CN111053825A - Callicarpa kwangtungensis extract and application thereof - Google Patents

Abstract

The invention relates to a Callicarpa kwangtungensis Chun extract, and discloses a Callicarpa kwangtungensis Chun total extract and a phenylethanoid glycosides compound enriched by the Callicarpa kwangtungensis Chun total extract through a macroporous resin column, which have the effect of resisting myocardial ischemia, and the content of LDH, CK and MDA in serum of the part can be reduced, the content of GSH in myocardial tissues can be increased, and the content of Na in the myocardial tissues can be increased⁺/K⁺-ATPase activity, reducing inflammation and apoptosis levels to protect rat myocardial damage, and the Callicarpa kwangtungensis Chun total phenylethanoid glycosides are obtained by Na⁺/K⁺-ATPase/Src/ERK1/2 signal pathway to exert myocardial protective effects.

Description

Callicarpa kwangtungensis extract and application thereof

Technical Field

The invention discloses a traditional Chinese medicine extract for protecting cardiac muscle, belonging to the field of traditional Chinese medicines.

Background

Myocardial ischemia is a condition in which the blood perfusion of the heart is reduced, which results in a reduction in oxygen supply to the heart and an abnormal energy metabolism of the heart muscle. Severely affects people's life and work, and severe people can cause myocardial infarction and sudden death. It is characterized by hypoxia, myocardial inflammation and apoptosis of myocardial cells, leading to a severe imbalance between myocardial oxygen supply and demand that leads to rapid development of myocardial necrosis. The major symptoms of myocardial ischemia are angina pectoris, myocardial infarction, ischemic cardiomyopathy, and sudden death. Cardiovascular diseases (CVDs) are a group of diseases that affect the heart and/or blood vessels, with hypertension and atherosclerosis in large part in cardiovascular patients. Hypertension and/or atherosclerosis can progress to arrhythmia, ischemic heart disease, myocardial infarction, and ultimately heart failure.

Callicarpa kwangtungensis (Callicarpa kwangtungensis) is perennial deciduous shrub of Callicarpa of Verbenaceae, and its dried stem, branch and leaf can be used as raw material, and its bitter and astringent taste is cool, and can be used for treating hematemesis, sore throat, traumatic hemorrhage, and pyocutaneous disease. The traditional Chinese medicine composition is clinically used for treating gynecological diseases such as cervicitis, pelvic inflammation and vaginitis. The callicarpa kwangtungensis extract has obvious effect of inhibiting inflammatory reaction on the exudation in the early stage of experimental inflammation, and has the advantages of small side effect, low price and wide source. The research aims to develop the traditional Chinese medicine of Callicarpa kwangtungensis Chun which has the myocardial protection activity and further elucidate the mechanism of the myocardial protection function.

Disclosure of Invention

The invention aims to provide a new application of a callicarpa kwangtungensis chun extract, namely, the extract has a myocardial protection effect.

Technical scheme

The callicarpa kwangtungensis extract is characterized by being obtained by the following steps: extracting dry stem and leaf of Callicarpa kwangtungensis Chun with 80% ethanol under heating and refluxing, and concentrating the filtrate to obtain extract; passing the extract of Callicarpa kwangtungensis Chun through macroporous resin column, eluting with 30% ethanol, collecting 30% ethanol eluate, and concentrating under reduced pressure to obtain extract, i.e. Callicarpa kwangtungensis Chun extract.

Specifically, the method comprises the following steps:

the callicarpa kwangtungensis extract is characterized by being obtained by the following steps: extracting dry stem and leaf of Callicarpa kwangtungensis Chun with 80% ethanol under heating and refluxing for 3 times, each time for 1 hr, mixing filtrates, and concentrating to obtain extract, i.e. Callicarpa kwangtungensis Chun total extract; loading the Callicarpa kwangtungensis total extract by dry method, passing through D101 macroporous resin column, sequentially eluting with 10%, 30%, 50%, 70%, and 90% ethanol, collecting 30% ethanol eluate, and concentrating under reduced pressure to obtain extract, i.e. Callicarpa kwangtungensis extract-Callicarpa kwangtungensis total phenylethanoid glycoside.

The Callicarpa kwangtungensis Chun extract and the Callicarpa kwangtungensis Chun total phenylethanoid glycosides are applied to the preparation of the myocardial protection medicament.

The invention further concretely discovers that the Callicarpa kwangtungensis Chun extract and the Callicarpa kwangtungensis Chun total phenylethanoid glycosides have the protection effect on myocardial ischemia injury and discovers that the Callicarpa kwangtungensis Chun total phenylethanoid glycosides are obtained by changing Na⁺/K⁺-ATPase activity exerts a cardioprotective effect, in particular by activating Na⁺/K⁺-ATPasw/Src/ERK1/2 signal path.

Further explaining the medicine of the present invention is obtained from the market, the extraction method is that 5.4kg of raw medicinal materials of Guangdong beautyberry are heated and refluxed for extraction for 3 times by 80% ethanol, each time is 1 hour, and after being combined, the raw medicinal materials are decompressed and concentrated to obtain a total extract, and the Guangdong beautyberry extract is obtained. Passing the total extract through D101 macroporous resin column, eluting with 30% ethanol, collecting eluate, and concentrating to obtain extract, i.e. Callicarpa kwangtungensis Chun total phenylethanoid glycosides.

In vitro activity screening of Callicarpa kwangtungensis Chun extract: the obtained Guangdong beautyberry total extract, namely Guangdong beautyberry total phenylethanoid glycosides, is subjected to myocardial oxidative damage resistant activity screening experiments, and the experiments show that the Guangdong beautyberry total phenylethanoid glycosides can resist H₂O₂H9c2 cells inducing oxidative damage have protective effect.

Screening the in vivo activity of the Callicarpa kwangtungensis Chun total phenylethanoid glycosides: preparing the Callicarpa kwangtungensis Chun total phenylethanoid glycosides into low, medium and high (100mg/kg,150mg/kg and 200mg/kg) concentrations for intragastric administration, and after 20 days of administration, beginning molding, wherein the molding method comprises the following steps:isoprenaline hydrochloride (ISO) induces SD rats to generate myocardial ischemia, cardiac color ultrasonography is performed after ISO administration for two days, and the heart function constant of rats in a blank group is normal, the short axis shortening rate of myocardial ejection fraction of a model group is reduced, the hypertrophy index is increased compared with that of the blank group, and the heart function is recovered after administration, wherein a specific chart is shown in figure 4. Callicarpa kwangtungensis extract for increasing myocardial tissue Na⁺/K⁺-ATPase activity: after each group of rats died, a certain amount of myocardial tissue is taken to measure Na in the tissue⁺/K⁺The activity of ATPase enzyme, and the discovery that the total phenylethanoid glycosides of Callicarpa kwangtungensis can improve Na in myocardial tissues⁺/K⁺-ATPase activity.

Advantageous effects

1. The total extract of the callicarpa kwangtungensis chun and the total phenylethanoid glycosides of the callicarpa kwangtungensis chun are found to have the myocardial protection activity for the first time.

2. The first discovery that the total phenylethanoid glycosides of Callicarpa kwangtungensis Chun can increase the Na on the surface of the myocardium of a rat^+/K⁺-ATPase activity.

3. The first discovery that the Callicarpa kwangtungensis Chun total phenylethanoid glycosides can regulate Na in myocardial tissues⁺/K⁺-ATPase expression, capable of activating Na⁺/K⁺The ATPase/Src/ERK1/2, and Bax/Bcl2 signaling pathways exert myocardial protective effects.

Drawings

FIG. 1: the liquid phase diagram of the total phenylethanoid glycosides of Callicarpa kwangtungensis Chun and Callicarpa kwangtungensis Chun is shown in the specification, wherein A is the total extract of Callicarpa kwangtungensis Chun, B is the total phenylethanoid glycosides of Callicarpa kwangtungensis Chun, C is the comparison diagram of the mixed standard of forsythoside B and poliumoside, and D and E are the standard curves of forsythoside B and poliumoside respectively.

FIG. 2: TOF-MS (time of flight-mass spectrometry) graph of Callicarpa kwangtungensis extract (A) and Callicarpa kwangtungensis total phenylethanoid glycoside (B)

FIG. 3: the screening picture of the heart muscle protecting activity of the Callicarpa kwangtungensis Chun extract and the Callicarpa kwangtungensis Chun total phenylethanoid glycosides. FIG. 4: callicarpa kwangtungensis Chun total phenylethanoid glycosides and forsythiaside B for inhibiting Na⁺/K⁺-map of ATPase activity screening results;

FIG. 5: rat heart color Doppler ultrasound A and heart function constant map, hypertrophy index (B), short axis shortening rate (C), systolic ventricular interval thickness (D), diastolic ventricular interval thickness (E), left ventricular end-systolic internal diameter (F), and diastolic end-left ventricular posterior wall thickness (G)

FIG. 6: the level of myocardial damage marker in rat serum, the content of lactate dehydrogenase in serum (A), and the content of creatine kinase in serum (B).

FIG. 7: the level of oxidative stress in rat serum and myocardial tissue, the content of Malondialdehyde (MDA) in the serum (B) and the content of Glutathione (GSH) in the myocardial tissue (A);

FIG. 8 shows the levels of inflammatory factors in rat serum and myocardial tissue, caspase3 activity in myocardial tissue (A) tumor necrosis factor in serum (TNF- α) (B), interleukins in serum (C), (D);

FIG. 9: pathological section of rat myocardial tissue;

FIG. 10: na in rat myocardial tissue⁺/K⁺-ATPase activity;

FIG. 11: immunohistochemical method for determination of Na in myocardial tissue⁺/K⁺ATPase expression, immunohistochemical staining (A), quantitative determination of Na⁺/K⁺-ATPase expression (B);

FIG. 12 immunoblotting of Src, p-Src protein expression in myocardial tissue, p-Src (A), Erk1/2, p-Erk1/2 protein expression using Src total protein as internal reference, Erk1/2 total protein as reference for quantification (B), Bcl2, Bax protein expression using β -actin as reference for quantification (C).

The specific implementation mode is as follows:

example 1 extraction and crude separation of Callicarpa kwangtungensis Chun

Crushing 5.4kg of raw callicarpa kwangtungensis Chun medicinal materials into powder, heating and refluxing the powder by using 80% ethanol for 3 times, extracting for 1 hour each time, filtering, combining filtrate, concentrating under reduced pressure at 50 ℃ to obtain a total extract, namely a callicarpa kwangtungensis Chun total extract, and passing the total extract through a D101 macroporous resin column. The content of forsythoside B and poliumoside is used to measure the total phenylethanoid glycoside content in the extract, and liquid phase analysis shows that the content of phenylethanoid glycoside in the Callicarpa kwangtungensis Chun is 33.7%, as shown in figure 1.

TABLE 1 quantification of phenethyl alcohol content in Callicarpa kwangtungensis Chun and Callicarpa kwangtungensis Chun Total phenethyl alcohol glycoside by forsythoside B and poliumoside content

Mass spectrometry conditions of Callicarpa kwangtungensis Chun and Callicarpa kwangtungensis Chun total phenylethanoid glycosides are as follows:

mobile phase A: 0.1% formic acid-water solution, B: methanol solution, elution conditions: 0-5min: 5% (B) -15% (B),5-8min: 15% (B) -16% (B),8-12min: 16% (B) -32% (B), 12-42min: 32% (B) -37% (B),42-62min: 37% (B) -47% (B),62-72min: 47% (B) -52% (B),72-82min: 52% (B) -95% (B), the flow rate is 1mL/min, the detection wavelength: 280 nm. Sample introduction volume: 10 μ L, the results are shown in FIG. 2.

TABLE 2 Structure inference of possible compounds from Callicarpa kwangtungensis Chun extract

TABLE 3 structural inference of possible compounds of Callicarpa kwangtungensis Chun total phenylethanoid glycosides

23.449	640.1963	campneoside
			24.607	180.0588	Caffeic acid
25.319	650.2147	Ribes burejense glycoside
			26.2	786.2570	Echinacoside
27.285	786.2567	β-OH-poliumoside
			28.099	654.2482	Isocampneoside
28.947	786.2566	β-OCH3-forsythoside
			31.422	756.2465	fosythoside B
33.829	624.2027	Acteoside
			36.644	756.2444	samioside
47.325	797.2561	2’-forsythoside
			56.615	811.2730	Brandioside
63.193	635.1312	Acacetin-7-O-diglucuroniside
			67.330	417.2561	syringaresind
68.177	462.242	decaffeoylacteoside

Medicine Na⁺-K⁺ATPase activity screen (affinity screen): to evaluate the drug pairs to be tested for Na⁺-K⁺Affinity of ATPase, usually by drugs inhibiting Na⁺-K⁺The ability of ATPase to hydrolyze ATP is characterized. Dissolving Callicarpa kwangtungensis extract in DMSO to obtain 100mg/mL mother solution, and precisely adding 15 μ L of medicinal solution and 15 μ L of Na into 1.5mL EP tube⁺-K⁺-ATPase solution to 70. mu.L reaction buffer and mix well. Experimental grouping conditions were: blank group, administration group, positive drug group (ouabain); two controls were made for each group. Incubating in water bath at 37 deg.C for 15min, and activating Na⁺/K⁺-ATPase followed by addition of 15 μ L ATP solution to EP tube and mixing; incubating in 37 deg.C water bath for 25min, adding 18 μ L protein precipitant, precipitating, centrifuging at 10000rpm for 5min, collecting supernatant, determining phosphorus content with phosphorus test kit (phosphomolybdic acid method) according to the instruction, and evaluating Na by absorbance⁺-K⁺The extent to which ATPase is inhibited. The results are shown in FIGS. 3 and 4, and the Callicarpa kwangtungensis Chun extract and Na are found⁺/K⁺The ATPase affinity is very good.

Animal experiments: selecting 250gSD rats as molding animals, pre-administering the Guangdong beautyberry total phenylethanoid glycosides for 3 weeks, performing intragastric administration, performing subcutaneous injection of Isoproterenol (ISO) at 20 days and 21 days, wherein the dose is 120mg/kg, performing echocardiogram examination on the rats two days after continuous molding (24 hours after the first subcutaneous injection), and finding that the rat cardiac muscle (mainly ventricle) is serious in hypertrophy and the cardiac muscle is seriously injured, which shows that the molding is successful, the rat hypertrophy phenomenon of an administration group is relieved and the injury degree is reduced.

LDH, CK and MDA kit for detecting factor expression of LDH, CK and MDA in serum

LDH, CK and MDA are labeled factors of myocardial ischemia injury, rat serum is taken and operated according to the specification of a reagent kit, the result of the reagent kit is shown in figure 6, and figure 7 shows that the levels of LDH, CK and MDA in a model group are obviously increased compared with a blank group, and the expression of LDH, CK and MDA in an administration group is obviously reduced compared with the model group, which shows that the Callicarpa kwangtungensis total phenylethanoid glycosides have protective effect on the myocardial ischemia injury.

And (3) detecting the activity of GSH:

taking a proper amount of myocardial tissues of each group, adding normal saline according to the proportion of 1:9, homogenizing, then 12000r/min, taking supernate, carrying out protein quantification, and carrying out GSH activity determination according to the kit instructions. The results of the reagent kit show that the results are shown in figure 7, and compared with the blank group, the GSH content of the rats in the model group is reduced, after the reagent kit is administrated, the GSH level in the tissues of the rats in each group is increased, and the Guangdong total phenylethanoid glycosides can increase the antioxidant capacity of myocardial tissues.

Inflammatory apoptosis-related factors

caspase3 activity assay: caspase3 is an important component involved in DNA maintenance and cell regulation, namely caspase3 activation, apoptosis. The results are shown in fig. 8(A) and provided according to the kit description, a method for extracting caspase3 protein in tissues is provided, and caspase3 activity is determined according to the specification method, and the results show that caspase3 in a model group has the strongest activity and the most serious apoptosis, and the apoptosis condition is weakened after the callicarpa bodinieri extract is given, which shows that the callicarpa kwangtungensis extract can increase the anti-apoptosis level of cardiac muscle so as to play a role in protecting cardiac muscle.

TNF- α -6 and IL-1 β activity detection shows that when cells are injured, an organism generates an inflammatory response which is a protective and defensive response of the organism to injury factors, TNF- α -6 and IL-1 β are endogenous inflammatory mediators and can regulate natural immunity, the activity of TNF- α -6 and IL-1 β is determined according to kit instructions, and the results are shown in figures 8(B), 8(C) and 8(D), and experimental results show that the content of TNF- α -6 and IL-1 β is remarkably increased after modeling, and the level of TNF- α -6 and IL-1 β is remarkably reduced after administration, which shows that Callicarpa kwangtungensis can play a myocardial protection role through anti-inflammation.

HE staining looked at the level of pathology: pathological section examination finds that the nucleus of the model group is solidified and shrunk, the myocardial fiber is swelled, the arrangement is disordered, local striations disappear, inflammatory cells are infiltrated, and part of myocardial cells are broken, necrotic and fused, so that pathological damage is obvious. The results are shown in fig. 9, the degree of myocardial fibroid swelling is reduced after the drug administration, inflammatory cell infiltration is reduced, and the pathological injury degree is reduced, which indicates that the callicarpa kwangtungensis extract has a protective effect on myocardial ischemia injury.

Myocardial tissue Na⁺-K⁺-ATPase activity assay:

taking appropriate amount of myocardial tissue of each group, adding normal saline at a ratio of 1:9, homogenizing, then 12000r/min, taking supernatant for protein quantification, the result is shown in figure 10, activity detection result shows that model group Na⁺-K⁺-decrease in ATPase activity, increase in activity after administration, Na⁺-K⁺The ATPase activity has protective effect on myocardial cells, which indicates that Callicarpa kwangtungensis Chun is probably increased by Na⁺-K⁺-ATPase activity to exert a cardioprotective effect.

Immunohistochemical staining detection of Na in rat myocardial tissue⁺-K⁺-ATPase expression:

dewaxing a paraffin section of rat myocardial tissue, heating the section for antigen retrieval, incubating the section with 3% H2O2 for 15-20min to remove endogenous catalase, adding citric acid-sodium citrate as a retrieval solution,5% BSA seals the section for 1h, adds primary antibody dropwise for incubation overnight, PBST is washed for 3 times and then incubates secondary antibody for 1h under the condition of room temperature, then adds SABC reagent for incubation for 30min, operates according to the kit and according to the instruction, dyes the section with DAB staining solution and counterstains hematoxylin, finally dehydrates and dewaxes the section, observes under a normal microscope, and quantifies with image J software. The results are shown in FIG. 11(A), and 11 (B) quantitative results show that: na in myocardial tissue after molding⁺-K⁺-ATPase expression is significantly reduced, Na in myocardial tissue after administration of Callicarpa kwangtungensis extract⁺-K⁺Increased expression of ATPase, indicating that Callicarpa kwangtungensis extract can increase Na⁺-K⁺-expression of ATPase to exert a myocardial protective effect.

Western Blot assay for the detection of Src, p-Src, Erk1/2, p-ERK1/2 expression in rat myocardial tissue 100mg of rat left ventricular tissue was taken and 1ml of RIPA lysate, 20. mu.L protease inhibitor and 20. mu.L phosphatase inhibitor were added, the tissue was crushed with a glass homogenizer on ice, lysed for 15 minutes on ice and centrifuged for 5 minutes at 12000rpm at 4 ℃ and the supernatant was collected and the protein concentration was quantified with BCA kit, after denaturation of the protein sample with BCA buffer, the protein was separated by polyacrylamide gel electrophoresis and transferred to PVDF membrane, which was then blocked with 5% (w/v) skim milk at room temperature for 2h, Src (1; 8000), p-Src (1; 1000), Erk1/2 (1; 1000), p-ERK1/2 (1; 8000) Bcl-2(1:800), Bax (1:1000) and β -actin (internal reference 1:10000), after incubation of the protein in the corresponding bands with light, the staining of the protein bands were analyzed by staining with 12. mu.12. after incubation of the protein bands with 5% (w/v) of the staining of the protein in the test, after staining the staining of the protein bands at room temperature, the staining of the protein bands with light, the test band was analyzed by a test, and the protein bands were analyzed by staining with 12. mu.12. mu.H, and incubated with 5. the test, and incubated with light, and the⁺-K⁺Reduced expression of ATPase, activation of Src, Erk1/2 protein, increased expression of the apoptotic protein Bax, and reduced expression of the anti-apoptotic protein Bcl2, after administration, Na, as compared to the model group⁺-K⁺Increased expression of ATPase, decreased activation of Src, Erk1/2 protein, decreased expression of apoptotic proteins. Shows that the total phenylethanoid glycosides of Callicarpa kwangtungensis Chun can regulate Na⁺-K⁺The ATPase/Src/Erk1/2 signal pathway and the Bax/Bcl2 signal pathway exert myocardial protective effects.

Claims (2)

1. The Callicarpa kwangtungensis extract is characterized by being obtained by the following steps: heating and reflux-extracting dry stem and leaf of Callicarpa kwangtungensis Chun with 80% ethanol, and concentrating the filtrate to obtain extract to obtain Callicarpa kwangtungensis Chun total extract; and (3) passing the total extract of the callicarpa kwangtungensis chun through a macroporous resin column, eluting with 30% ethanol, collecting 30% ethanol eluate, and concentrating under reduced pressure to obtain an extract, namely the callicarpa kwangtungensis chun extract.

2. Use of the extract of callicarpa kwangtungensis as claimed in claim 1 for the preparation of a medicament for the protection of the myocardium.

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