CN111840272A - New application of vitexin - Google Patents

Abstract

The invention relates to a new application of vitexin, in particular to an application of vitexin in preparing a medicament for preventing and treating hypertension or/and nephropathy induced by hypertension. The invention has the following beneficial effects: through a large number of experiments, the vitexin is found to be capable of preventing and treating hypertension and nephropathy caused by hypertension, and particularly, through verification on SHR hypertensive nephropathy model rats, the medicine can effectively prevent and treat hypertensive nephropathy.

Description

New application of vitexin

Technical Field

The invention relates to the technical field of chemical medicines, in particular to a new application of vitexin.

Background

The onset of hypertension is closely related to the kidney, which is not only an important organ for blood pressure regulation, but also one of the major target organs for hypertension damage. Hypertensive nephropathy, a kidney damage secondary to hypertension, is one of the most important complications of hypertension. Statistically, the incidence of hypertensive nephropathy is 42%, second only to cardiac complications, with 18% of hypertensive renal patients eventually experiencing chronic renal failure. The number of hypertensive nephropathy patients in China is 3 of renal failure peritoneal dialysis and hemodialysis patients, and the hypertensive nephropathy patients are second to chronic nephritis and diabetes, so that the life and the life quality of the hypertensive patients are seriously damaged.

The traditional clinical treatment measures for hypertension or hypertensive nephropathy mainly adopt antihypertensive drugs, such as: angiotensin converting enzyme inhibitors or angiotensin receptor blockers. However, studies have shown that the conventional therapeutic effect is far from sufficient, and about 4% to 16% of patients with abnormal urinary protein excretion occur in hypertensive patients who receive blood pressure lowering therapy. The incidence of chronic renal failure has increased at a rate of 9% per year in some countries over the last 10 years, with approximately 28% of the incidence due to hypertension, suggesting that the clinical use of traditional antihypertensive drugs is insufficient for adequate renal protection.

Vitexin (chemical name 5,7, 4-trihydroxyflavone-8-glucoside) and Vitexin (molecular formula C)₂₁H₂₀O₁₀CAS number 3681-93-4, which can be separated from Chinese medicinal materials such as fructus crataegi, folium crataegi, fructus Viticis negundo, and folium Bambusae. The chemical structural formula of the vitexin is as follows:

the vitexin has the effects of resisting oxidation, cancers, viruses, spasm and thyroid, protecting the heart, improving the neurological dysfunction and the like, but the application of the vitexin in medicaments for treating hypertension and hypertensive nephropathy is not reported.

Disclosure of Invention

Based on the above, the main purpose of the invention is to provide a new application of vitexin, mainly relating to the application of vitexin in preparing medicines for preventing and treating hypertension or/and hypertension-induced nephropathy.

The specific technical scheme is as follows:

the vitexin can be used for preparing medicine for preventing and treating hypertension or/and nephropathy induced by hypertension.

In some of these embodiments, the hypertension is essential hypertension.

In some embodiments, the medicament comprises an active ingredient vitexin in combination with a pharmaceutically acceptable carrier.

In some of these embodiments, the medicament comprises 0.1 wt% to 99.5 wt% of the vitexin.

In some of these embodiments, the medicament comprises 10.0 wt% to 90.0 wt% of the vitexin.

In some of these embodiments, the carrier is selected from at least one of diluents, wetting agents, binders, disintegrants, lubricants, color, flavor modifiers, solvents, solubilizers, solubilizing agents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, topical analgesics, pH modifiers, isotonic or isotonic modifiers. In some of these embodiments, the diluent is selected from at least one of starches, sugars, celluloses, and inorganic salts; or/and the wetting agent is selected from at least one of water and ethanol; or/and the adhesive is selected from at least one of starch slurry, dextrin, sugar, cellulose derivative, gelatin, povidone and polyethylene glycol; or/and the disintegrant is selected from at least one of dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, crospovidone, surfactant and effervescent disintegrant; or/and the lubricant is selected from at least one of talcum powder, calcium stearate, magnesium lauryl sulfate, superfine silica powder and polyethylene glycol; or/and the color, aroma and taste regulator is selected from at least one of pigment, spice, sweetener, mucilage and flavoring agent; or/and the solvent is at least one selected from water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil and ethyl acetate; or/and the solubilizer is selected from at least one of tween, maize, polyoxyethylene fatty alcohol ether, soap, sulfate and sulfonate; or/and the cosolvent is at least one selected from organic acids and salts thereof, amides and amine compounds, inorganic salts, polyethylene glycol, povidone and glycerol; or/and the emulsifier is selected from at least one of span, tween, maize, beneze, glycerin fatty acid ester, higher fatty acid salt, sulfate, sulfonate, acacia, tragacanth, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silicon dioxide and bentonite; or/and the suspending agent is at least one selected from glycerin, syrup, gum arabic, tragacanth, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol and thixotrope; or/and the antioxidant is at least one selected from sulfite, pyrosulfite, bisulfite, ascorbic acid, gallic acid and esters thereof; or/and the metal complexing agent is selected from one of disodium ethylene diamine tetraacetate and polycarboxylic acid compound; or/and the inert gas is selected from one of nitrogen and carbon dioxide; or/and the preservative is selected from at least one of parabens, organic acid and salt thereof, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols and volatile oil; or/and the local analgesic is selected from at least one of benzyl alcohol, chlorobutanol, lidocaine and procaine; or/and the pH regulator is at least one selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate and citrate; and/or, the isotonic or isotonic regulator is at least one selected from glucose, sodium chloride, sodium citrate, sorbitol and xylitol.

In some embodiments, the pharmaceutical formulation is a tablet, granule, pill, powder, or capsule.

In some embodiments, the pharmaceutical formulation is an injection, an oral liquid, an ophthalmic preparation or an external preparation.

In some embodiments, the route of administration of the drug is intravenous, intraperitoneal, intramuscular, subcutaneous, oral, or sublingual.

In some of these embodiments, the route of administration of the drug is nasal administration, or transdermal administration.

The invention has the following beneficial effects:

through a large number of experiments, the vitexin is found to be capable of preventing and treating hypertension and nephropathy caused by hypertension, and particularly, through verification on SHR hypertensive nephropathy model rats, the medicine can effectively prevent and treat hypertensive nephropathy.

Drawings

FIG. 1 is an electrophoretogram of ACE enzyme in example 3;

FIG. 2 is a fluorescent microscopic photograph (200) of the staining of HUVEC apoptotic AO-EB by vitexin in example 4;

FIG. 3 is a graph of the results of experiments on the effect of vitexin on the percentage of HUVEC apoptosis in example 4 (compared to AGEs:. about.p < 0.01);

in the figure, "Low", "Middle" and "High" indicate concentrations of vitexin of "5. mu. mol/L", "50. mu. mol/L" and "500. mu. mol/L", respectively.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The embodiment of the invention provides a new application of vitexin, and mainly relates to the application of vitexin in preparing a medicament for preventing and treating hypertension or/and nephropathy caused by hypertension.

The "nephropathy induced by hypertension" according to the embodiment of the present invention may also be referred to as "hypertensive nephropathy", or renal damage or renal function decline induced by hypertension.

Vitexin is a natural active ingredient separated from Chinese medicinal materials such as fructus crataegi and folium crataegi, and is safe and reliable.

Through a large number of experiments, the vitexin is found to be capable of preventing and treating hypertension and nephropathy caused by hypertension, and particularly, through verification on SHR hypertensive nephropathy model rats, the medicine can effectively prevent and treat hypertensive nephropathy.

Preferably, the hypertension is essential hypertension.

In the present invention, the vitexin improves blood pressure abnormality. The vitexin can improve abnormal blood pressure, which means that vitexin can regulate the blood pressure higher than normal of healthy people to normal or approximate to normal blood pressure. The vitexin inhibits angiotensinase activity. Said vitexin inhibits endothelial cell apoptosis. It is understood that the endothelial cell apoptosis may be endothelial cell apoptosis by AGEs. The vitexin can improve abnormal urine volume. Abnormal urine volume means that urine volume is higher than normal urine volume of a healthy person. The vitexin can improve abnormal urine volume, and means that vitexin can be used for regulating abnormal urine volume to normal urine volume or to approach normal urine volume as much as possible. The vitexin can improve trace albumin content abnormality. The abnormal content of the microalbumin means that the microalbumin content is higher than the microalbumin level contained in urine of healthy people. The vitexin for improving the trace albumin content abnormality means that the vitexin can be used for regulating the urine volume trace albumin abnormality to be at a normal trace albumin level or be as close to the normal trace albumin level as possible. The vitexin can improve abnormal blood sugar content. Abnormal urine volume means a higher blood sugar level than normal urine volume of a healthy person. The vitexin can improve abnormal blood sugar content, namely the vitexin can be used for regulating abnormal blood sugar content to normal blood sugar content or close to normal blood sugar content as far as possible.

Preferably, the medicament comprises vitexin and a pharmaceutically acceptable carrier.

Preferably, the medicament comprises 0.1 wt% to 99.5 wt% of the vitexin. Further, the medicine comprises 10.0 wt% -90.0 wt% of the vitexin. The dosage of the drug to be used may be adjusted accordingly depending on the particular administration mode, the severity of the condition, and the like. Generally, the clinical oral dose of vitexin is 20 μ g/kg body weight/day according to the minimum therapeutic effect in animal (rat) in vivo experiment, and the dose in human body is 4 μ g/kg body weight/day according to the conversion of body surface area.

Preferably, the carrier is selected from at least one of diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, topical analgesics, pH modifiers, isotonic or isotonic modifiers.

Further: diluents such as starch, sucrose, celluloses, inorganic salts, etc.; wetting agents such as water, ethanol, and the like; adhesives such as starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, and the like; disintegrants, such as starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium croscarmellose, crospovidone, surfactants, transpiration disintegrants, and the like; lubricants such as talc, calcium stearate, magnesium lauryl sulfate, colloidal silica, polyethylene glycol, and the like; color, flavor and taste modifiers such as pigment, perfume, sweetener, mucilage, and flavoring agent, specifically fuchsin and xylitol; solvents such as water, oil, ethanol, glycerin, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate, etc.; solubilizers such as tweens, maizes, polyoxyethylene fatty alcohol ethers, soaps, sulfides, sulfonates, and the like; cosolvents such as organic acids (e.g., citric acid) and salts thereof, amides and amines, inorganic salts, polyethylene glycol, povidone, glycerol, and the like; emulsifiers such as span, tween, maize, benze, glycerin fatty acid ester, higher fatty acid salt, sulfate, sulfonate, gum arabic, gum tragacanth, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silica, bentonite, etc.; suspending agents such as glycerol, syrup, acacia, tragacanth, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol, thixotrope, etc.; antioxidants such as sulfites, pyrosulfites, bisulfites, ascorbic acid, gallic acid and esters thereof, and the like; metal complexing agents such as disodium ethylenediaminetetraacetate, polycarboxylic acid compounds, and the like; inert gases such as nitrogen, carbon dioxide, and the like; antiseptics such as parabens, organic acids and salts thereof (such as sodium benzoate), quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, volatile oils, etc.; local analgesics such as benzyl alcohol, chlorobutanol, lidocaine, procaine and the like; pH regulators such as hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, citric acid, citrate, etc.; isotonic or isotonic regulator, such as glucose, sodium chloride, sodium citrate, sorbitol, xylitol, etc. It is understood that the diluents of the embodiments of the present invention may also be called bulking agents, and may function in the same manner in pharmaceutical formulations; the water in the embodiment of the invention is water meeting the requirements of medicaments, such as water for injection, purified water and the like, and the oil is oil for injection; the preservative provided by the embodiment of the invention can also be called as an antibacterial agent, and plays roles of inhibiting the growth of microorganisms, prolonging the shelf life and the like in a preparation; the lubricant of the embodiment of the invention contains glidant, anti-adhesive agent and the like; the sugar in the embodiment of the invention can be powdered sugar or syrup, and the type of the sugar is not limited to glucose; the perfume described in the embodiment of the present invention includes, but is not limited to, essence.

It is understood that the dosage form of the drug according to the embodiment of the present invention is not limited, and the drug may be a solid preparation, such as tablet, granule, pill, powder, capsule, etc., or a liquid preparation, such as injection, oral liquid, ophthalmic preparation, etc., or other preparations, such as external preparation, etc.

It is understood that the administration mode of the drug according to the embodiment of the present invention is not limited, and the administration route of the drug may be intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

The invention is further illustrated by the following specific examples.

Example 1 preparation of vitexin

The vitexin is prepared by extracting, separating and purifying the hawthorn leaves according to the following preparation method:

taking 1.0kg of hawthorn leaves, crushing into coarse powder, adding 50% ethanol for extraction twice (55-60 ℃), adding 10.0L for the first time and 8.0L for the second time when the temperature is 2 hours each time, filtering, combining filtrates, concentrating under reduced pressure to a proper volume, adding into D101 macroporous resin, eluting with water, then respectively eluting with 10% and 70% ethanol solutions, collecting 70% ethanol partial solution eluate, extracting with ethyl acetate, vacuum drying, and adding 1% acetic acid: dissolving acetonitrile (85: 15) solution, filtering with 0.45 μm microporous membrane, separating prepared liquid phase, collecting vitexin solution with corresponding retention time, concentrating under reduced pressure, vacuum drying to obtain 5.0mg yellow powder, identifying as vitexin, and detecting purity of 98% by HPLC.

Example 2 Effect of vitexin on intrarenal pressure

In the embodiment, a renal perfusion system is adopted to detect the change condition of intrarenal pressure (in vitro simulated blood pressure), valsartan is taken as a positive drug, the influence of vitexin on the change condition is observed, and whether the change condition has the effect of reducing blood pressure or not is examined.

The experimental contents are as follows:

1. test materials and instruments

SPF grade rats, purchased at southern medical university, license number SCXK (Guangdong) 2016-;

norepinephrine bitartrate, lot #: 1805191, Tianjin Jinyao amino acids, Inc.;

valsartan, lot #: SML0142, Sigma;

the other reagents are all domestic analytical purifiers.

Electronic analytical balance, BS224S/CB-25A, Sidolis Instrument systems, Inc., Beijing;

ex vivo renal perfusion device, TIS3852, Kent corporation;

four-channel recorder, MPA2000/CB-275, Olcott Biotechnology, Inc. of Shanghai.

2. Test method

2.1 preparation of reagents

Preparing Tyrode's perfusion fluid: according to 8.01g of NaCl, 0.2g of KCl and CaCl₂0.2g、MgCl₂·6H₂O0.214g、NaH₂PO₄·2H₂O 0.07g、NaHCO₃1.0g、Glu·H₂O 1.11g、EDTA-Na₂Weighing 0.2g of formula weighing reagent, placing the reagent in a 1000ml beaker, adding 1000ml of ultrapure water, stirring and fully dissolving, adjusting the pH value to 7.35-7.45, and storing at normal temperature.

2.2 preparation of perfusion model of isolated rat Kidney

Rats were fasted for 12h before surgery without water deprivation, weighed, and anesthetized by intraperitoneal injection of 10% chloral hydrate.

The model was prepared as follows:

fixing the back, cutting the abdominal wall, and separating mesenteric artery, abdominal aorta, left renal artery and vein and ureter.

The mesenteric artery is inserted into the renal artery, the catheter pretreated by heparin is inserted, the ligature and fixation are carried out, the kidney is taken out, perfusion liquid containing 2000U/L heparin is injected into the renal artery for lavage, and the residual blood in the kidney is removed until the venous effluent is transparent, colorless and clear.

Immediately starting peristaltic pump, transferring kidney into preheated 37 deg.C constant temperature container, adding 95% O₂With 5% CO₂Continuously perfusing perfusate pre-saturated with mixed gas, monitoring intrarenal perfusion pressure in real time, and regulating perfusion flow to stabilize perfusion pressure. Perfusion was continued for subsequent experiments.

2.3 in vitro Kidney perfusion

Perfusate (95% O) for isolated rat kidney₂With 5% CO₂Pre-saturation of mixed gas) to balance, adding a proper amount of liquid medicine to flow into the kidney after the intrarenal pressure signal reaches the baseline stability, perfusing with perfusion liquid, and after the baseline is stabilized again; adding a proper amount of liquid medicine again to flow into the kidney, perfusing with perfusion liquid, and after the baseline is stabilized again; the next drug addition was continued and the procedure was repeated until the kidneys were inactivated. The pressure signal atlas is recorded by the pressure monitor in the whole experiment and is stored.

2.4 data processing and results analysis

According to the monitored pressure signal map, the change of the intrarenal pressure of the isolated kidney before and after the action of the medicine is compared and analyzed, and the pressure signal is subjected to data statistical analysis.

3. Test results

Following Norepinephrine (NE) administration, intrarenal pressure rises rapidly; then valsartan is given, and the increased intrarenal pressure is rapidly reduced until the baseline level is recovered; the administration of norepinephrine followed by the administration of various doses of vitexin resulted in a rapid decrease in the intrarenal pressure to a level close to baseline, which is a dose-effect relationship indicating that vitexin is capable of counteracting the increase in the intrarenal pressure of the isolated kidney caused by norepinephrine, as detailed in table 1.

TABLE 1 Effect of vitexin on ex vivo renal simulated intrarenal pressure

Example 3 Effect of vitexin on ACE Activity

In this example, the influence of vitexin on the enzyme activity thereof was observed by using ACE derived from pig lung, and whether the enzyme had the effect of lowering blood pressure was examined.

The experimental contents are as follows:

1. test materials and instruments

Hippuric Acid (HA), Lot: f1212042, Aladdin;

equine ureyl histidyl leucine (HHL), Lot: SLBW5368, Sigma;

DEAE sephadex A-50, lot #: DEI702HA30, limited development of peking ruida constant brightness technology;

Triton x-100, lot #: 125346, Shanghai Jiebai Gene technology, Inc.;

dialysis bag, MW: 14000 +/-2000;

BCA kit, lot #: 041318180711, Biyun day;

Tris，lot#：0922S0715，Solarbio；

bovine serum albumin, BSA, lot #: SL27152202, Shanghai Liaoshu, Inc.;

the other reagents are all domestic analytical purifiers.

Fresh pig lungs were purchased from a pig breeding base.

pH meter, type: five Eosy, METTLER TOLEDO;

high-speed refrigerated centrifuge, model: GL-21M, changshan instrument centrifuge, ltd;

microplate reader, SpectraMax type 190, Molecular Devices;

electronic analytical balance, BS224S/CB-25A, Sidolis Instrument systems, Inc., Beijing;

nucleic acid protein assay, model: 6132, Ebende China Co., Ltd;

hplc, type 1260, Agilent.

2. Test method

2.1 preparation of reagents

Preparation of 0.05mol/L Tris-HCl solution (pH 7.4): weighing 6.055g of Tris into a 1000ml beaker, adding a proper amount of ultrapure water for dissolution, adding 4.3ml of HCl, uniformly mixing, transferring into a volumetric flask, fixing the volume to 1000ml, and storing at 4 ℃.

Preparation of 2mmol/L HHL solution: weighing HHL powder 8.67mg into a 10ml beaker, adding a proper amount of ultrapure water for dissolving, transferring into a volumetric flask to be constant volume to 10ml, and storing at 4 ℃.

Preparing a maleic acid mother solution: weighing 10.17mg of hippuric acid powder in a 100ml beaker, adding a proper amount of ultrapure water for dissolving, transferring the hippuric acid powder to a volumetric flask for metering the volume to 100ml to obtain 101.7 mu g/ml hippuric acid mother liquor, and storing the hippuric acid mother liquor at 4 ℃.

2.2 extraction and purification of ACE from pig Lung

Cleaning fresh pig lung normal saline, cutting into small pieces, homogenizing, adding appropriate amount of 0.5% Triton x-100, slowly stirring for 30min, filtering with gauze, centrifuging the filtrate at 4 deg.C and 3000rpm for 20min, collecting supernatant, adding 55% saturated ammonium sulfate, standing for 12h, centrifuging at 4 deg.C and 7000rpm for 10min, collecting precipitate, dissolving with appropriate amount of 0.1mol/L boric acid buffer solution with pH of 8.3, dialyzing at 4 deg.C until the dialysate has no SO, and collecting the supernatant₄ ^2-Obtaining the ACE crude extract. The crude extract was purified by DEAE sephadex A-50 gel column separation and identified as ACE by SDS-PAGE gel electrophoresis, as shown in FIG. 1.

2.3 enzyme Activity measurement

Taking ACE, and determining the protein concentration by a BCA kit. Taking a proper amount of ACE, adding 40 mu L of vitexin or purified water with each concentration, carrying out water bath at 37 ℃ for 10min, adding 25 mu L of HHL with the concentration of 2mmol/L, fully and uniformly mixing, carrying out water bath at 37 ℃ for 15min, adding 150 mu L of 0.2mol/L HCl, centrifuging at 12000rpm for 10min, taking supernatant, filtering at 0.22 mu m, carrying out HPLC sample injection analysis, and detecting the content of Hippuric Acid (HA) in a reaction solution.

2.4 data processing and results analysis

The ACE relative specific activity was calculated according to the formula ACE relative specific activity (U/mg) ═ hippuric acid content/(reaction time × protein concentration).

3. Test results

The administration of different dosages of vitexin significantly reduced the relative ACE activity, and had a dose-effect relationship, indicating that vitexin reduced ACE activity, as detailed in table 2.

TABLE 2 Effect of vitexin on ACE Activity

n＝3)

P <0.01, p <0.05vs control group.

Example 4 Effect of Vitexin on endothelial apoptosis

In this example, AGEs-induced human umbilical vein endothelial cell apoptosis model and vitamin E (vitE) as positive drugs were used to observe the effect of vitexin on it and examine its anti-endothelial cell apoptosis effect.

The experimental contents are as follows:

1. materials, reagents and apparatus

DMEM medium (GIBCO), Life Technologies inc;

fetal Bovine Serum (FBS), zhejiang hangzhong biotechnology, ltd;

polylysine and VitE are Sigma products;

acridine Orange (AO) and Ethidium Bromide (EB) dyes are all Biyuntian products;

human Umbilical Vein Endothelial Cells (HUVEC), american ATCC product;

vitexin is prepared by separation and purification of a preparation room of Kangchen nephropathy pharmaceutical research center, and the purity of the vitexin is more than 98 percent through High Performance Liquid Chromatography (HPLC);

BS-224S type electronic analytical balance, Beijing Saedodus Instrument systems, Inc.;

model LDZ5-2 medical centrifuge, beijing li medical centrifuge factory;

CO₂incubator, changsha changjin science and technology limited;

olympus IX71, BX51 microscope, olympus japan;

clean bench, Suzhou baishen science and technology network systems, Inc., Suzhou clean technology research institute;

SPECTRA max190 microplate reader, USA MD.

2. Experimental methods

2.1 cell culture

Preparing a single-cell suspension from cells in a logarithmic growth phase by using DMEM containing 10% FBS, inoculating the single-cell suspension into a 24-well plate which is placed into a cover glass coated with polylysine in advance by 1 mL/hole, culturing the cells by 1 multiplied by 105/hole at 37 ℃ and 5% CO2 for 24 hours, adding serum-free DMEM, and then incubating for 12 hours to synchronize the cells;

cell supernatants were aspirated, 3 wells at the same level, and DMEM 900. mu.L/well containing 10% FBS and 100. mu.L/well of each drug were added. Adding Phosphate Buffer Solution (PBS) with the same volume to a normal control group, and adding AGEs (the final concentration is 0.1g/L) to an AGEs model control group; adding AGEs (with final concentration of 0.1g/L) and VitE (with final concentration of 100 μmol/L) into the VitE positive control component; AGEs (with final concentration of 0.1g/L) and vitexin (with final concentrations of 5. mu. mol/L, 50. mu. mol/L and 500. mu. mol/L) were added into the experimental group with low, medium and high dosages of vitexin, respectively.

2.2 staining and visualization of apoptosis

Preparing an AO-EB dye solution: 0.20g each of AO and EB was weighed, dissolved completely in 2.0mL of ultrapure water, and charged into a 2mL brown EP tube, and stored at 4 ℃ for further use.

Dyeing and observing: taking out the cell slide, washing with PBS for 2 times, and spin-drying; dripping 15 mu L of AO-EB staining solution on the glass slide, covering the cell slide, and leading the cell face to be downward; standing for 30 seconds, placing under a fluorescence microscope, and observing and taking pictures. When shooting, the center position of each slide is taken.

2.3 data analysis and statistics

Each picture (orange + red partial cell area) and total cell area were analyzed with IPP image analysis software and the percentage of apoptosis for each sample was calculated as follows:

percent apoptosis (%) - (orange + red partial cell area)/total cell area x 100

All experimental data were analyzed by variance using a block design, processed with SPSS software, and the results were expressed in x ± s (mean ± variance).

3. Results of the experiment

As can be seen from FIGS. 2 and 3, the apoptosis percentage of vitexin in each experimental group is obviously reduced (p is less than 0.05, p is less than 0.01) compared with the AGEs model control group, and the vitexin has an obvious dose-effect relationship, which indicates that vitexin can effectively inhibit AGEs-induced endothelial apoptosis.

Experimental example 5 Effect of Vitexin on SHR hypertensive nephropathy model rats

In this example, SHR rats fed a high-fat and high-sugar diet were subjected to hypertensive renal disease model, and after administration of vitexin for 8 weeks, urine volume, urinary microalbumin, blood pressure, and blood sugar level were examined for pharmacological effects of vitexin on whole animals.

The experimental contents are as follows:

1. materials and reagents

80 SPF-grade SHR rats, 10 SD rats, with a weight of 130 + -20 g, purchased from Beijing Wittingle laboratory animal technology GmbH, quality certificate number: SCKX (Kjing) 2016-;

a glucose detection kit, a urine total protein detection kit and a creatinine detection kit which are all products of Nanjing institute of bioengineering;

roche glucometer and test paper, roche diagnostics china company;

electronic analytical balance (BS-224S), Beijing Saedodes Instrument systems, Inc.;

medical centrifuge (LDZ5-2), Beijing medical centrifuge Mill;

CODA noninvasive blood pressure monitor, Kent corporation, usa.

2. Experimental methods

2.1 establishment of hypertensive nephropathy model rat

80 SHR rats were given a high-fat, high-sugar diet; 10 SD rats were given normal diet; each animal had free access to water for 4 weeks. Keeping the animal breeding environment clear: dark time 12: for 12 hours.

2.2 grouping and administration

And (3) determining blood sugar after molding, taking the animals with blood sugar value of 16-30 mmol/L as qualified animals, and grouping the qualified animals according to the blood sugar, wherein each group comprises 15 animals, so that no significant difference exists among blood sugar value groups, and the grouping condition is as follows: a model group; irbesartan group (dose 13.5 mg/kg); the low, medium and high dose groups of vitexin (the dose is respectively 0.02mg/kg, 0.04mg/kg and 0.08 mg/kg); the normal control group, 10 rats, remained unchanged.

The administration groups are respectively administered with corresponding drugs with the volume of 0.5ml/220g by adopting the method of intragastric administration; normal control group and model group were given the same volume of physiological saline. Dosing was 6 days per week for 8 weeks.

2.3 index detection

Blood pressure, blood sugar, urine volume, and urine microalbumin content were measured before administration and at weeks 4 and 8 of administration.

2.4 data statistics

All experimental data were analyzed by variance in a group design, processed by SPSS software, and the results were obtained

(mean ± variance) is shown.

3. Results of the experiment

3.1 Effect on blood pressure and blood sugar of SHR hypertensive nephropathy model rat

As can be seen from Table 3, the blood sugar of the model group is significantly increased (p <0.01), and the blood sugar of the vitexin is significantly improved by 0.04mg/kg and 0.08mg/kg (p < 0.05).

As can be seen from Table 4, the blood pressure of the model group is obviously increased (p <0.01), and the blood pressure of the model group is obviously improved by irbesartan and 0.04 and 0.08mg/kg of vitexin in different periods (p <0.05 and p < 0.01).

TABLE 3 Effect of Mucardin on blood glucose in SHR hypertensive nephropathy model rats: (

n＝10)

vs is normal: # p < 0.01; a vs model: p < 0.05.

TABLE 4 Effect of Vitexin on blood pressure in SHR hypertensive nephropathy model rats: (

n＝10)

vs is normal: # p < 0.01; a vs model: p <0.05, p < 0.01.

3.2 Effect on urine volume and urine microalbumin of SHR hypertensive nephropathy model rat

As can be seen from Table 5, the urine volume of the model group at 24h is obviously increased (p <0.01), and the vitexin at 0.04mg/kg and 0.08mg/kg can obviously improve the condition at different periods (p <0.05 and p < 0.01).

As can be seen from Table 6, the increase of urinary microalbumin in 24 hours in the model group can obviously improve or reverse the situation (p is less than 0.05, and p is less than 0.01) by 0.04 and 0.08mg/kg of vitexin.

TABLE 5 influence of vitexin on 24h urine volume in SHR hypertensive nephropathy model rats: (

n＝10)

vs is normal: # p <0.05, # p < 0.01; a vs model: p <0.05, p < 0.01.

TABLE 6 Effect of vitexin on 24h microalbumin in urine of SHR hypertensive nephropathy model rats: (

n＝10)

vs is normal: # p <0.05, # p < 0.01; a vs model: p <0.05, p < 0.01.

The above examples show that vitexin can resist intrarenal pressure increase caused by norepinephrine, inhibit ACE activity, inhibit mesangial cell apoptosis caused by AGEs, and improve urine volume, microalbumin, blood pressure, and blood sugar abnormality of rats in SHR hypertensive nephropathy model, thereby also proving that vitexin has prevention and treatment effects on hypertension and hypertensive nephropathy.

Example 6 pharmaceutical granules for prevention and treatment of hypertensive nephropathy

The embodiment provides a pharmaceutical granule for preventing and treating hypertensive nephropathy, wherein each package of granules comprises the following components in parts by mass:

the vitexin is 5.0mg,

2497.5mg of starch, and

microcrystalline cellulose 2497.5 mg.

The preparation method of the medicine granules comprises the following steps:

mixing vitexin with starch and microcrystalline cellulose, sieving with 20 mesh sieve, granulating by dry method, grading with 1.2mm mesh sieve, and packaging.

Example 7 pharmaceutical tablet for prevention and treatment of hypertensive nephropathy

The embodiment provides a pharmaceutical tablet for preventing and treating hypertensive nephropathy, wherein each tablet comprises the following components in parts by mass:

the preparation method of the medicine tablet comprises the following steps:

mixing vitexin with lactose and corn starch, sieving with 20 mesh sieve, granulating by dry method, grading with 1.2mm mesh sieve, mixing with other excipients, and tabletting.

Example 8 pharmaceutical capsules for the prevention and treatment of hypertensive nephropathy

The embodiment provides a pharmaceutical capsule for preventing and treating hypertensive nephropathy, and each capsule comprises the following components in parts by mass:

the content of the vitexin is 199.0mg,

dextrin 1.0mg, and

Proper amount of hydroxypropyl methylcellulose.

The preparation method of the medicine capsule comprises the following steps:

sieving vitexin with 20 mesh sieve, mixing with dextrin by equivalent multiplication method, adding appropriate amount of hydroxypropyl methylcellulose, granulating by dry method, sieving with 1.2mm mesh sieve, and making into capsule.

EXAMPLE 9 pharmaceutical composition dropping pill for prevention and treatment of hypertensive nephropathy

The embodiment provides medicinal pills for preventing and treating hypertensive nephropathy, wherein each pill comprises the following components in parts by mass:

vitexin 15.0g, and

400015.0 g of polyethylene glycol.

The preparation method of the medicine dripping pill comprises the following steps:

weighing 15.0g of vitexin, micronizing, sieving with 200 mesh sieve to obtain fine powder, adding into melted 15.0g of polyethylene glycol 4000 matrix, stirring, adding dimethicone as coolant, making into dripping pill, and drying to obtain dripping pill.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (11)

1. The vitexin can be used for preparing medicine for preventing and treating hypertension or/and nephropathy induced by hypertension.

2. The use of claim 1, wherein the hypertension is essential hypertension.

3. The use according to claim 1 or 2, wherein the medicament comprises vitexin in combination with a pharmaceutically acceptable carrier.

4. The use according to claim 3, wherein said medicament comprises 0.1 wt% to 99.5 wt% of said vitexin.

5. The use according to claim 4, wherein said medicament comprises 10 wt% to 90 wt% of said vitexin.

6. Use according to claim 3, wherein the carrier is selected from at least one of diluents, wetting agents, binders, disintegrants, lubricants, colour, flavour modifiers, solvents, solubilisers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, topical analgesics, pH modifiers and isotonic or isotonic modifiers.

7. The use according to claim 6, wherein the diluent is at least one selected from the group consisting of starches, sugars, celluloses, and inorganic salts; or/and the wetting agent is selected from at least one of water and ethanol; or/and the adhesive is selected from at least one of starch slurry, dextrin, sugar, cellulose derivative, gelatin, povidone and polyethylene glycol; or/and the disintegrant is selected from at least one of dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, crospovidone, surfactant and effervescent disintegrant; or/and the lubricant is selected from at least one of talcum powder, calcium stearate, magnesium lauryl sulfate, superfine silica powder and polyethylene glycol; or/and the color, aroma and taste regulator is selected from at least one of pigment, spice, sweetener, mucilage and flavoring agent; or/and the solvent is at least one selected from water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil and ethyl acetate; or/and the solubilizer is selected from at least one of tween, maize, polyoxyethylene fatty alcohol ether, soap, sulfate and sulfonate; or/and the cosolvent is at least one selected from organic acids and salts thereof, amides and amine compounds, inorganic salts, polyethylene glycol, povidone and glycerol; or/and the emulsifier is selected from at least one of span, tween, maize, beneze, glycerin fatty acid ester, higher fatty acid salt, sulfate, sulfonate, acacia, tragacanth, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silicon dioxide and bentonite; or/and the suspending agent is at least one selected from glycerol, syrup, acacia gum, tragacanth gum, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol and thixotrope; and/or, the antioxidant is at least one selected from sulfite, pyrosulfite, bisulfite, ascorbic acid, gallic acid and esters thereof; or/and the metal complexing agent is selected from one of disodium ethylene diamine tetraacetate and polycarboxylic acid compound; or/and the inert gas is selected from one of nitrogen and carbon dioxide; or/and the preservative is at least one of nipagin, organic acid and salt thereof, quaternary ammonium compound, chlorhexidine acetate, alcohol, phenol and volatile oil; or/and the local analgesic is selected from at least one of benzyl alcohol, chlorobutanol, lidocaine and procaine; or/and the pH regulator is at least one selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate and citrate; and/or, the isotonic or isotonic regulator is at least one selected from glucose, sodium chloride, sodium citrate, sorbitol and xylitol.

8. The use according to any one of claims 1, 2 and 4 to 7, wherein the medicament is in the form of tablets, granules, pills, powders or capsules.

9. The use according to any one of claims 1, 2 and 4 to 7, wherein the medicament is in the form of an injection, an oral liquid, an ophthalmic preparation or an external preparation.

10. The use according to any one of claims 1, 2 and 4 to 7, wherein the medicament is administered by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration or sublingual administration.

11. Use according to any one of claims 1, 2 and 4 to 7, wherein the drug is administered by nasal or transdermal administration.

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