CN115671154A - Semen Lepidii flavone component, its extraction method and application in preparing medicine for treating or preventing heart failure - Google Patents
Semen Lepidii flavone component, its extraction method and application in preparing medicine for treating or preventing heart failure Download PDFInfo
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- CN115671154A CN115671154A CN202211516197.5A CN202211516197A CN115671154A CN 115671154 A CN115671154 A CN 115671154A CN 202211516197 A CN202211516197 A CN 202211516197A CN 115671154 A CN115671154 A CN 115671154A
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Abstract
The invention relates to the technical field of traditional Chinese medicines, and discloses a semen lepidii flavone component, an extraction method and application in preparation of medicines for treating or preventing diseases related to mitochondrial structural dysfunction such as heart failure, wherein the extraction process comprises the steps of preparing semen lepidii concentrated solution, eluting the concentrated solution in an adsorption column by using water and 15-60% ethanol, collecting 15-60% ethanol eluent, concentrating and drying to obtain the semen lepidii flavone component, and separating by liquid chromatography to obtain quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside. The invention firstly provides that the semen lepidii flavone component and the quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside can be respectively used as active components/components to obviously improve the ROS level, the cell activity, the mitochondrial membrane potential, the mitochondrial bioenergy and the malondialdehyde level in myocardial cells, and have certain development potential in the aspect of preparing medicaments for treating cardiovascular related diseases such as heart failure and the like.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to extraction and pharmacological activity research of active ingredients of traditional Chinese medicines, and specifically relates to a lepidium seed flavone component, an extraction method and application in preparation of a medicine for treating or preventing heart failure.
Background
Heart failure (Heart failure) is a long-standing and intractable public health problem. Typical clinical symptoms of heart failure are pulmonary congestion and peripheral edema caused by fluid retention and low cardiac output. During the progression of heart failure, structural and functional abnormalities of the myocardium are associated not only with abnormal activation of the sympathetic nervous system and renin-angiotensin aldosterone system, but also with overproduction of Reactive Oxygen Species (ROS). ROS originate primarily from the mitochondrial electron transport chain, and increased levels of ROS can cause excessive oxidative stress, characterized by DNA damage, protein oxidation, and lipid peroxidation, leading to cellular dysfunction. In fact, there is increasing evidence that oxidative stress caused by an imbalance in the intracellular excess of ROS and/or the endogenous antioxidant system is closely linked to the progression of heart failure.
The normal operation of the heart requires a large amount of energy, which is supplied by mitochondrial oxidative phosphorylation and also produces mitochondrial ROS (mROS). When oxidative stress occurs, there is an increase in the production of mROS, with excessive mitosis of the mitochondria, resulting in structural damage and dysfunction of the mitochondria. In other words, under excessive oxidative stress, damaged mitochondria accumulate, mitochondrial permeability transition pores open, resulting in calcium overload, decreased mitochondrial membrane potential, damage to mitochondrial bioenergy, and apoptosis. In particular, cardiomyocytes are energy-intensive cells and it is important to maintain the normal function of the cardiomyocytes intact with the structure and function of mitochondria. Therefore, targeting mROS within cardiomyocytes is considered a promising option for treating heart failure.
Various antioxidant systems exist within cardiomyocytes to protect against oxidative stress injury. Phosphatidylinositol 3 kinase (PI 3K) or protein kinase B (AKT) is involved in the cellular antioxidant defense system as an upstream signal of nuclear factor erythroid 2-related factor 2 (Nrf 2). Nrf2 transcription factors are important transcription factors for stress signaling, and bind to cytoplasmic adaptor protein (Keap 1) under physiological conditions. When stimulation such as oxidative stress occurs, nrf2 enters cell nucleus and combines with promoter region of antioxidant gene such as heme oxygenase-1 (HO-1), and HO-1 can express phase II detoxification enzyme of cell to remove toxic heme and generate biliverdin, iron and carbon monoxide. HO-1 and its products exert protective effects by regulating oxidative damage, apoptosis and inflammation. In heart failure, the cardioprotective role of HO-1 in relation to the regulation of mitochondrial function has been elucidated.
The traditional Chinese medicine has a unique theoretical system, can regulate the whole body of an organism and has low drug resistance. The semen Lepidii is derived from dried mature seed of Descurainia sophia (L.) Webb.ex Prantl) belonging to family Brassicaceae. Pungent and bitter in flavor and cold in nature. It enters lung and bladder meridians. Has the effects of purging lung, relieving asthma, promoting diuresis and relieving swelling. It is indicated for phlegm and saliva obstructing lung, cough and asthma with profuse sputum, fullness in chest and hypochondrium, inability to lie flat, difficult urination, etc. and is mainly used to treat various pulmonary inflammations and edema.
Semen lepidii is often used in combination with other traditional Chinese medicine components to play a certain role in treating heart failure, for example, CN103202879A discloses a traditional Chinese medicine composition for treating chronic heart failure and a preparation method thereof, wherein the traditional Chinese medicine composition comprises main traditional Chinese medicine materials such as astragalus, cassia twig, salvia miltiorrhiza, angelica, ginseng, safflower, motherwort, semen lepidii, tuckahoe, pseudo-ginseng and the like and medicinal auxiliary materials, and clinical symptoms of patients with heart failure are improved and myocardial contractility is improved by methods of tonifying qi, activating blood circulation to dissipate blood stasis and warming yang and promoting diuresis. In recent years, many cardiovascular pharmacological studies on semen lepidii are focused on animal models, and whether the semen lepidii can resist myocardial cell oxidative stress-induced mitochondrial damage is not reported in the study. The semen Lepidii contains complex components, and its main components are flavonoids, saccharides and glucosides. The research on the activity characteristics of certain single components is also shallow, quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is a characteristic component and an index component of a semen lepidii medicinal material, belongs to a flavonoid compound, and CN107602629A discloses a flavonoid compound extracted from semen lepidii and application thereof.
Moreover, the effective components of the pepperweed seed for resisting heart failure and other cardiovascular diseases are determined, and a foundation can be provided for the quality control of the pepperweed seed and the development of related new drugs. The effective components or the new developed medicine of the components of the semen lepidii can remove the ineffective components in the semen lepidii for treating related diseases, reduce the dosage of the medicine used by a patient, improve the quality control level of the related new medicine, and ensure that the related new medicine can exert stable and reliable clinical curative effect.
Disclosure of Invention
The invention provides application of a semen lepidii flavone component and quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside (F3) in preventing and/or treating cardiovascular related diseases such as heart failure, and finds that the flavone component and the F3 in the semen lepidii can obviously improve the ROS level, cell activity, mitochondrial membrane potential, mitochondrial bioenergy and malondialdehyde level in myocardial cells as active components/components for the first time, and the semen lepidii flavone component and the F3 have certain development potential in the aspect of preparing medicines for treating mitochondrial structure dysfunction related diseases such as heart failure.
In order to achieve the purpose, the invention adopts the technical scheme that:
a semen lepidii flavone component, a preparation method of the semen lepidii flavone component, comprises the following steps:
and 2, suspending the semen lepidii extract concentrated solution in water, loading the mixture to a macroporous adsorption resin column, eluting the mixture by using water and 15-60% ethanol in sequence, collecting 15-60% ethanol eluent, concentrating and drying the eluent to obtain the semen lepidii flavone component.
The invention provides a semen lepidii flavone component, which is extracted from semen lepidii, wherein in step 1, semen lepidii is crushed and then extracted in water or ethanol to obtain concentrated solution, and the ethanol can be any concentration; preferably, the extraction is performed 2 times, each for 1-2h.
The invention also provides a preparation method of the semen lepidii flavone component, which comprises the following steps:
and 2, suspending the semen lepidii extract concentrated solution in water, loading the mixture to a macroporous adsorption resin column, eluting the mixture by using water and 15-60% ethanol in sequence, collecting 15-60% ethanol eluent, concentrating and drying the eluent to obtain the semen lepidii flavone component.
Preferably, the step 2 is sequentially eluted by water, 20-30% ethanol and 30-55% ethanol, the 20-30% ethanol eluate and the 30-55% ethanol eluate are collected, and the semen lepidii flavone component is obtained by concentration and drying.
The ethanol concentration of the invention refers to volume fraction, for example, 20-30% ethanol represents 20-30% ethanol.
Further preferably, the flow rate of the sample liquid and the eluent of the macroporous adsorption resin is preferably 0.5-3 BV/h (column volume/hour), and the dosage of the eluent is 2-8BV (column volume), preferably 2-6 BV.
The invention provides application of the semen lepidii flavone component in preparing a medicine for treating or preventing heart failure. The semen lepidii contains a plurality of flavone components, but not all the flavone components have the effect of resisting myocardial oxidative stress, and the invention discovers the effect of resisting myocardial oxidative stress of the flavone components of the semen lepidii by regulating mROS, MMP, adenosine Triphosphate (ATP) and Malondialdehyde (MDA) levels and the like under the oxidative stress of rat embryonic cardiac muscle cells to relieve the oxidative damage of myocardial mitochondria, so that the semen lepidii can be applied to the preparation of medicaments for treating cardiovascular related diseases such as heart failure and the like.
The invention also provides application of quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside in preparation of a medicine for treating or preventing heart failure.
The content of quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside in semen Lepidii is higher, and the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in semen Lepidii is not less than 0.075% in Chinese medicine pharmacopoeia (2020 edition). In the prior art, the research on quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside is mainly focused on the content measurement, and the research on the biological activity of the quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside is limited. The inventor researches and discovers that the flavone component and quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside in the pepperweed seed can be used as active components/ingredients to obviously improve the ROS level, the cell activity, the mitochondrial membrane potential, the mitochondrial bioenergy and the malondialdehyde level in myocardial cells, and the clear component activity can remove ineffective ingredients in the pepperweed seed in treating related diseases, reduce the dosage of the medicaments used by patients, is easy to realize high content, is very beneficial to the preparation of new medicaments, can improve the quality control level of the related new medicaments, and enables the related new medicaments to exert stable and reliable clinical effects.
Preferably, the semen lepidii is semen lepidii.
The heart failure refers to any one or more symptoms of myocardial hypertrophy, myocardial fibrosis, ejection fraction reduction, left ventricular short axis shortening rate reduction, cardiac output reduction, myocarditis, arrhythmia, atherosclerosis, cardiac insufficiency, cardiorenal syndrome and the like.
The medicine for treating or preventing heart failure comprises any one of tablets, oral liquid, pills, granules, capsules, injections, slow-release agents and the like.
The invention provides a medicament for preventing or treating heart failure, wherein the active component of the medicament is the lepidium seed flavone component or quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside.
The medicament for preventing or treating heart failure, which has at least one of the following functions:
(1) Prevention and/or treatment of heart failure;
(2) Prevention and/or treatment of myocardial hypertrophy;
(3) Reducing the relative area of hypertrophic cardiomyocytes;
(4) Reducing or alleviating myocardial fibrosis;
(5) Improving cardiac output;
(6) Improving the cardiac ejection fraction;
(7) Improving the left ventricular short axis shortening rate;
(8) Improving myocarditis;
(9) Improving arrhythmia;
(10) Improving atherosclerosis;
(11) Improving cardiac insufficiency;
(12) Relieving or treating heart and kidney syndrome.
The invention takes rat embryo ventricular myocyte system H9c2 cells as experimental objects, and researches the protective effect of the draba flavone component and F3 on oxidative stress injury and apoptosis of the tert-butyl hydroperoxide (tBHP) -induced H9c2 cells. Mitochondrial oxidative damage indicators, such as mROS, mitochondrial Membrane Potential (MMP), mitochondrial morphology, and ATP content, were first examined. The oxidative status of the cells was then assessed using Malondialdehyde (MDA) and intracellular total Reactive Oxygen Species (ROS) levels. Finally, whether an AKT/Nrf2/HO-1 signal channel participates in the heart protection effect of TLZ is observed, and the application of the semen lepidii flavone component and quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside in treating cardiovascular diseases through the activity of resisting myocardial mitochondrial oxidative damage is found, so that a brand-new treatment means or method can be provided for cardiovascular related diseases such as heart failure and the like.
The content of the semen lepidii flavone component in the medicine is 2-2000mg/g; the content of quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside in the medicine is 1-1500mg/g.
The invention also provides a method for extracting quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside from semen lepidii, which comprises the following steps: separating the semen Lepidii flavone component by liquid chromatography to obtain quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside.
The liquid chromatographic separation conditions are as follows: and (3) chromatographic column: an Agilent Zorbax SB-C18 column; mobile phase: a phase of 0.1% formic acid water solution and B phase of acetonitrile; the flow rate is 0.5-10mL/min; the detection wavelength is 200 nm-400 nm;
the linear elution gradient was: 0-1min,2% B;3-5min,4-6% B;8-12min,10-14% of B; 14-1695in, 14-16% B;24-26min,24-26% B;28-32min,31-35% B;35min,42-46% B;44-46min,65-75% of B;48-55min,100% B; column temperature: 35 ℃ is carried out.
Preferably, the linear elution gradient: 0min,2% of B;4min,5% B;10min,12% B;15min,15% B;25min,25% B;30min,33% B;35min,45% B;45min,70% B;50min,100% B.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention firstly provides the application of the semen lepidii flavone component and the quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside in preventing and/or treating the diseases related to mitochondrial structure dysfunction such as heart failure, the semen lepidii flavone component and the quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside as active components/ingredients can obviously improve the ROS level, cell activity, mitochondrial membrane potential, mitochondrial bioenergy and malondialdehyde level in cardiac muscle cells, and the invention has certain development potential in the aspect of preparing the medicaments for treating the cardiovascular related diseases such as the heart failure.
(2) The extraction and preparation method of the semen lepidii flavone component and the quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside is simple, the semen lepidii serving as the raw material is a common Chinese medicinal material in Chinese medicinal materials, has wide sources, is safe and low in toxicity, is used as a new raw material for preparing medicaments for resisting cardiovascular related diseases such as heart failure and the like, and has the advantages of small dosage, remarkable curative effect and small toxic and side effects. Therefore, the semen lepidii flavone component and the quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside have potential medical value and good social benefit in preparing the medicaments for treating cardiovascular related diseases such as heart failure and the like.
(3) The invention provides a method for extracting quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside from semen lepidii, which has the advantages of high product purity and high yield, and has high content of quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside in the semen lepidii, wide raw material source and low price, thereby having more beneficial medical value.
Drawings
FIG. 1 shows the effect of the flavones and sugar components of semen Lepidii on the oxidative damage of myocardial cells to mitochondrial reactive oxygen species.
FIG. 2 shows the effect of the flavones and sugar components of semen Lepidii on the mitochondrial membrane potential of H9c2 cells due to oxidative damage.
FIG. 3 shows the effect of typical flavone components in pepperweed seed on oxidative damage of mitochondrial reactive oxygen species to cardiomyocytes.
FIG. 4 shows the effect of a representative flavone component in pepperweed seed on oxidative damage of mitochondrial reactive oxygen species to cardiomyocytes.
FIG. 5 shows the effect of the flavones fraction from Lepidium seed, lepidium seed and F3 on the AKT/Nrf2/HO-1 signaling pathway after oxidative stress of cardiac myocytes.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Those skilled in the art should understand that they can make modifications and substitutions without departing from the spirit and scope of the present invention.
The raw materials used in the following embodiments are all commercially available, and semen Lepidii (seeds of Descurainia sophia (L.) Webb ex Prantl) is collected from Shandong province. Quercetin-3-O- β -D-glucose (denoted F1) and isorhamnetin-3-O- β -D-glucose (denoted F2) were purchased from Shanghai-derived leaf Biotechnology Ltd, china, shanghai. The purity of the standard products is more than 98 percent. Ultrapure water was prepared using a Milli-Q system (Millipore, bedford, MAUSA).
DMEM medium was purchased from Corning (CA, USA). Fetal Bovine Serum (FBS), penicillin-streptomycin and 0.25% trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). 3- (4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) and tBHP were purchased from Sigma-Aldrich (Merk KGaA, darmstadt, germany). Bicinchoninic acid (BCA) protein assay kit, HEPES solution (1m, ph 7.3, for cell culture), active oxygen assay kit, lipid oxidation (MDA) detection kit, hurst fluorescent dye (Hoechst 33342), horseradish peroxidase-labeled goat anti-rabbit IgG (H + L), RIPA lysate (strong), and BeyoECL Star (ultra sensitive ECL chemiluminescence kit) were purchased from bi yunnan biotechnology limited (shanghai, china). Tetramethylrhodamine methyl ester perchlorate (TMRM) was purchased from Sammerfeishi scientific, massachusetts, USA. Cell counting kit-8 (CCK-8) was purchased from Mellon Biotechnology Inc. (Dalian, china). Mitochondrial fluorescent probes (MitoSOX red mitochondrial superoxide indicator) were purchased from saint next biotechnology limited (shanghai, china). Phosphate Buffered Saline (PBS) was purchased from Katy Biotechnology Inc. (Jiangsu, china).
Primary antibodies against Nrf2 (16396-1-AP) and HO-1 (10701-1-AP) were used, purchased from Proteintetech (Wuhan, china), against AKT (pan) (11E 7), against phospho-AKT (Ser 437) and against beta-actin (8H 10D 10) from Cell Signaling Technology (Danvers, MA, USA).
Luminophore viability assay (CTG) was purchased from Promega technologies ltd (beijing, china). All other chemicals and solvents were of analytical grade.
Example 1 extraction of flavone component from semen Lepidii
And 2, dissolving the semen lepidii concentrated solution in 100mL of water, and performing ultrasonic treatment for 30min to obtain a semen lepidii sample to be separated. Filling a proper amount of pretreated D101 macroporous resin into a column, loading a lepidium sub-band separation sample on the macroporous resin from the top end of the resin, washing with 2BV water after static adsorption, collecting eluent, concentrating and drying to serve as a sugar component of the lepidium sub-band, and serving as a later-stage control. Eluting with 2BV of 30% ethanol and 50% ethanol respectively, collecting eluates of 30% ethanol and 50% ethanol, mixing, concentrating, and drying to obtain semen Lepidii flavone component. Continuously eluting with 1BV of 70% ethanol and 95% ethanol in turn, and washing the macroporous resin.
EXAMPLE 2 preparation of Quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside
The semen lepidii flavone component prepared in example 1 is subjected to liquid chromatography, and the liquid chromatography column comprises the following components: an Agilent Zorbax SB-C18 column; mobile phase: phase A is 0.1% formic acid water solution, phase B is acetonitrile; the detection wavelength is 200 nm-400 nm; the flow rate is 0.5-10mL/min.
Linear elution gradient: 0min,2% of B;4min,5% B;10min,12% of B;15min,15% B;25min,25% B;30min,33% B;35min,45% B;45min,70% B;50min,100% B; column temperature: at 35 deg.c.
Collecting chromatographic peak at 9-10.5min, and recovering solvent under reduced pressure to obtain quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside.
Effect of application example 1 on mitochondrial oxidative stress in rat embryonic myocardium H9c2 cells
1. Cell culture and oxidative damage
H9c2 cells were purchased from Shanghai cell Bank of China scientific research institute, DMEM high-glucose (4.5 g glucose/liter) medium supplemented with 10% (v/v) FBS and 1% (v/v) diabody mixture (100 IU/mL penicillin and 100mg/mL streptomycin) as the cell culture-used complete medium. Cells were cultured at 37 ℃,5% CO2 and 95% air in an incubator environment. When the cell density is 80-90%, cell passage and plating are carried out.
According to the experimental requirements, the cells were divided into three groups, the control group was cultured in normal complete medium, and the model group and the administration group were incubated for 2 hours in complete medium containing 150. Mu.M tBHP with or without preincubation for 24 hours. Administration group cells were given pepperweed seed, the pepperweed seed flavone component, the pepperweed seed sugar component, and 3 pepperweed seed flavone representative compounds (F1, F2, and F3 prepared in example 2) prepared in example 1.
2. Cell viability assay
H9c2 cells at 5X 10 3 cells/well density in 96-well plate, incubator for 24h, using different concentrations of semen Lepidii, semen Lepidii flavone component, semen Lepidii sugar component prepared in example 1 to pre-incubate 24h, discarding supernatant, adding MTT solution (0.5 mg/mL, DMEM dilution), incubator dark incubation for 4h, using DMSO to replace MTT solution, cell culture plate shaking at room temperature for 15min, using enzyme labeling instrument (Tecan Infinite M1000 PRO, switzerland) to detect the absorbance at 580nm, and counting the absorbance of each group relative to the blank control group.
It is found that the lepidium seed extract, the lepidium seed flavone component and the lepidium seed sugar component do not show the influence on the cell viability under the concentration of 500 mu g/mL, so the subsequent test is carried out by using the concentration of 500 mu g/mL and below.
3. Detecting mitochondrial and total ROS levels in cells
Mitochondrial ROS levels were measured using a 5. Mu.M MitoSOX Red Superoxide Indicator (mitochondrial Superoxide fluorescent probe, which selectively detects Superoxide in mitochondria). Hoechst33342 is a nuclear staining reagent that penetrates the cell membrane and stains DNA. Briefly, H9c2 cells treated as designed were first labelled with Hoechst33342 and then cultured with MitoSOX in DMEM at 37 ℃ for 15min. Then, the cells are treated with Ca-containing solution 2+ And Mg 2+ Was washed three times with HBSS buffer, and then fluorescence image capturing and fluorescence intensity analysis were performed using an ImageXpress PICO imaging system (Molecular Devices, USA). And (4) counting the active oxygen level of mitochondria and the number of Hoechst33342 positive cells. DCFH-DA probe detected intracellular oxidative free radicals. Cardiomyocytes in 96-well plates were incubated with serum-free medium containing DCFH-DA (10. Mu.M) at 37 ℃ for 30min in the dark. Then, the cells are grouped and processed according to the method, and the detection parameters of the microplate reader are as follows: excitation wavelength488nm and emission wavelength of 525nm.
4. Detecting mitochondrial membrane potential and mitochondrial morphology
Tetramethylrhodamine methyl ester perchlorate (TMRM) is a red fluorescent probe that can localize at the cell mitochondria and reflect changes in the mitochondrial membrane potential, and is used in this study to evaluate mitochondrial structural changes. After tBHP stimulation, H9c2 cells were nuclear-labeled with Hoechst33342 (2. Mu.g/mL photophobic staining for 10 min). Subsequently, cells were labeled with HEPES-containing TMRM probe dye for mitochondria (200 nM incubation for 30min away from light). Fluorescence imaging and fluorescence analysis were performed using the ImageXpress PICO imaging system. The average integrated fluorescence intensity of the cells of the TRITC channel represents the mitochondrial membrane potential, and the average area of the cells is represented by the average area of the positive cells. Analysis of mitochondrial morphology-associated parameters was performed using Image Pro Plus software.
5. Evaluation of ATP and MDA levels in H9c2 cells
Cellular ATP and MDA levels were detected using CellTiter-Glo fluorescent cell viability assay kit and MDA assay kit, respectively, according to the instructions.
And detecting the expression of AKT/Nrf2/HO-1 protein in the myocardial cells. After the H9c2 cells in the 6-well plate were grouped and treated as described above, the cells were rinsed with pre-cooled PBS buffer, then RIPA lysate was added, 100. Mu.L/well, and incubated on ice for 10min. Cell lysates were collected, centrifuged at 12000rpm for 10min, and the protein concentration in the supernatants was determined using the BCA kit. Equal amounts of protein samples were electrophoresed through SDS-PAGE and transferred to PVDF (Millipore, MA, USA) membranes, which were blocked with 5% skimmed milk on a shaker at room temperature for 2h and then rinsed gently three times with TBST for 5min each. The PVDF membrane was incubated with the corresponding primary antibody (1. The following day, after rinsing 3 times with TBST, incubation with HRP-conjugated secondary antibody (1. Exposure was performed with a GelDoc XR system (BIO RAD). The fluorescence intensity of the bands was analyzed with ImageJ 1.8.0 (NIH, MD, ESA).
All values are expressed as mean ± standard deviation. Student's t-test was used to compare the differences between the two groups. More than three groups of differences were compared using one-way ANOVA. P values <0.05 are statistically significant for the differences.
During the experiment, H9c2 cells were preincubated for 24H with or without semen lepidii, semen lepidii flavone component (TLZ-F), semen lepidii sugar component (TLZ-S) and then exposed to 150. Mu.M tBHP for 2H. As shown in fig. 1, representative images of mROS in H9C2 cells (a) and related analyses (B and C), hoechst33342 fluorescent staining (blue); mitosox (red) at mitochondrial reactive oxygen levels. Cell viability test (D). (n =3, # P <0.05, # P <0.01, as compared to the control group, as indicated by mean ± standard deviation, # P <0.05, # P <0.01, as compared to the tBHP treatment group).
As shown in fig. 2, hoechst33342 was stained with fluorescence (blue); TMRM marks mitochondrial membrane potential (red). Representative images of mitochondrial membrane potential (a) and associated analyses (B and C). (D) ATP content of H9c2 cells. (E) representative images of mitochondrial morphology analysis. (F) quantitative analysis of mitochondrial morphology by different parameters. (n =3, # P <0.05, # P <0.01, as compared to the control group, as indicated by mean ± standard deviation, # P <0.05, # P <0.01, as compared to the tBHP treatment group).
As can be seen from fig. 1 and 2, compared with the sugar component of semen lepidii, the flavone component of semen lepidii can significantly improve the mitochondrial active oxygen level, the mitochondrial membrane potential, the ATP producing ability of mitochondria in cardiomyocytes, and improve the mitochondrial morphology.
H9c2 cells were preincubated for 24H with or without F1, F2 and F3 and then exposed to 150. Mu.M tBHP for 2H. As shown in fig. 3, representative images of mROS in H9C2 cells (a) and related analyses (B and C), hoechst33342 fluorescent staining (blue); mitosox (red) at mitochondrial reactive oxygen levels. Cell viability test (D). (F1 represents quercetin-3-O-beta-D-glucose, F2 represents isorhamnetin-3-O-beta-D-glucose, F3 represents quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, compared with a control group, represented by mean + -standard deviation, n =3, # P <0.05, # P <0.01, compared with a tBHP treatment group, # P <0.05, # P < 0.01).
As shown in fig. 4, hoechst33342 was stained with fluorescence (blue); TMRM marks mitochondrial membrane potential (red). Representative images of mitochondrial membrane potential (a) and associated analyses (B and C). (D) ATP content of H9c2 cells. (n =3, # P <0.05, # P <0.01, as compared to the control group, as indicated by mean ± standard deviation, # P <0.05, # P <0.01, as compared to the tBHP treatment group).
As is clear from fig. 3 and 4, quercetin-3-O- β -glucose-7-O- β -D-gentiobioside (denoted by F3) has stronger abilities to improve mitochondrial reactive oxygen levels, mitochondrial membrane potential, and mitochondrial ATP production in cardiomyocytes than the other flavone components (quercetin-3-O- β -D-glucose (denoted by F1), isorhamnetin-3-O- β -D-glucose (denoted by F2)) in lepidium seed. It is demonstrated that although many flavone components are contained in the pepperweed seed, not all the flavone components have the effect of improving the mitochondrial reactive oxygen level in the myocardial cells. F3 is flavonoid component with F1 and F2, and has similar structure, but F3 has better activity than F1 and F2 in improving mitochondrial active oxygen level, mitochondrial membrane potential, ATP production of mitochondria and the like in myocardial cells.
As shown in FIG. 5, H9c2 cells were incubated with TLZ, the tansymustard flavone fraction and F3 for 24 hours and then exposed to 150. Mu.M tBHP for 2 hours. (A) ROS production in H9c2 cells. (B) MDA levels in H9c2 cells. Gray-scale analysis of representative immunoblots for AKT, nrf2 and HO-1 (C, D and E). (values are expressed as mean ± standard deviation, n =3-5, # P <0.05, # P <0.01, compared to control group, # P <0.05, # P <0.01 compared to tBHP treatment group). As shown in figure 5, the flavone component of the pepperweed seed and the quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside can reduce the active oxygen level and the malondialdehyde level in the myocardial cells, and can be used for preparing the medicine for treating or preventing the anti-heart failure.
Claims (10)
1. A semen lepidii flavone component is characterized in that the preparation method of the semen lepidii flavone component comprises the following steps:
step 1, pulverizing semen lepidii, performing reflux extraction for 1-3 times in an ethanol solvent with the weight of 5-20 times, filtering, combining filtrates, concentrating and removing ethanol to obtain semen lepidii extract concentrated solution; the volume concentration of the ethanol solvent is 0-95%;
and 2, suspending the semen lepidii extraction concentrated solution in water, loading the solution to a macroporous adsorption resin column, eluting the solution with water and 15-60% ethanol in sequence, collecting the 15-60% ethanol eluent, concentrating and drying to obtain the semen lepidii flavone component.
2. The method for preparing the semen lepidii flavone component as claimed in claim 1, comprising the steps of:
step 1, pulverizing semen lepidii, then carrying out reflux extraction for 1-3 times in an ethanol solvent with the weight 5-20 times of that of the powder, filtering, combining filtrates, concentrating and removing ethanol to obtain semen lepidii extraction concentrated solution; the volume concentration of the ethanol solvent is 0-95%;
and 2, suspending the semen lepidii extraction concentrated solution in water, loading the solution to a macroporous adsorption resin column, eluting the solution with water and 15-60% ethanol in sequence, collecting the 15-60% ethanol eluent, concentrating and drying to obtain the semen lepidii flavone component.
3. The use of the semen Lepidii flavone component of claim 1 in the preparation of a medicament for treating or preventing heart failure.
4. Application of quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside in preparing medicine for treating or preventing anti-heart failure is provided.
5. The use of claim 3 or 4, wherein said semen Lepidii is semen Lepidii.
6. The use according to claim 3 or 4, wherein the heart failure comprises presentation of any one or more symptoms of myocardial hypertrophy, myocardial fibrosis, decreased ejection fraction, decreased left ventricular short axis shortening rate, decreased cardiac output, myocarditis, arrhythmia, atherosclerosis, cardiac insufficiency, cardiorenal syndrome;
and/or the medicine for treating or preventing heart failure comprises any one of tablets, oral liquid, pills, granules, capsules, injections and sustained and controlled release agents.
7. A medicament for preventing or treating heart failure, characterized in that the active ingredient of the medicament is the pepperweed seed flavone component or quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside of claim 1.
8. The agent for the prophylaxis or treatment of heart failure according to claim 7, wherein said agent has at least one of the following functions:
(1) Prevention and/or treatment of heart failure;
(2) Prevention and/or treatment of myocardial hypertrophy;
(3) Reducing the relative area of hypertrophic cardiomyocytes;
(4) Reducing or ameliorating myocardial fibrosis;
(5) Improving cardiac output;
(6) Improving the cardiac ejection fraction;
(7) Improving the left ventricular short axis shortening rate;
(8) Improving myocarditis;
(9) Improving arrhythmia;
(10) Improving atherosclerosis;
(11) Improving cardiac insufficiency;
(12) Relieving or treating heart and kidney syndrome.
9. A method for extracting quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside from semen lepidii is characterized by comprising the following steps: separating the semen Lepidii flavone component of claim 1 by liquid chromatography to obtain quercetin-3-O-beta-glucose-7-O-beta-D-gentiobioside.
10. The method for extracting quercetin-3-O- β -glucose-7-O- β -D-gentiobioside from pepperweed seed as claimed in claim 9, wherein the liquid chromatography separation conditions are as follows: a chromatographic column:
an Agilent Zorbax SB-C18 column; mobile phase: a phase is 0.1% formic acid water solution, B phase is acetonitrile; the flow rate is 0.5-10mL/min; the detection wavelength is 200 nm-400 nm;
the linear elution gradient was: 0-1min,2% by weight B;3-5min,4-6% by weight of B;8-12min,10-14% by weight B; 14-1695in, 14-16% by weight B;24-26min, 24-26%; 28-32min,31-35% by weight B;35min,42-46% B;44-46min,65-75% by weight B;48-55min,100% by weight B; column temperature: 35 ℃ is carried out.
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| CN106866753A (en) * | 2017-03-02 | 2017-06-20 | 河南中医药大学 | A kind of method that sulphur glycoside compound is extracted from lepidii,semen and its application |
| CN106928287A (en) * | 2017-03-02 | 2017-07-07 | 河南中医药大学 | A kind of method that phenolic glycoside compound is extracted from lepidii,semen and its application |
| CN113150052A (en) * | 2021-04-20 | 2021-07-23 | 烟台大学 | Method for purifying quercetin glycoside in semen lepidii |
| CN113759037A (en) * | 2021-08-26 | 2021-12-07 | 北京康仁堂药业有限公司 | Characteristic spectrum of formula granules of semen lepidii and/or semen lepidii as well as construction method and identification method thereof |
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2022
- 2022-11-29 CN CN202211516197.5A patent/CN115671154A/en active Pending
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| CN106866753A (en) * | 2017-03-02 | 2017-06-20 | 河南中医药大学 | A kind of method that sulphur glycoside compound is extracted from lepidii,semen and its application |
| CN106928287A (en) * | 2017-03-02 | 2017-07-07 | 河南中医药大学 | A kind of method that phenolic glycoside compound is extracted from lepidii,semen and its application |
| CN113150052A (en) * | 2021-04-20 | 2021-07-23 | 烟台大学 | Method for purifying quercetin glycoside in semen lepidii |
| CN113759037A (en) * | 2021-08-26 | 2021-12-07 | 北京康仁堂药业有限公司 | Characteristic spectrum of formula granules of semen lepidii and/or semen lepidii as well as construction method and identification method thereof |
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