CN112500384A - Synthetic method of flavonoid sulfate and application thereof under acidic condition - Google Patents
Synthetic method of flavonoid sulfate and application thereof under acidic condition Download PDFInfo
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Abstract
The invention provides a method for synthesizing flavonoid sulfate, which comprises the steps of uniformly mixing flavonoid, dicyclohexylcarbodiimide and tetrabutylammonium hydrogen sulfate according to a proportion, adding a proper amount of cosolvent, standing, carrying out reduced pressure distillation, fully removing the cosolvent, dissolving with a proper amount of methanol, and carrying out centrifugal filtration to obtain a methanol solution of the flavonoid sulfate tetrabutylammonium. Removing methanol from the obtained solution, re-dissolving the product with a small amount of methanol, centrifuging, filtering, adding saturated solution of potassium acetate or sodium acetate methanol into the filtrate, reacting to obtain flavonoid compound potassium sulfate and tetrabutylammonium acetate, dissolving tetrabutylammonium acetate in methanol, centrifuging, and filtering to obtain flavonoid compound potassium (sodium) sulfate. The invention also provides application of the flavonoid compound sulfate in the fields of hemostasis and blood coagulation under acidic conditions. The invention has simple synthesis process condition and lower cost, and can provide research and development directions for the preparation for treating traumatic hemorrhage or gastrointestinal tract hemorrhage by applying the preparation under the acidic condition.
Description
Technical Field
The invention relates to the field of research of biological pharmaceutical preparations, in particular to a synthesis method of flavonoid compound sulfate and application thereof under an acidic condition.
Background
The flavonoid compounds are formed by connecting two benzene rings (ring A and ring B) with phenolic hydroxyl groups through a central three-carbon atom (ring C), namely a series of compounds taking C6-C3-C6 as a basic skeleton. Flavonoids are widely present in various tissues and organs of plants and are important secondary metabolites of plants. The flavonoid compounds found at present are more than 10000, and are mainly divided into flavonoids, flavonols, flavanones (alcohols), isoflavones, chalcones, anthocyanidins, flavanols and the like according to the oxidation degree of the carbon chain of the middle C ring, whether the C ring is formed into a ring, the position (2-position or 3-position) connected with the B ring and other characteristics.
The flavonoid compounds have wide physiological activities, such as antioxidation, anti-tumor, anti-cardiovascular disease, antibiosis, antivirus, inhibition of peptic ulcer and inflammation, liver protection, depression resistance, anti-aging, diabetes, osteoporosis resistance, neuroprotection, immunoregulation, fatigue resistance and the like, and have wide application in the fields of food, medicine and health care.
However, the flavonoid compound has poor solubility and poor stability, and the biological efficacy of the flavonoid compound is greatly limited. Therefore, the structural modification of the flavonoid compound, the improvement of the solubility and other properties, the improvement of the biological activity and the increase of the biological function are important ways for developing and applying the flavonoid compound. Hydroxyl groups of the A ring and the B ring of the flavonoid compound can be subjected to esterification reaction under certain conditions to form compounds such as sulfate or phosphate of the flavonoid compound, and the compounds further react with NaCl, KCl, sodium acetate or potassium acetate to form sodium (potassium) sulfate or sodium (potassium) phosphate of the flavonoid compound, so that the water solubility of the flavonoid compound is improved. Flavonoid sulfate also exists in plants, such as Ecliptae herba which contains a large amount of luteolin-7-sulfate. The sulfate of various flavonoid compounds can be efficiently obtained by taking the flavonoid compounds as substrates and adopting artificial synthesis. The flavonoids compounds have the effects of dilating coronary vessels, inhibiting the aggregation of erythrocytes and platelets and the activity of related thrombin, thereby having the effects of anticoagulation and hemostasis. Research shows that quercetin-7-sodium sulfate and quercetin-7, 4' -disodium disulfate both have the capacity of inhibiting platelet aggregation, which indicates that the sulfate of flavonoid compound also has the anticoagulant effect.
In the process of researching the blood coagulation stopping active ingredients of the eclipta alba, the pH value is found to have important influence on the blood coagulation stopping activity of the extract (related researches such as the blood coagulation stopping activity of the eclipta alba and the like have applied for Chinese invention patents, patent application numbers are 201911322326.5 and 202010416759.3); further research shows that the main blood coagulation stopping matter in eclipta is a yellow compound, and through separation and identification, the main component of the yellow compound is luteolin-7-sulfate, and the yellow compound has strong activity of directly agglutinating erythrocytes under acidic conditions and can cause soluble fibrinogen to become insoluble floccule. Therefore we speculate that: sulfates of other flavonoids may also have the activity to directly agglutinate erythrocytes and cause soluble fibrinogen to become insoluble flocs under acidic conditions. The activity detection shows that the sulfate salt of the flavonoid compounds can not agglutinate red blood cells under neutral or alkaline conditions and has no obvious flocculation effect on fibrinogen, but under acidic conditions, the flavonoid compounds can agglutinate red blood cells directly and cause soluble fibrinogen to become insoluble floccules, so that the flavonoid compounds have the blood coagulation stopping activity. At present, no relevant report is found at home and abroad, and no relevant application is found.
Disclosure of Invention
The invention aims to further develop the biological function and the application range of the flavonoid compound, provides a synthesis method of the sulfate salt of the flavonoid compound, and also aims to provide the application of the sulfate salt of the flavonoid compound in the medical fields of hemostasis, blood coagulation and the like under the acidic condition.
The technical scheme adopted by the invention is as follows:
a method for synthesizing flavonoid compound sulfate comprises the following steps:
1) uniformly mixing three substances according to the proportion of flavonoid compound dicyclohexylcarbodiimide to tetrabutylammonium hydrogen sulfate = 1: 1-20: 1-10 (molar ratio), adding a proper amount of cosolvent, reacting at 4-50 ℃ for 1-72 hours, then carrying out reduced pressure distillation at 45-60 ℃, fully removing the cosolvent, dissolving with a proper amount of methanol, and carrying out centrifugal filtration to obtain a methanol solution of flavonoid compound tetrabutylammonium sulfate;
2) removing methanol in the obtained solution, re-dissolving the product with a small amount of methanol, centrifuging and filtering, further removing a small amount of precipitate impurities, adding a saturated solution of potassium acetate or sodium acetate methanol into the filtrate, reacting to generate flavonoid compound potassium sulfate or flavonoid compound sodium sulfate and tetrabutylammonium acetate, dissolving the tetrabutylammonium acetate in methanol, centrifuging and filtering, and washing the precipitate with methanol to obtain the product, namely the flavonoid compound potassium sulfate or the flavonoid compound sodium sulfate.
In the step 1), the cosolvent is pyridine.
The step 2) is that the method for removing the methanol comprises the following steps: and removing the methanol by decompression and distillation at the temperature of 45-60 ℃.
In the step 2), the obtained product flavonoid compound sulfate is flavonoid compound potassium sulfate or flavonoid compound sodium sulfate.
In the step 2), the conversion rate of the obtained product, namely the flavonoid compound sulfate salt, is 70-96%.
The application of flavonoid sulfate in the fields of hemostasis and blood coagulation under acidic conditions.
The acidic condition is the addition of one or more of tartaric acid, citric acid, oxalic acid, malic acid, succinic acid, malonic acid, ascorbic acid and maleic acid.
The hemostasis and coagulation fields comprise the fields of medicines, medical treatment, sanitary materials and medical appliances.
The invention has the technical effects that:
1. the purified flavonoid compound sulfate salt of the invention generally has the function of stopping blood coagulation under the acidic condition, and provides a definite direction and a wide space for developing a high-efficiency and low-toxicity therapeutic preparation for traumatic hemorrhage or gastrointestinal tract hemorrhage.
2. The synthesis process conditions of the flavonoid sulfate are simple, a plurality of flavonoids have extremely high content in plants and low price, and even the flavonoid can be produced by microbial fermentation, so the invention has great production potential when being applied to the preparation for treating traumatic hemorrhage or gastrointestinal tract hemorrhage.
Drawings
FIG. 1 shows the agglutination of erythrocytes by potassium sulfate mixture of flavonoid compounds. Wherein a is luteolin sulfate potassium salt mixture, b is quercetin sulfate potassium salt mixture, c is dihydroquercetin sulfate potassium salt compound, d is apigenin sulfate potassium salt mixture, e is myricetin sulfate potassium salt mixture, f is dihydromyricetin sulfate potassium salt mixture, g is kaempferol sulfate potassium salt mixture, and h is baicalein sulfate potassium salt compound.
FIG. 2 shows the agglutination of erythrocytes by part of potassium flavonoid-7-sulfate and dipotassium flavonoid-7, 4' -disulfate. Wherein, a is luteolin-7-sulfate potassium, b is luteolin-7, 4 '-disulfate dipotassium, c is quercetin-7, 4' -disulfate dipotassium, d is dihydroquercetin-7, 4 '-disulfate dipotassium, e is apigenin-7-sulfate potassium, f is apigenin-7, 4' -disulfate dipotassium, g is myricetin-7, 4 '-disulfate dipotassium, h is kaempferol-7-sulfate potassium, and i is kaempferol-7, 4' -disulfate dipotassium.
FIG. 3 shows the flocculation of human fibrinogen caused by the mixture of flavonoid sulfate salts. Wherein a is luteolin sulfate potassium salt mixture, b is quercetin sulfate potassium salt mixture, c is dihydroquercetin sulfate potassium salt compound, d is apigenin sulfate potassium salt mixture, e is myricetin sulfate potassium salt mixture, f is dihydromyricetin sulfate potassium salt mixture, g is kaempferol sulfate potassium salt mixture, and h is baicalein sulfate potassium salt compound.
FIG. 4 shows the flocculation of part of flavonoid-7-potassium sulfate and flavonoid-7, 4' -dipotassium disulfate on human fibrinogen. Wherein, a is luteolin-7-potassium sulfate, b is luteolin-7, 4 ' -dipotassium disulfate, c is apigenin-7-potassium sulfate, d is apigenin-7, 4 ' -dipotassium disulfate, e is quercetin-7, 4 ' -dipotassium disulfate, f is kaempferol-7-potassium sulfate, g is kaempferol-7, 4 ' -dipotassium disulfate, and h is myricetin-7, 4 ' -dipotassium disulfate.
Detailed Description
The present invention will now be described more clearly and fully with reference to the following examples.
Example 1
The synthesis of sulfate salt of flavonoid compound comprises the following test process:
uniformly mixing three substances according to the proportion of a flavonoid compound, namely Dicyclohexylcarbodiimide (DCC) and tetrabutylammonium hydrogen sulfate (TBAHS) = 1: 1-20: 1-10 (molar ratio), adding a proper amount of pyridine to dissolve the three substances, reacting at 4-50 ℃ for 1-72 hours, distilling under reduced pressure at 45-60 ℃, fully removing pyridine, dissolving with a proper amount of methanol, carrying out centrifugal filtration to obtain a methanol solution of the flavonoid compound tetrabutylammonium sulfate, carrying out reduced pressure distillation at 45-60 ℃ to remove methanol, re-dissolving a product with a small amount of methanol, carrying out centrifugal filtration to further remove a small amount of precipitated impurities, adding a saturated solution of potassium acetate or sodium acetate methanol into the filtrate, reacting to generate the flavonoid compound potassium sulfate or the flavonoid compound sodium sulfate and the tetrabutylammonium acetate, dissolving tetrabutylammonium acetate in methanol, centrifuging, filtering, and washing precipitate with methanol to obtain potassium sulfate or sodium sulfate.
Example 2
The separation and identification of the monomeric compounds of the flavonoid-7-sulfate salt and the flavonoid-7, 4' -disulfate dihydrochloride are carried out by the following test processes:
separating the flavonoid compound potassium sulfate or the flavonoid compound sodium sulfate mixture obtained in the example 1 by adopting a reversed phase medium pressure liquid chromatography, wherein the elution condition is deionized water methanol = 1: 0 → … → 0: 1, the detection wavelength is 254 nm, collecting corresponding components according to an absorption peak to obtain flavonoid compound-7-potassium sulfate (or sodium), flavonoid compound-7, 4 '-dipotassium disulfate (or sodium), and carrying out structure identification on the flavonoid compound-7-potassium sulfate (or sodium) and the flavonoid compound-7, 4' -dipotassium disulfate (or sodium) by adopting mass spectrum and nuclear magnetic resonance data.
Example 3
The test process of the agglutination activity of the flavonoid compound sulfate on erythrocytes under the acidic condition is as follows:
collecting venous blood of healthy human body by using a vacuum hemostix (containing anticoagulant sodium citrate), diluting with 0.9% sterile physiological saline by 50 times, preparing a sample solution with a certain concentration by using deionized water for a mixture of flavonoid compound sulfate ester salt in example 1, flavonoid compound-7-potassium (or sodium) sulfate and flavonoid compound-7, 4' -dipotassium (or sodium) disulfate monomer compound in example 2, and uniformly mixing 1 microliter of sample solution (1 microliter of physiological saline is used for replacing the sample solution in a blank control group), 1 microliter of tartaric acid solution (0.005M) and 3 microliter of diluted blood on a clean glass slide (the coating area is 1 cm)2) And immediately photographed under an optical microscope, the number of free and agglutinated erythrocytes was counted, 3 replicates were made for each concentration, and 10 fields were photographed at random for each replicate. Yunnan white drug powder (product batch No. ZFA 1615) with the same concentration was used as drug control group. Calculating by adopting a formula: erythrocyte agglutination rate =100 × (1-a)t/Ac) Wherein A istFor the number of free erythrocytes in the test group,Acthe number of red blood cells in the blank control group.
The test results show that the mixture of the flavonoid sulfuric ester salt in example 1, the flavonoid-7-potassium sulfate and the flavonoid-7, 4' -dipotassium disulfate in example 2 have no agglutination effect on erythrocytes under the neutral condition without adding acid.
But has good agglutination activity to erythrocytes under the acidic condition after adding acid, wherein the agglutination rate of part of flavonoid compounds with potassium sulfate reaches more than 95% at the concentration of 0.2 mg/ml (see figure 1 and figure 2). In addition, the activity of agglutinating erythrocytes by sulfate ester salts of different flavonoid compounds is different, wherein the activity of agglutinating erythrocytes by a mixture of sulfate potassium salts of liquiritigenin and daidzein is weaker, and the agglutination rate to erythrocytes is less than 90% at 2.4 mg/ml, but the agglutination rate to erythrocytes is more than 90% at the concentration of 0.8 mg/ml by daidzein-7-sulfate potassium and daidzein-7, 4' -dipotassium disulfate. After the Yunnan white powder is added into the blood cell suspension, no hemagglutination is seen no matter whether the condition is acidic or not, and the red blood cells are rapidly broken.
Example 4
The flocculation of the flavonoid compound sulfate on fibrinogen under the acidic condition comprises the following test processes:
278 mg of medical fibrinogen product (Boya biopharmaceutical group, Inc., approved article: national drug standard S20013006) was weighed and dissolved in 58 ml of physiological saline. 10 μ l of flavonoid potassium sulfate sample (3.6, 7.2, 10.8, 14.4, 18 mg/ml, prepared with normal saline, mixed with fibrinogen, etc. to final concentrations of 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/ml, respectively, 10 μ l of normal saline for blank control group, Yunnan white drug of the same concentration as drug control group), 100 μ l of 0.003M tartaric acid solution (equal volume of normal saline for acid-free sample test group and blank control group) and 250 μ l of fibrinogen solution were added to a 1.5 ml centrifuge tube, heated in 36 deg.C water bath for 5 min, centrifuged for 1 min at 7817 g, and 50 μ l of supernatant was taken, 1.65 ml of brilliant blue developer was added, and absorbance was measured at 595 nm (adjusted to zero with corresponding reference).
Blank control group reference: 360 mul of physiological saline is added into a 1.5 ml centrifuge tube; heating in water bath at 36 deg.C for 5 min, centrifuging at 7817 g for 1 min, collecting 50 μ l, adding 1.65 ml Coomassie brilliant blue, and mixing to obtain blank control.
Reference with acid sample: adding 250 ul of normal saline, 10 ul of sample solution and 100 ul of tartaric acid solution into a 1.5 ml centrifuge tube, heating in a 36 ℃ water bath for 5 min, centrifuging for 1 min under the condition of 7817 g, taking 50 ul, adding 1.65 ml of Coomassie brilliant blue, and uniformly mixing to obtain an acid sample reference.
Acid-free sample reference: adding 350 μ l of physiological saline and 10 μ l of sample solution into a 1.5 ml centrifuge tube, heating in 36 deg.C water bath for 5 min, centrifuging at 7817 g for 1 min, collecting 50 μ l, adding 1.65 ml Coomassie brilliant blue developer, developing, and mixing well to obtain acid-free sample reference.
Calculation of fibrinogen flocculation rate: fibrinogen flocculation rate (%) =100 × (1-a)t/Ac) Wherein A istAnd Ac is the absorbance of the test group and the absorbance of the blank control.
Test results show that the tested sulfate salt mixture or monomer compound of the flavonoid compound has no flocculation on fibrinogen or has weak flocculation (generally less than 20%) under the neutral condition without adding acid; but under the acidic condition, the fibrinogen is extremely obviously flocculated, and a part of compounds can form flocculent precipitate (shown in figures 3 and 4) of more than 90 percent of the fibrinogen under the concentration of 0.3-0.5 mg/ml, even a large amount of flocculent appears after the sample, the acid solution and the fibrinogen are mixed.
Meanwhile, the flocculation of the synthetic products of different flavonoids compounds on fibrinogen is different, the flocculation of daidzein and liquiritigenin on fibrinogen is the weakest, and the flocculation rate is less than 80% at the concentration of 2.4 mg/ml; the same dose of tartaric acid and fibrinogen mixed alone did not cause flocculation of fibrinogen, even after heating in a water bath, which remained dissolved and did not change the protein content of the solution. This shows that the sulfate salt of flavonoid compound can effectively convert fibrinogen into insoluble protein under acidic condition, thereby promoting the anti-coagulation activity. The Yunnan white drug powder has no flocculation effect on fibrinogen no matter under neutral or acidic condition.
The tests show that the flavonoid sulfate can stop blood coagulation through at least 2 routes under the acidic condition, has great potential in the development of preparations for treating traumatic hemorrhage or gastrointestinal hemorrhage, and has direct procoagulant and hemostatic effects in the gastrointestinal tract due to the fact that the gastrointestinal tract is an acidic environment.
Combinations not exemplified above should be within the scope of the invention as would be apparent to one of skill in the art in view of the present disclosure.
Claims (8)
1. A method for synthesizing flavonoid sulfate is characterized by comprising the following steps:
1) uniformly mixing three substances according to the proportion of flavonoid compound dicyclohexylcarbodiimide to tetrabutylammonium hydrogen sulfate = 1: 1-20: 1-10 (molar ratio), adding a proper amount of cosolvent, reacting at 4-50 ℃ for 1-72 hours, then carrying out reduced pressure distillation at 45-60 ℃, fully removing the cosolvent, dissolving with a proper amount of methanol, and carrying out centrifugal filtration to obtain a methanol solution of flavonoid compound tetrabutylammonium sulfate;
2) removing methanol in the obtained solution, re-dissolving the product with a small amount of methanol, centrifuging, filtering, further removing a small amount of precipitate impurities, adding saturated solution of potassium acetate or sodium acetate methanol into the filtrate, reacting to generate flavonoid compound potassium sulfate and tetrabutylammonium acetate, dissolving tetrabutylammonium acetate in methanol, centrifuging, filtering, and washing the precipitate with methanol to obtain the product of flavonoid compound potassium sulfate or flavonoid compound sodium sulfate.
2. The method for synthesizing the flavonoid sulfuric acid ester salt according to the claim, wherein the cosolvent in the step 1) is pyridine.
3. The method for synthesizing the flavonoid sulfuric ester salt according to the claim, wherein the method for removing the methanol in the step 2) comprises the following steps: and removing the methanol by decompression and distillation at the temperature of 45-60 ℃.
4. The method for synthesizing the flavonoid sulfuric ester salt according to the claim, wherein the flavonoid sulfuric ester salt obtained in the step 2) is flavonoid sulfuric ester potassium (sodium).
5. The method for synthesizing the flavonoid compound sulfate salt according to the claim, wherein the conversion rate of the flavonoid compound sulfate salt obtained in the step 2) is 70-96%.
6. The application of flavonoid sulfate in the fields of hemostasis and blood coagulation under acidic conditions.
7. The use of the sulfate salts of flavonoids according to claim 6 in the fields of hemostasis and blood coagulation under acidic conditions, wherein the acidic conditions are one or more of tartaric acid, citric acid, oxalic acid, malic acid, succinic acid, malonic acid, ascorbic acid and maleic acid.
8. The use of the sulfate salts of flavonoids according to claim 6 under acidic conditions in the fields of hemostasis and coagulation, which is characterized in that the fields of hemostasis and coagulation include the fields of medicines, medical treatment, sanitary materials and medical devices.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB704516A (en) * | 1951-07-12 | 1954-02-24 | Roche Products Ltd | A blood anticoagulating agent and process for the manufacture thereof |
| WO2012068539A1 (en) * | 2010-11-19 | 2012-05-24 | Limerick Biopharma, Inc. | Use and composition of quercetin-3'-o-sulfate for therapeutic treatment |
| CN110934899A (en) * | 2019-12-20 | 2020-03-31 | 九江学院 | Extraction method and application of blood coagulation and hemostasis active ingredients of eclipta alba |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB704516A (en) * | 1951-07-12 | 1954-02-24 | Roche Products Ltd | A blood anticoagulating agent and process for the manufacture thereof |
| WO2012068539A1 (en) * | 2010-11-19 | 2012-05-24 | Limerick Biopharma, Inc. | Use and composition of quercetin-3'-o-sulfate for therapeutic treatment |
| CN110934899A (en) * | 2019-12-20 | 2020-03-31 | 九江学院 | Extraction method and application of blood coagulation and hemostasis active ingredients of eclipta alba |
Non-Patent Citations (2)
| Title |
|---|
| DENIS BARRON等: "Synthesis of flavonoid sulfates:1.Stepwise sulfation of positions 3,7, and 4\' using N,N\'-dicyclohexylcarbodiimide and tetrabutylammonium hydrogen sulfate" * |
| 彭游等: "槲皮素化学修饰与体内转运过程研究进展" * |
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