WO2013063889A1

WO2013063889A1 - Use of multiflora glycoside in preparing anti-hypoxic drug

Info

Publication number: WO2013063889A1
Application number: PCT/CN2012/071738
Authority: WO
Inventors: 李灵芝; 张永亮; 龚海英; 王越; 顾军; 郭鹏; 段晓彦; 王舒
Original assignee: 中国人民武装警察部队后勤学院
Priority date: 2011-11-04
Filing date: 2012-02-28
Publication date: 2013-05-10
Also published as: CN102499932A; CN102499932B

Abstract

Provided is the use of multiflora glycoside in preparing an anti-hypoxic drug. A full body and cell level hypoxia model indicates that multiflora glycoside can significantly increase the survival time of a mouse with asphyxiant hypoxia, and has a significant protection effect on endothelial cell injury induced by hypoxia.

Description

Application of wild rosemary in preparation of anti-hypoxia drugs

The present invention relates to the use of a pharmaceutical preparation, and more particularly to the use of wild rosemary in the preparation of an anti-hypoxia drug. Background technique

Oxygen is an important condition for humans and many living things to survive. When the oxygen required for the life of the organism cannot be sufficiently supplied, it is called hypoxia or hypoxia (Hyp _0X ia). Hypoxia will cause abnormal changes in the body's metabolism, function, and even morphological structure. Severe or chronic hypoxia can cause serious harm to the body, which can eventually lead to death of vital organs such as the heart and brain of the body due to insufficient energy supply. Oxygen and hypoxia are important topics in the basic theory of life sciences. The formation of hypoxia can be divided into three categories: The first category is the reduction of the external environment oxygen content, so that normal physiological activities can not get enough oxygen, such as hypoxia caused by special environment such as plateau, diving and aviation; the second category refers to the cause Diseases and other causes that the normal oxygen level cannot reach the body, causing hypoxia in the heart, brain and respiratory system. The third type is that the oxygen consumption required by the body activity exceeds the physiological mobilization ability, resulting in insufficient relative oxygen supply. Strenuous exercise and excessive labor. Long-term hypoxia is an important hidden danger to human health. In severe cases, it can even be life-threatening. Therefore, prevention of hypoxic damage has become one of the major medical problems to be solved in the 21st century.

International research on hypoxia has focused on several important research directions such as hypoxic genetic adaptation, oxygen-sensing signaling, intermittent hypoxia, and prevention and treatment of high-grade diseases. On the one hand, the development of cell biology and molecular biology at the forefront of the life sciences, on the other hand, attention to the overall comprehensive research, for which a research situation in which multiple disciplines permeate and blend with each other emerges. The problems related to hypoxia and health have also attracted the attention of many researchers in China. The research work mainly includes hypoxia inducible factor (HIF), apoptosis and intermittent hypoxia, hypobaric hypoxia and other research hotspots. .

At present, most researches on anti-hypoxia focus on traditional Chinese medicine or traditional Chinese medicine, and have great potential for in-depth research on effective monomer compounds. In-depth exploration and research on the anti-hypoxia activity of natural product monomer compounds not only help to elucidate the material basis and mechanism of anti-hypoxia effect of traditional Chinese medicine, but also help to discover new chemical substances with anti-hypoxia effect, and then excavate and develop A new type of chemical drug with a true anti-hypoxia effect will have significant social and social value.

Wild rose glucoside is a triterpenoid component found in Rosaceae plants and has an alcohol glycoside structure. At present, there are few studies on the pharmacological activity of wild rose glucoside, and no research report on its anti-hypoxia activity has been reported. Summary of the invention

The object of the present invention is to provide a use of wild rosemary in the preparation of an anti-hypoxia drug.

The technical solution of the present invention is summarized as follows:

The application of wild rosemary in the preparation of anti-hypoxia drugs.

The present invention observes the anti-hypoxia effect of wild rosemary by the whole and cell level hypoxia model, and the results show that wild rosemary can significantly improve the survival time of asphyxiated hypoxic mice, and also has significant protection against endothelial cell hypoxia injury. effect. The use of wild rosemary in the preparation of anti-hypoxia drugs is provided. And has a good clinical application prospects. detailed description

The embodiments of the present invention are intended to enable those skilled in the art to better understand the present invention and are not intended to limit the invention.

Wild rosegium glycosides were purchased from Anhui Wuhu Kuerta Pharmaceutical Technology Co., Ltd., with a content of >98%. The following is an experimental description of the anti-hypoxia effect of wild rosemary:

Experiment 1 The experiment of wild rosemary in enhancing hypoxia tolerance in mice

1, method

100 Kunming mice (provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the People's Liberation Army, license number: SCK- (jun) 2002-01), weight (20 ± 2) g. They were randomly divided into the control group, the propranolol hydrochloride group, and the high-low-dose group of wild rosemary. The rats were administered by intragastric administration. Both propranolol hydrochloride and wild rosemary were treated with carboxymethyl fiber with a concentration of 0.3%. Prepared by aqueous sodium solution. The blank control group was given a concentration of 0.3% sodium carboxymethylcellulose (CMC-Na) at a dose of Zml.Kg, and the propranolol hydrochloride group was administered with propranolol at a dose of 20 mg.Kg- ¹ . the CMC-Na solution, respectively glycosides multiflora 20 mg.Kg- ^{^1,} 10 mg.Kg- ^1, administered dose of 5 mg.Kg ¹ CMC-Na solution containing multiflora glycosides, are by dosing volume of Zml.Kg- ¹ calculation. After 50 minutes of administration, the mice were placed in a 125 ml jar (pre-water corrected volume, 5 g of sodium lime was placed in the bottle), the stopper was capped, and the survival time of the mice was recorded with the respiratory arrest as a marker.

2, the result

Compared with the control group, wild rosemary lOmg.Kg^ ZOmg.Kg prolonged the survival time of mice under normal pressure tight conditions by 30.0% and 46.2%, respectively, and the difference was significant (Ρ<0.01) (Table 1). Table 1 Effect of wild rosemary on survival time of hypoxic mice s, «=20) Group dose death time extension rate

(mg.Kg ¹ ) (min) (%) Control group - 16.9 ± 2.9 ―

**

Propranolol hydrochloride group 20.0 25.2±2.5 49.2 wild rosemary (low) 5.0 18.8±3.1 11.2 wild rosemary (middle) 10.0 21.9±3.9 ^## 30.0 wild rosemary (high) 20.0 24.7±2.8 ^## 46.2

Note: ** P <0.01 control group; ^# P <0.05 model group, ^## P <0.01 model group. Experimental study on the protective effect of wild rose glucoside on hypoxia injury of EA.hy926 endothelial cells

1, method

(1) EA. hy926 endothelial cell hypoxia injury model established EA.hy926 endothelial cells with 10% FBS high glucose DMEM medium (culture medium containing 100 U/ml penicillin and 100 μ _§ /ιη1 streptomycin) in carbon dioxide The culture was carried out in an incubator, and the EA.hy926 cells grown into a single layer were continuously cultured for 12 hours in a serum-free and sugar-free DMEM medium, and then D-hanks which were previously saturated with a 95% N ₂ -5% CO ₂ mixture for 30 minutes. The medium was substituted for the normal medium, and then the plate was transferred to a mixed gas incubator (95% N ₂ , 5% C0 ₂ , 0 ₂ concentration < 1%), and cultured under anoxic conditions at 37 ° C for 2 h.

(2) Experimental group experiments: blank control group, hypoxia model group, hypoxia + wild rosemary group A (lx l0_ ¹⁽⁾ m _O l/L), hypoxia + wild rosemary B group (lx lO-Umol /L) and hypoxia + wild rosemary group C (I x l0_ ¹² mol / L), hypoxia + salidroside control group (lx lO ⁵ mol / L), a total of 6 groups, each group of 8 wells. Except the blank control group, the other groups were treated with hypoxia for 2 h, while the normal control group was incubated for 2 h at 37 ° C in a 5% CO ₂ incubator.

(3) MTT assay of endothelial cells with hypoxia injury. Take samples of each group, add 20μ1 每 (5g/L) per well, continue to incubate for 4h in 37V, 5%C0 ₂ incubator, terminate the culture, and pour the plate. The crystal was sufficiently dissolved by adding 15 (^1 DMS0 for 15 min), and the absorbance (A) value of each well was measured at a measurement wavelength of 570 nm and a reference wavelength of 630 nm.

MTT metabolic rate (%) = experimental group A value / normal control group A value X 100%

(4) Biochemical indicators were detected in 24-well plates of EA.hy926 endothelial cells, hypoxia injury model group and wild rosemary intervention group for 2h hypoxia treatment, normal control carbon dioxide incubator was incubated simultaneously, taking each group of cell culture supernatant 200 μl of liquid, the NO content, NOS activity and LDH activity were determined by colorimetric method according to the kit description. The intracellular MDA content was determined by thiobarbituric acid colorimetric method, and the intracellular SOD activity was measured by xanthine oxidase method.

2, the result When cardiomyocytes are damaged by hypoxia, mitochondrial function is abnormal, oxidative respiratory chain is impaired, electron transfer is blocked, and ATP production is reduced. Succinate dehydrogenase is one of the important complex enzymes in the oxidized respiratory chain of succinic acid. It can catalyze the dehydrogenation of succinic acid to fumaric acid, so that FAD accepts two hydrogen atoms to form FADH ₂ and then transfers hydrogen to C ₀ Q. Generate CoQ3⁄4 and continue the electron transfer process of the respiratory chain. Therefore, the activity of succinate dehydrogenase can reflect the degree of hypoxia injury of cardiomyocytes. The principle of MTT experiment is to use succinate dehydrogenase to catalyze the property of thiazole blue (MTT) to produce purple-red insoluble colored products, and reflect the cell viability of each group by measuring the absorbance. The results of this experiment showed that: EA.hy926 endothelial cells were hypoxic-induced for 2 h. Compared with the normal control group, the absorbance value of the hypoxic model group was significantly decreased, the cell metabolic activity was decreased, and the LDH activity in the culture medium was significantly increased, indicating that the cell membrane was deprived of oxygen. Injury, intracellular LDH leakage increases o

Force mouth. In the wild rosemary intervention group and the salidroside group, the absorbance values were increased compared with the hypoxic injury model group, and the LDH activity in the culture medium was significantly decreased, indicating that both wild bovine and salidroside can resist hypoxia against cardiomyocytes. Damage to acid dehydrogenase activity, maintain the respiratory function of hypoxic-damaged cells; When hypoxia leads to cell membrane damage, intracellular LDH will leak out, resulting in increased LDH activity in the medium. This test shows three doses of wild rosemary group. Compared with the model group, the activity of LDH in the medium was significantly decreased, indicating that wild rosemary can maintain the integrity of endothelial cell membrane under acute hypoxia, and the above effects of wild rosemary have a dose-dependent effect (Table 2).

Table 2 Effect of wild rosemary on metabolic activity and extracellular LDH activity of EA.hy926 cells in hypoxia injury

(Λ: ± S, n=8)

Group dose A value LDH

( mol. I" ¹ ) (u. r ¹ ) normal control group 0.5424±0.0125 11.519±3.721 hypoxia model group 0.2478±0.0091 ** 78.937±5.339** hypoxia + salidroside group 0.4025±0.0132 ^## 36.104 hypoxia ± 5.547 ^## + multiflora glycosides ^{(high) 0.4653 ± 0.0151 ## 13.485 ± 5.267} ## + multiflora hypoxia glycosides ^{(in) lx lO "11 0.3718 ± 0.0143} ## 39.354 ± 6.279 ## hypoxia + wild rosemary group (low) 0.3344±0.0087 ^## 46.333±8.415 ^##

Note: **p<0.0\ vs control group; ^# <0.01 vs model group

SOD reflects the ability of cells to scavenge free radicals. MDA reflects the degree of lipid peroxidation damage in cell membrane. Compared with normal control group, SOD activity decreased and MDA content increased significantly (^<0.01). Three doses of wild rosemary intervention group could increase intracellular SOD activity and reduce LDH leakage and MDA production (Ρ<0.01) (Table 3), indicating that wild rosemary can enhance the ability of endothelial cells to scavenge free radicals. Resistance to oxidative damage to cell membranes caused by hypoxia. Table 3 Effect of wild rosemary on SOD activity and MDA content in EA.hy926 cells under hypoxia injury (x ± s, n=S) Group dose SOD MDA

(mg.ml" ¹ ) ( nmol. mgprot" ¹ ) Normal control group 50.38±3.80 13.25±1.34 Hypoxic model group 21.44±2.55** 20.07±0.86** Hypoxia + salidroside group 36.53±4.20 ^## 11.64±0.71 ^##氧氧+野蔷蔷苷组(高) o 34.61±5.87 ^## 10.91±2.69 ^#氧氧+野蔷蔷苷组(中) Ι χ ΙΟ" ¹¹ 31.27±7.41 ^# 11.53±0.72 ^## o

Hypoxia + wild rosemary group (low) 32.30±4.63 ^## 11.99±2.76 ^#

Note: ** <0.01 vs control group; #P<0.05 vs model group; ##P<0.01 vs model group

Nitric oxide (NO) has protective effects on ischemia and hypoxia injury of tissue cells, which can dilate blood vessels, increase blood flow, regulate vascular tone, maintain blood pressure, reduce damage of vascular endothelium, and protect the integrity of vascular endothelial cells. It is speculated that it can improve the ability of vascular endothelial cells to resist hypoxia injury to some extent. Compared with the normal control group, the NO content of EA.hy9266 endothelial cells decreased, and the activity of NOS decreased significantly (P<0.01). However, the dose of wild rosemary increased the NO content and NOS activity (P<0.01). Dose-dependent effect. (Table 4 )

Table 4 Effect of wild rosemary on NO content and NOS activity in EA.hy926 cells induced by hypoxia (x ± s, n=8) Group dose (mg.mr ¹ ) ΝΟ(μιηο1丄^-1 ) NOSiU.ml" ¹ ) Normal control group 12.83±1.45 1.937±0.078 Hypoxic model group 4.26±0. 98 ** 0.669±0.152** Salidroside group 38.58±1.26 ^## 1.201±0.122 ^##氧氧+野蔷薇苷组(高51.72±2.80 ^## 2.542±1.082 ^##氧氧+野蔷苷组 (中) Ι ΙΟ ΙΟ" ¹¹ 30.42±4.19 ^## 1.352±0.067 ^##氧氧+野蔷蔷苷组(低) 16.71±2.09 ^{# #} 1.115±0.054 ^## Note: ** <0.01 vs. control group; ^## Ρ<0.01 vs model group experiment Three wild rose glucosides protective effect on neuronal hypoxia injury

1, method

(1) Establishment of neuron hypoxia injury model The hippocampal neurons of newborn SD rats were cultured in 37 °C, 5% CO ₂ incubator for 7 days, and then inoculated into 24-well plates in a mixed gas incubator (95%). N ₂ , 5% C0 ₂ , 0 ₂ concentration < 1%) Medium 37

°0 hypoxia culture for 3h.

(2) Experimental group experiments were performed with blank control group, hypoxia model group, hypoxia + wild rosemary A group ( 1 X 10-1 ⁽ ) 1 / L), hypoxia + wild rosemary B group ( 1 X 10 - "mol/L" and hypoxia + wild rosemary group C (1 X ^10-12 mol/L), hypoxia + red Glycosides day control group ^{(1 X 10- 5 mol / L} ), a total of 6 groups, 8 wells each. Except the blank control group, the other groups were treated with hypoxia for 3 h, while the normal control group was incubated for 3 h at 37 ° C in a 5% CO ₂ incubator.

(3) MTT assay of hypoxic-injured neurons. Samples of each group were taken, 20 μl MTT (5 g/L) was added to each well, and incubation was continued for 4 h at 37 °C in a 5% CO ₂ incubator. The culture was terminated and inverted. The crystal was sufficiently dissolved by adding 150 μl of DMS0 for 15 minutes, and the absorbance (A) of each well was measured at a measurement wavelength of 570 nm and a reference wavelength of 630 nm.

(4) Biochemical indicators were detected in hippocampal neurons in 24-well plates. Except for the blank control group, all other groups were treated with hypoxia for 3 h, while the normal control group was incubated at 37 ° C in a 5% CO ₂ incubator. h. After the culture is finished, take the supernatant and press

Ο Ο o

The kit describes the determination of LDH activity; cells plus ο 0.25% trypsin digestion, serum suspension, centrifugation 1000r / min, lOmin, 0. 01M PBS lml dissolved precipitate into a cell suspension, ultrasonic pulverization, detection of SOD according to the kit instructions Activity and MDA content.

(5) Hippocampal neurons trypan blue stained hippocampal neurons in each experimental group were added lml 0. 04% trypan blue, mixed, observed under light microscope, count the number of blue-stained cells, and the total number of cells As a mortality rate, a statistical analysis of mortality was performed.

2, the result

The hippocampal neurons were hypoxic-induced for 3 h, and the absorbance values were significantly lower than the normal control group (0.01). Multiflora glycosides and three dose groups hypoxia model group increased absorbance values (<0.01, 0.05) ₀ compared with normal control group, model group, hypoxia hippocampal neurons increased LDH leakage (<0 01), the leakage of LDH in neurons of the three doses of wild rosemary group decreased (0.01) (Table 5).

Effects of wild rosemary on neuronal metabolic activity and extracellular LDH activity in hypoxic injury (X ± s, n=6) group dose devaluation LDH(U.L)

(mol.L" ¹ )

Normal control group 0.5241±0.0105 195.221±19.521 hypoxia model group 0.1908±0.0385"521.854±58.352" hypoxia + salidroside group 0.3025±0.0351 ^## 288.214±31.245 ^##氧氧+野蔷薇苷组(高) 0.3102 ±0.0431 ^## 261.358±30.871 ^##) Hypoxia + wild rosemary group (middle) Ι χ ΙΟ" ¹¹ 0.2645±0.0395 ^## 291.132±21.025 ^##氧氧+野蔷蔷苷组(低) 0.2342±0.0287 ^# 329.185 ±23. 512 ^## Note: **Ρ<0.01 control group; ^## Ρ<0.01 vs model group, #P<0.05 vs model group.

Compared with the normal control group, the intracellular SOD activity of hippocampal neurons in the hypoxia injury model group decreased (P<0.01). Compared with the hypoxia model group, each dose of wild rosemary could reduce the LDH leakage caused by hypoxia injury ( Ρ <0.01), increased intracellular SOD activity, reduced MDA production (Ρ <0.01). (Table 6) Table 6 Effect of wild rosemary on the SOD activity and MDA content of wild hypothyroidism neurons (X ± s, n=6) group dose SOD MDA

(mg.ml" ¹ ) ( nmol. mgprot" ¹ ) Normal control group 27.735±2.804 0.167 ± 0.015 Hypoxia model group 8.682±1.941" 0.403 ± 0.026" Hypoxia + salidroside group 18.476±1.633 ^## 0.168 ± 0.018 ^##缺氧+野蔷薇苷组(高) 19.421±2.176 ^## 0.155 ± 0.017 ^##氧氧+野蔷蔷苷组(中) Ι χ ΙΟ" ¹¹ 16.583±2.965 ^## 0.192 ± 0.013 ##

Ο Ο o

Hypoxia + wild rosemary group (low) ο 12.390±1.642 ^## 0.223 ± 0.019 ^##

Note: **Ρ<0.01 vs control group, ##Ρ<0.01 vs hypoxia model group

Trypan blue staining stains dead cells blue, while living cells do not. In the control group, no blue-stained cells were observed. In the model group, large cells were necrotic and blue-stained, the cells were disconnected, and the cells began to flaky, leaving only a single cell in some areas. After the intervention of wild rose, the high-concentration group only saw individual dead cells. Blue staining, cell-free detachment; blue-stained cells in the middle concentration group were scattered, compared with the model group, the number was significantly reduced, but the number of blue-stained cells was slightly higher than that in the high-concentration group; The reduction was nearly 1/2; the individual cells in the salidroside group were blue-stained, and the number was not significantly different from the medium-concentration group. After one year of animal experiment observation, no obvious side effects were observed on the animals by using the drug of the present invention.

It can be seen from the above that wild saponin has significant anti-hypoxia damage effect, and animal experiments have confirmed that the drug has low toxicity and can be used for preparing drugs for preventing and treating hypoxia injury.

The following are examples of the preparation of pharmaceutical preparations using wild rosemary:

Example 1

a tablet made from wild rosemary

Wild rosemary 20mg

Starch 25mg

15% starch slurry

Magnesium stearate

The above formulation was made into tablets by conventional processing.

Example 2

Capsules made from wild rosemary

Wild rosemary 20mg

Starch 70mg

Prepared according to the traditional pharmaceutical process, filled with No. 2 capsules. Example 3

Oral syrup made from wild rosemary:

20mg

200mg

50% ethanol lml

5mg

Sodium benzoate 3mg

Add water to 10ml

Add the above components to the required amount in water, mix and bottle, then Example 4

Granules made from wild rosemary:

Wild rosemary 20mg

Sucrose 300mg

Orange Flavor 3mg

Food coloring

The above components are mixed to form granules and packaged.

Claims

right

1. The application of wild rosemary in the preparation of anti-hypoxia drugs.