Preparation process of baicalein derivative

Abstract

The invention relates to the field of medicines, and provides a preparation process of a baicalein derivative, which comprises the following steps: step 1, preparation of baicalin: sequentially carrying out a baicalin extraction process, a baicalin purification process and a baicalin refining process to obtain refined baicalin; the method comprises the following steps: 1, preparing baicalein, 2, preparing baicalein to obtain refined baicalein, and 3, preparing baicalin phosphate to obtain a mixture of 5, 6, 7-trihydroxy sodium phosphate or sodium phosphate or sodium phosphate of 5, 6, 7-trihydroxy of baicalein. Baicalin is modified through a series of chemical and physical methods, the baicalin is structurally modified to change the physical and chemical properties of the baicalin, and the baicalin is prepared into sodium phosphate with higher fat solubility, so that the medicine effect is improved, the stability is improved, the solubility is improved, the absorption is promoted, and the oral bioavailability is improved; the pharmacological activity can be better exerted. Therefore, the compound with higher physiological activity and higher practical value can be obtained.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a preparation process of a baicalein derivative. Background Art

[0002] Baicalin is the main active ingredient extracted from the traditional Chinese medicine Scutellaria baicalensis. It is a flavonoid compound with a molecular formula of C21H18O11. It has antiviral, anti-inflammatory, anti-tumor and immunomodulatory effects. It is clinically used for acute, chronic persistent and chronic active hepatitis, and can also be used to treat nephritis, pyelonephritis, acute biliary infection, lead poisoning and allergic diseases. Baicalin has a very broad prospect as a clinical anti-thrombotic, antihypertensive, and treatment of cardiovascular and cerebrovascular diseases such as diabetes and coronary heart disease. However, on the other hand, its aglycone is affected by fat solubility, which affects its absorption and activity. Therefore, by modifying baicalin through some chemical and physical methods, the physical and chemical properties of baicalin can be changed, the efficacy can be improved, the stability can be increased, the solubility can be improved, and the absorption can be promoted, which is conducive to the further development and utilization of baicalin and the search for new drugs, and also to find new directions for the application of traditional Chinese medicine. Based on references to relevant domestic and foreign literature, this study modified the structure of baicalin and prepared it into a more lipid-soluble sodium phosphate salt in order to improve its oral bioavailability and better exert its pharmacological activity.

[0003] At present, baicalin is mainly administered in the form of Chinese medicine decoctions, etc. Baicalin is easily oxidized, thus affecting the efficacy of the drug. Therefore, researchers have developed new dosage forms such as baicalin oral tablets and dripping pills. Although these dosage forms have improved stability to a certain extent, due to the poor water solubility and fat solubility of baicalin, poor stability, poor oral absorption and low bioavailability, this greatly affects the clinical application and efficacy of these baicalin oral preparations. In addition, the half-life of baicalin is very short and the bioavailability is low, so it requires a large dosage and frequent administration. Therefore, baicalin is structurally modified by appropriate physical and chemical methods to improve the physicochemical properties of baicalin and improve its affinity and permeability with tissues, which is of great significance for the expanded application of baicalin. Therefore, a preparation process of baicalin derivatives is needed. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a preparation process of baicalin derivatives, by which baicalin is structurally modified by appropriate physical and chemical methods, the physicochemical properties of baicalin are improved, and its affinity and permeability to tissues are increased.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] A preparation process of baicalein derivatives comprises the following steps:

[0007] Step 1, preparation of baicalin: preferentially performing a baicalin extraction process, dissolving baicalin into a decoction by boiling in water, adjusting the pH of the extract, filtering, precipitating, washing with water, and drying to obtain a light yellow powder, taking the light yellow powder for a baicalin purification process, adding ethanol to the baicalin of the light yellow powder for reflux extraction, performing multiple pH adjustments, precipitation, and water bath heating on the water solution after evaporation of the ethanol, filtering, precipitating, washing with water, and drying to obtain a purified light yellow powder, taking the purified light yellow powder for a baicalin refining process, adding water to the baicalin of the light yellow powder and stirring evenly, sequentially performing pH adjustment, activated carbon adsorption, and ethanol dissolution, and then washing and drying the precipitated precipitate to obtain refined baicalin;

[0008] Step 2, preparation of baicalin: first, prepare crude enzyme powder of Scutellaria baicalensis, according to the mass ratio, take fresh Scutellaria baicalensis and pH 4-6 buffer solution, add a small amount of quartz sand, grind, centrifuge and filter to obtain crude enzyme solution of Scutellaria baicalensis, filter the crude enzyme solution, dialyze, and vacuum freeze-dry to obtain crude enzyme powder of Scutellaria baicalensis, dissolve the baicalin prepared in the step 1 in a low eutectic solvent, perform enzymatic hydrolysis of baicalin, add buffer solution with pH 4-6 and crude enzyme powder of Scutellaria baicalensis in sequence, mix well uniformly, heated to 40-50°C, kept warm and stirred for 2.0-4.0 hours, cooled to room temperature, filtered, and the precipitate was recovered. The filtrate can be used again for hydrolysis of baicalin, and the precipitate obtained above is dissolved in 95% ethanol, filtered, and the insoluble matter is discarded, and a baicalin purification process is performed, 5-6 times the amount of deionized water is added to the filtrate, stirred evenly, allowed to stand, baicalin is precipitated, filtered to obtain a precipitate, and the precipitate is washed with deionized water, filtered, and dried to obtain refined baicalin;

[0009] Step 3, preparation of baicalin phosphate: 9 g of baicalin was dissolved in 50 mL of pyridine, cooled with ice water, and phosphorus oxychloride was slowly added under stirring for reaction, and then added to 10-20 mL of water, pyridine and water were evaporated under reduced pressure, 300-400 mL of 1 mol / L HCL was added to the residue, the precipitate was separated and washed with water, and then suspended in a small amount of water, and the pH was adjusted to 7 with 1 mol / L NAOH, the insoluble matter was filtered out, washed with water, the filtrates were combined, concentrated to 15 ml, and then 300 mL of ethanol was added to obtain a yellow precipitate, which was separated and dried to obtain 2 g of the product, which is a mixture of 5,6,7-trihydroxy mono-, di- or tri-phosphate sodium salts of baicalin;

[0010] Step 4, performing structural identification of baicalin phosphate;

[0011] Step 5: obtaining baicalin phosphate derived from baicalein.

[0012] Preferably, the specific steps of the baicalin extraction process in step 1 are:

[0013] S1. Take 50g of Scutellaria baicalensis, add ten times the amount of water for the first time and boil for one hour, then add eight times the amount of water for the second time and boil for one hour, stir with a glass rod while boiling to accelerate dissolution, and combine the two decoctions after heating;

[0014] S2. Adjust the pH to 1.5-2.0 with hydrochloric acid, heat in a water bath to 80°C, keep warm for half an hour and then let stand for 24 hours, filter, add appropriate amount of water to the precipitate and stir, adjust the pH to 7.0 with 40% NaOH,

[0015] S3, add an equal amount of ethanol to dissolve and filter, and adjust the pH of the filtrate to 1.5-2.0 with hydrochloric acid for the second acid precipitation

[0016] S4. Heat the extract to 60°C in a water bath, keep warm for half an hour, let it stand, filter, wash the precipitate with appropriate amount of water, and dry to obtain a light yellow powder.

[0017] Preferably, the specific steps of the baicalin purification process in step 1 are:

[0018] S1. Take 50 g of Scutellaria baicalensis, reflux with 60% ethanol for two hours and extract twice, filter, and recover ethanol by rotary evaporation of the filtrate until there is no alcohol taste;

[0019] S2, the water solution is heated to 80°C in a water bath, the pH is adjusted to 1.5-2.0 with hydrochloric acid, the temperature is kept for 30 minutes, the solution is allowed to stand at room temperature for 24 hours, and the precipitate is centrifuged;

[0020] S3. Add ten times the amount of water to the precipitate and stir to form a suspension. Then adjust the pH to 7.0 with 20% NOH, centrifuge it and take the supernatant and adjust the pH to 1.5-2.0 with hydrochloric acid.

[0021] S4. Continue heating in a water bath to 80°C for 30 minutes, place at room temperature, filter, wash the precipitate with appropriate amount of water, and dry to obtain a light yellow powder.

[0022] Preferably, the specific steps of the baicalin refining process in step 1 are:

[0023] S1. Weigh crude baicalin, add 10 times the amount of water and stir evenly, add 20% NaOH to adjust the pH to 6.0-7.0;

[0024] S2. After all baicalin is dissolved, add an appropriate amount of activated carbon and mix well. Heat to 80℃ in a water bath as above for 30 minutes.

[0025] S3, remove the activated carbon by suction filtration, and adjust the pH of the filtrate to 1.5-2.0 with concentrated hydrochloric acid;

[0026] S4. Add an equal volume of 95% ethanol, keep warm in a 50°C water bath for 30 minutes, precipitate, leave overnight, filter, wash the precipitate with a small amount of ethanol, drain, and dry below 60°C to obtain refined baicalin.

[0027] Preferably, in step 2, the mass ratio of fresh Scutellaria baicalensis to buffer solution is 1:1.5-2.5.

[0028] Preferably, the baicalin content used in step 2 is ≥80%.

[0029] Preferably, in step 2, the mass ratio of baicalin, low eutectic solvent, buffer and crude scutellaria baicalenzyme powder is 1:40-60:150-250:0.025-0.1.

[0030] Preferably, the number of times of particle removal water washing in step 2 is at least 3 times.

[0031] Preferably, the amount of phosphorus oxychloride added in step three is 3.5 ml.

[0032] Preferably, the reaction time after adding phosphorus oxychloride is 2 hours.

[0033] The present invention provides a preparation process of baicalein derivatives, which has the following beneficial effects:

[0034] 1. The present invention modifies baicalin through a series of chemical and physical methods, modifies the structure of baicalin, changes the physical and chemical properties of baicalin, and prepares it into a more fat-soluble sodium phosphate salt, in order to improve the efficacy, increase stability, improve solubility, promote absorption, increase its oral bioavailability, and better exert its pharmacological activity. This helps to obtain compounds with stronger physiological activity and greater practical value, enables the further development and utilization of baicalin and the search for new drugs, and also finds new directions for the application of traditional Chinese medicine.

[0035] 2. In the present invention, the baicalin derivatives prepared by this process have different degrees of antibacterial effects on a variety of common bacteria in vitro, and can effectively inhibit the growth of bacteria in vitro.

[0036] 3. In the present invention, the baicalin derivatives prepared by the process can effectively inhibit the growth of bacteria on the premise of antibacterial effects on a variety of common bacteria in vivo, greatly reducing the harm of bacteria to the body, thereby ensuring the effectiveness of the baicalin derivatives. DETAILED DESCRIPTION

[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0038] Example:

[0039] The embodiment of the present invention provides a preparation process of a baicalein derivative, comprising the following steps:

[0040] Step 1, preparation of baicalin: the baicalin extraction process is preferentially performed, the baicalin is dissolved in water to make a decoction, the extract is pH adjusted, filtered, precipitated, washed, and dried to obtain a light yellow powder, wherein the specific steps of the baicalin extraction process are as follows:

[0041] S1. Take 50g of Scutellaria baicalensis, add ten times the amount of water for the first time and boil for one hour, then add eight times the amount of water for the second time and boil for one hour, stir with a glass rod while boiling to accelerate dissolution, and combine the two decoctions after heating;

[0042] S2. Adjust the pH to 1.5-2.0 with hydrochloric acid, heat in a water bath to 80°C, keep warm for half an hour and then let stand for 24 hours, filter, add appropriate amount of water to the precipitate and stir, adjust the pH to 7.0 with 40% NaOH,

[0043] S3, add an equal amount of ethanol to dissolve and filter, and adjust the pH of the filtrate to 1.5-2.0 with hydrochloric acid for the second acid precipitation

[0044] S4. Heat the extract to 60°C in a water bath, keep warm for half an hour, let it stand, filter, wash the precipitate with appropriate amount of water, and dry to obtain a light yellow powder.

[0045] The light yellow powder is taken for baicalin purification process, the light yellow powder baicalin is added with ethanol for reflux extraction, the water solution after evaporation of ethanol is subjected to multiple pH adjustment, precipitation and water bath heating, and then filtered, precipitated, washed and dried to obtain the purified light yellow powder, wherein the specific steps of the baicalin purification process are:

[0046] S1. Take 50 g of Scutellaria baicalensis, reflux with 60% ethanol for two hours and extract twice, filter, and recover ethanol by rotary evaporation of the filtrate until there is no alcohol taste;

[0047] S2, the water solution is heated to 80°C in a water bath, the pH is adjusted to 1.5-2.0 with hydrochloric acid, the temperature is kept for 30 minutes, the solution is allowed to stand at room temperature for 24 hours, and the precipitate is centrifuged;

[0048] S3. Add ten times the amount of water to the precipitate and stir to form a suspension. Then adjust the pH to 7.0 with 20% NOH, centrifuge it and take the supernatant and adjust the pH to 1.5-2.0 with hydrochloric acid.

[0049] S4. Continue heating in a water bath to 80°C for 30 minutes, place at room temperature, filter, wash the precipitate with appropriate amount of water, and dry to obtain a light yellow powder.

[0050] The purified light yellow powder is subjected to a baicalin refining process, wherein the light yellow powder of baicalin is added with water and stirred evenly, and pH adjustment, activated carbon adsorption and ethanol dissolution are performed in sequence, and then the precipitated precipitate is washed and dried to obtain refined baicalin, wherein the specific steps of the baicalin refining process are as follows:

[0051] S1. Weigh crude baicalin, add 10 times the amount of water and stir evenly, add 20% NaOH to adjust the pH to 6.0-7.0;

[0052] S2. After all baicalin is dissolved, add an appropriate amount of activated carbon and mix well. Heat to 80℃ in a water bath as above for 30 minutes.

[0053] S3, remove the activated carbon by suction filtration, and adjust the pH of the filtrate to 1.5-2.0 with concentrated hydrochloric acid;

[0054] S4. Add an equal volume of 95% ethanol, keep warm in a 50°C water bath for 30 minutes, precipitate, leave overnight, filter, wash the precipitate with a small amount of ethanol, drain, and dry below 60°C to obtain refined baicalin.

[0055] The solubility of baicalin in methanol and ethanol is 1.569 mg / ml and 1.458 mg / ml respectively. The solubility of baicalin in ethanol is lower than that in methanol, so ethanol is selected as the solvent for recrystallization.

[0056] Step 2, preparation of baicalin: first, prepare crude enzyme powder of Scutellaria baicalensis, take fresh Scutellaria baicalensis and buffer solution with a pH of 4 to 6 at a mass ratio of 1:1.5 to 2.5, add a small amount of quartz sand, grind, centrifuge and filter to obtain crude enzyme solution of Scutellaria baicalensis, filter the crude enzyme solution, dialyze, and vacuum freeze-dry to obtain crude enzyme powder of Scutellaria baicalensis, dissolve the baicalin with a content of at least 80% prepared in step 1 in a low eutectic solvent, prepare enzymatic hydrolysis of baicalin, add buffer solution with a pH of 4 to 6 and crude enzyme powder of Scutellaria baicalensis in sequence, mix well, heat to 40 to 50°C, keep warm and stir for 2.0 ~ 4.0 hours, cooled to room temperature, filtered, and the precipitate was recovered. The filtrate can be used again for hydrolysis of baicalin, wherein the mass ratio of baicalin, low eutectic solvent, buffer, and scutellaria crude enzyme powder is 1:40~60:150~250:0.025~0.1, and the precipitate obtained above is dissolved in 95% ethanol, filtered, and the insoluble matter is discarded, and a baicalin purification process is performed, 5 to 6 times the amount of deionized water is added to the filtrate, stirred evenly, and allowed to stand to precipitate baicalin, and the precipitate is filtered to obtain the precipitate, and the precipitate is washed with deionized water at least 3 times, filtered, and dried to obtain refined baicalin;

[0057] Step 3, preparation of baicalin phosphate: 9 g of baicalin was dissolved in 50 mL of pyridine, cooled with ice water, and 3.5 mL of phosphorus oxychloride was slowly added under stirring to react for 2 h, and then added to 10-20 mL of water, pyridine and water were evaporated under reduced pressure, 300-400 mL of 1 mol / L HCL was added to the residue, the precipitate was separated and washed with water, and then suspended in a small amount of water, and the pH was adjusted to 7 with 1 mol / L NAOH, the insoluble matter was filtered out, washed with water, the filtrates were combined, concentrated to 15 ml, and then 300 mL of ethanol was added to obtain a yellow precipitate, which was separated and dried to obtain 2 g of the product, which is a mixture of 5,6,7-trihydroxy mono-, di- or tri-phosphate sodium salts of baicalin;

[0058] Step 4: Structural identification of baicalin phosphate:

[0059] UV: There should be strong absorption at wavelengths of 274mm and 324m.

[0060] IR (cm-): 3450, 2950, ​​2650, (boad) 1663, 1624, 1590, 1450, 1380, 165, 11192, 931, 765, 681.

[0061] MS detection:

[0062] Chromatographic conditions:

[0063] Chromatographic column: YMC-Triart C18 (100mm×2.0mm, 1.9m): Mobile phase: methanol-water (containing 0.1% formic acid), gradient 010min:15%100%, 12-12.1min:100-15%, 14min:15%, flow rate 220uL+min1, column temperature 45℃, injection volume: 8uL.

[0064] Mass spectrometry conditions:

[0065] Ion Source:ESI, Capillary Temperature:350C, Vaporizer Temperature:250C, Sheath Gas Pressure:206.85KPa, Aux Gas Pressure:4.5L'min, Spray Voltage:3.5kV, Posi-tive Polarity, Monitoring Mode: Selected Reaction Monitoring (SRM), Monitoring Ion:mz463.000→m / 2286.944, Collision Energy:27eV;

[0066] Step 5: obtaining baicalin phosphate derived from baicalein.

[0067] The baicalein derivative prepared by the preparation process of a baicalein derivative in the above embodiment was used for the experiment:

[0068] Experimental Example 1:

[0069] Objective: To observe and evaluate the antibacterial effect of baicalein phosphate in vitro.

[0070] Methods: Eight representative bacterial species were selected in this experiment, mainly common pathogens and conditional pathogens of respiratory tract infections, including Staphylococcus aureus (26112), beta-hemolytic Streptococcus (32210), Escherichia coli (44113), Pseudomonas aeruginosa (10211), Proteus, Klebsiella pneumoniae (ATCC25922), alpha-hemolytic Streptococcus (32209), and Streptococcus pneumoniae (31001). The broth dilution method was used to detect the minimum inhibitory concentration (MIC) of baicalein phosphate against the test bacteria with turbidity as an indicator.

[0071] Results: Baicalein phosphate had different degrees of antibacterial activity against Staphylococcus aureus, beta-hemolytic Streptococcus, alpha-hemolytic Streptococcus, Streptococcus pneumoniae, and Pseudomonas aeruginosa, with MICs of 0.0125, 0.05, 0.1, 0.05, and 0.4 g / ml, respectively. However, it showed no antibacterial activity against Escherichia coli, Proteus, and Klebsiella pneumoniae within a concentration of 0.4 g / ml.

[0072] Conclusion: Baicalein phosphate has certain antibacterial effect in vitro.

[0073] 1. Purpose of the test

[0074] To observe and evaluate the antibacterial effect of baicalein phosphate in vitro.

[0075] 2. Test drug

[0076] 2.1 Usage and Dosage of the Test Drug

[0077] 2.1.1 Test drugs

[0078] Baicalein phosphate powder, provided by the Pharmacy Department of Xi'an Traditional Chinese Medicine Hospital, production batch number: 210501. Purity: 86.2%.

[0079] 2.1.2 Clinical Usage and Dosage: Daily oral dose of Scutellaria baicalensis: 100g per day, twice a day. Each gram of Scutellaria baicalensis contains about 0.09g of baicalin. For an adult with a body weight of 60kg, 50g / time, 2 times / day, the clinical daily dose is 0.09g baicalin × 50g / time × 2 times / day ÷ 60kg = 0.15g / kg body weight.

[0080] 2.2 Sterilization drug concentration design

[0081] During the experiment, baicalein phosphate was prepared in nutrient broth to prepare 9 drug concentrations per ml, including 0.4g, 0.2g, 0.1g, 0.05g, 0.025g, 0.0125g, 6.25×10-3, 3.125×10-3, and 1.56×10-3. The total volume in each tube was 1 ml and sterilized by circulating steam.

[0082] 3. Test strains and culture medium

[0083] Bacterial species: Staphylococcus aureus (26112), beta-hemolytic Streptococcus (32204), Escherichia coli (44113), Pseudomonas aeruginosa (10211), Klebsiella pneumoniae (ATCC25922), alpha-hemolytic Streptococcus (32209), Streptococcus pneumoniae (31001), Proteus was isolated from intestinal specimens by the bacteriological laboratory, and the remaining bacterial species were purchased from the China Pharmaceutical and Biological Products Research Institute, Beijing.

[0084] Culture medium: nutrient broth medium, 1% glucose broth medium, 10% rabbit serum broth medium, the above nutrient media are routinely prepared by the laboratory.

[0085] 4. Main instruments

[0086] 1900 type sterile operating table; ultra-low temperature refrigerator; biochemical incubator; high-pressure steam sterilizer; electric constant temperature drying oven; electric furnace, inoculation loop, alcohol lamp, test tube, gauze and other conventional experimental instruments and equipment.

[0087] 5. Experimental methods

[0088] 5.1 Preparation of drug solution

[0089] Take 3g of baicalein phosphate powder, add distilled water to 25ml, stir and mix well to make baicalein phosphate solution.

[0090] 5.2 Preparation of test bacterial solution

[0091] The various test bacteria were subcultured on nutrient agar slants at 37°C for 24 h. One loop of each bacterium was picked out for further enrichment in broth and cultured at 37°C for 6 h. Then, the fresh culture of each test bacteria was made into a bacterial suspension with a concentration equivalent to 5×106 cfu·ml.

[0092] 5.3 Determination of MIC (broth two-fold dilution method)

[0093] 5.3.1 Preparation of sterilization solution

[0094] The drug solution was prepared with nutrient broth to a total of 9 drug concentrations (numbered 1-9 tubes), 1 ml per tube, and sterilized by circulating steam. For the test on Streptococcus, 1% glucose was added to the sterilized drug solution, and for Pneumococcus, 10% rabbit serum was added. The strain control was a medium without drug plus the test bacteria, and the drug control was a drug solution without the test bacteria.

[0095] 5.3.2 Adding bacteria and culturing

[0096] 0.1 ml of 5×106 cfu·ml test bacterial solution was added to each concentration tube of baicalein phosphate solution and the bacterial control tube, and cultured in a constant temperature incubator at 37°C for 24 h.

[0097] 5.3.3 Results and observations

[0098] The presence or absence of bacterial growth in each group was observed with the naked eye using turbidity as an indicator, and the lowest concentration without bacterial growth was taken as the minimum inhibitory concentration (MIC).

[0099] 6. Experimental results

[0100] Table 1. In vitro inhibitory effects of baicalein phosphate on various bacteria

[0101]

[0102] Note: The bacterial strain control showed normal growth of each bacterium; the drug control showed sterile growth.

[0103] The results in Table 1 show that baicalein phosphate is not sensitive to Escherichia coli, Proteus, and Klebsiella pneumoniae, with MIC>0.4g / ml, but has different degrees of antibacterial effect on other bacteria, with the minimum inhibitory concentration ranging from 0.0125 to 0.4g / ml. The MICs for Staphylococcus aureus, beta-hemolytic Streptococcus, alpha-hemolytic Streptococcus, Streptococcus pneumoniae, and Pseudomonas aeruginosa are 0.0125g / ml, 0.05g / ml, 0.1g / ml, 0.05g / ml, and 0.4g / ml, respectively.

[0104] 8. Conclusion

[0105] The broth dilution method was used to observe the antibacterial effect of baicalein phosphate in vitro. The results showed that baicalein phosphate had different degrees of antibacterial effect on Staphylococcus aureus, beta-hemolytic Streptococcus, alpha-hemolytic Streptococcus, Streptococcus pneumoniae, and Pseudomonas aeruginosa.

[0106] Experimental Example 2:

[0107] Experimental purpose: To study the inhibitory effect of baicalein phosphate on Staphylococcus aureus, beta-hemolytic Streptococcus, and Pseudomonas aeruginosa in mice, and to evaluate the antibacterial effect of baicalein phosphate in vivo.

[0108] Methods: Amoxicillin was used as the positive drug, and the in vivo infection model was established by intraperitoneal injection of the strain into KM mice. The mice were observed for 3 days after administration, and the mortality rate was recorded and the differences among the groups were compared.

[0109] Results: Compared with the blank control group, there was a significant difference in the amoxicillin group (P<0.05), and there was a significant difference in the high-dose baicalein phosphate group (P<0.05); there was no significant difference in the number of mouse deaths between the medium-dose and low-dose baicalein phosphate groups (P>0.05).

[0110] Conclusion: Baicalein phosphate has different degrees of inhibitory effect on Staphylococcus aureus, beta-hemolytic Streptococcus, and Pseudomonas aeruginosa strains in mice

[0111] 1. Purpose of the test

[0112] To study the inhibitory effect of baicalein phosphate on Staphylococcus aureus, beta-hemolytic Streptococcus, and Pseudomonas aeruginosa in mice and to evaluate the antibacterial effect of baicalein phosphate in vivo.

[0113] 2. Test drug

[0114] 2.1.1 Test drugs

[0115] Baicalein phosphate powder, provided by the Pharmacy Department of Xi'an Traditional Chinese Medicine Hospital, production batch number: 210501. Purity: 86.2%.

[0116] 2.1.2 Clinical Usage and Dosage: Daily oral dose of Scutellaria baicalensis: 100g per day, twice a day. Each gram of Scutellaria baicalensis contains about 0.09g of baicalin. For an adult with a body weight of 60kg, 50g / time, 2 times / day, the clinical daily dose is 0.09g baicalin × 50g / time × 2 times / day ÷ 60kg = 0.15g / kg body weight.

[0117] 2.2 Experimental dosage design

[0118] The experiment designed three dose groups of baicalein phosphate, high, medium and low. The dosage was calculated by body weight, which was 40, 20 and 10 times the clinical dosage, equivalent to 6g / kg, 3g / kg and 1.5g / kg of crude drug, respectively. The administration volume was 10ml / kg body weight, and the drug concentrations in the high, medium and low dose groups were 0.6g / ml, 0.3g / ml and 0.15g / ml, respectively, based on the weight of crude drug.

[0119] 3. Experimental control and positive drug selection

[0120] 3.1 Set up a blank control group

[0121] An equal volume of normal saline was administered by gavage.

[0122] 3.2 Positive Control

[0123] 3.2.1 Positive drugs

[0124] Amoxicillin Capsules: Specification: 0.25g.

[0125] Dosage and Administration: Adults take 0.5g once, once every 6 to 8 hours, and the daily dose should not exceed 4g. That is, the daily dose is: 0.5g / time ╳ 4 times / day / 60kg = 0.033g / kg / day.

[0126] 3.2.2 Selection of positive drugs

[0127] Amoxicillin Capsules: Amoxicillin is a penicillin antibiotic that has good antibacterial activity against Streptococcus such as Streptococcus pneumoniae and hemolytic Streptococcus, aerobic Gram-positive cocci such as non-penicillinase-producing Staphylococci and Enterococcus faecalis, aerobic Gram-negative bacteria such as Escherichia coli, Proteus mirabilis, Salmonella, Haemophilus influenzae, Neisseria gonorrhoeae, and Helicobacter pylori. Amoxicillin exerts its bactericidal effect by inhibiting the synthesis of bacterial cell walls, which can quickly dissolve and rupture bacteria into spherical bodies.

[0128] 3.3 Experimental dose

[0129] The dosage for mice was designed to be 10 times the clinical dosage, which was 0.33 g / kg of the drug prepared with distilled water to 0.033 g / ml, and the dosage volume was 10 ml / kg body weight.

[0130] 4. Experimental Animals

[0131] SPF grade Kunming mice, weighing 18-22g, half male and half female.

[0132] 5. Main reagents and materials

[0133] Staphylococcus aureus (26112) (provided by the Institute of Biological Products, Ministry of Health, Beijing);

[0134] Beta-hemolytic Streptococcus (32204) (provided by the Institute of Biological Products, Ministry of Health, Beijing);

[0135] Pseudomonas aeruginosa (10211); (provided by the Institute of Biological Products, Ministry of Health, Beijing);

[0136] 0.9% Sterile Sodium Chloride Injection.

[0137] 6. Main instruments

[0138] Electronic balance; 1 / 10,000 analytical balance

[0139] 7. Methods and Results

[0140] 7.1 Inhibitory effect of baicalein phosphate on Staphylococcus aureus, beta-hemolytic Streptococcus, and Pseudomonas aeruginosa in mice

[0141] 7.1.1 Preliminary experiment

[0142] (1) Staphylococcus aureus infection concentration

[0143] The preserved Staphylococcus aureus was inoculated into the solid culture medium, and then inoculated again in the same way after culturing in a constant temperature box for 24 hours. Then, the bacteria were peeled off with sterile saline on a sterile operating table, and the concentration of the bacterial solution was adjusted to 2×109 / ml and 3×109 / ml by turbidimetry. 5% dry yeast solution was added to the bacterial solution, and 0.1 ml of dry yeast solution was added to 1 ml of the bacterial solution.

[0144] Thirty-five healthy Kunming mice weighing 18-22g were randomly divided into seven groups, namely, 2×109 / ml-0.2ml / mouse, 2×109 / ml-0.4ml / mouse, 2×109 / ml-0.6ml / mouse, 3×109 / ml-0.2ml / mouse, 3×109 / ml-0.4ml / mouse, 3×109 / ml-0.6ml / mouse of Staphylococcus aureus, and a 5% dry yeast saline control group. There were 5 mice in each group, regardless of gender. Each mouse was intraperitoneally injected with diluted bacterial solution and 5% dry yeast saline, and the mortality rate of the mice was observed after 3 days. The dilution concentration with an infection mortality rate of 80% to 100% was selected as the infection bacterial solution for the formal experiment.

[0145] There was no animal death in the 5% dry yeast saline control group, and the mortality rate of mice in the 2×109 / ml-0.4ml / mouse group was 80%. 2×109 / ml-0.4ml / mouse was determined to be the dosage of Staphylococcus aureus liquid for infection in this experiment.

[0146] (2) Concentration of beta-hemolytic streptococcal infection

[0147] The preserved beta-hemolytic streptococci were cultured in a solid culture medium in a constant temperature incubator for 24 hours and then inoculated again using the same method. The bacteria were then peeled off with sterile saline on a sterile operating table and the concentration of the bacterial solution was adjusted to 2×109 / ml by turbidimetry. 5% dry yeast solution was added to the bacterial solution, and 0.1 ml of dry yeast solution was added to 1 ml of the bacterial solution.

[0148] Thirty healthy Kunming mice weighing 18-22g were randomly divided into 6 groups, namely, 2×109 / ml-0.1ml / mouse, 2×109 / ml-0.2ml / mouse, 2×109 / ml-0.4ml / mouse, 1×109 / ml-0.1ml / mouse, 5×108 / ml-0.1ml / mouse, and a 5% dry yeast saline control group. There were 5 mice in each group, regardless of gender. Each mouse was intraperitoneally injected with diluted bacterial solution and 5% dry yeast saline, and the mortality rate of the mice was observed after 3 days. The dilution concentration with an infection mortality rate of 80% to 100% was selected as the infection bacterial solution for the formal experiment.

[0149] No animals died in the 5% dry yeast saline control group, and the mortality rate of mice in the 2×109 / ml-0.2ml / mouse group was 80%. 2×109 / ml-0.2ml / mouse was determined to be the bacterial liquid dosage for infection in this experiment.

[0150] (3) Pseudomonas aeruginosa infection concentration

[0151] The preserved Pseudomonas aeruginosa was cultured in a solid culture medium in a constant temperature incubator for 24 hours and then inoculated again using the same method. The bacteria were then peeled off with sterile saline on a sterile operating table and the concentration of the bacterial solution was adjusted to 2×108 / ml and 3×108 / ml by turbidimetry. 5% dry yeast solution was added to the bacterial solution, and 0.1 ml of dry yeast solution was added to 1 ml of the bacterial solution.

[0152] Thirty-five healthy Kunming mice weighing 18-22g were randomly divided into seven groups, namely, 3×108 / ml-0.2ml / mouse, 2×108 / ml-0.2ml / mouse, 1×108 / ml-0.2ml / mouse, 1 / 2×108 / ml-0.2ml / mouse, 1 / 4×108 / ml-0.2ml / mouse, 1 / 8×108 / ml-0.2ml / mouse of Pseudomonas aeruginosa, and a 5% dry yeast saline control group. Each group had 5 mice, regardless of gender. Each mouse was intraperitoneally injected with diluted bacterial solution and 5% dry yeast saline, and the mortality rate of the mice was observed after 3 days, and the dilution concentration with an infection mortality rate of 80% to 100% was selected as the infection bacterial solution for the formal experiment.

[0153] No animals died in the 5% dry yeast saline control group, and the mortality rate of mice in the 3×108ml-0.2ml / mouse group was 80%. 3×108ml-0.2ml / mouse was determined to be the bacterial liquid dosage for infection in this experiment.

[0154] 7.1.2 Formal Experiment

[0155] 75 healthy Kunming mice, weighing 18-22g, half male and half female, were randomly divided into a blank group, an amoxicillin positive control group, and high, medium, and low doses of baicalein phosphate groups, with 15 mice in each group. The mice were given oral administration for 1 day in advance, once in the morning and once in the afternoon. On the second day, the bacterial solution concentration (2×109 / ml) selected in the pre-experiment was taken for the experiment, and 0.4mL / mouse was injected intraperitoneally into each group of mice. The mice were given oral administration once at 1 and 6 hours after the injection. The blank control group was given an equal volume of distilled water by oral administration. The mice were observed for 3 consecutive days, and the number of deaths of mice was recorded daily, and the mortality rate of mice in each group was calculated.

[0156] 7.2 Statistical methods: SPSS statistical software was used for data processing, and the chi-square test was used to compare the differences between the groups.

[0157] 7.3 Results

[0158] Table 1 Effect of baicalein phosphate on the inhibition of Staphylococcus aureus in mice

[0159]

[0160] Note: Compared with the blank control group, *p<0.05, **p<0.01; compared with the amoxicillin group, #p<0.05, ##p<0.01.

[0161] Table 2 Effect of baicalein phosphate on the inhibition of beta-hemolytic streptococci in mice

[0162]

[0163]

[0164] Note: Compared with the blank control group, *P<0.05, **P<0.01; compared with the amoxicillin group, #P<0.05, ##P<0.01.

[0165] Table 3 Effect of baicalein phosphate on the inhibition of Pseudomonas aeruginosa in mice

[0166]

[0167] Note: Compared with the blank control group, *P<0.05, **P<0.01; compared with the amoxicillin group, #P<0.05, ##P<0.01.

[0168] The results show:

[0169] Compared with the blank control group, amoxicillin can significantly inhibit the death of mice infected with Staphylococcus aureus (P<0.05), indicating that the model was successfully established and the experimental method was feasible. Compared with the blank control group, high-dose baicalein phosphate can significantly inhibit the death of mice infected with Staphylococcus aureus (P<0.05), and there is no significant difference between the medium and low-dose groups (P>0.05). Compared with the amoxicillin group, there is a significant difference between the medium and low-dose groups of baicalein phosphate (P<0.05); there is no significant difference in the high-dose group (P>0.05), indicating that its protective effect on the death of mice caused by Staphylococcus aureus is equivalent to that of amoxicillin.

[0170] Compared with the blank control group, amoxicillin can significantly inhibit the death of mice infected with beta-hemolytic streptococcus (P<0.01), indicating that the model was successfully established and the experimental method was feasible. Compared with the blank control group, all doses of baicalein phosphate groups can significantly inhibit the death of mice infected with beta-hemolytic streptococcus (P<0.01). Compared with the amoxicillin group, there was no significant difference in the high, medium and low dose groups of baicalein phosphate (P>0.05), indicating that its protective effect on the death of mice caused by beta-hemolytic streptococcus is equivalent to that of the amoxicillin group.

[0171] Compared with the blank control group, amoxicillin can significantly inhibit the death of mice infected with Pseudomonas aeruginosa (P<0.01), indicating that the model was successfully established and the experimental method was feasible. Compared with the blank control group, the high dose of baicalein phosphate can significantly inhibit the death of mice infected with Pseudomonas aeruginosa (P<0.01), and the medium dose of baicalein phosphate significantly inhibits the death of mice infected with Pseudomonas aeruginosa (P<0.05). The number of deaths of mice in the low-dose group was reduced compared with the blank control group, but there was no significant difference (P>0.05). Compared with the amoxicillin group, there was no significant difference in each dose group of baicalein phosphate (P>0.05), indicating that it has a significant protective effect on the death of mice caused by Pseudomonas aeruginosa.

[0172] 8. Conclusion

[0173] 8.1 The strain-infected mouse model was used. Compared with the blank control group, amoxicillin could significantly inhibit the death of infected mice (P<0.05), indicating that the model was successful and the experimental method was feasible.

[0174] 8.2 High doses of baicalin phosphate have a significant protective effect against the death of mice caused by Staphylococcus aureus, beta-hemolytic Streptococcus, and Pseudomonas aeruginosa.

[0175] The results obtained from the above two experimental examples prove that the baicalin derivatives prepared by this preparation method have excellent properties in various aspects. This preparation method modifies baicalin through a series of chemical and physical methods, modifies the structure of baicalin to change the physical and chemical properties of baicalin, and prepares it into a more fat-soluble sodium phosphate salt, in order to improve the efficacy, increase stability, improve solubility, promote absorption, improve its oral bioavailability, and better exert its pharmacological activity. This helps to obtain compounds with stronger physiological activity and greater practical value, so that baicalin can be further developed and utilized and new drugs can be found, and it also finds new directions for the application of traditional Chinese medicine.

[0176] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for preparing a baicalein derivative, characterized in that: The following steps are involved: Step 1, preparation of baicalin: preferentially performing a baicalin extraction process, dissolving baicalin into a decoction by boiling in water, adjusting the pH of the extract, filtering, precipitating, washing with water, and drying to obtain a light yellow powder, taking the light yellow powder for a baicalin purification process, adding ethanol to the baicalin of the light yellow powder for reflux extraction, performing multiple pH adjustments, precipitation, and water bath heating on the water solution after evaporation of the ethanol, filtering, precipitating, washing with water, and drying to obtain a purified light yellow powder, taking the purified light yellow powder for a baicalin refining process, adding water to the baicalin of the light yellow powder and stirring evenly, sequentially performing pH adjustment, activated carbon adsorption, and ethanol dissolution, and then washing and drying the precipitated precipitate to obtain refined baicalin; Step 2, preparation of baicalin: first, prepare crude enzyme powder of Scutellaria baicalensis, according to the mass ratio, take fresh Scutellaria baicalensis and pH 4-6 buffer solution, add a small amount of quartz sand, grind, centrifuge and filter to obtain crude enzyme solution of Scutellaria baicalensis, filter the crude enzyme solution, dialyze, and vacuum freeze-dry to obtain crude enzyme powder of Scutellaria baicalensis, dissolve the baicalin prepared in the step 1 in a low eutectic solvent, perform enzymatic hydrolysis of baicalin, add buffer solution with pH 4-6 and crude enzyme powder of Scutellaria baicalensis in sequence, mix well uniformly, heated to 40-50°C, kept warm and stirred for 2.0-4.0 hours, cooled to room temperature, filtered, and the precipitate was recovered. The filtrate can be used again for hydrolysis of baicalin, and the precipitate obtained above is dissolved in 95% ethanol, filtered, and the insoluble matter is discarded, and a baicalin purification process is performed, 5-6 times the amount of deionized water is added to the filtrate, stirred evenly, allowed to stand, baicalin is precipitated, filtered to obtain a precipitate, and the precipitate is washed with deionized water, filtered, and dried to obtain refined baicalin; Step 3, preparation of baicalin phosphate: 9 g of baicalin was dissolved in 50 mL of pyridine, cooled with ice water, and phosphorus oxychloride was slowly added under stirring for reaction, and then added to 10-20 mL of water, pyridine and water were evaporated under reduced pressure, 1 mol / L HCL300-400 mL was added to the residue, the precipitate was separated and washed with water, and then suspended in a small amount of water, and the pH was adjusted to 7 with 1 mol / L NAOH, the insoluble matter was filtered out, washed with water, the filtrate was combined, concentrated to 15 ml, and then 300 mL of ethanol was added to obtain a yellow precipitate, which was separated and dried to obtain 2 g of the product, which is a mixture of 5,6,7-trihydroxy mono-, di- or tri-phosphate sodium salts of baicalin; Step 4, performing structural identification of baicalin phosphate; Step 5: obtaining baicalin phosphate derived from baicalein.

2. The preparation process of a baicalein derivative according to claim 1, characterized in that: The specific steps of the baicalin extraction process in step 1 are: S1. Take 50g of Scutellaria baicalensis, add ten times the amount of water for the first time and boil for one hour, then add eight times the amount of water for the second time and boil for one hour, stir with a glass rod while boiling to accelerate dissolution, and combine the two decoctions after heating; S2. Adjust the pH to 1.5-2.0 with hydrochloric acid, heat in a water bath to 80°C, keep warm for half an hour and then let stand for 24 hours, filter, add appropriate amount of water to the precipitate and stir, adjust the pH to 7.0 with 40% NaOH, S3, add an equal amount of ethanol to dissolve and filter, and adjust the pH of the filtrate to 1.5-2.0 with hydrochloric acid for the second acid precipitation S4. Heat the extract to 60°C in a water bath, keep warm for half an hour, let it stand, filter, wash the precipitate with appropriate amount of water, and dry to obtain a light yellow powder.

3. The preparation process of a baicalein derivative according to claim 1, characterized in that: The specific steps of the baicalin purification process in step 1 are: S1. Take 50 g of Scutellaria baicalensis, reflux with 60% ethanol for two hours and extract twice, filter, and recover ethanol by rotary evaporation of the filtrate until there is no alcohol taste; S2, the water solution is heated to 80°C in a water bath, the pH is adjusted to 1.5-2.0 with hydrochloric acid, the temperature is kept for 30 minutes, the solution is allowed to stand at room temperature for 24 hours, and the precipitate is centrifuged; S3, add ten times the amount of water to the precipitate and stir to form a suspension, then adjust the pH to 7.0 with 20% NOH, centrifuge it and take the supernatant and adjust the pH to 1.5-2.0 with hydrochloric acid; S4. Continue heating in a water bath to 80°C for 30 minutes, place at room temperature, filter, wash the precipitate with appropriate amount of water, and dry to obtain a light yellow powder.

4. The preparation process of a baicalein derivative according to claim 1, characterized in that: The specific steps of the baicalin refining process in step 1 are: S1. Weigh crude baicalin, add 10 times the amount of water and stir evenly, add 20% NaOH to adjust the pH to 6.0-7.0; S2. After all baicalin is dissolved, add an appropriate amount of activated carbon and mix well. Heat to 80℃ in a water bath as above for 30 minutes. S3, remove the activated carbon by suction filtration, and adjust the pH of the filtrate to 1.5-2.0 with concentrated hydrochloric acid; S4. Add an equal volume of 95% ethanol, keep warm in a 50°C water bath for 30 minutes, precipitate, leave overnight, filter, wash the precipitate with a small amount of ethanol, drain, and dry below 60°C to obtain refined baicalin.

5. The preparation process of a baicalein derivative according to claim 1, characterized in that: In the step 2, the mass ratio of fresh Scutellaria baicalensis radix to buffer solution is 1:1.5-2.

5.

6. The process for preparing a baicalein derivative according to claim 1, characterized in that: The baicalin content used in step 2 is ≥80%.

7. The process for preparing a baicalein derivative according to claim 1, characterized in that: In the step 2, the mass ratio of baicalin, low eutectic solvent, buffer and scutellaria crude enzyme powder is 1:40-60:150-250:0.025-0.

1.

8. The process for preparing a baicalein derivative according to claim 1, characterized in that: The number of times of washing with deionized water in the step 2 is at least 3 times.

9. The process for preparing a baicalein derivative according to claim 1, characterized in that: The amount of phosphorus oxychloride added in step 3 is 3.5 ml.

10. The process for preparing a baicalein derivative according to claim 1, characterized in that: The reaction time after adding phosphorus oxychloride was 2 h.

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