac muscular tissue changes
Become and take a picture.
1.3 Myocardial cell lysate ion ([Ca2+] i) concentration measure
Reference pertinent literature (such as：Ke Jun, Chen Feng, Xiao Xing, Dai Musen, Wang Xiaoping, mass troops, Chen Min, Zhang Cuntai calcium tune
Influence Chinese Journal of Emergency Medicine of the protein kinase II inhibitor to hypertrophied ventricular myocytes, 2012, doi：10.3760/
Cma.j.issn.1671-0282.2012.02.011) method separates left room cardiac muscle cell, will load the cardiac muscle cell of fura-3
(PTll048, Japan) is placed under fluorescence microscope, and the excitation wavelength of calcium fluorescence is 340/380nm, launch wavelength 510nm.It is glimmering
Optical signal is handled through Felix special-purpose softwares.
The protein expression of 1.4Western blot detection ANP, β-MHC
Rat heart muscle tissue is taken, extracts tissue protein with Trizol reagents, and protein is measured with Bradford methods
Content.The equal protein matter of extraction is added into 3 × sample loading buffer, loading after 3min is boiled, in the SDS polypropylene prepared
Protein, is then transferred on nitrocellulose filter by acrylamide gel vertical electrophoresis, according to the Western blot sides of standard
Method is incubated 2h jointly with ANP, β-MHC antibody respectively, then elutes, and diluted secondary antibody is added after closing rinsing.Hybridize at room temperature
1h, chemiluminescent agent warm bath 1min post-exposure, develop and be fixed, and finally carries out absorbance scanning analysis to result.
1.5 statistical method
All data mean ± standard deviationTo represent, analyzed using SPSSl7.0 statistical softwares.Multigroup
Compare and use one-way analysis of variance, two comparison among groupss are examined with t, think statistically significant with P ＜ 0.05.
2 results
The influence of 2.1 couples of rat SBP
Compared with myocardial hypertrophy group, 120mg/kg CE groups SBP does not significantly reduce [(25.05 ± 1.70) kPa vs
(24.74 ± 1.68) kPa, P>0.05], but 480mg/kg CE groups, Valsartan group SBP are reduced obvious [(18.38 ± 1.36)
KPa, (19.07 ± 1.52) kPa vs (24.49 ± 1.64) kPa, P<0.01], illustrate that heavy dose CE has the work for reducing SBP
With heavy dose of 480mg/kg CE effects are similar to Valsartan group.
The influence of 2.2 pairs of interventricular septums, left ventricular posterior wall thickness, cardiac muscle fibre diameter (MFD), left ventricular mass index (LVMI)
The interventricular septum of CE groups and Valsartan group, left ventricular posterior wall thickness are higher than control group (P<0.05), but it is less than myocardial hypertrophy
Group (P<0.01)；The chamber interval thickness of CE groups is higher than Valsartan group (P<0.05) 2, are shown in Table.
The MFD of CE groups and Valsartan group is substantially less than myocardial hypertrophy group (P<0.01), higher than control group (P<0.05).Carry
Show that CE and Valsartan play the role of suppressing Hypertensive disease.And CE groups MFD is higher than Valsartan group (P<0.05) work of CE groups, is illustrated
With not as Valsartan group it is obvious, be shown in Table 2.
The LVMI of CE groups is significantly lower than myocardial hypertrophy group (P<0.05), prompting CE plays the role of suppressing left ventricular hypertrophy.But
Compared with Valsartan group, the LVMI of CE groups is of a relatively high, shows that its effect is weaker than Valsartan, is shown in Table 2.
Change (the n=8 of each experimental group interventricular septum of 2 rat of table, left ventricular posterior wall thickness and myocardial pathology；)
Compared with the control group：*P<0.05,**P<0.01；Compared with myocardial hypertrophy group：#P<0.05。
2.3 myocardial pathologicals change
HE dyeing displays, control group diameter of myocytes is without significantly increasing, cardiac muscle cell's arranged regular；Myocardial hypertrophy group can
See that diameter of myocytes significantly increases, volume is loose, and cardiac muscle cell is disorganized.CE groups and Valsartan group cardiac muscle cell are more myocardium
Plumpness group cardiac muscle cell Histopathology, which changes, to be mitigated, and sees Fig. 7.
2.4 cardiac muscle cell [Ca2+] i change
The intracellular [[Ca of myocardial hypertrophy group2+] i [(161.52 ± 11.54) nmoL/L] is apparently higher than control group, 120mg/kg
CE groups, 480mg/kg CE groups and Valsartan group [(108.32 ± 9.76,135.28 ± 13.79,119.16 ± 9.83,126.14
± 9.62) nmol/L, P<0.01]；Statistically significant (the P of difference between CE groups and control group<0.05)；Valsartan group remains above
480mg/kg CE groups and control group (P<0.05).As it can be seen that 480mg/kg CE reduce cardiac muscle cell [Ca2+] i concentration effect it is strong
In Valsartan (P<0.05).
Influence of 2.5 Flos Chrysanthemi extracts to abdominal aorta constriction hypertrophic myocardium tissue ANP, β-MHC
The immunoblot results of ANP, β-MHC show and (are shown in Table 3, Fig. 8)：Compared with control group, the ANP of myocardial hypertrophy group,
β-MHC protein expressions substantially increase (P<0.01) in myocardial hypertrophy ANP, β-MHC, is prompted to take part in the pathology mistake of myocardial hypertrophy
Journey, ANP, β-MHC play certain role in the pathogenesis of myocardial hypertrophy.ANP, β-MHC of CE groups and Valsartan group
Expression is substantially less than myocardial hypertrophy group (P<0.01), downward effects of the CE to ANP, β-MHC protein expressions is weaker than Valsartan.Cause
This, CE is likely to be the plumpness for slowing down and preventing cardiac muscle by acting on ANP, β-MHC.
Change (the n=8 of 3 each group abdominal aorta constriction hypertrophic myocardium tissue ANP, β-MHC of table expression；)
Compared with the control group：*P<0.05,**P<0.01；Compared with myocardial hypertrophy group：#P<0.05。
Conclusion：
1st, heavy dose CE has the function that to reduce SBP；
2nd, AAC induced rats myocardial hypertrophy, diameter of myocytes significantly increase, and cell arrangement is disorderly；CE and Valsartan are controlled
After treatment, myocardium markers Histological change can be alleviated, CE has myocardium protecting action to AAC postoperative myocardial plumpness rats；
3rd, CE resists the postoperative heart hypertrophy in rats of AAC, may be with suppressing cardiac muscle cell [Ca2+] i, suppress cardiac muscle cell ANP,
β-MHC protein expressions are related.
Embodiment 3：The heart hypertrophy in rats and renin angiotensin aldehyde that Flos Chrysanthemi extract induces pressure load are consolidated
1 materials and methods of influence of ketone system
1.1 main agents and instrument
Flos Chrysanthemi extract (CE, general flavone mass fraction 36%), Zhejiang Huzhou Sheng Shi bio tech ltd are yellowish
Color powder；Feritin (PRA) detection kit, purchased from Shanghai miracle Bioisystech Co., Ltd；(Ang II, aldehyde are solid for angiotensins
Ketone ALD) and Hydroxyproline assay kit, the production of biotechnology Co., Ltd is built up in Nanjing；Masson staining kits
Purchased from Shanghai Lian Ke Bioisystech Co., Ltd；XSP-C type light microscopes, one factory of Shanghai optical instrument；DU-600 types are ultraviolet
Spectrophotometer, Beckman Coulter Inc. of the U.S.；5415D supercentrifuges, German Eppendorf companies, echocardiogram
(GE companies of the U.S., VIVID7 models) and 13MHz high frequencies line traffic control pop one's head in (GE companies of the U.S.).
1.2 experimental animals and packet
Male SD rat (238 ± 19g of weight), replicates myocardial hypertrophy model, by 40 after 8w with constriction of abdominal aorta
Rat model is randomly divided into model group and the high, medium and low dosage groups of CE (each n=10).The high, medium and low dosage groups of CE are carried with FLOS CHRYSANTHEMI ALBA from Haizhou of China
Thing 480,240,120mg/kg gavages is taken to handle, 1 time/d, continuous 8w.Choose 10 at the same time and only open abdomen and the rat of not all right ligation
As sham-operation group, sham-operation group and model group are daily with equivalent distilled water gavage.All rats are purchased from Shanghai Si Laike public affairs
Department, raises, manual control illumination during modeling and later observation in SPF grades of experimental animal rooms, and light and shade alt time is 12:
12, room temperature (24 ± 1) DEG C, humidity 60%~70%.
The foundation of 1.3 rat cardiac hypertrophy models
Rat adaptability raising 1w is bought back, with reference to pertinent literature Methods of Preparing Myocardial hypertrophy model：Yellow Jackets abdominal cavity
Injection, xiphoid-process downlink median abdominal incision, after successively opening abdominal cavity, dissociate abdominal aorta more than renal artery branch, by No. 7 syringe needles
With abdominal aorta it is arranged in parallel after both are ligatured, close abdominal cavity after syringe needle is extracted out.Sham-operation group only row opens abdomen without ligaturing,
Remaining operation is identical.Penicillin (30U/500g) intramuscular injection is given in postoperative 1w to prevent to infect.
1.4 echocardiography
Echocardiography is used after treatment 8w, after 10% chloraldurate intraperitoneal anesthesia, preserved skin after dorsal position is taken, adopts
Continuously detected for 3 cardiac cycle with M types echocardiogram, record left ventricular end diastolic diameter (LVEDD), left room end-systole respectively
Internal diameter (LVESD), left ventricular ejection fraction (LVEF) and fractional shortening of the ventricular minor semi axis (LVFS)
1.5 myocardial hypertrophy Indexs measures
Rat will be put to death after 8w, chest is opened and takes out heart, surface liquid is weighed after blotting, and is had a common boundary along atrioventricular junction and two ventricles
Cut off, after cutting off atrium and right ventricle successively, left ventricle is separated and weighed, with cardiac mass and the ratio calculation heart of weight
Dirty index (CI), with left ventricular mass and the ratio calculation Left ventricular massindex (LVMI) of weight.By left ventricle vertical section
Paraffin embedding is carried out, film-making, after Masson dyeing, each cardiac muscle sample randomly selects 5 visuals field and measures, with myocardial collagen
The ratio of area and the visual field gross area is myocardial collagen fraction by volume (CVF), with area of collagen and artery around petty action vessel lumen
Lumen Area is collagen volume fraction ratio (PVCA).5 rats separately are taken, take 50mg left ventricular tissues through being homogenized, disappearing
After change, supernatant is left and taken using kit detection hydroxyproline content.
1.6 renin angiotensin aldosterone system indexs of correlation detect
Chest after rat anesthesia, will be opened after 8w and take aortic blood 3ml, be not added with anti-coagulants, centrifuged after standing 2h with 4000r/min
Supernatant is taken after 10min, blood-serum P RA, Ang II is detected using kit and ALD is horizontal, concrete operations illustrate fully according to kit
Book carries out.
1.7 statistical procedures
All data are analyzed with SPSS17.0 statistical softwares.As a result with mean ± standard deviationRepresent, multigroup
Compare and use one-way analysis of variance, two comparison among groupss are examined with t, are considered statistically significant with P ＜ 0.05.
2 results
2.1 each group rat echocardiogram results contrasts
Compared with sham-operation group, LVEDD, LVESD of model group are raised, and LVEF, LVFS are reduced (P ＜ 0.05)；CE
High, medium and low dosage group LVEDD, LVESD is below model group (P ＜ 0.05), and LVEF, LVFS are above model group (P ＜
0.05) 4, are shown in Table.
4 each group rat of table echocardiogram results contrast (N=10)
Compared with sham-operation group：* P ＜ 0.05；Compared with model group：#P ＜ 0.05；##P ＜ 0.01.
The index of correlation of 2.2 each group heart hypertrophy in rats is horizontal relatively
Compared with sham-operation group, CI, LVMI, CVF, PVCA of model group are raised (P ＜ 0.05)；The high, medium and low dosage of CE
Group CI, LVMI, CVF, PVCA are below model group (＜ 0.05), are shown in Table 5.
The index of correlation of 5 each group heart hypertrophy in rats of table compare (N=10)
Compared with sham-operation group：* P ＜ 0.05；Compared with model group：#P ＜ 0.05；##P ＜ 0.01.
2.3 each group rat left ventricle cardiac muscular tissue hydroxyproline contents compare
The hydroxyproline content of model group is (0.64 ± 0.21) μ g/mg, higher than (0.29 ± 0.07) μ g/ of sham-operation group
Mg (P ＜ 0.05)；The hydroxyproline content of the high, medium and low dosage groups of CE be followed successively by (0.25 ± 0.08), (0.37 ± 0.17) and
(0.46 ± 0.14) μ g/mg, are below model group (P ＜ 0.05)；In CE, the hydroxyproline content of low dose group be higher than sham-operation
Group (P ＜ 0.05).
The comparison of 2.4 each group rat RASS system indexs of correlation
Compared with sham-operation group, PRA, Ang II and ALD of model group are raised (P ＜ 0.05)；The high, medium and low dosage groups of CE
PRA, Ang II and ALD levels be below model group (P ＜ 0.05), it is but still variant with sham-operation group, be shown in Table 6.
6 each group rat RASS system indexs of correlation of table comparison (N=10)
Compared with sham-operation group：* P ＜ 0.05；Compared with model group：#P ＜ 0.05；##P ＜ 0.01.
Conclusion：
1st, CE can improve the Cardiac Function in Rat and myocardial hypertrophy of pressure load induction.
2nd, CE acts on rat heart muscle, and it is related to improve renin angiotensin aldosterone system activation with it, and with
Upper improvement result is related to its dosage.
Claims (9)
1. application of the Flos Chrysanthemi extract in the medicine of prevention myocardial hypertrophy or the disease based on myocardial hypertrophy is prepared, institute
Flos Chrysanthemi extract is stated to extract to obtain through drying, 80% ethanol water by raw material FLOS CHRYSANTHEMI ALBA from Haizhou of China.
2. application as claimed in claim 1, it is characterised in that the disease based on myocardial hypertrophy is：Hypertension, wind
Moist heart disease, pulmonary heart disease, diabetes, dilated cardiomyopathy, hypertrophic cardiomyopathy, coronary heart disease, miocardial infarction or anaemia.
3. application as claimed in claim 1, it is characterised in that the Flos Chrysanthemi extract is made as follows：
Fresh FLOS CHRYSANTHEMI ALBA from Haizhou of China is placed in air dry oven, 45 DEG C of dryings to constant weight, crushed, crossed 100 mesh sieves, obtain FLOS CHRYSANTHEMI ALBA from Haizhou of China powder
End, solid-liquid ratio 1 is pressed by gained FLOS CHRYSANTHEMI ALBA from Haizhou of China powder：15 add in 80% ethanol water, and ultrasonic extraction 30min, is collected by filtration filter
Liquid, filter residue repeat extraction 2 times, merge gained filtrate every time, are concentrated under reduced pressure, vacuum freeze-drying obtains the Flos Chrysanthemi extract.
4. application as claimed in claim 1, it is characterised in that the dosage of the Flos Chrysanthemi extract is 120~480mg/kg/
d。
5. application as claimed in claim 1, it is characterised in that the route of administration of the Flos Chrysanthemi extract is oral or injection
Administration.
6. application as claimed in claim 1, it is characterised in that the medicine system of following form is made in the Flos Chrysanthemi extract
Agent：Flos Chrysanthemi extract piece, Flos Chrysanthemi extract sustained release tablets or Flos Chrysanthemi extract capsule.
7. application as claimed in claim 6, it is characterised in that the pharmaceutical preparation is calculated as 120 with Flos Chrysanthemi extract~
240mg/ unit dosages.
8. Flos Chrysanthemi extract officinal salt is in the medicine of prevention myocardial hypertrophy or the disease based on myocardial hypertrophy is prepared
Application.
9. the drug regimen of Flos Chrysanthemi extract or Flos Chrysanthemi extract officinal salt pharmaceutic adjuvant composition acceptable with human body
Application of the thing in the medicine of prevention myocardial hypertrophy or the disease based on myocardial hypertrophy is prepared.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7125573B2 (en) * | 2000-07-12 | 2006-10-24 | Kao Corporation | Preventive, alleviative or remedy for hypertension |
| CN106963803A (en) * | 2017-03-31 | 2017-07-21 | 浙江工业大学 | Application of the gynostemma pentaphyllum total flavones in the medicine for preparing preventing and treating myocardial hypertrophy |
-
2017
- 2017-12-25 CN CN201711417355.0A patent/CN108030797A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7125573B2 (en) * | 2000-07-12 | 2006-10-24 | Kao Corporation | Preventive, alleviative or remedy for hypertension |
| CN106963803A (en) * | 2017-03-31 | 2017-07-21 | 浙江工业大学 | Application of the gynostemma pentaphyllum total flavones in the medicine for preparing preventing and treating myocardial hypertrophy |
Non-Patent Citations (2)
| Title |
|---|
| 邝春林等: ""杭白菊不同溶剂提取物的抗氧化活性研究"", 《食品工业科技 》 * |
| 高莉萍: ""杭白菊提取物对异丙肾上腺素诱导心肌肥厚大鼠notch1信号通路的影响"", 《中国优秀硕士学位论文全文数据库》 * |
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