WO2023028730A1

WO2023028730A1 - Use of aloesone in preparation of product for resisting oxidative stress injury

Info

Publication number: WO2023028730A1
Application number: PCT/CN2021/115228
Authority: WO
Inventors: 王燕; 李友宾; 谭银丰; 王雪松; 徐俊裕; 曾婷婷; 崔雪
Original assignee: 海南医学院
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2023-03-09

Abstract

Disclosed is the use of aloesone in the preparation of a product for resisting oxidative stress injury. The aloesone has anti-oxidation activity of directly scavenging DPPH free radicals, and can scavenge ROS to inhibit macrophage apoptosis and thus resist oxidative stress injury, and therefore can be used to prepare a drug for preventing and treating diseases induced by oxidative stress injury.

Description

Application of aloe vera pine in the preparation of anti-oxidative stress damage products

technical field

The invention relates to the field of antioxidants, in particular to the application of aloe vera pine in the preparation of anti-oxidative stress damage products.

Background technique

Modern research shows that free radicals are constantly produced in human life activities. One is the endogenous free radicals produced by various metabolic reactions in the body; the other is the exogenous free radicals produced by the homolysis of covalent bonds of organic molecules in organisms due to high temperature, ultraviolet rays, photolysis, ionizing radiation, chemical drugs, and environmental pollution. free radicals. Free radicals are mainly oxygen free radicals, and the content of oxygen free radicals accounts for about 95%, such as superoxide anion radicals (·O ₂ ^- ), alkoxyl groups (RO·), hydroperoxyl groups (HOO·), alkane Oxygen (ROO·), etc. In addition, there are many oxygen-containing compounds that are easy to lose an electron and become free radicals although the electrons are paired, such as: singlet oxygen (excited oxygen ¹ O ₂ ), hydrogen peroxide Active oxygen radicals such as (H ₂ O ₂ ), ozone (O ₃ ), and other oxygen-containing substances with the reaction characteristics of oxygen radicals are collectively called reactive oxygen species (ROS). Followed by non-oxygen free radicals, such as hydrogen free radicals (H ), organic free radicals (R ), reactive nitrogen (RNS) and reactive chlorine (RCS) and so on.

Under normal physiological conditions, the body’s production and elimination of oxygen free radicals are in a dynamic balance, but when the dynamic balance of the body’s production and elimination of free radicals is broken and the level of free radicals in the body increases, because the free radicals are active, there will be extreme Strong oxidative ability can destroy the structure and function of cells through a series of oxidation reactions, causing the body to produce irreversible oxidative damage, thereby inducing various pathological injuries, such as atherosclerosis, cardiovascular disease, diabetes, tumors, etc. . If excessive oxidative free radicals can be eliminated, many free radical-induced and aging-related diseases can be prevented. So finding safe and effective antioxidants from plants has become one of the research hotspots.

Aloe is a medicinal and edible plant, which is a perennial evergreen succulent herb of the genus Aloe of the family Liliaceae. Aloe vera has pharmacological effects such as anti-inflammatory, anti-oxidation, promotion of wound healing, liver protection, hypoglycemia, hemostasis, anti-tumor and anti-virus. Traditionally, anthraquinones are believed to be the antioxidant active components of aloe. Aloe pine (aloesone) is the aglycon of aloin, and it is also a unique chemical component in the epidermis of aloe. The content of aloe pine in aloe is low and there are few studies on the relevant pharmacological activities.

Aloe pine structure 3D map:

Chinese name: Aloe pine, molecular formula C ₁₃ H ₁₂ O ₄ ; molecular weight: 232.23198; English name: aloesone; other names: 2-acetonyl-7-hydroxy-5-methylchromone base-chromone), 2-acetonyl-7-hydroxy-5-methyl-chromen-4-one (2-acetonyl-7-hydroxyl-5-methyl-chromen-4-one), 2-(2 -oxopropyl)-7-hydroxy-5-methyl-4H-1-benzopyran-4-one(2-(2-oxopropyl)-7-hydroxy-5-methyl-4H-1-benzopyran- 4-keto), 7-hydroxy-5-methyl-2-(2-oxopropyl)chromen-4-one(7-hydroxy-5-methyl-2-(2-oxopropyl)chromen-4-one ).

Contents of the invention

In view of this, the present invention proposes the application of aloe vera pine in the preparation of anti-oxidative stress damage products.

Technical scheme of the present invention is realized like this:

The aloe vera pine is used in the preparation of anti-oxidative stress damage products, and the aloe vera pine inhibits macrophage apoptosis by clearing ROS to resist oxidative stress damage.

Further, the anti-oxidative stress damage product is applied to the preparation of drugs for preventing or treating diseases induced by oxidative stress damage.

Further, the medicine also includes pharmaceutically acceptable auxiliary materials.

Compared with prior art, the beneficial effect of the present invention is:

The present invention finds that aloe vera pine has the anti-oxidative activity of directly removing active oxygen, can inhibit the generation of active oxygen (ROS) in RAW264. Therefore, aloe vera pine can be used in the preparation of anti-oxidative stress damage products, especially in the preparation of drugs for the prevention and treatment of diseases induced by oxidative stress damage. Moreover, compared with the hepatotoxicity of anthraquinone compounds, which are the antioxidant active ingredients of aloe, the chromone compound aloe vera pine has better safety, and is more conducive to the preparation of safe anti-oxidative stress damage products.

Description of drawings

Figure 1. Aloe pine reduces lipopolysaccharide-induced ROS levels in macrophages.

A) DCF fluorescence images under different objective lenses in each group (×50,×100);

B) Mean fluorescence intensity (flow cytometry, *compared with normal control group, #compared with lipopolysaccharide treated group)

Figure 2. Aloe pine inhibits lipopolysaccharide-induced macrophage apoptosis.

Detailed ways

In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.

The materials and reagents used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

1. Synthesis of aloe vera pine

1.1 Aloe pine synthetic route

1.2 The synthetic method of aloe vera pine:

(1) Mix 3,5-dihydroxytoluene with acetic acid and boron trifluoride ether at a molar ratio of 10:8:20, and react with magnetic stirring in an oil bath at 80°C for 12 hours. After the reaction was completed, cool to room temperature, extract with ethyl acetate and water, and extract three times, take the ethyl acetate layer, add it to a saturated NaHCO solution, add 3,5 _- dihydroxytoluene to the saturated NaHCO solution to make the volume _1L , The mixture was stirred for 10 min to completely react the acetic acid in the solution. Then extract with ethyl acetate and water, extract three times, take the ethyl acetate layer, concentrate under reduced pressure to remove the solvent, obtain a concentrate, add ethyl acetate to dissolve the concentrate, add an appropriate amount of petroleum ether, stand for recrystallization, and filter with suction Compound 1-1 was obtained.

(2) Compound 1-1, dichloromethane and DIPEA (N,N-diisopropylethylamine) are mixed, and the volume of dichloromethane added per mole of compound 1-1 is 3.5L, and the moles of compound 1-1 and DIPEA The ratio is 1:6.5; after the reaction is heated to 0°C with magnetic stirring in an ice bath, MEM-Cl (2-methoxyethoxymethyl chloride) is added, and the molar ratio of compound 1-1 and MEM-Cl is 1:1; Magnetic stirring was carried out at room temperature for 12 h. After the reaction is completed, extract with ethyl acetate and water, extract three times, take the ethyl acetate layer, concentrate under reduced pressure to remove the solvent, and obtain a concentrate that is purified by column chromatography (petroleum ether: ethyl acetate = 6: 1, v/v) Compound 1-2, namely compound C, was obtained.

(3) Mix compound C, malic acid, 4-dimethylaminopyridine and dichloromethane, the molar ratio of compound C, malic acid and 4-dimethylaminopyridine is 1:1.0:1.0, compound C and dichloromethane The molar volume ratio mol/L is 1:4.0; after stirring the reaction in an ice bath to 0°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir the reaction at 25°C 6.0h; After the reaction, extract with dichloromethane and water, take the dichloromethane layer, concentrate under reduced pressure to remove the solvent, and obtain a concentrate, which is purified by column chromatography, and the solvent of the column chromatography is 6.0 by volume. : Prepared by mixing petroleum ether and ethyl acetate in 1 to obtain compound 1-3, namely compound B;

(4) Mix compound B, sodium hydroxide and dimethyl sulfoxide, the mol ratio of compound B and sodium hydroxide is 1:2.4, and the molar volume ratio mol/L of compound B and dimethyl sulfoxide is 1: 4.0, stirred and reacted at 25°C for 8.0h, extracted with ethyl acetate and water after the reaction, took the ethyl acetate layer, concentrated under reduced pressure to remove the solvent, and obtained a concentrate, which was purified by column chromatography, the column chromatography The solvent is prepared by mixing petroleum ether and ethyl acetate with a volume ratio of 5.0:1 to obtain compound 1-4, namely compound A;

(5) Mix compound A, hydrogen chloride solution and isopropanol, the molar ratio of compound A to hydrogen chloride is 1:4.0, the molar ratio of isopropanol to compound A is 32.7:1, heat to 45°C and stir for 1.5h; react Afterwards, the solvent was concentrated under reduced pressure to obtain a concentrate, which was purified by column chromatography. The solvent for column chromatography was prepared by mixing petroleum ether and ethyl acetate with a volume ratio of 5:1; after purification and drying, compound 1 was obtained. -5, the target product aloe vera pine.

Aloe pine NMR data:

White solid powder, ¹ H NMR(400MHz,D ₆ )δ10.61(s,1H),6.62(s,1H),6.60(s,1H),6.03(s,1H),3.85(s,2H), 2.65(s,3H),2.21(s,3H); ¹³ C NMR(100MHz,D ₆ )δ202.87,178.22,161.09,160.62,159.27,141.65,116.70,114.39,112.92,100.58,47.58,29.89.22.

2. Application test

1) Test method

1. DPPH method to measure the scavenging rate of aloe vera pine to free radicals

Dissolve 2 mg of DPPH solid in 20 mL of absolute ethanol to a concentration of 0.1 mg/ml, sonicate for 5 min, shake fully, the absorbance is between 0.6-1.0, and store in the dark. Aloe vera pine was dissolved in absolute ethanol, and diluted to 5 concentrations (2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL). Add 100uL of different concentrations of aloe vera pine and DPPH solutions to each well of a 96-well plate, mix well and incubate at 37°C in the dark for 30min, measure the absorbance of the well plate at 517nm, and use ascorbic acid standard solution as a positive control (0.05mg/mL) . Taking the concentration of aloe vera pine as the abscissa and the free radical scavenging rate as the ordinate, draw a standard curve and calculate the IC ₅₀ .

Clearance %=[1-(A ₁ -A ₂ )/A ₃ ]*100%

A ₁ = absorbance of aloe vera pine and DPPH

A ₂ = absorbance of aloe vera pine and ethanol

A ₃ = absorbance of DPPH and ethanol

2. DCFH-DA method to detect the scavenging effect of aloe vera pine on the reactive oxygen species (ROS) in macrophages induced by lipopolysaccharide

Macrophage RAW264.7 was cultured in 5% CO2 incubator at 37°C in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. RAW264.7 cells were plated (24-well plate) at 1.0-2.0×10 ⁵ cells/well for 24 hours and treated with aloe pine (0.1 μM, 1 μM, 10 μM and 100 μM) for 2 hours. Subsequently, LPS (1 μg/mL) was applied for 24 hours. After the experiment, the cell supernatant was collected, washed three times with PBS, and then added with 1:1000 diluted DCFH-DA. Protect from light and incubate for 20 minutes in a 37°C incubator. Subsequently, the cells were washed 3 times with PBS to fully remove the DCFH-DA that did not enter the cells. Observation and photographing were carried out by an inverted fluorescence microscope (Zeiss X-Cite), and the average fluorescence intensity was measured by flow cytometry with FITC channel.

3. Annexin V-FITC/PI detects the effect of aloe vera pine on macrophage apoptosis induced by lipopolysaccharide

After macrophages were treated with different concentrations of aloe vera pine, they were washed twice with PBS, lysed with trypsin, and collected by centrifugation. Add 500 μL binding buffer, then add 5 μL Annexin V-FITC and PI staining solution in sequence, incubate at room temperature for 5 minutes, and analyze the proportion of early and late apoptotic cells in macrophages by flow cytometry (Agilent NovoCyte).

2) Test results

1. Aloe pine has DPPH scavenging activity

DPPH is a stable organic free radical with a strong absorption peak at 517nm. Antioxidants can pair with their single electrons to make their absorption gradually disappear. The change of light absorption value has a quantitative relationship with the number of electrons they accept. The ability to scavenge DPPH can indicate the strength of its antioxidant activity. The experimental results show that aloe vera pine can scavenge DPPH free radicals very well, and its IC ₅₀ is 0.73, which is equivalent to the antioxidant activity of natural antioxidant tea polyphenols.

Table 1. Aloe pine in vitro DPPH free radical scavenging activity

2. Aloe pine reduces lipopolysaccharide-induced macrophage ROS level

DCFH-DA is a general indicator of oxidative stress. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to generate DCFH (2',7'-Dichlorodihydrofluorescein, DCFH). However, DCFH cannot permeate the cell membrane, so the probe can be easily loaded into the cell. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to produce fluorescent DCF. The fluorescence of DCF was detected to determine the level of reactive oxygen species in the cells. In this study, DCFH-DA was used to observe the effect of aloe vera pine on macrophage reactive oxygen species induced by lipopolysaccharide. The results showed that, as shown in Figure 1, LPS treatment of RAW264.7 cells induced ROS accumulation compared with normally cultured macrophages. Aloe pine can significantly reduce the ROS level of RAW264.7 cells induced by lipopolysaccharide, and the inhibitory effect is dose-dependent.

3. Aloe pine inhibits lipopolysaccharide-induced macrophage apoptosis

In the early stage of apoptosis, the asymmetry of the cell membrane disappears, the phosphatidylserine in the membrane is transferred to the outside of the membrane, and the FITC-labeled annexin (Annexin V) can be combined with phosphatidylserine to indicate the early stage of apoptosis; while another staining The agent PI can diffuse into the nucleus of apoptotic and necrotic cells and combine with DNA to indicate the late stage of apoptosis.

The results of this study showed that, as shown in Figure 2, compared with normal macrophages, lipopolysaccharide-induced macrophages had more early and late apoptotic cells, and the intervention of 10 μM and 100 μM aloe vera pine could significantly reduce the early apoptotic cells. Proportion of macrophages in apoptosis (43.5% and 28.6%) and late apoptosis (58.9% and 47.4%).

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection.

Claims

Aloe pine is used in the preparation of anti-oxidative stress damage products.
The application according to claim 1, characterized in that the aloe vera pine inhibits macrophage apoptosis by clearing ROS to resist oxidative stress damage.
The use according to claim 1 or 2, characterized in that the anti-oxidative stress damage product is used in the preparation of drugs for the prevention or treatment of diseases induced by oxidative stress damage.
The use according to claim 3, characterized in that the medicament further comprises pharmaceutically acceptable auxiliary materials.