CN111206062A

CN111206062A - Preparation of isoquercitrin derivative for removing free radicals

Info

Publication number: CN111206062A
Application number: CN202010054797.9A
Authority: CN
Inventors: 张才来
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-05-29

Abstract

The invention provides a preparation method of isoquercitrin derivatives for scavenging free radicals; the preparation method of the isoquercitrin derivative comprises the following steps: isoquercitrin derivatives with high purity, high solubility and high bioavailability can be prepared by connecting isoprenyl on the 5' position of isoquercitrin and then processing the isoprenyl isoquercitrin by using biological enzyme; the isoquercetin derivatives have the effects of scavenging free radicals, resisting inflammation and inhibiting bacteria, and are 3-5 times of that of isoquercetin; the effect of the medicine is better than isoquercitrin in the aspects of protecting cardiovascular and cerebrovascular, reducing blood fat and lowering blood pressure.

Description

Preparation of isoquercitrin derivative for removing free radicals

Technical Field

The invention relates to preparation of an isoquercitrin derivative for removing free radicals, and application of the isoquercitrin derivative in removing free radicals, protecting cardiovascular and reducing blood fat, belonging to the field of biological medicines.

Technical Field

The results of relevant pharmacological experiments show that the pharmaceutical value of the isoquercitrin is extremely high, and the pharmacological activity of the isoquercitrin is far higher than that of Rutin (royal Kan, fish red flashing, and gold phoenix Rutin- α -rhamnosidase separation and purification and the enzymatic properties thereof, the university of Dalian Industrial science reports 2004,23(1):30), therefore, the isoquercitrin is a hot new medicine compound in the current pharmaceutical kingdom and is always a high-grade food additive, an auxiliary medicine or a medicine effective component which is competitively developed by various pharmaceutical food companies internationally.

Prenyl has an important role in biosynthesis. The existence of the isoamylene side chain increases the lipophilicity and biological guidance of the flavonoid compound, so that the flavonoid compound is easier to permeate cell membranes in organisms to reach an action target, and the medicinal value of the flavonoid compound is greatly improved.

However, the solubility of isoquercetin in water and buffer solution is low, so that the bioavailability of isoquercetin is low, and the effect is not obvious. Therefore, the groups which are easy to dissolve in water are connected on the isoquercetin to increase the solubility of the isoquercetin, and the fat-soluble groups are connected to increase the application of the isoquercetin in organisms, thereby improving the bioavailability of the isoquercetin.

Disclosure of Invention

Isoquercitrin has various medicinal values, but the monomer is difficult to dissolve in water, and when the isoquercitrin is directly used as a medicament, the bioavailability in a human body is low, and the biological function of the isoquercitrin is difficult to be fully exerted.

In order to solve the defects, the invention adds isoprenyl groups on isoquercitrin, increases the treatment efficacy of isoquercitrin flavone compounds in reducing atherosclerosis caused by cholesterol and enhancing the dilation of capillary vessels. Then under the action of biological enzyme, glycosyl group is added to isoquercitrin which has been prenylated, so that the isoquercitrin derivative with high purity, high quality and high biological activity is obtained. The solubility of the isoquercetin derivatives is improved by more than 100 times compared with that of quercetin and rutin, and the isoquercetin derivatives can be widely applied to the fields of food, medicines, cosmetics and the like.

The synthesis steps of the isoquercitrin derivative (I) of the invention are as follows:

1) reacting 2g of isoquercitrin, 2-2.4 times equivalent of acetic anhydride and 2-4 times equivalent of piperidine at 100-140 ℃ for 2-24h to obtain a product IIa with the yield of 92.4%;

2) 2g of IIa, 1-5 times of thiophenol, 0.1-1 time of imidazole and N-methylpyrrolidone as a solvent are reacted for 5-25h at 0 ℃, and the yield of the product IIb is 89.2%;

3) reacting IIb with 1-5 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine serving as a catalyst in a THF solution at the temperature of-20-0 ℃ for 12 hours to obtain IIc with the yield of 95.6 percent;

4) 2g of IIc was added to 2 equivalents of acetic anhydride and 10 times of ammonium acetate and refluxed in methanol for 24 hours to give the product IId in 93.1% yield.

The reaction equation is:

further preparing isoquercitrin derivative (I) by biological enzyme from IId, wherein the structural formula is shown as the formula (I):

wherein: n is 1-5; r ═ Glu, Rha.

The enzymatic method of the present invention comprises: one or a combination of glucosyltransferase and rhamnosyltransferase; the added glycosyl is glucose, UDP-glucose, rhamnose, UDP-rhamnose.

The reaction conditions of the invention are as follows: disodium hydrogen phosphate-sodium citrate buffer solution with the mass fraction of the reaction buffer solution being 0.05-1% or NaCl buffer solution with the mass fraction of 0.9%; and (3) controlling the pH value of the buffer solution to be 6-10, controlling the reaction temperature to be 35-75 ℃, controlling the reaction time to be 3-15 hours, and after the reaction is finished, performing suction filtration and drying to obtain the isoquercitrin derivative.

The preparation steps of the isoquercetin derivatives of the invention are preferably as follows:

1) 2g of isoquercitrin, 2-3 times of equivalent weight of acetic anhydride and 2-3 times of piperidine react for 10-15 h at 100-110 ℃, and the product is IIa.

2) 2g of IIa, 3-5 times of thiophenol, 0.5-1 time of imidazole and N-methylpyrrolidone as a solvent react for 5-15 hours at 0 ℃, and the product is IIb.

3) And reacting IIb with 3-5 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine in a THF solution at-5-0 ℃ for 12 hours to obtain IIc.

4) 2g of IIc is added with 2 times of equivalent of acetic anhydride and 10 times of ammonium acetate, and refluxed in methanol for 24 hours to obtain a product IId.

5) The structural formula of the isoquercitrin derivative prepared by treating IId with biological enzyme is shown as the formula (I-1):

wherein: n is 1-3; r ═ Glu.

The raw materials of the isoquercetin derivative of the invention are rutin, quercetin and isoquercetin plant extracts or/and derivatives transformed by biological enzyme.

The isoquercetin derivative of the invention solves the problem of poor solubility of rutin and quercetin, improves the bioavailability of the isoquercetin derivative and increases the curative effect of the isoquercetin derivative.

An object of the present invention is to provide a process for the preparation of isoquercetin derivatives.

Another object of the present invention is to provide the use of isoquercitrin derivatives in the prevention and treatment of hypertension and vasodilation.

Another object of the present invention is the use of isoquercitrin derivatives for cardiovascular protection and for lowering blood lipids.

Another object of the invention is to provide the application of the isoquercitin derivative in bacteriostasis.

The invention provides a pharmaceutical composition, which comprises the compound and pharmaceutically acceptable salts, carriers, excipients, diluents, vehicles or the combination thereof.

Has the advantages that: after adding isoquercitrin, the invention adds glycosyl group to isoquercitrin prenylated under the action of glycosyl transferase to obtain isoquercitrin derivative, and increases the solubility of isoquercitrin derivative.

The isoquercetin derivative of the invention not only solves the problem of poor solubility of isoquercetin, but also enhances the efficacy of isoquercetin, is 3-5 times of isoquercetin in free radical removal, anti-inflammation and bacteriostasis, and improves the bioavailability of the isoquercetin derivative.

Drawings

FIG. 1HPLC method determines the solubility of isoquercitrin derivatives in water.

FIG. 2 the ability of derivatives of isoquercetin derivatives to scavenge free radicals.

FIG. 3 is an HPLC chart of isoquercitrin derivatives.

The present disclosure is specifically described with reference to specific embodiments, but the scope of the present disclosure is not limited to the following embodiments.

EXAMPLE 16 preparation of prenyl-substituted isoquercitrin derivatives

1) 2g of isoquercitrin, 3-4 times equivalent of acetic anhydride and 1-2 times of pyridine react for 15h at the temperature of 140 ℃ under the condition of 100 ℃, the product is IIIa, and the yield is 90.5 percent;

2) 2g of IIIa, 3 times of thiophenol, 0.1-1 time of imidazole and N-methylpyrrolidone as a solvent are reacted for 4-20h at 0 ℃, and the yield of the product IIIb is 85.1%;

3) reacting IIIb with 2-4 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine in a THF solution at-5-0 ℃ for 12h to obtain IIIc with the yield of 92.1%;

4) add IIIc 2g 2 equiv of acetic anhydride and 10 times of acetic anhydride to methanol and reflux 24h to give product IIId in 88.0% yield.

Example 2 glycosylation of prenylated isoquercitrin derivatives

6-prenyl substituted isoquercitrin derivatives are used as raw materials, a reaction buffer solution is a disodium hydrogen phosphate-sodium citrate buffer solution with the mass fraction of 0.05%, the pH value of the buffer solution is 8, the reaction temperature is 65 ℃, and the reaction time is controlled to be 18-24 hours. And adding glucosyltransferase into the reaction system, wherein the mass fraction of the glucosyltransferase is 25-30% of that of the 6-prenyl substituted isoquercetin derivative, so as to obtain the isoquercetin derivative. In the reaction system, the molar yield of the isoquercitrin derivative is 95.8 percent, and after the reaction is finished, the isoquercitrin derivative is obtained by pumping filtration and drying. The HPLC of isoquercitrin derivatives is shown in FIG. 3.

EXAMPLE 3 determination of solubility and dissolution Rate of Isoquercetin derivatives

1. Determination of solubility

The size of the compound's solvency directly affects the use of the drug in solution systems and cell systems. Since isoquercetin derivatives are stable in aqueous solutions, we used UV spectrophotometry to determine the solubility value of saturated aqueous solutions of isoquercetin. In the experiment, 10mg, 50mg, 100mg, 200mg and 1000mg phloretin are precisely measured respectively, placed in a 100mL volumetric flask, diluted to a scale by adding DMSO, shaken uniformly to obtain a series of phloretin standard solutions with the concentrations of 0.1mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL and 10mg/mL respectively, analyzed by HPLC, the characteristic peak in a 283nm interval is integrated, and the peak area is recorded. The phloretin concentration was plotted on the ordinate and the peak area on the abscissa, and linear regression was performed.

Standard curve equation 1: y is 0.00264A+0.01025,R²0.9925 is a phloretin fit curve.

And then adding excessive isoquercitrin derivative into 2mL of water phase, placing the water phase on a constant temperature oscillator at 25 +/-1 ℃ for continuous oscillation for 72h, taking out the isoquercitrin derivative, transferring the isoquercitrin derivative into a centrifuge tube, centrifuging the isoquercitrin derivative for 15min at 8000r/min, taking supernatant, filtering the supernatant by using a 0.45-micrometer microporous membrane, diluting the supernatant to be within a linear range by using methanol, and measuring the solubility of the isoquercitrin derivative in the water by adopting an HPLC method. The results are shown in FIG. 1.

The standard curve equation is that y is 6579.256A-267.19, R²0.9859. When the saturated solution was diluted 100 times, the value of the integrated absorbance was 856.38 and the concentration was 2.317g/L, the solubility of the isoquercitrin derivative was 76.46 g/L.

The results show that: the solubility of the isoquercetin derivatives is more than 100 times of that of isoquercetin.

Example 4 pharmacological experiments on swelling of mouse ears by Isoquercetin derivatives p-xylene

SPF-grade NIH mice, male, 60. The test results were randomized to 10 samples per group, namely a model control group, a positive control group (aspirin), and an isoquercetin derivative. Each group of mice was administered by gavage at 20ml/kg body weight for 1 time/day for 2 consecutive days. After 0.5h from the last administration, 0.05 ml/mouse was coated with xylene on the right ear, and distilled water was administered to the right ear. After 2h, the mice were sacrificed, the left and right ear pieces were cut off, round ear pieces were punched at the same positions of the left and right ear pieces with a punch having a diameter of 9mm, and the difference in weight between the two ear pieces was measured as swelling degree.

TABLE 1 Effect of isoquercetin derivatives on mouse ear swelling test

Group of	Dosage (mg/kg)	Quantity (only)	Swelling ear (mg)	Inhibition (%)
					Model control group	-	10	22.56±1.69	-
Aspirin	200	10	16.18±1.23	28.28％
					Isoquercitrin derivatives
	400	10	15.01±1.55	33.47％

The positive drug (aspirin) and the isoquercetin derivative have obvious inhibition effect (P is less than 0.01-0.05) on ear swelling caused by dimethylbenzene. The experiment shows that the isoquercitrin derivative has obvious anti-inflammatory activity.

Example 5 Effect of Isoquercetin derivatives on glycolipid metabolism in Nutrition obese C57BL/6J mice

1.1 Experimental materials

1.1.1 Experimental animals and rearing environments

C57BL/6J mice, male, SPF grade, 4 weeks old, supplied by the Experimental animals center of university of Zhongshan. License number: SCXK (yue) 2009-. Feeding conditions are as follows: the temperature is 20-25 ℃, the humidity is 55 +/-10%, the light is dark for 12h/12h for circulation, and the drinking water is freely eaten and drunk for 24 h.

1.1 Experimental methods

1.1. preparation, grouping and administration of animal models

C57BL/6J male mice, 10 of which were selected at random as normal control group, were fed with normal standard diet (diet formulation: crude protein 18.5%, crude fat 4.5%, carbohydrate 61%. energy ratio: crude protein 21%, crude fat 11%, carbohydrate 68%, total energy 3.5Kcal/g), and the remaining mice were fed with high-sugar high-fat diet (diet formulation: crude protein 27%, crude fat 24%, carbohydrate 37%, energy ratio: protein 23%, fat 46%, carbohydrate 31%, total energy 4.7 Kcal/g). After feeding for 12 weeks, selecting qualified obese mice according to standard that the body weight is more than the average body weight of a normal group +/-1.78 times standard deviation and more than 20% of the normal body weight, and dividing the obese mice into a model group, a positive drug simvastatin group, an isoquercetin derivative group and an isoquercetin dosage group according to the principle of uniform body mass, wherein each group comprises 10 animals, and the animals in the groups are respectively marked. The normal control group was given normal diet, and the rest groups were continued to be fed with high-sugar and high-fat diet. The dose of isoquercitrin administered: the dose group of isoquercetin derivatives is 150mg/kg body weight, the dose group of isoquercetin is 100mg/kg, the required concentration is prepared by distilled water, the existing preparation is used, the administration is carried out 1 time per day, and the continuous administration is carried out for 14 weeks.

The dosage of simvastatin is 3mg/kg body weight, is ground by a mortar, is prepared into the required concentration by distilled water, is prepared immediately before use, is administered 1 time a day, and is continuously administered for 14 weeks.

The model control group and the normal control group were administered 1 time a day for 14 weeks by instilling an equal volume of physiological saline daily.

1.1.2 measurement of blood glucose and blood lipid

After 14 weeks of administration, the animals were fasted for 12h, one drop of blood was taken from the tail vein, and fasting blood glucose was measured with a glucometer; then, blood is taken from orbital venous plexus, the orbital venous plexus is kept still at room temperature for more than 30min, the orbital venous plexus is centrifuged at 3000rpm for 15min, serum is separated, and the serum total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein and free fatty acid levels are measured by adopting a kit.

1.2 results of the experiment, body weight change during administration of the groups of mice

TABLE 2 weight changes in the groups of mice during the administration period

The above results show that: the body weight of the model group mice was significantly increased compared to the normal group (P < 0.01). The weights of the simvastatin, isoquercitrin derivative and isoquercitrin dose groups of mice begin to decrease from 9 weeks of administration, and the weights of the simvastatin, isoquercitrin derivative and isoquercitrin dose groups of mice are remarkably reduced compared with the model group after the administration (P is less than 0.01). Wherein the isoquercetin derivative dosage group has effects of reducing blood lipid with the most weight loss in mice.

Claims

1. A preparation of isoquercitrin derivatives for scavenging free radicals, characterized by: the structural formula of the isoquercitrin derivative is shown as the formula (I):

wherein: n is 1-5; r ═ Glu, Rha;

the isoquercetin derivatives are prepared from rutin, quercetin and isoquercetin plant extracts;

the reaction steps of the isoquercetin derivatives are as follows:

1) 2g of isoquercitrin, 2-5 times of equivalent of acetic anhydride and 2-4 times of piperidine react for 2-24h at the temperature of 100-120 ℃, and the product is IIa;

2) 2g of IIa, 1-5 times of thiophenol, 0.1-1 time of imidazole and N-methylpyrrolidone as a solvent react for 5-25h at 0 ℃, and the product is IIb;

3) reacting IIb with 1-5 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine in a THF solution at-20-0 ℃ for 12h to obtain IIc;

4) adding 2g of IIc into 2 times of equivalent of acetic anhydride and 10 times of ammonium acetate, and refluxing in methanol for 24 hours to obtain a product IId;

5) treating IId with biological enzyme to prepare isoquercitrin derivative;

the biological enzyme comprises: rhamnosidase, glucosidase, glucoside transferase.

2. The preparation of an isoquercetin derivative for scavenging free radicals as claimed in claim 1, characterized in that: the preparation steps of the isoquercetin derivatives are preferably as follows:

1) reacting 2g of isoquercitrin, 2-3 times of equivalent of acetic anhydride and 2-3 times of piperidine at 100-110 ℃ for 10-15 h to obtain a product IIa;

2) reacting 2g of IIa with 3-5 times of thiophenol, 0.5-1 time of imidazole and N-methylpyrrolidone as a solvent at 0 ℃ for 5-15 hours to obtain a product IIb;

3) reacting IIb with 3-5 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine serving as catalysts in a THF solution at the temperature of-5-0 ℃ for 12 hours to obtain IIc;

wherein: n is 1-3; r ═ Glu.

3. The preparation of an isoquercetin derivative for scavenging free radicals according to claim 1 or 2, characterized in that: the conditions for treating IId with the biological enzyme are as follows: disodium hydrogen phosphate-sodium citrate buffer solution with the mass fraction of the reaction buffer solution being 0.05-1% or NaCl buffer solution with the mass fraction of 0.9%; and (3) controlling the pH value of the buffer solution to be 6-10, controlling the reaction temperature to be 35-75 ℃, controlling the reaction time to be 3-15 hours, and after the reaction is finished, performing suction filtration and drying to obtain the isoquercitrin derivative.

4. The preparation of an isoquercetin derivative for scavenging free radicals according to claim 1 or 2, characterized in that: the isoquercitrin derivative can be used for scavenging free radicals, protecting cardiac vessels and reducing blood fat.

5. The preparation of an isoquercetin derivative for scavenging free radicals according to claim 1 or 2, characterized in that: the isoquercitrin prenyl derivatives can be used in pharmaceutically acceptable salts, carriers, excipients, diluents, vehicles or pharmaceutical compositions thereof.