CN110693928B - Use of apocynum venetum leaf polyphenol - Google Patents
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Abstract
The invention relates to an application of apocynum venetum leaf polyphenol, wherein the apocynum venetum leaf polyphenol is obtained by extracting, purifying and refining the apocynum venetum leaves, and in-vitro enzyme activity screening and cell tests show that: apocynum venetum leaf polyphenol for treating retinopathy caused by high-sugar injury, can inhibit aldose reductase activity in vitro and remarkably increase Na in human retinal pigment epithelial cells+‑K+ATP enzyme activity, reduction of intracellular sorbitol content, regulation of polyol pathways, and treatment of retinopathy caused by high glucose damage; the apocynum venetum leaf polyphenol can be used for preparing various preparations with the function of improving retinopathy caused by high-sugar injury.
Description
Technical Field
The invention belongs to the field of biological activity of traditional Chinese medicine plant polyphenol, relates to an application of apocynum venetum leaf polyphenol, and particularly relates to an application of apocynum venetum leaf polyphenol in preparation of a medicine or a health-care product for treating retinopathy caused by high-sugar injury. The cell experiment proves that the medicine has the function of improving retinopathy caused by high-sugar injury.
Background
Apocynum venetum L, Apocynum genus of Apocynum of Apocynaceae family, also called tea flower, wild hemp, Japanese St.John's wort, cutch, tea berberis, kenaf, safflower, beef tea, etc. according to the records of Chinese plant records, is distributed in provinces such as Xinjiang, Qinghai, Gansu, Shanxi, Henan, Hebei, Jiangsu, Shandong, Liaoning and inner Mongolia in China.
Apocynum venetum L is a natural plant in China, has wide medicinal value in folk, is recorded in Ben Cao Ben and has the functions of relieving palpitation, stopping dizziness, eliminating asthma and relieving cough, strengthening heart and promoting urination, and is also recorded in the first 211 page of 2015 edition Chinese pharmacopoeia, with sweet, bitter and cool taste. It enters liver meridian. Pacify liver, tranquilize mind, clear heat and induce diuresis. Can be used for treating liver-yang vertigo, palpitation, insomnia, edema and oliguria.
According to the international diabetes union, about 4.25 million adults 20 to 79 years old worldwide have diabetes, and it is expected that 6.28 million people worldwide, i.e., 1 out of 10 adults, will have diabetes in 2045 years. More and more diabetics are affected by complications arising from diabetes, some affecting daily activities and quality of life, and others severe to the point of being life threatening.
Diabetic Retinopathy (DR) is a highly specific microvascular complication of diabetes that may not be apparent at an early stage but may lead to visual impairment and blindness. Diabetes can cause two types of retinopathy, proliferative and nonproliferative, with diabetic retinopathy being one of the major blinding eye diseases. The basic pathological changes include: selective loss of pericytes, thickening of the basement membrane, formation of microangiomas, endothelial cell proliferation, and neovascularization. It is estimated that 1 out of every 3 diabetic patients is affected by the disease, which is one of the major causes of blindness in the 20 to 65 year old population.
Polyol pathway, also called sorbitol pathway, is an important pathway for glucose metabolism of tissue cells, and abnormal pathway is one of important pathogenesis of diabetic retinopathy. It is composed of Aldose Reductase (AR) and Sorbitol Dehydrogenase (SDH). Aldose reductase is mainly present in tissues such as crystalline lens, retina, nerve, kidney and the like, AR takes NADPH as coenzyme, catalyzes the reduction reaction of hexose, converts glucose into corresponding reduction product sorbitol, and then sorbitol is oxidized into fructose under the action of sorbitol dehydrogenase and coenzyme I (NAD +). When the blood sugar concentration is increased, hexokinase is saturated by glucose, AR is activated and expressed, the sorbitol level is increased, excessive glucose in cells is converted into sorbitol through AR, and accumulation of sorbitol and fructose in the cells is caused, so that cell damage is caused, and diabetic retinopathy is induced.
Diabetes mellitusRetinopathy has various degrees of retinal epithelial cell ARPE-19 damage, polyol pathway abnormality, aldose reductase activity enhancement, intracellular Na+-K+Decreased ATPase activity and accumulation of sorbitol. Epalrestat, an aldose reductase inhibitor, is currently marketed, but is sometimes accompanied by allergic symptoms such as erythema, blisters, vomiting, and the like, and side effects such as stomach discomfort, and the patient cannot take it for a long time. At present, the activity research of the apocynum venetum leaf polyphenol is more in activity research of antioxidation, blood pressure reduction and depression resistance, and no report about the apocynum venetum leaf polyphenol in diabetic retinopathy is found.
The invention utilizes the apocynum venetum leaf polyphenol obtained in the preparation method and the application of the apocynum venetum leaf polyphenol 20130509440.5 to research retinopathy caused by high-sugar injury.
Disclosure of Invention
The invention aims to provide the application of apocynum venetum leaf polyphenol, the apocynum venetum leaf polyphenol is obtained by extracting, purifying and refining the apocynum venetum leaf polyphenol, and in-vitro cell experiments show that the apocynum venetum leaf polyphenol has the capability of improving retinopathy caused by high-sugar injury and can remarkably increase Na of a high-sugar induced high-sugar injury human retinal pigment epithelium (ARPE) -19 cell model+-K+ATPase activity, reducing sorbitol levels, has been shown to modulate the polyol pathway, thus ameliorating retinopathy resulting from high sugar damage.
The invention relates to an application of apocynum venetum leaf polyphenol, in particular to an application of the apocynum venetum leaf polyphenol in preparing a medicine for treating retinopathy caused by high glucose damage.
An application of apocynum venetum leaf polyphenol in preparing health care products for treating retinopathy caused by high sugar injury.
The apocynum venetum leaf polyphenol disclosed by the invention is a preparation method and application of the apocynum venetum leaf polyphenol based on the name of patent 20130509440.5, and the obtained apocynum venetum leaf polyphenol is specifically operated according to the following steps:
a. heating dry folium Apocyni Veneti with 10-30 times of 30-70% ethanol water bath at 40-80 deg.C for 1-3 times, each for 1-3 hr, and collecting extractive solution;
b. concentrating the extract under reduced pressure to a concentration of 6mg/mL, purifying with macroporous resin, adsorbing, washing with water to remove impurities, wherein the volume of the washing with water is 2-5BV, and then eluting with 30-70% ethanol, wherein the elution amount is 2-4 BV;
c. collecting ethanol eluate, and vacuum drying to obtain folium Apocyni Veneti polyphenol.
The invention relates to an application of apocynum venetum leaf polyphenol, in particular to an application of apocynum venetum leaf polyphenol in preparing a medicine for treating retinopathy caused by high-sugar injury, and the adopted technical scheme is as follows:
the invention adopts high-sugar induction to construct a human retinal pigment epithelial cell (ARPE-19) high-sugar damage model; detection of intracellular Na+-K+ATP enzyme activity and sorbitol content, combined with statistical analysis, and intracellular Na of ARPE-19 in control group, high sugar injury group and administration group+-K+-ATPase activity and sorbitol content change, and deep analysis of improvement effect of folium Apocyni Veneti polyphenol on diabetic retinopathy; the results show that: folium Apocyni Veneti polyphenol has no obvious effect on the viability of human retinal pigment epithelial cells (ARPE-19), and the Na in the polyol pathway of the human retinal pigment epithelial cells (ARPE-19) with high sugar injury+-K+The activity of ATP enzyme is obviously reduced, the content of sorbitol is obviously increased, and the intervention of apocynum venetum leaf polyphenol on a human retina pigment epithelial cell (ARPE-19) high-sugar damage model shows that the apocynum venetum leaf polyphenol has obvious protective effect on diabetic retinopathy and can be applied to the medicines for preventing and treating the diabetic retinopathy.
According to the invention, through the intervention treatment effect of the apocynum venetum leaf polyphenol, the apocynum venetum leaf polyphenol has a remarkable protection effect on high-sugar injury induced retinopathy, a new way is provided for the high-sugar injury induced retinopathy, and the apocynum venetum leaf polyphenol has important significance and value for further deep research on the apocynum venetum leaf function and has wide potential market and application.
Drawings
FIG. 1 shows high sugar damage according to the present inventionHuman retinal pigment epithelial cell (ARPE-19) Na+-K+-map of influence of ATPase activity;
FIG. 2 is a graph showing the effect of high carbohydrate damage to sorbitol in human retinal pigment epithelial cells (ARPE-19) in accordance with the present invention;
FIG. 3 is a graph showing the effect of different concentrations of apocynum venetum leaf polyphenol on the viability of human retinal pigment epithelial cells (ARPE-19) according to the present invention;
FIG. 4 shows the effect of different concentrations of apocynum venetum leaf polyphenol on high-sugar-content damaged human retinal pigment epithelial cell (ARPE-19) Na+-K+-map of influence of ATPase activity;
FIG. 5 is a graph showing the effect of different concentrations of apocynum venetum leaf polyphenol on sorbitol of high-sugar-content damaged human retinal pigment epithelial cells (ARPE-19) in accordance with the present invention, wherein: p <0.05vs. model set, P <0.01vs. model set.
Detailed Description
Example 1
a. Taking 25g of dry apocynum venetum leaves, heating and refluxing the dry apocynum venetum leaves by using 30% ethanol with the mass volume ratio of 25 times, extracting for 3 times for 2 hours at the water bath temperature of 50 ℃, collecting extract, and recovering the ethanol under reduced pressure;
b. concentrating the extracting solution in the step a to 6.0mg/mL of total polyphenol by adopting a vacuum rotary evaporator at the temperature of 45 ℃, purifying the concentrated solution by using HPD-450 macroporous resin, wherein the adsorption rate of the sample loading solution is 1BV/h, then washing with water, the eluting volume of the water is 3BV, the flow rate of the water washing is 2BV/h, then eluting with 40 percent ethanol, the eluting amount of the ethanol is 4BV, and the eluting speed of the ethanol is 2 BV/h;
c. and c, collecting the ethanol eluent obtained in the step b, and drying in vacuum to obtain the apocynum venetum polyphenol.
Example 2
a. Taking 25g of dry apocynum venetum leaves, heating and refluxing the dry apocynum venetum leaves by using 50% ethanol with the mass volume ratio of 10 times, extracting for 3 times for 2 hours at the water bath temperature of 70 ℃, collecting extract, and recovering ethanol under reduced pressure;
b. concentrating the extracting solution in the step a to 6.0mg/mL of total polyphenol by adopting a vacuum rotary evaporator at the temperature of 45 ℃, purifying the concentrated solution by using HPD-300 macroporous resin, wherein the adsorption rate of the sample loading solution is 1BV/h, then washing with water, the eluting volume of the water is 4BV, the flow rate of the water washing is 3BV/h, then eluting with 50 percent ethanol, the eluting amount of the ethanol is 4BV, and the eluting speed of the ethanol is 2 BV/h;
c. and c, collecting the ethanol eluent obtained in the step b, and drying in vacuum to obtain the apocynum venetum polyphenol.
Example 3
a. Taking 25g of dry apocynum venetum leaves, heating and refluxing the dry apocynum venetum leaves by using 70% ethanol with the mass volume ratio of 30 times, extracting for 3 times for 2 hours at the water bath temperature of 70 ℃, collecting an extracting solution, and recovering the ethanol under reduced pressure;
b. concentrating the extracting solution in the step a to 6.0mg/mL of total polyphenol by adopting a vacuum rotary evaporator at the temperature of 45 ℃, purifying the concentrated solution by using HPD-600 macroporous resin, wherein the adsorption rate of the sample loading solution is 1BV/h, then washing with water, the eluting volume of the water is 5BV, the flow rate of the water washing is 4BV/h, then eluting with 70 percent ethanol, the eluting amount of the ethanol is 3BV, and the eluting speed of the ethanol is 2 BV/h;
c. and c, collecting the ethanol eluent obtained in the step b, and drying in vacuum to obtain the apocynum venetum polyphenol.
Example 4
a. Taking 25g of dry apocynum venetum leaves, heating and refluxing the dry apocynum venetum leaves by using 50% ethanol with the mass volume ratio of 10 times, extracting for 3 times for 2 hours at the water bath temperature of 70 ℃, collecting extract, and recovering ethanol under reduced pressure;
b. concentrating the extracting solution in the step a to 6.0mg/mL of total polyphenol by adopting a vacuum rotary evaporator at the temperature of 45 ℃, purifying the concentrated solution by using HPD-300 macroporous resin, wherein the adsorption rate of the sample loading solution is 1BV/h, then washing with water, the eluting volume of the water is 4BV, the flow rate of the water washing is 3BV/h, then eluting with 50 percent ethanol, the eluting amount of the ethanol is 4BV, and the eluting speed of the ethanol is 2 BV/h;
c. and c, collecting the ethanol eluent obtained in the step b, and drying in vacuum to obtain the apocynum venetum polyphenol.
Example 5
And (3) screening the biological activity of the application of the apocynum venetum polyphenol obtained in the examples 1 to 4: using a reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and DL-glyceraldehyde mixed solution as a substrate, using an enzyme labeling instrument to perform high-throughput screening of an Aldose Reductase inhibitor by using a positive drug quercetin as a control, using DL-glyceraldehyde as the substrate according to the existence of quantitative and excessive Nicotinamide Adenine Dinucleotide Phosphate (NADPH), and reflecting the activity of Aldose Reductase (Aldose Reductase, AR) by detecting the reduction of the Nicotinamide Adenine Dinucleotide Phosphate (NADPH), wherein an enzyme reaction system comprises the following components: phosphate buffered saline (pH7.4), aldose reductase, Nicotinamide Adenine Dinucleotide Phosphate (NADPH), DL-glyceraldehyde, specific inhibitor quercetin (6.25 μ g/mL) of aldose reductase, after the reaction system is mixed uniformly, the mixture is placed for 30min in a closed light mode, the mixture is placed on a colorimeter to measure an absorption value (A) under the condition of 340 wavelengths, and the enzyme activity is calculated after the background value is subtracted from the measurement result;
inhibition (I%) [ (OD)340blank-OD340sample)/OD340Blank space]×100％
And (4) screening results:
and (4) conclusion: the experimental result shows that the apocynum venetum polyphenol has the effect of inhibiting the activity of aldose reductase.
Example 6
Cell culture and construction of human Retinal Pigment epithelial cell (ARPE-19) high-sugar injury model: human retinal pigment epithelial cell (ARPE-19) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)/F12 Medium (D-glucose concentration: 4.5mmol/L) containing 10% fetal bovine serum, 100U/mL penicillin and 100U/mL streptomycin at 37 ℃ and 5% CO2Culturing under the condition, taking 2-8 generations of human retinal pigment epithelial cells (ARPE-19) for experiments, and carrying out high-sugar damage model conditions: the normal group adopts 10% fetal calf serum and 4.5mmol/L D-glucose DMEM/F12 culture medium, the high-glucose group adopts 10% fetal calf serum and 30mmol/L D-glucose DMEM/F12 culture medium, the culture is placed in an incubator for culturing for 48 hours, cells are collected, and Na in the cells is detected according to a kit method+-K+-ATPaseViability, sorbitol content (fig. 1, fig. 2).
High sugar damage to cell Na+-K+-atpase activity results:
results of high sugar damage on cellular sorbitol content:
the results showed that human retinal pigment epithelial cells (ARPE-19) had Na after high sugar damage+-K+Significant decrease in ATP enzyme activity, significant increase in sorbitol content, Na+-K+The ATP enzyme activity normal group vs. the model group has very significant difference (P)<0.01), sorbitol normal group vs. model group has significant difference (P)<0.05), cellular polyol pathway end products accumulated after high sugar damage, inducing cellular damage (fig. 1, fig. 2).
Example 7
Effect of Apocynum venetum Polyphenol obtained in examples 1-4 on human retinal pigment epithelial cell (ARPE-19) viability: inoculating human retinal pigment epithelial cells into a 96-well plate, wherein each hole is 5000, culturing for 12 hours to ensure that the cells adhere to the wall, replacing a culture medium to establish a high-glucose injury model, and the conditions are as follows: adopting a culture medium of 10% fetal calf serum and 4.5mmol/L D-glucose DMEM/F12 in a normal group, adopting a culture medium of 10% fetal calf serum and 30mmol/L D-glucose DMEM/F12 in a high-glucose group, simultaneously adding dogbane leaf polyphenol with different concentrations in an administration group under the condition of the high-glucose group, and detecting the cell viability by a 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide salt colorimetric Method (MTT) after 24 hours; the experimental group was checked for statistical differences from the dosing group using Graphpad Prism statistical analysis and studies showed: after 24 hours of treatment of ARPE-19 cells under high-sugar injury conditions by the apocynum venetum leaf polyphenol, compared with a control group, the cell viability is around 100%, and the result proves that the apocynum venetum leaf polyphenol has no toxic effect on the ARPE-19 cells (figure 3).
Cell viability results:
and (4) conclusion:
research shows that after apocynum venetum leaf polyphenol is used for treating human retinal pigment epithelial cells (ARPE-19) under high-sugar damage conditions for 24 hours, compared with a control group, the cell viability is near 100% in all concentrations, and the result proves that the apocynum venetum leaf polyphenol has no toxic effect on the human retinal pigment epithelial cells (ARPE-19).
Example 8
Screening of cells for the use of apocynum venetum polyphenols obtained in examples 1 to 4: inoculating human retinal pigment epithelial cells into a 6-well plate, culturing for 12 hours, adhering to the wall, replacing a culture medium, and carrying out conditions as follows: culturing normal group with 10% fetal calf serum +4.5mmol/L D-glucose DMEM/F12 culture medium for 48 hr, stimulating high-glucose group with 10% fetal calf serum +30mmol/L D-glucose DMEM/F12 culture medium for 48 hr, stimulating administration group with high-glucose for 24 hr, adding folium Apocyni Veneti polyphenol of different concentrations, culturing for 24 hr, collecting cells, and detecting intracellular Na according to kit method+-K+-atpase activity, sorbitol content;
different concentrations of apocynum venetum leaf polyphenol to cell Na+-K+-atpase activity results:
after 24 hours of administration of different concentrations of apocynum venetum leaf polyphenol, Na+-K+The ATP enzyme activity is obviously enhanced, and the 25 mu g/mL dose group is vs<0.01), 12.5 mug/mL dose group vs. model group has significant difference (P)<0.05), 6.25 mug/mL dose group vs. model group has significant difference (P)<0.05), which shows that the apocynum venetum leaf polyphenol has the protection function on polyol pathway abnormality caused by high sugar damage, has the functions of protecting cells against high sugar damage and improving retinopathy caused by high sugar damage (figure 4);
the results of different concentrations of apocynum venetum leaf polyphenol on the content of cell sorbitol are as follows:
after 24 hours of administration of the apocynum venetum leaf polyphenol with different concentrations, the content of sorbitol is obviously reduced, the dosage group of 50 mu g/mL has a significant difference in terms of v and model (P is less than 0.05), and the dosage group of 25 mu g/mL has a significant difference in terms of v and model (P is less than 0.05), which indicates that the apocynum venetum leaf polyphenol has the effects of inhibiting accumulation of sorbitol, a polyol pathway end product, in cells, caused by high sugar damage, protecting the cells against high sugar damage and improving retinopathy caused by the high sugar damage (figure 5);
treatment of Na in human retinal pigment epithelial cell (ARPE-19) under high sugar injury condition based on apocynum venetum leaf polyphenol+-K+The result of ATP enzyme activity and sorbitol shows that the apocynum venetum leaf polyphenol has the effects of preventing and treating related diseases caused by high-sugar damage.
Claims (1)
1. The usage of apocynum venetum leaf polyphenol is characterized in that the apocynum venetum leaf polyphenol is used for preparing a medicine for treating retinopathy caused by high-sugar damage, and the preparation of the apocynum venetum leaf polyphenol comprises the following specific operation steps:
a. heating dry folium Apocyni Veneti with 10-30 times of 30-70% ethanol water bath at 40-80 deg.C for 1-3 times, each for 1-3 hr, and collecting extractive solution;
b. concentrating the extract under reduced pressure to a concentration of 6mg/mL, purifying with macroporous resin, adsorbing, washing with water to remove impurities, wherein the volume of the washing with water is 2-5BV, and then eluting with 30-70% ethanol, wherein the elution amount is 2-4 BV;
c. collecting ethanol eluate, and vacuum drying to obtain folium Apocyni Veneti polyphenol.
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| CN103520233A (en) * | 2013-10-24 | 2014-01-22 | 中国科学院新疆理化技术研究所 | Preparation method and application of apocynum venetum leaf total polyphenol |
| CN107412295A (en) * | 2017-04-24 | 2017-12-01 | 新疆金骏阳光生物科技有限公司 | A kind of apocynum venetum health care tea and preparation method thereof |
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| CN106692667A (en) * | 2016-11-17 | 2017-05-24 | 中国人民解放军第二军医大学第二附属医院 | Composition with effects of reducing blood pressure, blood fat and blood glucose and use of composition |
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| CN103520233A (en) * | 2013-10-24 | 2014-01-22 | 中国科学院新疆理化技术研究所 | Preparation method and application of apocynum venetum leaf total polyphenol |
| CN107412295A (en) * | 2017-04-24 | 2017-12-01 | 新疆金骏阳光生物科技有限公司 | A kind of apocynum venetum health care tea and preparation method thereof |
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