CN111206061A

CN111206061A - Preparation of diosmin derivative for treating varicosity

Info

Publication number: CN111206061A
Application number: CN202010054812.XA
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 and application of a diosmin derivative for treating varicosity; the diosmin derivative is prepared by connecting a prenyl group to diosmin to obtain prenyl diosmin, and the prenyl diosmin and biological enzyme are subjected to glycosylation reaction in a buffer solution to prepare the diosmin derivative with high solubility; the diosmin derivative is superior to diosmin in the application of treating relevant symptoms of varicose insufficiency such as hemorrhoids/chilblain and the like, promoting blood circulation to remove blood stasis and resisting inflammation.

Description

Preparation of diosmin derivative for treating varicosity

Technical Field

The invention relates to a preparation method of a diosmin derivative for treating varicosity and varicosity for treating hemorrhoids/chilblain and the like, belonging to the field of medicines.

Technical Field

Diosmin is a flavonoid drug, such as: the product name of Aimailang (Schveya (Tianjin) pharmaceutical Co., Ltd.) and diosmin tablet (Mayilong pharmaceutical industry group Co., Ltd.) were used for the following indications: 1. various symptoms associated with venous lymphatic insufficiency (heavy legs, pain, swelling discomfort in the morning, etc.); 2. various symptoms associated with the onset of acute hemorrhoids.

On the one hand, diosmin has various medicinal values, but the monomer is difficult to dissolve in water, and when the diosmin is directly used as a medicament, the bioavailability in a human body is low, and the curative effect of the medicament is difficult to fully exert. On the other hand, diosmin has significant drawbacks in clinical applications, such as: low biological utilization, limited curative effect and the like, and the application of diosmin is severely limited. At present, no report is made on the improvement of the performance and use of diosmin by linking prenyl and glycosyl groups to diosmin.

The existence of the prenyl increases the lipophilicity and biological guidance of the flavonoid compound, so that the flavonoid compound can reach an action target through a cell membrane more easily in an organism, and the medicinal value of the flavonoid compound is greatly improved.

Disclosure of Invention

According to the invention, through adding isoprenyl groups on diosmin, isoprenyl diosmin is obtained, and then glycosylation conversion is carried out on the isoprenyl diosmin, so that diosmin derivatives with high purity, high quality and high biological activity are obtained, the problem of low content of isoprenyl in flavone compounds is solved, and the effect of diosmin is obviously improved. The solubility of the diosmin derivative is improved by 300-500 times compared with that of diosmin, and the diosmin derivative can be widely applied to the fields of medicines, cosmetics and the like, so that the application range of diosmin is expanded.

The synthesis steps of the diosmin derivative are as follows:

1) reacting 2g of diosmin, 2-2.4 times of equivalent of TsCl and 2-4 times of piperidine at 30-40 ℃ for 2-12 hours to obtain a product IIa with the yield of 86-91%;

2) reacting 2g of IIa with 1-3 times of thiophenol, 0.1-1 time of imidazole and N-methylpyrrolidone as a solvent at 0 ℃ for 5-15 hours to obtain a product IIb with the yield of 81-85%;

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 12 hours to obtain IIc, wherein the yield is 75-80%;

4) adding 2 times of equivalent TsCl and 8 times of ammonium acetate into IIc 2g, and refluxing in methanol for 20 hours to obtain a product IId, wherein the yield is 89-91%.

5) Treating IId with biological enzyme to prepare diosmin derivative (I).

Wherein: r₁、R₂Is OH, OCH₃Or OCH₂CH₃；

R₃Glu, Rha, Xyl or Gal, n is 1-5;

the reaction equation is:

wherein: r₃Glu, Rha, Xyl or Gal, n is 1-5;

according to the invention, after adding prenyl on diosmin, glycosyl is added to prenyl diosmin under the action of glycosyl transferase, so that glycosylated diosmin derivatives are obtained, and the solubility of the diosmin derivatives is increased.

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: the method comprises the following steps of preparing a dipotassium hydrogen phosphate-potassium citrate buffer solution with the mass fraction of a reaction buffer solution being 0.05-1% or a KCl buffer solution with the mass fraction of 0.9%, controlling the pH of the buffer solution to be 8-10, controlling the reaction temperature to be 35-75 ℃, controlling the reaction time to be 1-24 hours, and after the reaction is finished, performing suction filtration and drying to obtain the diosmin derivative.

The synthesis steps of the diosmin derivative of the invention are preferably as follows:

1) 2g of diosmin, 1-2 times of equivalent of TsCl and 2-4 times of piperidine react at 30-40 ℃ for 2-12 hours to obtain IIa.

2) 2g of IIa, 1-3 times of thiophenol, 0.5-1 time of imidazole and N-methylpyrrolidone as a solvent react for 8-10 hours at 0 ℃, and the product is IIb.

3) And reacting IIb with 2-5 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 was added to 2 equivalents of TsCl and 8 times of ammonium acetate and refluxed in methanol for 20h to give the product IId.

5) Treating IId with a biological enzyme to prepare a diosmin derivative (I-1) having the formula:

wherein: r₃Glu, Rha, Xyl or Gal, n is 1-5.

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

The invention also aims to provide a diosmin derivative for treating and preventing diseases related to hemorrhoids/chilblain and the like caused by venous lymphatic insufficiency.

The invention also aims to provide the function of the diosmin derivative in promoting blood circulation by removing blood stasis and resisting inflammation.

The invention also provides a pharmaceutical composition comprising the compound and pharmaceutically acceptable salts, carriers, excipients, diluents, vehicles or combinations thereof.

Has the advantages that: the invention obtains the diosmin derivative with high purity, high quality, high solubility and high biological activity by carrying out biological enzyme conversion on the isoprenyl diosmin and carrying out biological synthesis methods such as glycosylation, deglycosylation and the like, meets the market demand and improves the curative effect of the diosmin.

Drawings

Figure 1 diosmin derivative solubility fit curves.

Figure 2 is a comparison graph of solubility of diosmin derivatives and diosmin in water determined by HPLC method.

Figure 3 HPLC profile of diosmin derivatives.

The specific implementation example is as follows:

the present invention will be described in detail with reference to specific embodiments, but the scope of the invention is not limited to the following embodiments

EXAMPLE 16 preparation of prenyl-substituted Diosmin derivatives

1) Reacting 2g of diosmin, 2-2.4 times of equivalent of TsCl and 2-4 times of piperidine at 30-40 ℃ for 2-12 hours to obtain a product IIa with the yield of 88.3%;

2) reacting 2g of IIa with 1-5 times of thiophenol, 0.1-1 time of imidazole and N-methylpyrrolidone as a solvent at 0 ℃ for 5-15 hours to obtain a product IIb, wherein the yield is 82.6%;

3) reacting IIb with 1-3 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine in a THF solution at-20-0 ℃ for 12 hours to obtain IIc, wherein the yield is 78.4%;

4) 2g of IIc is added with 2 times of equivalent of TsCl and 8 times of ammonium acetate, and the mixture is refluxed for 20 hours in methanol to obtain the product 6-isoprenyl substituted diosmin derivative (IId), wherein the yield is 90.1%.

Example 2 preparation of Diosmin derivatives

The method is characterized in that 6-prenyl substituted diosmin derivatives are used as raw materials, a reaction buffer solution is 0.05% of dipotassium hydrogen phosphate-potassium citrate buffer solution in percentage by mass, the pH value of the buffer solution is 8.5, the reaction temperature is 60 ℃, and the reaction time is controlled to be 12-18 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 diosmin derivative, so as to obtain the diosmin derivative. In the reaction system, the molar yield of the diosmin derivative is 91.6%, and after the reaction is finished, the diosmin derivative is obtained by suction filtration and drying. The HPLC of the oxramification is shown in figure 3.

Example 3 solubility determination of Diosmin derivatives

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

Standard curve equation 1: 0.00212A +0.00913, R²0.9934 is a curve fitted to diosmin derivatives.

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

The results show that: the solubility of the diosmin derivative is about 450 times that of diosmin.

Example 4 Diosmin derivative swelling test

1. Experimental Material

Experimental animals 40 female Balb/c mice of SPF grade 5-6 from the center of Experimental animals in Guangdong province. Diosmin derivative, TPA-99.99%, acetone, dichloromethane, DMSO and ibuprofen.

2. The experimental method comprises the following steps:

1) 40 Balb/c mice were randomly divided into 4 groups, acetone blank, TPA control, diosmin derivative and ibuprofen, each group of 1 mouse, and repeated 3 times.

2) The acetone blank and TPA control were each treated with 15 μ L of self-prepared solution (dichloromethane: acetone 80: 20) the medicine is uniformly applied to two ears of a mouse, and the nano micelle coated diosmin is uniformly applied to two ears of the mouse.

3) After 6min, the acetone blank was evenly spread over both ears of the mice with acetone: the TPA control group and the diosmin group wrapped by the nano-micelle are uniformly coated on two surfaces of two ears of a mouse by 15 mu L of TPA respectively to cause inflammation.

4) After 6h, the mice were sacrificed, then the ears of the mice were cut off in time, and the round ear pieces were punched at the same positions respectively by a puncher with the diameter of 6 mm.

5) And weighing, taking an acetone blank group as a negative, and taking the difference between the weight of the lug of the experimental group and the weight of the lug of the blank group as the swelling degree.

6) The inhibition ratio was (1-degree of swelling in the administration group/degree of swelling in the control group) × 100%.

3 experimental results, inhibition of TPA-induced auricle swelling in mice by diosmin derivatives.

To investigate the inhibitory effect of diosmin derivatives on TPA-induced ear swelling in mice, a working concentration of 0.75 μ M of diosmin derivatives was selected. Through the inhibition experiment research on the diosmin derivative on the mouse auricle swelling, the result that the average weight of the mouse auricle is increased from 7.5mg to 13.3mg due to the fact that the mouse auricle swelling is induced by simply smearing TPA, the diosmin derivative has inhibition effects on the mouse auricle swelling in different degrees, and the inhibition rate range of the diosmin derivative is 84.2-95.9%.

The diosmin derivative has obvious inhibiting effect on TPA-induced mouse auricle swelling. The animal experiment result shows that the inhibition rate of ibuprofen is 81.4%.

Example 5 Effect of Diosmin derivatives on acute haemorrhoids in rats caused by croton oil

Wistar rats, male. The blank control group, the positive drug horse hemorrhoid ointment control group and the tested drug diosmin derivative group are arranged in the low, medium and high concentration groups. After molding, animals were divided into groups of 10 animals each according to the perianal swelling degree.

The inflammation-causing agent is prepared by 1 part of distilled water, 4 parts of pyridine, 5 parts of diethyl ether and 10 parts of 6% croton oil diethyl ether solution. Then, a cotton ball soaked with 0.16mL of croton oil mixed solution is inserted into the anus of a rat of 6 weeks old for 10s, and a croton oil swelling model of the anus of the rat is established. The next day, the blank matrix control group, the positive drug horse hemorrhoid ointment control group and the tested drug diosmin derivative are divided into groups according to the swelling degree, the diosmin derivative group is respectively coated with the blank matrix, the horse hemorrhoid ointment and the escin sodium gel once a day, the coating amount is 1.0g/kg.bw (0.2 g/piece), the groups are continuously coated for 7 days, 30min after the last administration, rats in each group are killed, rectal tissues 15mm from the hair margin of the anal skin are cut, and the rectal tissues are cleaned by cold physiological saline and then are dried by filter paper. Randomly selecting 5 animals in each group, fixing tissues with 10% formaldehyde, embedding in paraffin, slicing, HE staining, and performing pathological histological observation; the remaining tissue was used to determine anal swelling in each group of rats. The therapeutic effect of the diosmin derivative on the anal swelling of rats caused by croton oil was observed by using the disease detection results and the anorectal swelling coefficient as indexes.

The results show that the medium and high concentration groups of the medicine can obviously improve the anus swelling and inflammatory reaction of rats caused by croton oil. Compared with a blank control group, the difference has significance (P is less than 0.05).

TABLE 2 Effect of diosmin derivatives on anal swelling in rats by croton oil

Group of	Dosage (mg/kg)	Coefficient of rectal anal swelling
			Blank control	-	55.6±6.3
Mayinglong pile ointment	100	46.5±5.7
			Low dose diosmin derivative group	10	43.1±3.3
Intermediate dose diosmin derivative group	20	40.5±3.9
			High dose diosmin derivative group	40	38.3±3.8

Note: p < 0.05 compared to the blank control group.

Example 6 Effect of Diosmin derivative vasodilation

SD rats 60, male, 150-: yue Yi Xue Dy's word No. 20090043. Animals were divided into 3 groups of 20 animals each using a stratified randomization method. The ordinary feed A, the feed B containing 1g/kg of diosmin and the feed C containing 1g/kg of diosmin derivative are fed for one month in the experimental environment, and then A, B, C groups of SD rats are fed with the ordinary feed for one week.

The thoracic aorta was removed from the SD rat and transected into a vascular ring, each segment being about 1.0mm in length. Placing in a blood vessel tensiometer with constant temperature of 37 ℃, and continuously introducing mixed gas of 5% CO2 and 95% O2. After the arterial ring is arranged, 2mN load is given, the solution is changed once in 20min, and the solution is continuously stabilized for 1.0 h. The activity of the arterial loop was then examined with K + -Kreb's solution (K +60mM) and the difference in the amplitude of two contractions < 10% was used for subsequent experiments. The method comprises the steps of pre-contracting by 1.0 mu M phenylephrine, adding medicines FCCP (1.0 mu M), Valinomycin (1.0 mu M), Nigericin (1.0 mu M) and Monesin (1.0 mu M) which interfere mitochondrial function after the tension rises and is stable, detecting the change of the vascular tension of rats, finding that abdominal aorta contraction is most obvious 30min after the diosmin derivative is administered, the abdominal aorta contraction begins to weaken after 70min, and the abdominal aorta contraction returns to be normal 360min after the administration; abdominal aorta contraction is most evident 30min after diosmin administration, contraction begins to weaken after 50min and returns to normal 240min after administration; the abdominal aorta contracted most significantly 30min after glucose administration, beginning to decrease after 40min and returning to normal 120min after administration.

Experiments show that the diosmin derivative is obviously better than diosmin in promoting blood circulation, and can be used as an alleviator for treating acute hemorrhoid/chilblain and activating blood circulation to dissipate blood stasis.

Claims

1. A preparation of diosmin derivatives for the treatment of varicose veins, characterized in that: the structural formula of the diosmin derivative is shown as the formula (I):

wherein: r₁、R₂Is OH, OCH₃Or OCH₂CH₃；

R₃Glu, Rha, Xyl or Gal, n is 1-5;

the diosmin derivative is prepared from plant extracts of diosmin, hesperetin, hesperidin and hesperetin-7-O-glucoside as raw materials;

the synthesis steps of the diosmin derivative are as follows:

1) reacting 2g of diosmin, 1-3 times of equivalent of TsCl and 2-4 times of piperidine at 30-40 ℃ for 2-12 hours to obtain a product IIa;

2) reacting 2g of IIa with 1-3 times of thiophenol, 0.1-1 time of imidazole and N-methylpyrrolidone as a solvent at 0 ℃ for 5-15 hours to obtain a product IIb;

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

5) Treating IId with biological enzyme to prepare diosmin derivative (I);

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

2. The preparation of diosmin derivatives for use in the treatment of varicose veins according to claim 1, characterized in that: the synthesis steps of the diosmin derivative are preferably as follows:

1) reacting 2g of diosmin, 1-2 times of equivalent of TsCl and 2-4 times of piperidine at 30-40 ℃ for 2-12 hours to obtain a product IIa;

2) reacting 2g of IIa with 1-3 times of thiophenol, 0.5-1 time of imidazole and N-methylpyrrolidone as a solvent at 0 ℃ for 8-10 hours to obtain a product IIb;

3) reacting IIb with 2-5 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) adding 2g of IIc into 2 times of equivalent TsCl and 8 times of ammonium acetate, and refluxing in methanol for 20 hours to obtain a product IId;

wherein: r₃Glu, Rha, Xyl or Gal, n is 1-5.

3. Preparation of a diosmin derivative for use in the treatment of varicose veins according to claim 1 or 2, characterized in that: the conditions for treating IId with the biological enzyme are as follows: the method comprises the following steps of preparing a dipotassium hydrogen phosphate-potassium citrate buffer solution with the mass fraction of a reaction buffer solution being 0.05-1% or a KCl buffer solution with the mass fraction of 0.9%, controlling the pH of the buffer solution to be 8-10, controlling the reaction temperature to be 35-75 ℃, controlling the reaction time to be 1-24 hours, and after the reaction is finished, performing suction filtration and drying to obtain the diosmin derivative.

4. Preparation of a diosmin derivative for use in the treatment of varicose veins according to claim 1 or 2, characterized in that: the diosmin derivative can be used for treating varicose veins caused by hemorrhoids/chilblain first pass.

5. Preparation of a diosmin derivative for use in the treatment of varicose veins according to claim 1 or 2, characterized in that: the diosmin derivative compounds can be used in pharmaceutically acceptable salts, carriers, excipients, diluents, vehicles or pharmaceutical compositions thereof.