CN106963803B

CN106963803B - Application of gynostemma pentaphylla total flavone in preparing medicine for preventing and treating cardiac hypertrophy

Info

Publication number: CN106963803B
Application number: CN201710208389.2A
Authority: CN
Inventors: 李乐; 叶雨婷; 尚好
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2017-03-31
Filing date: 2017-03-31
Publication date: 2020-10-09
Anticipated expiration: 2037-03-31
Also published as: CN106963803A

Abstract

The invention discloses an application of gynostemma pentaphylla total flavone in preparing a medicament for preventing and treating myocardial hypertrophy or diseases based on the myocardial hypertrophy; the invention utilizes the spontaneous hypertension rat myocardial hypertrophy model, beta receptor stimulant isoproterenol to stimulate and induce the mouse myocardium, and copies the mouse myocardial hypertrophy model; the abdominal aorta constriction is utilized to induce the myocardial hypertrophy of the rat, a rat myocardial hypertrophy model is copied, the traditional Chinese medicine active ingredient gynostemma pentaphylla total flavone with different doses is applied, and a positive treatment effect is obtained through pharmacodynamic whole animal experiment research, so that a foundation is provided for new medicine screening.

Description

Application of gynostemma pentaphylla total flavone in preparing medicine for preventing and treating cardiac hypertrophy

(I) technical field

The invention relates to a new application of gynostemma pentaphylla total flavonoids, in particular to an application of gynostemma pentaphylla total flavonoids in preparing a medicament for preventing and treating myocardial hypertrophy.

(II) background of the invention

Gynostemma pentaphylla, also known as Heptaculum glaucescens, is mainly a plant of the genus Gynostemma belonging to the family Cucurbitaceae. Has the effects of cold nature, sweet taste, tranquilization, tonifying qi and reducing blood pressure, so that the Chinese medicinal herb is used as a miraculous 'long-life herb' in folk and is the first of the famous and precious Chinese medicinal materials. The basic pharmacodynamics research result shows that the medicine has obvious pharmacological effects of resisting inflammation, resisting immunity, relieving pain, tranquilizing, relieving cough, reducing blood fat, resisting arrhythmia, promoting leukocyte increase, etc.

The gynostemma pentaphylla total flavone has a protective effect on hypoxia/reoxygenation injury of myocardial cells of suckling mice, is related to reduction of generation of tumor necrosis factor (TNF-a), down-regulation of Fas/FasL protein expression and apoptosis inhibition (Lele, high Xiao Li, Dingbaoxing, Yuan. The anti-myocardial ischemia effect of the gynostemma pentaphylla total flavonoids may be related to the down-regulation of the expression of myocardial cell p38MAPK, the inhibition of the generation of TNF-alpha, the reduction of the damage degree of myocardial free radicals and the like (Lele, high profit and Yuan. Oxidative stress, myocardial apoptosis, cytokine-triggered inflammatory responses, p38MAPK overexpression, etc. may be closely related to the development and outcome of myocardial hypertrophy. However, the application of the gynostemma total flavone in treating related diseases based on myocardial hypertrophy is not reported in research at home and abroad.

Disclosure of the invention

The invention aims to provide the application of the gynostemma pentaphylla total flavone extract in preparing the medicines for preventing and treating myocardial hypertrophy or related diseases based on the myocardial hypertrophy.

The technical scheme adopted by the invention is as follows:

the application of the gynostemma total flavone extract in preparing the medicine for preventing and treating myocardial hypertrophy or diseases based on the myocardial hypertrophy is characterized in that the gynostemma total flavone extract is obtained by extracting gynostemma stem and leaf fragments with ethanol as an extraction solvent.

Further, the extraction method of the gynostemma total flavone extract comprises the following steps:

(1) cleaning stem and leaf of herba Gynostemmatis, drying, pulverizing, sieving, defatting with petroleum ether, drying to obtain residue, ultrasonic extracting the residue in 65% ethanol solvent, concentrating under reduced pressure to obtain extract, and vacuum drying to obtain crude extract of herba Gynostemmatis total flavonoids; the adding amount of the ethanol solvent is 25-40 ml/g based on the mass of the gynostemma pentaphylla particles;

(2) dissolving the crude product of the gynostemma total flavone extract in the step (1) in water, separating the filtrate by AB-8 type macroporous adsorption resin, eluting with ethanol, and distilling under reduced pressure to obtain a concentrated solution A;

(3) and (3) separating the concentrated solution A obtained in the step (2) by using polyamide resin, eluting by using ethanol, distilling under reduced pressure to obtain a concentrated solution B, and freeze-drying the concentrated solution B to obtain the gynostemma total flavone extract.

Still further, the disease based on cardiac hypertrophy is hypertension, rheumatic heart disease, pulmonary heart disease, hyperthyroidism, dilated cardiomyopathy, hypertrophic cardiomyopathy, coronary heart disease or anemia.

Furthermore, the dosage of the gynostemma total flavone extract is 100-400 mg/kg/d, preferably 150-300 mg/kg/d, more preferably 100-200 mg/kg/d, and most preferably 150mg/kg/d, based on the gynostemma total flavone.

Furthermore, the herba Gynostemmatis total flavone extract can be made into pharmaceutical preparations such as herba Gynostemmatis total flavone tablet, herba Gynostemmatis total flavone capsule, herba Gynostemmatis total flavone sustained release tablet, or herba Gynostemmatis total flavone injection.

The invention also aims to provide application of medicinal salt of traditional Chinese medicine gynostemma pentaphylla flavone in preparing a medicament for preventing and treating myocardial hypertrophy or diseases based on myocardial hypertrophy.

Further, the medicinal salt of the gynostemma total flavone is hydrochloric acid gynostemma total flavone.

The invention also aims to provide application of the traditional Chinese medicine gynostemma pentaphylla flavone and/or a traditional Chinese medicine gynostemma pentaphylla flavone medicinal salt and a human body acceptable medicinal auxiliary material in preparing a medicine for preventing and treating myocardial hypertrophy or diseases based on the myocardial hypertrophy.

Furthermore, the dosage of the traditional Chinese medicine gynostemma pentaphylla medicine composition is 100-400 mg/kg/d, preferably 150-300 mg/kg/d, more preferably 100-200 mg/kg/d, and most preferably 150mg/kg/d calculated by total flavonoids of gynostemma pentaphylla.

When the medicinal preparation of the gynostemma total flavonoids is recommended to be a gynostemma total flavonoids tablet, a gynostemma total flavonoids slow-release tablet or a gynostemma total flavonoids capsule, the medicinal preparation of the gynostemma total flavonoids takes the gynostemma total flavonoids as 100-300 mg/tablet; when the gynostemma pentaphylla total flavone sustained-release tablets or the pharmaceutical preparation of the gynostemma pentaphylla total flavone are capsules, the pharmaceutical preparation of the gynostemma pentaphylla total flavone is 25-50 mg/capsule based on the gynostemma pentaphylla total flavone.

The invention utilizes the spontaneous hypertension rat myocardial hypertrophy model, beta receptor stimulant isoproterenol to stimulate and induce the mouse myocardium, and copies the mouse myocardial hypertrophy model; the abdominal aorta constriction is utilized to induce the rat myocardial hypertrophy, a rat myocardial hypertrophy model is copied, and the traditional Chinese medicine gynostemma pentaphylla total flavone with different doses is applied to obtain a positive treatment effect through pharmacodynamic whole animal experiment research.

The invention has the following beneficial effects: the invention provides application of gynostemma pentaphylla Total Flavonoids (TFG) in preparing a medicament for preventing and treating myocardial hypertrophy or related diseases based on the myocardial hypertrophy, and provides a foundation for new drug screening.

(IV) description of the drawings

FIG. 1 is the effect of TFG on the histopathological histology of SHR myocardium in example 1: (

n＝8；HE,×400)A:WKY；B:SHR；C:SHR+Cap；D:SHR+TFG-L；E:SHR+TFG-H

FIG. 2 protein expression of ACE and AT1 in rat myocardial tissues of various groups (,

n＝8)

a is WKY; SHR is used as B; SHR + Cap; SHR + TFG-H; SHR + TFG-L (P <0.01 or P < 0.05).

FIG. 3 shows the changes of LDH activity, SOD activity of myocardial tissue and MDA content in the serum of each group of mice in example 2;

n 15, compared to the control group,^**P<0.01; and IsoThe comparison of the model sets is carried out,^#P<0.05，^##P<0.01。

FIG. 4-I is the pathological section of the mouse cardiomyocytes in each group of mice in example 2: (

n-15, HE staining, × 400);

a: a control group; b: the Iso model group; c: iso + TFG100mg/kg/d treatment group; d: iso + TFG 300mg/kg/d treatment group.

FIG. 4-II is the pathological section of the mouse cardiomyocytes in each group of mice in example 2: (

n 15 Masson staining, × 400)

FIG. 5 is a quantitative analysis of myocardial interstitial fibrosis in each group of mice in example 2: (

n 15), compared to the control group,^**P<0.01; in comparison with the set of Iso models,^#P<0.05，^##P<0.01。

FIG. 6-I shows the change in the expression of ERK1 protein (ERK1, X400) in the myocardial cells of the mice of each group in example 2;

FIG. 6-II shows the change in the expression of ERK2 protein (ERK2, X400) in the myocardial cells of the mice of each group in example 2;

FIG. 7 shows the quantitative determination of protein expression change of ERK1/2 in myocardial cells of mice of each group in example 2 (((C))

n-15) compared to the control,^**P<0.01; in comparison with the set of Iso models,^#P<0.05，^##P<0.01。

FIG. 8 shows histological changes of myocardium of rats in each group of example 3: (

n-8) (HE staining, × 400);

a: a control group; b: an AAC model group; c: AAC + TFG100mg/kg/d treatment group; d: AAC + TFG 300mg/kg/d treatment group; e: AAC + Valsartan 10mg/kg/d treatment group.

Fig. 9 shows the change in the expression of ANP, &ttttranslation = β "&gtttβ &ltt/t &gtt-MHC in each group of abdominal aortic constrictive hypertensive rats of example 3 (n ═ 8;

),

a: a control group; b: an AAC model group; c: AAC + TFG100mg/kg/d treatment group; d: AAC + TFG 300mg/kg/d treatment group; e: AAC + Valsartan 10mg/kg/d treatment group. Comparison with the control group:^*P<0.05，^**P<0.01; comparison with AAC model group:^#P<0.05,^##P<0.01.

(V) detailed description of the preferred embodiments

The invention is further described below by means of specific examples, without restricting its scope to these.

Example 1: preliminary study on reduction of spontaneous hypertension rat myocardial hypertrophy by using gynostemma pentaphylla total flavonoids and mechanism

1 Material

1.1 raw materials and medicine herba Gynostemmatis total flavonoids (total flavonoids of Gynostemma pentaphylum (Thunb)

Makinor, TFG); captopril (Cap), specification: 25 mg/tablet, batch No. 20151214, Guangdong Shantou gold Shi pharmaceutical general factory.

1.2 reagents and instruments

1.2.1 Primary reagent iodine [ 2 ]¹²⁵I]Angiotensin II (II: (II) (II))¹²⁵I-AngiotensinⅡ,¹²⁵I-Ang II) radioimmunoassay kit, lot number 150132; iodine [ 2 ]¹²⁵I]Aldosterone¹²⁵Ⅰ-Aldosterone,¹²⁵I-Ald) radioimmunoassay kit, batch number 150084; are purchased from the institute of biotechnology in northern Beijing; TRIzol Invitrogen, lot No. 15596018; a total RNA extraction kit and a cDNA first strand synthesis kit (Hangzhou Bori science and technology, Inc.); fluorescent PCR kit (Dalian biological engineering Co., Ltd.), lot No. C28025-032; SYBR Selective cocktail ABI, lot # 4472920; BCA protein analysis kit comprises Nanjing Biotech, lot number 3445667, goat anti-rabbit IgG, (H + L), anti-angiotensin converting enzyme antibody (rabbit), Abcam, lot number ab 108252; anti-Angiotensin Converting Enzyme, ACE1 antibody (murine): Abcam, lot No. ab 11734; anti-angiotensin II type 2 antibody (rabbit): Abcam, lot No. ab 18801. ELISA detection kit (Brilliant bioengineering Co., Ltd.) for tumor necrosis factor (TNF-a, lot No. 150213) and interleukin 6(IL-6, lot No. 150121).

1.2.2 Main instrumentation

MPA heart function analysis system (Shanghai Oercott Biotechnology, Inc.), NIS-Elements Nikon image processing software (Nikon, Japan), Ge multifunctional microplate reader (Berton instruments, USA), fluorescence quantitative PCR analyzer (ABI, USA), chemiluminescence imaging system Chemiscope 3000 (Shanghai Xiang scientific instruments, Inc.); electrophoresis apparatus DYCZ-24DN (six instruments, Beijing), Image Lab5.0 imaging analysis System (BIO-RAD, U.S.A.).

1.3 SHR rats 48 animals, WKY rats 8, female, 12 weeks old, body weight 254 + -18 g, SPF grade, purchased from Shanghai Slek laboratory animals Ltd, license number SCXK 2015-. All animals were kept in the SPF barrier system of animal laboratories of Zhejiang university of industry, 4 animals/cage, constant temperature (24 + -2) deg.C, constant humidity (60 + -5)%, artificial lighting for 16h every day, and free access to food and water.

2 method

2.1 preparation of TFG the stem and leaf of Gynostemma pentaphyllum is cleaned, dried by blowing at 50 deg.C for 48h, crushed and sieved, degreased with petroleum ether for 2h, the solvent is volatilized from the dregs of a decoction, and dried in vacuum. Ultrasonic extracting 200g of dried residue with 65% ethanol 3000ml for 3 times, each time for 30min, mixing filtrates, concentrating under reduced pressure to obtain extract, and vacuum drying to obtain crude TFG. Dissolving in water, filtering, separating the filtrate with AB-8 type macroporous adsorbent resin, eluting with ethanol, collecting eluate, and distilling under reduced pressure to obtain concentrated solution A. Separating with polyamide resin, eluting with ethanol, collecting eluate, distilling under reduced pressure to obtain concentrated solution B, freeze drying to obtain refined TFG product with total flavone content of 56.73%.

2.2 grouping and administration of experimental animals WKY (hypertensive rat control strain) groups, namely 8 WKY rats are subjected to intragastric administration of 10ml/kg of distilled water; 32 SHR rats were randomly divided into 4 groups of 8 per group, model group (SHR) administered by gavage at 10 ml/kg; TFG low dose group (SHR + TFG-L,100 mg/kg; TFG high dose group (SHR + TFG-H,300 mg/kg; Captopril group (SHR + Cap). The rats in each group, 50 mg/kg. mg/gavage, were gavage 1 time daily for 4 weeks with a gavage volume of 10 ml/kg., weight 1 time per week and the dose was adjusted.

2.3 specimen Collection and index measurement

2.3.1 preparation of plasma or serum after the last administration, pentobarbital sodium solution ip anesthetized rats (30mg/kg), immediately 2mL of blood was taken from the abdominal aorta to the blood collection tube (containing 0.1g/L heparin anticoagulant), and centrifuged at 3000r/min for 10 min. Respectively sucking the supernatant, subpackaging into 1.5mLEP tubes, and freezing at-70 deg.C in a refrigerator. After blood is collected from another rat, the rat is kept still for 15min, serum is separated after centrifugation (3000r/min, 10min), and the level of IFN-a and IL-6 in the serum is detected by an ELISA method.

2.3.2 left ventricular hypertrophy index determination after blood sampling, open chest to pick up heart, flushing with physiological saline, filter paper blotting and weighing, calculating heart index (heart weight/body weight x 100%). The heart was cut at the ventricular interval, and the left and right ventricles were separated, blotted dry with filter paper, weighed separately, the heart weight and body weight ratio (HW/BW), left ventricular weight and body weight ratio (LVM/BW) were calculated, and left ventricular hypertrophy was analyzed.

2.3.3 histopathological examination after weighing, a portion of the heart tissue was fixed in 100mg/g neutral formalin solution, paraffin embedded, sectioned at 5 μm thickness, HE stained, and the myocardial histopathological changes were observed. Measuring the diameter of cardiac ventricular cardiomyocyte (cardiac myocyte) under 400 times of optical microscope by HMIAS-2000 high-definition color medical image-text analysis software

Diameter, CD) and cardiomyocyte cross-sectional area (CSA). 3 fields were randomly selected for each section, and 20 cells were counted each time and averaged.

2.3.4 preparation of tissue homogenate part of the heart tissue was taken, rinsed repeatedly in ice-cold physiological saline, wiped dry with filter paper, weighed, added with 0.9% cold physiological saline at a ratio of 1: 10 as the homogenate medium, the tissue was cut into pieces with ophthalmic scissors, poured into a glass homogenizer, homogenized with a tissue homogenizer for 10 s/time, 30 s/time, 4-6 times (all operations were carried out in an ice-water bath), then placed in a 4 ℃ low temperature centrifuge, centrifuged for 10min at 3000r/min, the supernatant was taken and distributed to 1.5mL EP tubes, frozen at-70 ℃ in a refrigerator for testing.

2.3.5 plasma or tissue Ang II and Ald content and serum IFN-a, IL-6 level determination: determining Ang II and Ald content in plasma or tissue homogenate strictly according to the kit instruction; the serum IFN-a and IL-6 levels were determined following the protocol of the ELISA kit.

2.3.6RT-PCR method for detecting the expression level of ACE and AT1 (angiotensin receptor 1) mRNA:

the total RNA is extracted by a TRIzol method, and the concentration and the purity (both are between 1.8 and 2.2) of the total RNA are measured by an enzyme-labeling instrument. Total RNA, cDNA is synthesized by using a cDNA first strand synthesis kit, Primer 5.0 software assists in designing a Primer, an internal reference beta-actin Primer has an upstream of 5-CCCATCTAT-GAGGGTTACGC-3 and a downstream of 5-TTTAATGTCACG-CACGATTTC-3, and the length of an amplification product is 165 bp; the ACE primer has upstream of 5-ATTGCAGCCGGGCAACTT-3 and downstream of 5-CTCCGTGATGTTGGTGTCGT-3', and the length of the product is 147 bp; the AT1 primer has an upstream of 5 '-CTCTGCCAATTCCCT-GAGTT-3' and a downstream of 5 '-TTGGGGCAGTCATCTTG-GA-3', and the product length is 231 bp. RT-PCT cycling conditions: pre-denaturation at 95 ℃ for 30 s; circulating for 40 times at 95 ℃ for 5s and 60 ℃ for 30 s; the melting point curve is formed at 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 15 s. And (3) taking each amplification product to perform agarose gel electrophoresis, analyzing the target gene by an Image Lab imaging analysis system, representing the expression quantity of the corresponding gene by using an average gray value, and dividing the average gray value by the average gray value of beta-actin to obtain a numerical value serving as the relative expression quantity of mRNA of each amplification product.

2.3.7Western blot method for detecting ACE and AT1 protein expression of myocardial tissues of SHR rats, protein samples are mixed with a loading buffer solution, 10% SDS-PAGE electrophoresis is carried out after boiling, protein transfer is carried out by adopting semi-dry transfer, primary antibodies (beta-actin, ACE and AT1) are dyed and sealed according to requirements and incubated for 24 hours AT 4 ℃, and secondary antibodies (Pierce Goat Anti-mouse IgG, Pierce Goat Anti-Rabbit IgG) are incubated for 1 hour AT room temperature. And after the incubation of the secondary antibody is finished, ECL display treatment is carried out, a chemiluminescence method is used for exposing with a film in a darkroom, photographing is carried out, the integral optical density value of the target protein is calculated, and the expression level of the target protein is calculated by comparing the integral optical density value with beta-actin.

2.4 statistically analyzing all data measurements to,

statistical analysis shows that the SPSS17.0 software One-Way ANOVA method is adopted to compare the average number of a plurality of groups of samples, and the LSD test is adopted for the comparison between the two groups. P<A difference of 0.05 is statistically significant.

3 results

3.1 comparing the left ventricular hypertrophy index with the WKY group, the HW, HW/BW, LVM and LVM/BW of the SHR group rats are obviously increased (P is less than 0.01); compared with the SHR group, the HW, HW/BW, LVM and LVM/BW of the SHR + TFG-L and SHR + TFG-H groups are reduced to different degrees (P <0.01 or P <0.05), and the SHR + TFG-H group is changed more remarkably (P < 0.01). The results are shown in Table 1.

Table 1 effect of TFG on SHR myocardial hypertrophy parameters (,

n＝8)

# P <0.05, # P <0.01vs WKY group; p <0.05, P <0.01vs SHR group

3.2 influence of TFG on histopathological change of SHR left ventricular myocardium HE staining shows that myofibers of myocardial cells in WKY group are arranged regularly and nuclei are obvious; compared with the WKY group, SHR myocardial cells are hypertrophic, gaps are widened, nuclei are increased and irregularly arranged, the diameter of myocardial fibers is increased, the cross-sectional area of the myocardial cells is increased, and part of myocardial nuclei are hypertrophic. The myocardial lesions of the treatment groups of the medicaments are relieved to different degrees, the arrangement of local myocardium is disordered, and the hypertrophy degree of myocardial cells is reduced compared with that of an SHR group. The results are shown in FIG. 1, Table 2.

TABLE 2TFG effects on CD and CSA of SHR cardiomyocytesSounding sound (sound,

n＝8)

# P <0.01vs WKY group; p <0.05, P <0.01vs SHR group

3.3 the experimental results of the influence of TFG on the Ang II and Ald contents in SHR heart and blood and IFN-a and IL-6 in serum show that the Ang II and Ald contents in the SHR heart and blood are both obviously increased (P <0.01) compared with WKY group. Compared with the SHR group, the Ang II and Ald contents of the administration groups are reduced to different degrees, wherein the variation difference of the SHR + Cap group and the SHR + TFG-H group has statistical significance (P <0.01 or P <0.05), and the results are shown in Table 3. In addition, compared with the WKY group, the SHR group has obviously increased IFN-a and IL-6 (P < 0.01); the SHR + TFG-H group was significantly reduced compared to the SHR group (P < 0.01). See Table 4

Table 3TFG versus Ang ii ALD content in SHR heart and blood (,

n＝8)

# P <0.01vs WKY group; p <0.05, P <0.01vs SHR group

TABLE 4 Effect of TFG on IFN-a, IL-6 levels in SHR sera (,

n＝8)

# P <0.01vs WKY group; p <0.05, P <0.01vs SHR group

3.4 the influence of TFG on mRNA expression quantity of ACE and AT1 of the myocardial tissue of SHR is compared with that of WKY group, and the mRNA expression quantity of ACE and AT1 of the myocardial tissue of SHR is obviously up-regulated; the expression level of ACE and AT1mRNA was down-regulated to different extents in myocardial tissues of each group compared with that of SHR group, and the results are shown in Table 5.

3.5 influence of TFG on ACE and AT1 protein expression of myocardial tissues of SHR compared with WKY group, ACE and AT1 protein expression of myocardial tissues of SHR group is obviously increased (P < 0.01); the protein expression of ACE and AT1 was reduced to different degrees in myocardial tissues of each group compared with that of SHR group. The results are shown in FIG. 2.

Table 5 relative expression amounts of ACE and AT1mRNA in myocardial tissues of rats in each group (,

n＝8)

# P <0.01vs WKY group; p <0.05, P <0.01vs SHR group

And (4) conclusion:

1. the research result shows that TFG can reduce cardiac index and left ventricular hypertrophy index, and the histopathological research shows that TFG can reduce the hypertrophy degree of myocardial cells, which shows that TFG has the function of inhibiting the occurrence and development of myocardial hypertrophy of hypertensive rats.

2. The experimental result shows that TFG can reduce the content of TNF-a and IL-6 in serum, and suggests that TFG has protective effect on myocardial hypertrophy by inhibiting TNF-a and IL-6.

3. The experimental results further show that TFG can not only reduce the content of AngII and Ald in plasma, but also reduce the content of AngII and Ald in myocardial tissues, which indicates that TFG can inhibit the activity of circulating and heart local Renin Angiotensin Aldosterone System (RAAS).

The relative expression of ACE and AT1mRNA and protein was shown to be reduced by TFG as shown by RT-PCR and Western blot results, indicating that TFG may act AT the gene transcription and translation levels, thereby inhibiting RAAS activity it is speculated that TFG may reduce AngII synthesis by inhibiting ACE and reduce the level of AngII binding by reducing the expression of AT 1.

Example 2: effect of TFG in inhibiting isoproterenol-induced myocardial hypertrophy of mice and mechanism research of TFG

1 materials and methods

1.1 Experimental animals:

clean-grade Kunming mouse, male and female, weight (20 + -2) g, provided by Zhejiang province animal center, and qualification number: SCXK (Zhe) 2016-.

1.2 drugs and reagents:

gynostemma pentaphylla total flavonoids ((total flavonoids of Gymostemma pentaphylum (Thunb) Mak, TFG), self-made (see example 1), isoproterenol hydrochloride (Iso) injection (Shanghai Secale pharmaceutical Co., Ltd., batch No. 160008), Lactate Dehydrogenase (LDH), Malondialdehyde (MDA), total superoxide dismutase (T-SOD) test kit, and protein quantification test kit were all purchased from Nanjing Bioengineered Co., Ltd. Rabbit anti-ERK 1/2 antibody was purchased from Santa Cruz Co., Ltd., U.S., and horseradish peroxidase goat anti-mouse antibody IgG was purchased from cell signaling Co., Ltd., U.S.).

1.3 Main instruments:

ultra pure water systems available from Millipore corporation, usa; low temperature centrifuges and low temperature refrigerators (Heraeus products, Germany); -70 ℃ low temperature refrigerator (product of Forma corporation, usa); an ultraviolet imaging scanner (Bio-Rad, USA); an image analyzer manufactured by Foeodyne corporation of America; PCY Instrument 9600 (Perkinelmer, USA); microplate reader (U.S. Bio-Rad 550); a voltage-stabilizing current-stabilizing timing electrophoresis apparatus (Shanghai precision instrument factory); qwin image analysis software (Leica, Inc., Germany); 722N visible light spectrophotometer (shanghai precision scientific instruments ltd); model MP200A electronic balance (shanghai second balance instrument factory).

1.4 animal grouping and modeling:

taking 48 healthy Kunming mice, wherein the male and female mice are divided into two parts: blank (control) group, model + TFG low dose treatment group, model + TFG high dose treatment group 4 groups of 12 individuals each. Except for the blank group, mice in each group were injected subcutaneously with Iso once daily at a dose of 40mg/kg on the first day, 20mg/kg on the second day, 10mg/kg on the third day, and 10mg/kg on the fourth day for 14 consecutive days. Free feeding and water supply are carried out, a myocardial hypertrophy model is established, and the blank group is injected with the same amount of physiological saline subcutaneously every day as a contrast.

1.5 administration:

the administration and the modeling are carried out simultaneously, and after injecting Iso 4h every day in the morning, the low and high dose TFG100mg/kg and 300mg/kg are respectively administered for intragastric administration once a day. The blank and model groups were gavaged with an equal amount of saline once a day for 4 consecutive weeks.

1.6 cardiac weight index determination:

after the mice are subjected to last administration, the mice are fasted for 12h without water prohibition, the weight of the mice is weighed (body weight, BW), eyeballs are subjected to blood taking and killed, the chest cavity is opened quickly, the heart is taken out, residual blood is washed out by precooled physiological saline, the Heart Weight (HW) is weighed after filter paper is sucked dry, then the left ventricle is separated and weighed (LVW), and the weight of the left ventricle is weighed (HW/BW) and the weight index of the left ventricle (LVWI ═ LVW/BW) are respectively calculated. And preparing a histopathological section from myocardial tissue, cutting off great vessels, atria and the free wall of the right ventricle along the atrioventricular ring, and fixing the rest ventricular septum and the free wall of the left ventricle in 10% formalin for 24 hours. Taking out the left ventricle specimen in the stationary liquid, washing with running water, dehydrating with gradient ethanol, transparentizing, embedding with conventional paraffin, continuously cutting 5 μm thick slices along the long axis of the left ventricle at intervals of 1mm cross section, observing under HE and Masson staining optical microscope, and taking a picture. Observing the degree of myocardial hypertrophy and fibrosis, analyzing the degree of myocardial fibrosis by Qwin image analysis software, and analyzing and comparing the histopathological changes of the myocardium of each group.

1.7 determination of serum LDH activity, myocardial tissue SOD activity and MDA content:

after blood is taken from mouse eyeballs, the mouse eyeballs are centrifuged at 4 ℃ and 3000r/min for 10 minutes, serum is separated, and the LDH activity is determined according to the strict operation of an LDH kit. Taking mouse myocardial tissues, manually preparing tissue homogenate, centrifuging at 4 ℃ and 3000r/min for 10 minutes, taking supernate to determine SOD activity and MDA content, and strictly operating according to the kit instructions.

1.8 immunohistochemical detection of mouse cardiomyocyte ERK1/2 protein expression

Immunohistochemical staining, wherein ERK1/2 protein expression adopts an SP method, and the main steps are as follows: dewaxing and hydrating a paraffin section, soaking in 3% hydrogen peroxide for 20min, incubating goat serum for 10min, sequentially adding an ERK1/2 antibody primary antibody at room temperature for 60min, a goat anti-mouse IgG secondary antibody at room temperature for 30min, and a streptavidin-peroxidase solution at room temperature for 10min, performing DAB color development, performing hematoxylin counterstaining, dehydrating and sealing, and observing under a microscope. Quantitative analysis of positive expression in immunohistochemical stained cytoplasm of tissue sections was performed using Qwin image analysis software, and the optical density under the microscope was calculated.

1.9 statistical analysis:

all data were analyzed using SPSS17.0 statistical software. Results are in mean. + -. standard deviation

It is shown that the comparison between groups is analyzed by one-way variance, and the comparison between two groups is tested by t, and P is less than 0.05, which is considered to be statistically significant.

2 results

2.1 influence of TFG on Iso-induced cardiac quality parameters of mice:

compared with the blank group, after continuous subcutaneous injection of Iso, the weight indexes of the whole heart and the left ventricle of the mice in the model group are obviously increased (P is less than 0.01), and the weight indexes accord with the standard of the myocardial hypertrophy model in the second edition of pharmacological experimental methodology, which indicates that the preparation of the myocardial hypertrophy model is successful. After treatment with TFG100, 300mg/kg, both HWI and LVWI were reduced compared to the model group, with statistical differences (P <0.05), whereas the 300mg/kg Sin group changed more significantly. The results are shown in Table 1.

Table 1 comparison of the whole heart and left ventricle weight indices for each group of mice (mg/g,

)

in comparison with the blank set, the results,^*p is less than 0.01; in comparison with the set of models,^#P＜0.05，^##P＜0.01

2.2 influence of TFG on indicators of oxidative damage in mouse myocardial hypertrophy tissue by Iso:

after continuous subcutaneous injection of Iso, the serum LDH and the MDA content in myocardial tissues of the model mice are obviously increased, the SOD activity is obviously reduced, and the model mice have obvious difference (P is less than 0.01) compared with a blank group; after TFG100 and TFG 300mg/kg treatment, LDH and MDA values of each treatment group are obviously reduced, compared with a model group, SOD is obviously increased, and the differences are obvious (P is less than 0.05); the effect was more pronounced in the 300mg/kg TFG group, and the results are shown in FIG. 3.

2.3 pathological section examination of myocardial tissue

The section of each group of mice under the optical microscope shows that the myocardial cells of the normal control group are red, are arranged regularly and compactly, the cytoplasm is uniformly colored, and the interstitial cells are not proliferated. The Iso model group cells show myocardial hypertrophy change, the volume of the myocardial cells is increased and is circular or similar to the circular shape, the cell nucleus volume is increased and the staining is deepened, and blue fibrosis increase and inflammatory cell infiltration can be seen between blood vessels and the myocardial cells. After the high-dose TFG treatment, the myocardial damage condition is reduced compared with a model group, the cell arrangement is relatively regular, the hypertrophy is not obvious, the inflammatory cell infiltration is reduced, the cytoplasm is obviously colored, and the interstitial fibrosis is obviously reduced. See FIG. 4-I, FIG. 4-II, FIG. 5.

2.4 Change in expression of ERK1/2 protein in mice in groups

FIG. 7, FIG. 6-I, and FIG. 6-II show that the relative expression level of total ERK1/2 protein in myocardial tissue of the model group is significantly increased compared with the blank control group, and the difference is significant compared with the blank control group (mean P < 0.05). After TFG treatment, the protein expression of total ERK1/2 in myocardial tissues is obviously reduced, and compared with a model group, the difference is obvious (the average P is less than 0.05).

And (4) conclusion:

1. the volume of the myocardial cells of the mouse in the Iso-induced myocardial hypertrophy model group is increased and is circular or similar to a circle, the cell nucleus volume is increased and dyed deepens, and blue fibrosis increase and inflammatory cell infiltration can be seen between blood vessels and the myocardial cells; in the model group, the serum LDH level, the myocardial MDA content and the myocardial SOD activity are increased, and meanwhile, the expression of the ERK1/2 protein is increased, and the difference is obvious compared with that of a control group;

2. by applying TFG treatment, the fibrosis change of the mouse myocardial hypertrophy induced by Iso can be obviously improved, the serum LDH level and the myocardial MDA content of the mouse myocardial hypertrophy model induced by Iso are reduced, the myocardial SOD activity is increased, the expression of ERK1/2 protein is inhibited, a good myocardial protection effect is shown, and the effect of large dose TFG is strong.

3. TFG has protective effect on mouse myocardial hypertrophy induced by Iso, the generation mechanism of TFG is probably related to inhibiting the expression of ERK1/2 protein, reducing the level of serum LDH and MDA content of myocardium and increasing the level of myocardial SOD activity, TFG can eliminate oxygen free radical and resist lipid peroxidation, and improve the oxidation resistance of organism.

Example 3: the protective effect of TFG on rat myocardial hypertrophy induced by abdominal aortic constriction and the mechanism thereof are researched.

Materials and methods

1.1 materials

1.1.1 Primary reagents and instruments

Gynostemma total flavonoids ((total flavonoids of Gymostemma Pentaphyllum (Thunb) Mak, TFG), homemade (see example 1), Valsartan, Wash. C.J.pharmaceutical Co., Ltd., batch number: 201512008, respectively; fluorescence detection kit (fura-3/AM) for intracellular calcium ion concentration, Shanghai Baoman Biotech limited; goat anti-mouse Atrial Natriuretic Peptide (ANP) and beta-myosin heavy chain (beta-myosin heavy chain, beta-MHC) polyclonal antibody, horseradish enzyme labeled rabbit anti-goat IgG, warhan doctor biotechnology limited; HP Sonos5500 color ultrasound diagnostic apparatus, hewlett packard, usa; rat sphygmomanometer, shanghai yuyan scientific instruments ltd; fluorescence microscopy, PTll048, japan; ultraviolet spectrophotometer, Ultmspec-2000 model, Pharmacia; electrophoretic data processing and analysis system, Kodak corporation.

1.1.2 Experimental animals

50 healthy SD rats, both male and female, with body weight (212 + -19) g, provided by the Experimental animals center in Zhejiang province, and the qualification number: SCXK (Zhe) 2016 + 0009, for animal feeding and experiments in the central barrier environment of laboratory animals in Zhejiang province.

1.2 Experimental methods

1.2.1 preparation of myocardial hypertrophy model and Experimental grouping

50 rats underwent Abdominal Aortic Coarctation (AAC), preoperatively fasted for 12h, with free access to water. After anesthesia by intraperitoneal injection of 3% sodium pentobarbital at 30mg/kg, the limbs are fixed in the supine position. Under aseptic condition, a longitudinal incision is made at a position 0.5cm below left costal margin and 0.5cm beside abdominal midline, and tissue is separated layer by layer and enters abdominal cavity. The abdominal aorta sheath was bluntly dissected and the abdominal aorta was dissected away. A1.0-gauge surgical suture was threaded under the left and right renal artery branches, a 7-gauge needle was placed in the running direction of the blood vessel and ligated together with the abdominal aorta (8 of them were threaded only and not ligated as a sham control group), and then the needle was withdrawn. A proper amount of penicillin (40 wumu/mL) is dripped into the abdominal cavity, and then the abdomen is closed. Closing the cage for feeding, and eating normally. 37 rats survived in abdominal aortic constriction surgery, and 8 rats survived in sham surgery control group; the cause of death is mainly related to bleeding and anesthesia caused by the injury of the inferior vena cava during the operation. The abdominal aorta constriction surgery rat tail artery blood pressure is measured 4 weeks after surgery, 33 rat blood pressures (SBP) are more than or equal to 140mm Hg, and the model is taken as a successfully-copied hypertension model, and 32 random extractions are divided into: myocardial hypertrophy group (normal saline l ml/d, ig), TFG group (100mg/kg/d/, ig), TFG group (300mg/kg/d, ig) and valsartan group (10mg/kg/d, ig), 8 of each group. Sham operation control group rats 8 (physiological saline 1ml/d, ip).

1.2.2 treatment of animal models

After 8 weeks of drug treatment, body weight and tail artery pressure were measured again; measuring the thickness of the posterior wall of the left ventricle and the ventricular septum at the end of diastole by the heart color Doppler ultrasound; the rats were then sacrificed and left ventricular mass wet (including ventricular septum) was measured for Left Ventricular Mass Index (LVMI). Fixing one part of left ventricular tissue in 10% formaldehyde buffer solution for 12-24 h, conventionally dehydrating, transparentizing, waxing, embedding, slicing, measuring the diameter of left ventricular Cardiomyocytes (CD) by HMIAS-2000 high-definition color medical image-text analysis software under a 400-fold optical microscope, analyzing the pathological morphological change of the myocardial tissue, and taking pictures.

1.3 free calcium ion ([ Ca ") in myocardial cells²⁺]i) Determination of concentration

Left ventricular cardiomyocytes were isolated by reference to relevant literature (e.g., Corjun, Chenfeng, Shouxin, Daissen, Wangxianping, Cheng soldier, Chen Ming, Zhangtai. influence of calmodulin kinase II inhibitors on hypertrophic cardiomyocytes. China journal of Emergency medicine, 2012, doi: 10.3760/cma.j.issn.1671-0282.2012.02.011), and the fura-3-loaded cardiomyocytes were placed under a fluorescence microscope (PTll048, Japan) with excitation wavelength of calcium fluorescence of 340/380nm and emission wavelength of 510 nm. The fluorescence signal is processed by Felix special software.

1.4 detection of protein expression of ANP and beta-MHC by Western blot

Rat myocardial tissue was taken, tissue protein was extracted using Trizol reagent, and protein content was measured using the Bradford method. Adding the extracted equivalent protein into a 3 Xloading buffer solution, boiling for 3min, then loading, performing vertical electrophoresis on the prepared SDS polyacrylamide gel, then transferring the protein onto a nitrocellulose filter membrane, respectively incubating with ANP and beta-MHC antibodies for 2h according to a standard Western blot method, then eluting, carrying out closed rinsing, and then adding a diluted secondary antibody. Hybridizing for 1h at room temperature, carrying out exposure, development and fixation after carrying out warm bath on a chemiluminescence agent for 1min, and finally carrying out absorbance scanning analysis on the result.

1.5 statistical methods

All data are mean. + -. standard deviation

To show that the analysis was performed using the spssl7.0 statistical software. The multiple comparisons were analyzed by one-way anova, and the two comparisons were examined by t-test, with P <0.05 considered statistically significant.

2 results

2.1 Effect on rat SBP

Compared with the myocardial hypertrophy group, SBP of the 100mg/kg TFG group is not significantly reduced [ (25.49 +/-1.86) kPa vs (24.99 +/-1.68) kPa, P >0.05], but SBP of the valsartan group is obviously reduced [ (18.54 +/-1.52) kPa, (19.04 +/-1.45) kPa vs (24.99 +/-1.68) kPa, P <0.01] by the 300mg/kg TFG group, which indicates that the large dose TFG has the function of reducing SBP, and the large dose of 300mg/kg TFG is similar to the valsartan group.

2.2 Effect on ventricular septum, left ventricular posterior wall thickness, Cardiomyocyte Diameter (CD), Left Ventricular Mass Index (LVMI)

The interventricular septum, left ventricular posterior wall thickness of the TFG group and valsartan group was higher than that of the control group (P <0.05), but lower than that of the myocardial hypertrophy group (P < 0.01); the interventricular septal thickness was higher for the TFG group than for the valsartan group (P <0.05), whereas the difference between the left ventricular posterior wall thickness was not statistically significant for the groups (P >0.05), see table 1.

The CD of both TFG group and valsartan group was significantly lower than that of the cardiac hypertrophy group (P <0.01) and higher than that of the control group (P < 0.05). Suggesting that TFG and valsartan have the function of inhibiting left ventricular hypertrophy. The TFG group has higher CD than the valsartan group (P <0.05), which indicates that the TFG group has less obvious effect than the valsartan group, and is shown in Table 1.

The LVMI of TFG group is obviously lower than that of myocardial hypertrophy group (P <0.05), suggesting that TFG has the function of inhibiting left ventricular hypertrophy. However, compared to the valsartan group, the LVMI of the TFG group was relatively high, showing a weaker effect than valsartan, see table 1.

Table 1 changes in the ventricular interval, left ventricular posterior wall thickness and myocardial histopathology for each experimental group of rats (n-8;

)

comparison with the control group:^aP<0.05，^bP<0.01; comparison with myocardial hypertrophy group:^CP<0.05。

2.3 myocardial tissue morphological changes

HE staining shows that the diameter of the myocardial cells of the control group is not obviously increased, and the arrangement of the myocardial cells is regular; the myocardial hypertrophy group shows that the diameter of myocardial cells is obviously increased, the volume is hypertrophic, and the arrangement of the myocardial cells is disordered. The myocardial cells of the TFG group and the valsartan group are less pathologically and histologically changed than those of the myocardial hypertrophy group, and the figure 8 shows that the myocardial cells of the TFG group and the valsartan group are more stable.

2.4 cardiac muscle cells [ Ca ]²⁺]Variation of i

Myocardial hypertrophy group intracellular [ Ca ]²⁺]i[(161.08±13.42)nmoL/L]Is obviously higher than that of a control group, a 100mg/kg TFG group, a 300mg/kg TFG group and a valsartan group [ (100.12 +/-10.23, 137.23 +/-13.23, 114.15 +/-10.12, 122.89 +/-10.41) nmol/L, P<0.01](ii) a The difference between the TFG group and the control group is statistically significant (P)<0.05); the valsartan group is still higher than the TFG group and the control group (P) of 300mg/kg<0.05). As can be seen, 300mg/kg TFG reduced cardiomyocytes [ Ca ]²⁺]The concentration of i is stronger than that of valsartan (P)<0.05)。

2.5 Effect of TFG on ANP, beta-MHC in myocardial tissue of hypertensive rats with abdominal aortic constriction

The results of immunoblotting of ANP, β -MHC show (see figure 9): compared with a control group, the ANP and beta-MHC protein expression of the myocardial hypertrophy group is obviously increased (P is less than 0.01), which indicates that the ANP and the beta-MHC participate in the pathological process of the myocardial hypertrophy when the myocardial hypertrophy is carried out, and the ANP and the beta-MHC play a certain role in the pathogenesis of the myocardial hypertrophy. The expression of ANP and beta-MHC of the TFG group and the valsartan group is obviously lower than that of the myocardial hypertrophy group (P <0.01), and the down regulation effect of TFG on the expression of ANP and beta-MHC proteins is weaker than that of valsartan. Thus, TFG may slow and prevent myocardial hypertrophy by acting on ANP, β -MHC.

And (4) conclusion:

1. large doses of TFG have SBP-reducing effects;

2. AAC induces rat cardiac hypertrophy, the diameter of cardiac muscle cells is obviously increased, and the cell arrangement is disordered; after TFG and valsartan are treated, the myocardial pathological and histological change can be relieved, and TFG has a myocardial protection effect on AAC postoperative myocardial hypertrophy rats;

3. TFG antagonizes myocardial hypertrophy of AAC postoperative rats and may inhibit myocardial cells [ Ca ]²⁺]i, inhibiting cardiomyocyte ANP, &lTtT transfer = β "&gTt β &lTt/T &gTt-MHC protein expression is relevant.

Claims

1. The application of the gynostemma pentaphylla total flavone extract in preparing a medicament for preventing and treating myocardial hypertrophy or diseases based on the myocardial hypertrophy is disclosed, wherein the extraction method of the gynostemma pentaphylla total flavone extract comprises the following steps:

(1) cleaning stem and leaf of herba Gynostemmatis, drying, pulverizing, sieving, defatting with petroleum ether, drying to obtain residue, ultrasonic extracting the residue in 65% ethanol solvent, concentrating under reduced pressure to obtain extract, and vacuum drying to obtain crude extract of herba Gynostemmatis total flavonoids; the adding amount of the ethanol solvent is 25-40 ml/g based on the mass of the gynostemma pentaphylla extract;

(3) separating the concentrated solution A obtained in the step (2) by polyamide resin, eluting by ethanol, distilling under reduced pressure to obtain a concentrated solution B, and freeze-drying the concentrated solution B to obtain a gynostemma total flavone extract;

the diseases based on myocardial hypertrophy are hypertension, rheumatic heart disease, cor pulmonale, hyperthyroidism, dilated cardiomyopathy, hypertrophic cardiomyopathy, coronary heart disease or anemia.

2. The use of claim 1, wherein the gypenosides extract is used in an amount of 100-400 mg/kg/d based on gypenosides.

3. The use of claim 1, wherein the extract of total flavonoids of gynostemma pentaphyllum is formulated into tablet, capsule or injection.

4. The application of medicinal salt of Chinese medicinal herba Gynostemmatis total flavone in preparing medicine for preventing and treating myocardial hypertrophy or diseases based on myocardial hypertrophy is provided, the extraction method of herba Gynostemmatis total flavone extract comprises:

5. The use of claim 4, wherein the pharmaceutically acceptable salt of total flavonoids of gynostemma pentaphyllum is total flavonoids of gynostemma pentaphyllum hydrochloride.

6. An application of a traditional Chinese medicine gynostemma pentaphylla medicinal composition consisting of traditional Chinese medicine gynostemma pentaphylla total flavonoids and/or a medicinal salt of the traditional Chinese medicine gynostemma pentaphylla total flavonoids and a medicinal auxiliary material acceptable to a human body in preparing a medicament for preventing and treating myocardial hypertrophy or diseases based on the myocardial hypertrophy is disclosed, wherein the extraction method of the gynostemma pentaphylla total flavonoids extract comprises the following steps:

7. The application of claim 6, wherein the dosage of the traditional Chinese medicine gynostemma pentaphylla pharmaceutical composition is 100-400 mg/kg/d calculated by total flavonoids of gynostemma pentaphylla.