CN103145657B - 17-hydrogen-9-dehydrogenation rographolide compound, preparation method and prepare pharmaceutical use - Google Patents

Abstract

The invention discloses 17-hydrogen-9-dehydroandrographolide or its salt, its preparation method, and its antipyretic, anti-inflammatory and anti-viral uses. The 17-hydrogen -9-dehydroandrographolide or its salt can completely not change the chemical properties of andrographolide, and the 17-hydrogen-9-dehydroandrographolide or its salt used in the present invention has good water solubility, thermal The characteristics of high stability and small hemolysis ensure the pharmacological activity of the pure natural drug of andrographolide to the greatest extent, and medical and pharmaceutical researchers cannot know in advance that 17-hydrogen-9 - Sodium dehydroandrographolide has good uses as mentioned above.

Description

17-Hydro-9-dehydroandrographolide, preparation method and pharmaceutical use thereof

technical field

The invention relates to a 17-hydrogen-9-dehydroandrographolide compound, a preparation method and a medicine preparation application thereof.

Background technique

Andrographis paniculata is the dry aerial part of Andrographis paniculata (Burm.f.) Nees of the Acanthaceae plant. Chemical composition and pharmacological experiments show that the active ingredients of Andrographis paniculata are mainly diterpenoids represented by andrographolide [National Traditional Chinese Medicine Administration Bureau. Chinese Materia Medica Volume 7. Shanghai: Shanghai Science and Technology Press, 1999: 439], the structure of andrographolide is:

Molecular formula: C ₂₀ H ₃₀ O ₅ , a colorless square crystal, mp230-232°C, [α] ₀ ²⁰ -126° (c0.2, H ₂ O). Very bitter taste, soluble in methanol, ethanol, propanol, pyridine, slightly soluble in chloroform, ether, hardly soluble in water and petroleum ether. Therefore, oral preparations usually make tablets, capsules, dropping pills, soft capsules; because andrographolide is insoluble in water, it brings difficulties to the preparation of liquid preparations. At present, existing multiple methods are used to convert andrographolide into Various derivatives to improve the water solubility of andrographolide. Generally, after extracting andrographolide, it is prepared into injections of various water-soluble derivatives, such as adding sulfuric acid with sodium sulfite or adding reaction with sodium bisulfite, to obtain water-soluble sulfonate, sodium bisulfite andrographolide (Lianbizhi Injection), monopotassium succinate half ester, refined from andrographolide through esterification, dehydration, and salt formation 14-dehydroxy-11,12-didehydroandrographolide-3,19- Potassium disuccinate half ester sodium salt (Yanhuning injection) or 14-dehydroxy-11,12-didehydroandrographolide-3,19-disuccinate half ester monopotassium salt, but the above salts are in water Solubility and stability are not particularly good.

Contents of the invention

The object of the present invention is to provide a compound represented by general formula (I) or its salt, its chemical name is 17-hydrogen-9-dehydroandrographolide,

Another object of the present invention is to provide a method for preparing a compound of general formula (I) or a salt thereof, comprising the following steps:

[1] Add a solvent to the reaction kettle, add andrographolide to dissolve it, then add a sulfonating agent for sulfonation reaction, adjust the temperature of the reaction kettle at 4.5-36.5°C, and perform the sulfonation reaction for 0.5-29.5 hours;

[2] The andrographolide solution is added to the sulfonating agent by slowly dripping, spraying or ventilating while stirring, and when the sulfonating agent is added by slowly dripping or spraying, the addition speed is controlled at 1.25 ml ~ 4.65ml/min/1g andrographolide, when the sulfonating agent is added by feeding gas, the feeding speed is controlled at 0.026 liters to 0.27 liters/min/1g andrographolide, and the chemical name is obtained after the sulfonation reaction It is a mixture of reactants and unreacted substances of 17-hydrogen-9-dehydroandrographolide;

[3] The mixture is subjected to solvent extraction and crystallization to obtain the final 17-hydrogen-9-dehydroandrographolide; or

Put the mixture into a saturated saline solution and crystallize to obtain the final 17-hydrogen-9-dehydroandrographolide or its salt; or

The mixture was adjusted to pH 7 with alkaline solution, separated by macroporous adsorption resin, ODS or SephadexLH-20 column chromatography, using water: ethanol or methanol as eluent, gradient elution, collected eluent components, crystallized, Obtain final 17-hydrogen-9-dehydroandrographolide or its salt; or

The mixture is successively processed together by the aforementioned two or more steps to obtain the final 17-hydrogen-9-dehydroandrographolide or its salt.

Preferably, the solvent added in the reaction kettle is one or both of acetic anhydride or glacial acetic acid, 1g of andrographolide is dissolved with 3g to 12g of the solvent, preferably, 1g of andrographolide is dissolved with 4g to 9g of dissolved in the above solvent.

Preferably, the solvent is acetic anhydride and glacial acetic acid, and the acetic anhydride and glacial acetic acid are made of the following weight ratios: 80% to 20% of acetic anhydride and 20% to 80% of glacial acetic acid. Preferably, acetic anhydride Anhydride 62%, glacial acetic acid 38%.

Preferably, the sulfonating agent adopts concentrated sulfuric acid and glacial acetic acid, and 1 g of andrographolide is sulfonated with 1 g to 10 g of concentrated sulfuric acid glacial acetic acid, and the concentrated sulfuric acid and glacial acetic acid are prepared by the following weight ratio: concentrated sulfuric acid 80 %～20%, glacial acetic acid 20%～80%, preferably, 1g andrographolide uses 1.5g～5g concentrated sulfuric acid glacial acetic acid, and the weight ratio of concentrated flowing acid and glacial acetic acid is 1:1.

Preferably, the sulfonating agent is sulfur trioxide, 1g of andrographolide is passed through 0.2 liters to 5 liters of sulfur trioxide with a volume concentration of 1% to 3% for sulfonation, preferably, 1g of andrographolide is passed through 0.3 1 to 3 liters of sulfur trioxide with a volume concentration of 1.5% to 2.5% is used for sulfonation.

Preferably, the sulfonating agent is chlorosulfonic acid, 1g of andrographolide is sulfonated with 0.2g-5g of chlorosulfonic acid, preferably, 1g of andrographolide is sulfonated with 0.3g-3.5g of chlorosulfonic acid.

Preferably, the saturated salt solution is sodium chloride or potassium chloride saturated solution, and the alkaline solution is 5% to 50% sodium hydroxide or potassium hydroxide or less than 25% ammonia solution.

Preferably, the temperature of the reaction kettle is 9-25°C, and the sulfonation reaction time should be controlled within 1-3 hours.

Preferably, the sulfonating agent is concentrated sulfuric acid glacial acetic acid or chlorosulfonic acid, which is slowly added dropwise and sprayed, and the adding speed is controlled at 1.8ml-3.6ml/min/1g andrographolide.

Preferably, the sulfonating agent is sulfur trioxide, and it is added by passing gas, and the feeding speed is controlled at 0.05 liters to 0.12 liters/min/1g of andrographolide.

Preferably, in the gradient elution, the proportion of water is 80-20%, and the proportion of ethanol or methanol is 20-80%.

Another object of the present invention is to provide a method for assaying a compound of general formula (I) or a salt thereof, which uses high performance liquid chromatography to measure the 17-hydrogen-9-dehydroandrographolide or a salt thereof, The measurement conditions are as follows:

Filler with octadecylsilane bonded silica gel;

The detection wavelength is 225nm;

The column temperature is 25°C;

The number of theoretical plates calculated by 17-hydro-9-dehydroandrographolide or its salts is not less than 10,000;

Preparation of Reference Substance Solution The 17-hydrogen-9-dehydroandrographolide or its salt was prepared as a reference substance through purity inspection and content standardization, and dried in a phosphorus pentoxide vacuum desiccator, and accurately weighed Appropriate amount, add water to make a solution with the required concentration;

Preparation of the test solution Precisely weigh 5mg of the sample, put it in a 50ml measuring bottle, add water to dilute to the mark, shake well, and get final product;

For elution, acetonitrile is used as mobile phase A, and potassium dihydrogen phosphate buffer is used as mobile phase B. The potassium dihydrogen phosphate buffer is 1.361 g of potassium dihydrogen phosphate and 1.0 g of sodium heptane-1-sulfonate per 1000 ml of water. g, carry out gradient elution according to the following conditions, and run for 70 minutes;

From 0 to 10 minutes, the proportion of acetonitrile is 8.0%, and the proportion of potassium dihydrogen phosphate buffer is 92.0%;

From 10 to 60 minutes, the proportion of acetonitrile increased from 8.0% to 35.0%, and the proportion of potassium dihydrogen phosphate buffer decreased from 92.0% to 65.0%;

At 60-62 minutes, the proportion of acetonitrile increased from 35.0% to 70.0%, and the proportion of potassium dihydrogen phosphate buffer decreased from 65.0% to 30.0%;

At 62 to 70 minutes, keep acetonitrile: potassium dihydrogen phosphate buffer solution at a ratio of 70.0%: 30.0% for elution;

Under the above conditions, 17-hydrogen-9-dehydroandrographolide or its salt has a chromatographic peak at a retention time of about 53 minutes.

Determination method Precisely draw the reference substance solution and the test solution respectively, inject it into the liquid chromatograph, measure it, and get it.

Another object of the present invention is to provide a preparation of 17-hydrogen-9-dehydroandrographolide or its salt, which is the combination of 17-hydrogen-9-dehydroandrographolide or its salt in pharmaceutical acceptable carrier.

Another object of the present invention is to provide the use of the 17-hydrogen-9-dehydroandrographolide or its salt in the preparation of antipyretic drugs.

Preferably, the antipyretic effect of 17-hydro-9-dehydroandrographolide or its salt on endotoxin-induced pyrexia.

Preferably, the antipyretic effect of 17-hydrogen-9-dehydroandrographolide or its salt on dry yeast pyrexia.

Another object of the present invention is to provide the use of the 17-hydrogen-9-dehydroandrographolide or its salt in the preparation of anti-inflammatory drugs.

Preferably, the drug effect of 17-hydrogen-9-dehydroandrographolide or its salt on sepsis.

Preferably, the anti-inflammatory effect of 17-hydrogen-9-dehydroandrographolide or its salt on mouse auricle swelling induced by xylene.

Preferably, the anti-inflammatory effect of 17-hydro-9-dehydroandrographolide or its salt on carrageenan-induced paw swelling in rats.

Another object of the present invention is to provide the use of the 17-hydrogen-9-dehydroandrographolide or its salt in the preparation of antiviral drugs.

Preferably, 17-hydro-9-dehydroandrographolide or a salt thereof is used to inhibit neuraminidase.

Preferably, 17-hydro-9-dehydroandrographolide or a salt thereof is used to inhibit influenza virus.

Preferably, 17-hydro-9-dehydroandrographolide or a salt thereof is used to inhibit influenza virus FM1.

The 17-hydrogen-9-dehydroandrographolide or its salt provided by the present invention has very good solubility in water and high stability, is very suitable for practical application, and can be applied to various common forms, such as tablets, capsules, Soft capsules, dispersible tablets, oral liquids, granules, chewable tablets, orally disintegrating tablets, dropping pills, sustained-release tablets, controlled-release tablets, sustained-release capsules, controlled-release capsules. Of course, it can also be made into liquid preparations such as syrups, injections, etc., especially the preparation of injections overcomes the shortcoming of low bioavailability of oral medicines.

On the other hand, the preparation method of 17-hydrogen-9-dehydroandrographolide or its salt provided by the present invention is simple and convenient, with mild conditions and high productivity, and can easily prepare 17-hydrogen-9-dehydroandrographolide esters or their salts.

More importantly, the present invention has finally determined a suitable solvent, an inventive sulfonating agent and the important process parameters of the sulfonating reaction thereof, such as the processing mode of the sulfonating agent and The determination of the complex relationship between the addition rates, and the determination of the appropriate numerical range, etc., so that the desired reactants are finally obtained. On this basis, further use of creative purification technology for purification, thus the present invention provides the 7-hydrogen-9-dehydroandrographolide-14-sulfate or its salt.

The comparison between the present invention and the prior art shows that: the 17-hydrogen-9-dehydroandrographolide or its salt formed by the preparation method of the present invention can not change the original chemical properties of andrographolide; and the present invention adopts Compared with the prior art, 17-hydrogen-9-dehydroandrographolide or its salt has the characteristics of good water solubility, high thermal stability, and small hemolytic effect. The pharmacological activity of the pure natural medicine of andrographolide is guaranteed to the greatest extent.

After comparing the experimental data, it can be seen that after endotoxin was administered for 1 hour, the average body temperature of healthy rabbits in the blank control group rose, and continued to rise for 3 hours, and then gradually began to decline. Compared with the blank control group, 50mg·kg ^-1 17-hydrogen-9-dehydroandrographolide sodium in the low-dose group showed obvious inhibitory effect on the temperature rise in rabbits for ¹ to 2 hours; Group 200mg·kg ^-1 17-hydrogen-9-dehydroandrographolide sodium showed a strong effect of inhibiting the rise of rabbit body temperature within 1 to 2 hours, and also showed the effect of inhibiting the rise of rabbit body temperature at 4 hours; the three drug experimental groups Among them, the antipyretic effect is the best with a dose of 100 mg·kg ^-1 or more. It shows that 50-200 mg·kg ^-1 17-hydrogen-9-dehydroandrographolide sodium has a cooling effect on the increase of rabbit body temperature caused by endotoxin, and an unexpected technical effect has been obtained.

After comparing the experimental data, it can be seen that the body temperature of the healthy rats in the blank control group continued to rise for the 6th hour when the dry yeast was given for 1 hour. Compared with the blank control group, 17-hydrogen-9-dehydroandrographolide sodium 100, 200 mg·kg ^-1 significantly inhibited the increase in body temperature of rats within 1 to 4 hours, and achieved unexpected technical effects.

After comparison of experimental data, it can be seen that both andrographolide and 17-hydro-9-dehydroandrographolide can significantly improve the survival rate of LPS-induced sepsis mice, and reduce TNF-α in the serum of sepsis mice in a time- and dose-dependent manner. The content of IL-1β, ALT, and AST can inhibit the increase of mRNA levels of inflammatory factors in liver tissue. Moreover, 17-hydrogen-9-dehydroandrographolide acts faster than andrographolide, and the effect is better. The experimental results show that after the modification of andrographolide into a sulfonate, its water solubility is better, the administration takes effect quickly, and the improvement effect on mouse sepsis is also enhanced, achieving unexpected technical effects.

After comparison of experimental data, it can be seen that the ear swelling degree of mice in each treatment group of 17-hydro-9-dehydroandrographolide sodium and dexamethasone group was significantly smaller than that of the control group, and unexpected technical effects were achieved.

Through comparison of experimental data, it can be seen that compared with the control group, each dose group of 17-hydrogen-9-dehydroandrographolide sodium and the dexamethasone group all have a significant inhibitory effect on the rat foot swelling caused by carrageenan, and 17- When hydrogen-9-dehydroandrographolide sodium was administered at 200 mg/kg, obvious inhibitory effect occurred in 30 minutes and lasted for 5 hours. The effect of 100 mg/kg was equivalent to that of dexamethasone. 17-hydrogen-9-dehydroandrographolide There is a quantitative-effect relationship between the three dosage groups of sodium ester sodium on swelling at the same time point, and an unexpected technical effect has been achieved.

After comparison of experimental data, it can be seen that 17-hydrogen-9-dehydroandrographolide sodium can extract effective neuraminidase inhibitory components; and 17-hydrogen-9-dehydroandrographolide sodium varies with the dosage, Its ability to inhibit neuraminidase activity, that is, the neuraminidase inhibition rate also changes accordingly and is positively correlated. 17-hydrogen-9-dehydroandrographolide sodium can inhibit the influenza virus surface neuraminidase, thereby inhibiting the influenza virus from entering the cell, inhibiting the replication and proliferation of the influenza virus that has entered the cell, thereby reducing the impact of the influenza virus on the cell. Infection, growth, and prevention and treatment of influenza and its complications have achieved unexpected technical effects.

After comparison of experimental data, it can be seen that 17-hydrogen-9-dehydroandrographolide sodium has a significant inhibitory effect on influenza virus at 0.075～0.6g/L (P<0.05), and the _ED50 is 0.0918g±0.0052g/L, The TI was 59.88±1.96. The IC50 of 17-hydro-9-dehydroandrographolide sodium was low and the TI was high. The inhibitory effect of 17-hydrogen-9-dehydroandrographolide sodium on the cytopathic effect of FM1 influenza virus was enhanced with the increase of drug dose. The correlation analysis of the drug dose and the drug's inhibition rate of CPE showed that there was a significant positive correlation between the dose of 17-hydrogen-9-dehydroandrographolide sodium and the drug's inhibition rate of CPE.

Medical and pharmaceutical researchers cannot know in advance that 17-hydro-9-dehydroandrographolide sodium has the above-mentioned good uses without performing relevant experiments.

Description of drawings

Figure 1: 17-hydrogen-9-dehydroandrographolide H NMR spectrum

Figure 2: Carbon NMR spectrum of 17-hydrogen-9-dehydroandrographolide

Figure 3: Mass Spectrum of 17-Hydro-9-Dehydroandrographolide

Figure 4: 17-Hydro-9-dehydroandrographolide linear relationship diagram.

Detailed ways

Example 1

Get andrographolide, add 5.3 times the amount of acetic anhydride (62%) and glacial acetic acid (38%) to dissolve, and under stirring, add 4.0 times the amount of sulfuric acid in equal proportions by spraying at a rate of 1.8ml per minute for 1g andrographolide Glacial acetic acid, mix well, adjust the temperature of the reaction kettle at 16°C, place it for 90 minutes for sulfonation, add an equal amount of purified water, then pour it into saturated sodium chloride solution, and then go through macroporous adsorption resin, SephadexLH-20 column chromatography Separation, gradient elution with water: methanol, the proportion of water is 80-20%, the proportion of methanol is 20-80%, the eluate is collected in sections, the same components are combined, and crystallized to obtain 17-hydrogen-9-de Hydroandrographolide, molecular formula C ₂₀ H ₃₀ O ₅ , molecular weight: 350.

Reaction example:

17-Hydro-9-dehydroandrographolide physical and chemical properties and spectral data:

17-Hydro-9-dehydroandrographolide: molecular formula C ₂₀ H ₃₀ O ₅ , white powder; ESIMSm/z: 351[M+H] ⁺ , 373[M+Na] ⁺ , 701[M+Na] ⁺ , 723 [2M+Na] ⁺ , HRESIMS: m/z 349.2029 [MH] ^- (cald. for C ₂₀ H ₂₉ O ₅ , 349.2015); ¹ HNMR and ¹³ CNMR data.

¹ NMR (DMSO, 600MHz) δ: 1.04～1.07(m, 1H, H-1α), 1.58～1.75(m, 1H, H-1β), 1.58～1.75(m, 2H, H-2), 3.18( m, 1H, H-3), 1.11 (brd, J=12.6Hz, 1H, H-5), 1.43 (m, 1H, H-6α), 1.58～1.75 (m, 1H, H-6β), 1.92 ~2.02 (m, 2H, H-7), 2.99 (dd, J=17.4, 7.8Hz, 1H, H-11α), 3.14 (dd, J=17.4, 5.6Hz, H-11β), 6.46 (ddd, J=7.8, 5.6, 1.4Hz, 1H, H-12), 4.96(brs, 1H, H-14), 4.03(dd, J=9.8, 2.3Hz, 1H, H-15α), 4.44(dd, J =9.8, 6.0Hz, 1H, H-15β), 1.52(s, 3H, H-17), 1.08(s, 3H, H-18), 3.28(dd, J=10.8, 6.0Hz, 1H, H- 19α), 3.84 (d, J=10.8Hz, 1H, H-19β), 0.92 (s, 1H, H-20), 4.96 (brs, 1H, 3-OH), 5.73 (brs, 1H, 14-OH ), 4.06 (brd, J=6.01H, 19-OH)

¹³ C-NMR (DMSO, 150MHz) δ: 35.0 (C-1), 28.1 (C-2), 78.8 (C-3), 42.6 (C-4), 51.8 (C-5), 19.5 (C- 6), 34.5(C-7), 128.6(C-8), 137.4(C-9), 38.6(C-10), 27.9(C-11), 146.3(C-12), 128.9(C-13 ), 65.2(C-14), 74.7(C-15), 170.5(C-16), 19.7(C-17), 23.4(C-18), 63.3(C-19), 20.6(C-20) .

Example 2

Get andrographolide, add 4.5 times the amount of acetic anhydride (57%) and glacial acetic acid (43%) to dissolve, and under stirring, slowly add 4.5 times the amount of sulfuric acid at a rate of 1.9ml per minute for 1g andrographolide (52%) and glacial acetic acid (48%), mix evenly, adjust the temperature of the reactor at 20°C, and place it for 90 minutes for sulfonation. Add an equal amount of purified water, stir well, adjust the pH to about 7.0 with 45% NaOH, add 95% ethanol to make the alcohol content reach more than 82%, recover ethanol under reduced pressure, and separate the aqueous solution through macroporous adsorption resin and ODS column chromatography. Gradient elution with water: ethanol, the proportion of water is 80-20%, the proportion of ethanol is 20-80%, the eluate is collected in sections, the same components are combined, and crystallized to obtain 17-hydrogen-9-dehydroandrographis paniculata Sodium lactone-3-sulfate.

Example 3

Get andrographolide, add 5.6 times the amount of acetic anhydride to dissolve, and under stirring, add 4.3 times the amount of sulfuric acid (52%) and glacial acetic acid (48%) by atomization at a rate of 2.5 ml per minute for 1 g of andrographolide , Mix well, adjust the temperature of the reactor at 22°C, and place it for 80 minutes for sulfonation. The reactant was poured into a saturated sodium chloride solution, the precipitate was washed with a saturated sodium chloride solution and water respectively, the solid was refluxed with chloroform, the insoluble was dissolved by adding absolute ethanol, the alcohol insoluble was removed, the ethanol was recovered, and crystallized to obtain 17-Hydro-9-dehydroandrographolide.

Example 4

Get andrographolide, add 5.5 times of amount of acetic anhydride (58%) and glacial acetic acid (42%) to dissolve, and under stirring, slowly add 4.5 times of sulfuric acid dropwise at a rate of 2.3ml per minute for 1g of andrographolide (51%) and glacial acetic acid (49%), mix evenly, adjust the temperature of the reactor at 22°C, and place it for 90 minutes for sulfonation. Add 95% ethanol to make the alcohol content reach more than 83%, reclaim ethanol under reduced pressure, and the aqueous solution is separated by macroporous adsorption resin and ODS column chromatography, and eluted with water:methanol gradient, the ratio of water is 80-20%, the ratio of methanol The ratio is 20-80%. The eluate components are collected, the same components are combined, and crystallized to obtain 17-hydrogen-9-dehydroandrographolide.

Example 5

Get andrographolide, add 5.4 times the amount of acetic anhydride (62%) and glacial acetic acid (38%) to dissolve, and under stirring, slowly add 4.5 times the amount of sulfuric acid at a rate of 2.2ml per minute for 1g andrographolide (53%) and glacial acetic acid (47%), mix evenly, adjust the temperature of the reactor at 20°C, and place it for 100 minutes for sulfonation. Add an equal amount of purified water, stir well, adjust the pH to about 7.0 with 32% KOH, add 95% ethanol to make the alcohol content reach more than 84%, recover ethanol under reduced pressure, and separate the aqueous solution through macroporous adsorption resin and ODS column chromatography. Gradient elution with water: ethanol, the proportion of water is 80-20%, the proportion of ethanol is 20-80%, the eluate is collected in sections, the same components are combined, and crystallized to obtain 17-hydrogen-9-dehydroandrographis paniculata Potassium lactone-19-sulfate.

Example 6

Get andrographolide, add 5.0 times of amount of acetic anhydride (62%) and glacial acetic acid (38%) to dissolve, under stirring, slowly add 3.8 times of sulfuric acid dropwise at a rate of 2.5ml per minute for 1g of andrographolide (53%) and glacial acetic acid (47%), mix evenly, adjust the temperature of the reactor at 16°C, and place it for 80 minutes for sulfonation. Add an equal amount of purified water, stir well, use 25% NH ₄ OH to adjust the pH to about 7.0, add 95% ethanol to make the alcohol content reach more than 80%, recover ethanol under reduced pressure, and the aqueous solution is chromatographed by macroporous adsorption resin and ODS column Separation, gradient elution with water: ethanol, the proportion of water is 80-20%, the proportion of ethanol is 20-80%, the eluate is collected in sections, the same components are combined, and crystallized to obtain 17-hydrogen-9-de Ammonium hydroandrographolide-14-sulfate.

Example 7

Get andrographolide, add 6.2 times the amount of acetic anhydride (62%) and glacial acetic acid (38%) to dissolve, and under stirring, feed 1.5 liters of 1.6% sulfur trioxide at a rate of 0.06 liters per minute for 1 g of andrographolide , adjust the temperature of the reactor at 20°C, and place it for 100 minutes for sulfonation. Add an equal amount of purified water, stir well, adjust the pH to about 7.0 with 32% NaOH, add 95% ethanol to make the alcohol content reach more than 82%, recover ethanol under reduced pressure, and separate the aqueous solution through macroporous adsorption resin and ODS column chromatography. Gradient elution with water: methanol, the proportion of water is 80-20%, the proportion of methanol is 20-80%, the eluate is collected in sections, the same components are combined, and crystallized to obtain 17-hydrogen-9-dehydroandrographis paniculata Sodium lactone-3-sulfate.

Example 8

Get andrographolide, add 5.6 times the amount of acetic anhydride (60%) and glacial acetic acid (40%) to dissolve, and under stirring, feed 1.5 liters of 1.6% sulfur trioxide at a rate of 0.08 liters per minute with 1g andrographolide , adjust the temperature of the reactor at 16°C, and place it for 100 minutes for sulfonation. The reactant was poured into a saturated sodium chloride solution, the precipitate was washed with a saturated sodium chloride solution and water respectively, the solid was refluxed with chloroform, the insoluble was dissolved by adding absolute ethanol, the alcohol insoluble was removed, the ethanol was recovered, and crystallized to obtain Sodium 17-hydro-9-dehydroandrographolide-19-sulfate.

Example 9

Get andrographolide, add 5.8 times the amount of acetic anhydride (55%) and glacial acetic acid (45%) to dissolve, and under stirring, feed 1.8 liters of 1.6% sulfur trioxide at a rate of 0.10 liters per minute with 1g andrographolide , adjust the temperature of the reactor at 18°C, and place it for 120 minutes for sulfonation. Add 95% ethanol to make the alcohol content reach more than 82%, reclaim ethanol under reduced pressure, and the aqueous solution is separated by macroporous adsorption resin and SephadexLH-20 column chromatography, and eluted with water: ethanol gradient, the ratio of water is 80-20%, The ratio of ethanol is 20-80%. The eluate is collected in sections, the same components are combined, and crystallized to obtain 17-hydrogen-9-dehydroandrographolide.

Example 10

Get andrographolide, add 4.7 times the amount of acetic anhydride (57%) and glacial acetic acid (43%) to dissolve, and under stirring, feed 1.7 liters of 2.4% sulfur trioxide at a rate of 0.0885 liters per minute with 1g andrographolide , adjust the temperature of the reactor at 16°C, and place it for 100 minutes for sulfonation. Add an equal amount of purified water, stir well, adjust the pH to about 7.0 with 33% KOH, then separate through macroporous adsorption resin and ODS column chromatography, and elute with water: ethanol gradient, the ratio of water is 80-20%, ethanol The ratio is 20 to 80%. The eluate is collected in sections, the same components are combined, and crystallized to obtain 17-hydrogen-9-dehydroandrographolide-3,14-potassium disulfate.

Example 11

Get andrographolide, add 5.5 times the amount of acetic anhydride (60%) and glacial acetic acid (40%) to dissolve, and under stirring, feed 1.9 liters of 2.0% sulfur trioxide at a rate of 0.065 liters per minute with 1g andrographolide , adjust the temperature of the reactor at 20°C, place it for 60 minutes for sulfonation, add purified water, and stir well. The reactant was poured into saturated potassium chloride solution, the precipitate was washed with saturated potassium chloride solution and water respectively, the solid was refluxed with chloroform, the insoluble was dissolved by adding absolute ethanol, the alcohol insoluble was removed, the ethanol was recovered, and crystallized to obtain Potassium 17-hydro-9-dehydroandrographolide-14-sulfate.

Example 12

Get andrographolide, add 5.0 times of amount of acetic anhydride (60%) and glacial acetic acid (40%) to dissolve, and under stirring, feed 1.5 liters of 1.7% sulfur trioxide at a rate of 0.08 liters per minute for 1 g of andrographolide , adjust the temperature of the reaction kettle at 23°C, let it stand for 70 minutes for sulfonation, add an equal amount of purified water, and stir well. Use 20% NH ₄ OH to adjust the pH to about 7.0, add 95% ethanol to make the alcohol content reach more than 83%, recover ethanol under reduced pressure, separate the aqueous solution through macroporous adsorption resin and ODS column chromatography, and elute with water: ethanol gradient , the proportion of water is 80 to 20%, the proportion of ethanol is 20 to 80%, the eluate is collected in sections, the same components are combined, and crystallized to obtain 17-hydrogen-9-dehydroandrographolide-3,19- ammonium disulfate.

Example 13

Get andrographolide, add 4.8 times the amount of acetic anhydride (55%) and glacial acetic acid (45%) to dissolve, and under stirring, slowly add 2.5 times the amount of chlorosulfur at a rate of 2.0ml per minute for 1g andrographolide Acid, mix well, adjust the temperature of the reactor at 16°C, place it for 100 minutes for sulfonation, add an equal amount of purified water, stir well, adjust the pH to about 7.0 with 45% NaOH, add 95% ethanol to make the alcohol content reach 82% Above, ethanol is reclaimed under reduced pressure, and the aqueous solution is separated by macroporous adsorption resin and ODS column chromatography, and is eluted with water: ethanol gradient, the ratio of water is 80-20%, and the ratio of ethanol is 20-80%. Deliquify, combine the same components, and crystallize to obtain 17-hydrogen-9-dehydroandrographolide-3-sulfate sodium.

Example 14

Get andrographolide, add 5.8 times the amount of acetic anhydride (56%) and glacial acetic acid (44%) to dissolve, under stirring, add 2.6 times the amount of chlorosulfonic acid by atomization at a speed of 1.8ml per minute for 1g andrographolide , mix well, mix well, adjust the temperature of the reaction kettle at 18°C, place it for 80 minutes for sulfonation, pour the reactant into saturated sodium chloride solution, wash the precipitate with saturated sodium chloride solution and water respectively, and wash the solid with chloroform Reflux, add absolute ethanol to dissolve insoluble matter, remove alcohol insoluble matter, recover ethanol, and crystallize to obtain 17-hydrogen-9-dehydroandrographolide-19-sulfate sodium.

Example 15

Get andrographolide, add 5.2 times the amount of acetic anhydride (62%) and glacial acetic acid (38%) to dissolve, and under stirring, slowly add 2.7 times the amount of chlorosulfur at a rate of 2.0ml per minute for 1g andrographolide acid, mix well, adjust the temperature of the reaction kettle at 18°C, place it for 80 minutes for sulfonation, add the same amount of purified water, stir well, add 95% ethanol to make the alcohol content reach more than 85%, recover ethanol under reduced pressure, and the aqueous solution Pore adsorption resin, ODS column chromatography separation, gradient elution with water: ethanol, the ratio of water is 80-20%, the ratio of ethanol is 20-80%, the eluate is collected in sections, the same components are combined, crystallized, In 17-hydrogen-9-dehydroandrographolide.

Example 16

Take andrographolide, add 5.2 times the same proportion of acetic anhydride and glacial acetic acid to dissolve, under stirring, slowly add 3.4 times the amount of chlorosulfonic acid dropwise at a rate of 2.2ml per minute for 1g of andrographolide, mix well, Adjust the temperature of the reaction kettle at 22°C, place it for 90 minutes for sulfonation, add an equal amount of purified water, stir well, adjust the pH to about 7.0 with 33% KOH, add 95% ethanol to make the alcohol content reach more than 85%, and recover under reduced pressure Ethanol, the aqueous solution is separated by macroporous adsorption resin and ODS column chromatography, eluted with water: ethanol gradient, the ratio of water is 80-20%, the ratio of ethanol is 20-80%, the eluate is collected in sections, and the same Components, crystallization, in 17-hydrogen-9-dehydroandrographolide-3.19-potassium disulfate.

Example 17

Get andrographolide, add 5.5 times the amount of acetic anhydride (58%) and glacial acetic acid (42%) to dissolve, and under stirring, slowly add 3.0 times the amount of chlorosulfur at a rate of 2.4ml per minute for 1g andrographolide acid, mix well, adjust the temperature of the reaction kettle at 23°C, place it for 80 minutes for sulfonation, add an equal amount of purified water, stir well, adjust the pH to about 7.0 with 18% NH ₄ OH, add 95% ethanol to make the alcohol content reach More than 84%, ethanol is recovered under reduced pressure, the aqueous solution is separated by macroporous adsorption resin and ODS column chromatography, and eluted with water: ethanol gradient, the ratio of water is 80-20%, the ratio of ethanol is 20-80%, segmented Collect the eluate, combine the same components, and crystallize to obtain 17-hydrogen-9-dehydroandrographolide-14-ammonium sulfate.

Example 18

Get andrographolide, add 4.7 times the amount of acetic anhydride (55%) and glacial acetic acid (45%) to dissolve, and under stirring, slowly add 2.5 times the amount of chlorosulfur at a rate of 2.5ml per minute for 1g of andrographolide acid, mix well, adjust the temperature of the reaction kettle at 25°C, place it for 80 minutes for sulfonation, pour the reactant into saturated potassium chloride solution, wash the precipitate with saturated potassium chloride solution and water respectively, and reflux the solid matter with chloroform, Add absolute ethanol to the insoluble matter to dissolve, remove the alcohol insoluble matter, recycle the ethanol, and crystallize to obtain 17-hydrogen-9-dehydroandrographolide-3-sulfate potassium.

17-hydrogen-9-dehydroandrographolide was used in the following experiments to take the samples obtained by the preparation method in Example 1.

Experimental data 1: Determination of 17-hydrogen-9-dehydroandrographolide content

1. Instruments and reagents

Instruments: Agilent1100 quaternary low-pressure gradient pump series, Chemstation chemical workstation, DAD detector; Shimadzu LC-2010A high performance liquid chromatography, dual-channel ultraviolet variable wavelength detector; Sartoriuscp211D one hundred thousandth electronic balance.

Chromatographic column: DiamonsilC ₁₈ column (250mm×4.6mm, 5μm);

Reagents: Acetonitrile is chromatographically pure, water is ultrapure water prepared by Millipore, and other reagents are analytically pure.

The reference substance of 17-hydrogen-9-dehydroandrographolide sodium is self-made, and the standardized content of purity is 99.82%.

2. Preparation of reference substance solution and test solution

2.1 Purity inspection and content standardization of the reference substance: Take the sample obtained by the preparation method of Example 1, respectively use n-butanol-glacial acetic acid-water (6: 1.5: 1.5), chloroform-methanol-water-glacial acetic acid (7.5: 2.5:1:0.5) for thin-layer chromatography purity check, the sample volumes were 5, 10, 15, 20, 25 μg respectively, and the results were all one spot;

With high performance liquid chromatography, adopt the area normalization method, respectively select chromatographic column: DiamonsilC ₁₈ (250mm * 4.6mm, 5 μ m); Mobile phase: phosphate buffer solution (potassium dihydrogen phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-acetonitrile (83:17) and mobile phase: phosphate buffer (potassium dihydrogen phosphate 1.36g/L+sodium heptanesulfonate 1g/L)-methanol (77:23); flow rate: 1ml/min ; Detection wavelength: 225nm; Column temperature: 25°C. 10μl and 20μl of each group of mobile phases were injected and withdrawn once respectively, and samples were injected 4 times in total, and the average content of the 4 times was measured to be 99.82%.

2.2 Preparation of reference substance solution: Accurately weigh 21.46 mg of the above-mentioned standardized 17-hydrogen-9-dehydroandrographolide sodium reference substance, put it in a 10ml measuring bottle, add water to dissolve and dilute to the mark, shake well, and obtain as The mother liquor of the reference substance is used for methodological research.

2.3 Preparation of the test solution Precisely weigh 50 mg of the sample of this embodiment, put it in a 500ml measuring bottle, add water to dilute to the mark, shake well, and you get it;

3. Chromatographic conditions and system suitability test

Octadecylsilane bonded silica gel is used as filler; the detection wavelength is 225nm; the column temperature is 25°C; the number of theoretical plates is not less than 10,000 based on 8-epi-isoandrographolide-19-sulfate or its salt;

Acetonitrile was used as mobile phase A, and potassium dihydrogen phosphate buffer was used as mobile phase B. The potassium dihydrogen phosphate buffer was added with 1.361 g of potassium dihydrogen phosphate and 1.0 g of sodium heptane-1-sulfonate per 1000 ml of water. Carry out gradient elution according to the following conditions, run for 70 minutes:

From 0 to 10 minutes, the proportion of acetonitrile is 8.0%, and the proportion of potassium dihydrogen phosphate buffer is 92.0%;

From 10 to 60 minutes, the proportion of acetonitrile increased from 8.0% to 35.0%, and the proportion of potassium dihydrogen phosphate buffer decreased from 92.0% to 65.0%;

At 60-62 minutes, the proportion of acetonitrile increased from 35.0% to 70.0%, and the proportion of potassium dihydrogen phosphate buffer decreased from 65.0% to 30.0%;

When 62～70 minutes, keep acetonitrile: Potassium dihydrogen phosphate buffer solution is eluted with 70.0%: 30.0% ratio; There is a chromatographic peak at 53 minutes.

4. Investigation of linear relationship

Each precision draws 1ml of the above-mentioned mother solution of the reference substance, puts it into 100ml, 50ml, 25ml, 10ml, and 5ml measuring bottles, respectively, and dilutes with water to the following concentrations: 0.02146mg/ml, 0.04292mg/ml, 0.08584mg/ml, 0.17168mg/ml ml, 0.34336mg/ml. Precisely draw 10 μl of the solution and inject it into the liquid chromatograph. According to the peak area of the chromatographic conditions under the 3 chromatographic conditions and the system suitability test, the integral value of the peak area of 17-hydrogen-9-dehydroandrographolide sodium is used as the ordinate respectively, and the respective comparisons The injection volume of the product is the abscissa, draw the standard curve, the regression equation of 17-hydrogen-9-dehydroandrographolide sodium is y=2380.2X-67.64, R ² =0.999995, 17-hydrogen-9-dehydroandrographolide The injection volume of sodium was between 0.2146 and 3.4336 μg, showing a good linearity. The results are shown in Table 1 and Figure 4.

Table 1.1 7-hydrogen-9-dehydroandrographolide sodium linear relationship results

5. Precision test

Take 17-hydrogen-9-dehydroandrographolide sodium reference substance solution, repeat injection 6 times, the result is 17-hydrogen-9-dehydroandrographolide sodium peak area RSD is 0.42%, the results are shown in Table 2.

Table 2 Results of precision test of reference substance

6. Stability test

Get need testing solution, measure according to the chromatographic condition under 3 chromatographic conditions and system suitability test item, inject once at regular intervals, the result needs testing solution 17-hydrogen-9-dehydroandrographolide sodium in 24 hours There was no significant change in the inner peak area, and the RSDs of the peak areas were 0.55%. The results are shown in Table 3.

.Table 3 Stability test results

7. Repeatability test

Get sample of the present invention, add water and make 6 parts of need testing solution, measure according to chromatographic condition under 3 chromatographic conditions and system suitability test item, 17-hydrogen-9-dehydroandrographolide sodium average content is 98.60% in the sample, RSD = 0.58%. The results are shown in Table 4.

Table 4.1 7-hydrogen-9-dehydroandrographolide sodium repeatability test results

8. Recovery test

Adopt sample addition recovery test, accurately draw the sample solution of the present invention in the 50ml volumetric flask, totally 6 parts, respectively accurately add 17-hydrogen-9-dehydroandrographolide sodium (expressed as dehydroandrographolide sodium in the table) contrast Product solution (0.962mg/ml) 2ml, dilute to the mark with water, shake well. Measure according to the method under 3 chromatographic conditions and system suitability test, calculate the recovery rate, and the results are shown in Table 5.

Table 5 Recovery test result

Experimental data 2: Effect of 17-hydrogen-9-dehydroandrographolide on pyrexia induced by endotoxin

Take Japanese big-eared white rabbits, weighing 1.8-2.3kg, both male and female. One day before the experiment, select rabbits whose body temperature is between 38.0 and 39.4°C, and whose body temperature does not change more than 0.4°C during the day as experimental rabbits. On the day of the experiment, take the above-mentioned qualified rabbits, measure the basal body temperature before modeling, inject the bacterial endotoxin physiological saline solution into the ear vein of the rabbits, the dose is 1mL/kg (10EU/mL), observe the temperature changes of the rabbits, and record once every 30min . Rabbits whose body temperature rose by more than 0.5°C 1 hour after injection were selected and randomly divided into 5 groups, with 8 rabbits in each group. 0.9% sodium chloride solution, 17-hydro-9-dehydroandrographolide low dose group 50mg·kg ^-1 , middle dose group 100mg·kg ^-1 , high dose group 200mg·kg ^-1 and Arginine aspirin for injection was 100 mg·kg ^-1 , and the body temperature of each rabbit was measured at 1, 2, 3, 4, 5, and 6 hours after administration, respectively. Taking the average body temperature before administration as the base, calculate the rabbit temperature change value at each measurement time. See Table 6.

Table 6. Changes in drug temperature after endotoxin-induced fever, °C,

Note: Compared with the empty control group ^※ P＜0.05 ^※※ P＜0.01

It can be seen from Table 6 that after endotoxin was administered for 1 hour, the average body temperature of healthy rabbits in the blank control group rose, and continued to rise for 3 hours before gradually beginning to drop. Compared with the blank control group, 50mg·kg ^-1 17-hydrogen-9-dehydroandrographolide in the low-dose group 1-2h significantly inhibited the temperature rise in rabbits, 100mg·kg ^-1 in the middle-dose group, high-dose group 200mg·kg ^-1 17-hydrogen-9-dehydroandrographolide showed a strong effect of inhibiting the rise of rabbit body temperature within 1～2h, and also showed the effect of inhibiting the rise of rabbit body temperature at 4h; The antipyretic effect is the best at doses above 100 mg·kg ^-1 . It shows that 50～200mg·kg ^-1 17-hydrogen-9-dehydroandrographolide has a cooling effect on the increase of rabbit body temperature caused by endotoxin.

Experimental data 3: Effect of 17-hydrogen-9-dehydroandrographolide on fever in rats induced by dry yeast

The body temperature of the rats used in the experiment was measured for 3 days in advance, and the measured value was the basal body temperature of the rat on the day of the experiment. Animals whose body temperature did not change by more than 0.3°C were screened and randomly divided into 5 groups, 8 animals in each group. Subcutaneous injection of 20% dry yeast suspension 5mL/kg, intraperitoneal injection of 0.9% sodium chloride solution after pyrexia, 50mg·kg ^-1 of 17-hydro-9-dehydroandrographolide low-dose group, 100mg·kg-1 of middle-dose group kg ^-1 , high-dose group 200mg·kg ^-1 and aspirin 100mg·kg ^-1 , measure the body temperature of the rats 1 to 6 hours after administration, once an hour, and take the difference between the body temperature and the base value at different time points as Observation indicators, the results are shown in Table 7.

Table 7. Changes in drug temperature after heating by dry yeast, °C,

Note: Compared with the empty control group ※P＜0.05※※P＜0.01

It can be seen from Table 7 that when the dry yeast was given for 1 hour, the body temperature of the healthy rats in the blank control group continued to rise for the 6th hour. Compared with the blank control group, 17-hydrogen-9-dehydroandrographolide 100, 200 mg·kg ^-1 showed a significant effect of inhibiting the increase in body temperature of rats within 1 to 4 hours.

Experimental data 4: Effect of 17-hydrogen-9-dehydroandrographolide on ear swelling in mice induced by xylene

50 mice were randomly divided into 5 groups. Give 17-hydro-9-dehydroandrographolide 50mg·kg ^-1 to the low-dose group, 100mg·kg ^-1 to the middle-dose group, and 200mg·kg ^- 1 to the high-dose group twice a day for 3 consecutive days, plus dexamethasone Sodium phosphate injection is administered only before inflammation. One hour after the last administration, each mouse was drip-coated with 0.1 ml of xylene on the inside and outside of the left auricle to induce inflammation, and the right ear was used as a control. Two hours after the inflammation, the mice were killed by taking off the neck cone, and the two ears of the mice were cut off along the baseline of the auricles, and the ears were punched in the same parts of the left and right ears with a 6mm diameter punch, and weighed on an electronic balance and recorded data . The degree of swelling was expressed by the weight difference between the left and right ear pieces.

Table 8. Effect of p-xylene on pinna swelling

Note: Compared with the empty control group ^※ P＜0.05 ^※※ P＜0.01

Table 8 shows that the ear swelling degree of the mice in the 17-hydrogen-9-dehydroandrographolide groups and the dexamethasone group was significantly smaller than that in the control group.

Experimental data 5: Effect of 17-hydrogen-9-dehydroandrographolide on paw swelling of rats induced by carrageenan

40 rats were randomly divided into 5 groups. Give 17-hydro-9-dehydroandrographolide 50mg·kg ^-1 to the low-dose group, 100mg·kg ^-1 to the middle-dose group, and 200mg·kg ^- 1 to the high-dose group twice a day for 3 consecutive days, plus dexamethasone Sodium phosphate injection is administered only before inflammation. After the last administration, each rat was intragastrically given 8ml of normal saline, and 30 minutes later, 0.15ml of 1% carrageenan solution was subcutaneously injected into the middle of the paw of the rat, and 30min, 1h, 2h, 3h, 4h, At 5 hours, the thickness of the left hind paw was measured with a micrometer as an indicator of the swelling degree of the rat paw.

Table 9 Effects of Carrageen on Paw Swelling in Rats

Note: Compared with the empty control group ^※ P＜0.05 ^※※ P＜0.01

The results in table 9 show that, compared with the control group, each dosage group of 17-hydrogen-9-dehydroandrographolide and the dexamethasone group all have obvious inhibitory effect on rat foot swelling caused by carrageenan, and 17-hydrogen-9-dehydroandrographolide When 9-dehydroandrographolide was administered at 200 mg/kg, there was an obvious inhibitory effect within 30 minutes of administration and lasted for 5 hours. The effect of 100 mg/kg was equivalent to that of dexamethasone. There is a quantitative-effect relationship between the dose groups in the same time point in the inhibition of swelling.

Experimental Data 6: Inhibition of Neuraminidase Activity by 17-Hydro-9-Dehydroandrographolide

Get 17-hydrogen-9-dehydroandrographolide, add an appropriate amount of water to dissolve it, and use a neuraminidase inhibitor identification kit to measure the inhibitory effect of 17-hydrogen-9-dehydroandrographolide on neuraminidase (N1). See Table 10 for titers.

(1). Standard curve preparation: a. Add 70 μl of neuraminidase detection buffer to each well of a 96-well fluorescent microplate; b. Add 0, 1, 2, 5, 7.5, 10 μl of H5N1 neuraminidase to each well Acidase; c. Add 0-20 μl Milli-Q water to each well.

(2). Preparation for sample detection: a. Add 70 μl of neuraminidase detection buffer to each well of a 96-well fluorescent microplate; b. Add 10 μl of H5N1 neuraminidase to each well; c. Add 0 μl to each well. ~10 μl of 17-hydrogen-9-dehydroandrographolide sample; d. Add 0~10 μl of Milli-Q water to each well.

(3). Detection steps:

a. Shake and mix for about 1 min;

b. Incubate at 37°C for 2 minutes to fully interact with the inhibitor and H5N1 neuraminidase, and incubate the samples for the standard curve together;

c. Add 10 μl neuraminidase fluorescent substrate to each well;

d. Vibrate and mix for about 1 min;

e. After incubating at 37°C for 20-30 minutes, measure the fluorescence. The excitation wavelength is 360nm and the emission wavelength is 440nm.

(4). Calculate the inhibition percentage of the sample for H5N1 neuraminidase according to the standard curve, and calculate the IC50 of 17-hydrogen-9-dehydroandrographolide for H5N1 neuraminidase after making the concentration curve. 17-hydrogen -9-Dehydroandrographolide achieved an IC50 of 0.34g/L for neuraminidase inhibition. See Table 10.

Table 10.17-Hydro-9-dehydroandrographolide inhibits neuraminidase activity

According to the above experimental results, it can be clearly seen that:

(1). 17-hydrogen-9-dehydroandrographolide can extract effective neuraminidase-inhibiting components;

(2).17-Hydro-9-dehydroandrographolide varies with the dosage, its ability to inhibit neuraminidase activity, that is, the neuraminidase inhibition rate also changes accordingly, and is positively correlated;

(3). It can be seen from the above experiments that 17-hydrogen-9-dehydroandrographolide can inhibit the influenza virus from entering the cell by inhibiting the neuraminidase on the surface of the influenza virus, and inhibit the replication and proliferation of the influenza virus that has entered the cell. , thereby reducing the infection and growth of cells by influenza virus, as well as the prevention and treatment of influenza and its complications.

Medical and pharmaceutical researchers cannot know in advance that 17-hydrogen-9-dehydroandrographolide has the ability to prevent and treat influenza virus infection without performing experiments on inhibiting influenza virus infection, replication, or inhibiting neuraminidase. Good effect on colds.

Experimental data 7: Inhibitory effect of 17-hydrogen-9-dehydroandrographolide on chicken embryo infection by influenza virus

17-Hydro-9-dehydroandrographolide was used to identify 17-Hydro-9-dehydroandrographolide inhibiting the replication of FM1 influenza virus in chicken embryos by using influenza virus subtype A murine lung-adapted strain (FM1) (H1N1) and the ability to suppress.

(1). Inoculate FM1 influenza virus liquid into the allantoic cavity of 10d-old specific pathogen-free chicken embryos, 0.2ml per embryo, and incubate at 37°C for 72h, observe and calculate the infection rate (EID50) of half of the chicken embryos.

(2). Toxic effect of 17-hydrogen-9-dehydroandrographolide on chicken embryos. Sterile physiological saline was used to serially dilute 17-hydrogen-9-dehydroandrographolide and then inoculated at 10 days without specific pathogens. In the allantoic cavity of chicken embryos, 0.2ml per embryo, 6 embryos were inoculated at each concentration, incubated at 37°C, the growth and development of chicken embryos were observed, and the maximum concentration of chicken embryos that could survive for 96 hours was used as the TD of the drug.

(3). The inhibitory effect of 17-hydrogen-9-dehydroandrographolide on influenza virus in chicken embryos is used. Mix 0.1ml of influenza virus liquid with different dilutions of 17-hydrogen-9-dehydroandrographolide , acted at 37°C for 2h, inoculated in the allantoic cavity of 10d-old specific pathogen-free chicken embryos, inoculated 6 embryos in each group, and incubated at 37°C for 72h. Virus challenge amount is 50EID50, set up virus contrast, sterile saline normal control simultaneously, calculate the half effective dose (ED50) of 17-hydrogen-9-dehydroandrographolide to virus inhibition.

(1). The virulence of FM1 influenza virus to chicken embryos was calculated by Reed-Muench method, and its EID50 was 10 ^-4.75 .

(2). After the 17-hydrogen-9-dehydroandrographolide was inoculated into the chicken embryo, its growth and development were basically the same as that of the normal control group. 96h chick embryos survived. Chicken embryos were given 17-hydrogen-9-dehydroandrographolide stock solution and no death of chicken embryos was observed.

So it can be considered that TD0 is 2.40g/L.

(3). The inhibitory effect of 17-hydrogen-9-dehydroandrographolide on influenza virus in chicken embryos is shown in Table 11.

Table 11.17-Hydrogen-9-dehydroandrographolide's inhibitory effect on influenza virus infection of chicken embryos

Compared with the virus control group: ^* P<0.05

As can be seen from Table 11, 17-hydrogen-9-dehydroandrographolide has significant inhibitory effect on influenza virus at 0.0875～0.7g/L (P＜0.05), _ED50 is 0.1005g±0.0042g/L, TI is 58.47±2.27.

Experimental data 8: Effect of 17-hydrogen-9-dehydroandrographolide on FM1 influenza virus

17-hydrogen-9-dehydroandrographolide was used to identify the ability of 17-hydrogen-9-dehydroandrographolide to inhibit the virulence of FM1 influenza virus by using influenza subtype A mouse lung-adapted strain (FM1) (H1N1).

(1). The virulence of FM1 on dog kidney passage cells (MDCK) was determined by the half cell infectious dose (TCID50) micromethod.

(2). The toxicity of 17-hydrogen-9-dehydroandrographolide to MDCK cells was measured by using serum-free DMEM to make serial dilutions of 17-hydrogen-9-dehydroandrographolide and inoculate it on MDCK that had formed a monolayer. In the cell wells, 100 μl per well, 4 wells were repeated for each dilution, and a normal cell control was set at the same time. Place the culture plate in a 37°C, 5% _CO2 incubator for culture, observe the cytopathic changes (CPE) every day for 3 days, record the results as "+~++++", and calculate half of the drug poisoning according to the Reed-Muench method Concentration (TD50) and maximum non-toxic concentration (TD0).

(3). Determination of 17-hydrogen-9-dehydroandrographolide's inhibitory effect on FM1 influenza virus: 5×10 ⁵ /ml of MDCK cells, 100 μl per well, cultured in 96-well plates at 37°C and 5% CO ₂ Cultivate in the box, absorb the culture medium in the wells the next day, add 100TCID50 influenza virus solution, 100μl per well, absorb at 37°C for 1 hour, and then absorb the supernatant. Wash twice with phosphate buffered saline (PBS), take TD0 of the drug as the first well, then serially dilute 17-hydrogen-9-dehydroandrographolide with serum-free DMEM solution, add the above-mentioned infected virus respectively In the cells, a virus control group and a normal control group were set at the same time, cultured in a 37°C, 5% CO ₂ incubator, and the characteristics of MDCK cell lesions produced by influenza virus were observed every day, that is, monolayer cells degeneration and rounding, etc., for 3 consecutive days, calculated The 50% inhibitory concentration (IC50) and therapeutic index (TI) of the drug were calculated. Calculation of TI: TI=TD50/IC50, the greater the TI value, the greater the safety range of the drug. Kruskal-Walis and Mann-Whitney tests were used to compare the differences in cytopathic changes between the test group and the virus control group, and to analyze the correlation between the drug dose and the inhibition rate of virus-infected cells to avoid cytopathic (CPE) to determine whether there is a dose effect. Response relationship.

(4). The toxicity of FM1 influenza virus to MDCK cells was calculated by Reed-Muench method, and its TCID50 was 10 ^-4.96 . (2). TD0 of 17-hydrogen-9-dehydroandrographolide MDCK cells was 1.24g±0.048g/L respectively. (3). After serially diluting 17-hydrogen-9-dehydroandrographolide, 100 TCID50 influenza virus was subjected to an inhibition test, and the IC50 and TI value of the half effective dose of the drug were calculated. The results are shown in Table 12.

IC ₅₀ (g/L) and TI (x±s) of table 12.17-hydrogen-9-dehydroandrographolide to FM1 influenza virus

It can be seen from Table 12 that the IC50 of 17-hydro-9-dehydroandrographolide is low and the TI is high. The effects of 17-hydrogen-9-dehydroandrographolide on inhibiting the cytopathic effect of FM1 influenza virus were enhanced with the increase of drug dose. The correlation analysis of the drug dose and the drug's inhibition rate of CPE showed that there was a significant positive correlation between the dose of 17-hydro-9-dehydroandrographolide and the drug's inhibition rate of CPE.

Experimental data 9: Effect of 17-hydrogen-9-dehydroandrographolide on spleen index and lung index of mice infected with influenza virus FM1 strain

Taking 17-hydrogen-9-dehydroandrographolide and using influenza virus subtype A murine lung-adapted strain (FM1) (H1N1) to identify the effect of 17-hydrogen-9-dehydroandrographolide on mice infected with influenza virus FM1 strain death protection.

(1) Influenza virus strain FM1 was diluted 10 times and inoculated into 10 BALB/C mice in each group, half male and half male. After mild ether anesthesia, each group was given different dilutions of the virus, and each mouse was inoculated with 20 μl nasally. The death of the mice was observed on the 10th day, and the LD50 calculated by the Reed-Muench method was 10 ^-1.52 . Therefore, it is determined that the modeling concentration used in the experiment is 10LD50.

(2) The protective effect of 17-hydrogen-9-dehydroandrographolide on the death of mice infected with influenza virus FM1 strain: normal control group, influenza virus FM1 strain virus control group, 17-hydrogen-9-dehydroandrographolide Esters 0.125g/L, 0.25g/L, 0.5g/L, 1.0g/L dosage groups were administered orally respectively, and the intragastric volume was 0.4ml/monkey. Three days later, mice in each group except the normal control group were infected with 10LD50 influenza virus FM1 strain 20μl/mouse under light ether anesthesia. At the same time, the normal control group was given the same volume of normal saline. The 4 groups that were given medicine continued to take medicine, and the normal control group and the influenza virus FM1 strain virus control group were given normal saline for 8 days, and the dosage was the same as above. Observe the disease of animals and record the number of deaths every day for a total of 14 days, and calculate the death rate (mortality=number of deaths in each group/total number of mice in each group×100%), and the results are shown in Table 13.

(3) Effect of 17-hydrogen-9-dehydroandrographolide on lung index of mice infected with influenza virus FM1 strain: normal control group, influenza virus FM1 strain virus control group, 17-hydrogen-9-dehydroandrographolide Esters 0.125g/L, 0.25g/L, 0.5g/L, 1.0g/L dosage groups were administered orally respectively, and the intragastric volume was 0.4ml/monkey. Three days later, except the normal control group, mice were infected with 1.0 LD50 influenza virus FM1 strain nasally at 20 μl/mouse under light ether anesthesia. At the same time, the normal control group was given the same volume of normal saline. The 4 groups that were given medicine continued to take medicine, and the normal control group and the influenza virus FM1 strain virus control group were given normal saline for 8 days, and the dosage was the same as above. The mice were killed on the 8th day after virus infection, body weight was weighed, the lungs were taken to weigh the lungs, and the lung index was calculated (lung index=lung mass/body mass×100%); in addition, the spleen was taken to weigh the spleen, and the spleen index (spleen Index=spleen mass/body mass×100%), the results are shown in Table 14.

Table 13.17-Hydrogen-9-dehydroandrographolide protects against death of influenza virus FM1 strain in mice

Note: ※※P＜0.01 VS influenza virus model group ※P＜0.05VS influenza virus model group

Table 14.17-Hydrogen-9-dehydroandrographolide affects the spleen index and lung index of mice infected with influenza virus FM1 strain

Note: #P＜0.05VS normal control group Note: ※※p＜0.001VS influenza virus model group※p＜0.05VS influenza virus model group

(1). It can be seen from Table 13 that 17-hydrogen-9-dehydroandrographolide at 0.25-1.0 g/L has a significant protective effect on mice infected with influenza virus (p<0.01).

(2). It can be seen from Table 14 that 17-hydrogen-9-dehydroandrographolide at 0.5-1.0 g/L has a significant effect on the lung index inhibition rate of influenza virus-infected mice (p<0.01).

Experimental data 10: Effect of 17-hydrogen-9-dehydroandrographolide on sepsis in mice

1.17-Hydro-9-dehydroandrographolide significantly improved the survival rate of septic mice

Andrographolide is because of extremely poor water solubility, so adopt 0.9% sodium carboxymethylcellulose aqueous solution to be mixed with suspension and carry out intragastric administration (30mg/kg), sulfonate is directly made into clear solution with PBS and carries out tail vein injection (10mg/kg). kg). Inject 5mg/kg LPS intraperitoneally to establish a model, observe the number of surviving mice within 60 hours, and calculate the survival rate. The mice in the model group died at 16 hours after modeling, and the survival rate after 60 hours was 25% (as shown in Table 15), and the survival rate of the andrographolide administration group was 50%. The survival rate of the ester administration group was 62.5%. In comparison, the effect of improving the survival rate of 17-hydrogen-9-dehydroandrographolide (abbreviated in the following table: dehydroandrographolide) is better than that of the andrographolide group. Slightly better, but at a lower dose.

Table 15.17-Hydrogen-9-dehydroandrographolide on the survival rate of LPS-induced sepsis mice

2.17-Hydrogen-9-dehydroandrographolide significantly inhibited the increase of inflammatory factors in the serum of septic mice BALB/C mice were intraperitoneally given 30 mg/kg of andrographolide, and 17-hydrogen-9-dehydroandrographolide was injected into the tail vein Andrographolide 1, 3, 10 mg/kg, and intraperitoneal injection of 5 mg/kg LPS at the same time, 3 mice were sacrificed at 2, 5, 8, and 12 hours after the modeling and administration, and blood was taken from the orbit to measure the inflammation in the serum The contents of factors TNF-α and IL-1β are shown in Table 16. After 2 hours of LPS injection, the contents of TNF-α and IL-1β in the serum of the mice in the model group reached the peak, which were 4815pg/ml and 391pg/ml respectively, and 17-hydrogen-9-dehydroandrographolide was administered for 2 hours. Significantly inhibited TNF-α to 3699pg/ml, and significantly inhibited the release of IL-1β at 5h. The effect of andrographolide for 8 hours can significantly inhibit the increase of TNF-α. The experimental results showed that compared with andrographolide, the inhibitory effect of the sulfonate on the inflammatory factors in the serum of septic mice was quicker, and the inhibitory effect was more significant.

Table 16.17-Hydrogen-9-dehydroandrographolide time- and dose-dependent reduction of inflammatory factors in serum of LPS-induced sepsis mice

TNFα (pg/ml)

IL1β(pg/ml)

n=3, ^* p<0.05vs model group, #p<0.05vsandrographolide group.

3.17-Hydro-9-dehydroandrographolide significantly inhibited liver injury in septic mice

Sepsis is a disease that causes damage to multiple organs, and the liver is one of the main organs damaged. BALB/C mice were given 30 mg/kg of andrographolide by intragastric administration, 10 mg/kg of 17-hydrogen-9-dehydroandrographolide was injected into the tail vein, and 5 mg/kg of LPS was injected intraperitoneally at 2 hours, 5 hours, 8 hours and 12 hours respectively. Blood was collected to determine the levels of ALT and AST in serum. As shown in Table 17, after the administration of LPS, ALT and AST in the serum of the model mice continued to increase, and after 2 hours of administration of 17-hydro-9-dehydroandrographolide, the content of ALT/AST decreased to some extent, and after 12 hours, it was the same as The ALT/AST of the model group had a significant difference, and the inhibitory effect of andrographolide on ALT/AST was slightly worse than that of 17-hydrogen-9-dehydroandrographolide. RT-PCR assayed the mouse RNA levels of various inflammatory factors in the liver tissue and found that ifn-γ, il-6, tnf-α, il-β, cox-2 mRNA were all significantly increased under LPS stimulation, and 17-hydrogen-9- The increase of ifn-γ, il-6, tnf-α, il-β, cox-2 mRNA can be significantly inhibited by the administration of dehydroandrographolide and andrographolide for 5h and 8h. The experimental results also showed that compared with andrographolide, the inhibitory effect of sulfonate on liver injury in septic mice was quicker, and the inhibitory effect was more significant.

Table 17.17-Hydrogen-9-dehydroandrographolide inhibits liver injury in LPS-induced sepsis mice

AST (karmen units)

ALT (karmen units)

n=3, ^* p<0.05, vs model group.

4. Summary

Both andrographolide and 17-hydrogen-9-dehydroandrographolide can significantly improve the survival rate of LPS-induced sepsis mice, time- and dose-dependently reduce serum levels of TNF-α, IL-1β, ALT, The content of AST can inhibit the increase of inflammatory factor mRNA level in liver tissue. 17-Hydro-9-dehydroandrographolide acts faster than andrographolide, and the effect is better. The experimental results show that after the modification of andrographolide into a sulfonate, its water solubility is better, the onset of administration is quick, and the improvement of sepsis in mice is also enhanced.

Claims (25)

1. a preparation method of a compound represented by general formula (I) or a salt thereof, the chemical name of the compound or a salt thereof is 17-hydrogen-9-dehydroandrographolide or a salt thereof, The preparation method of described compound or salt thereof, comprises the following steps: (1) Add a solvent to the reaction kettle, add andrographolide to dissolve it, then add a sulfonating agent to carry out the sulfonation reaction, adjust the temperature of the reaction kettle at 4.5-36.5°C, and perform the sulfonation reaction for 0.5-29.5 hours; (2) andrographolide solution adopts the mode of slowly dripping, spraying or ventilating to add sulfonating agent under the situation of stirring, and described sulfonating agent is concentrated sulfuric acid glacial acetic acid, chlorosulfonic acid or sulfur trioxide, when described When concentrated sulfuric acid and glacial acetic acid are used as the sulfonating agent, 1g of andrographolide is sulfonated with 1g to 10g of concentrated sulfuric acid and glacial acetic acid, and the concentrated sulfuric acid and glacial acetic acid are made of the following weight ratio: 80% to 20% , Glacial acetic acid 20%～80%; When described sulfonating agent adopts chlorosulfonic acid, 1g andrographolide is sulfonated with 0.2g～5g chlorosulfonic acid; When described sulfonating agent adopts sulfur trioxide, 1g andrographolide Add 0.2 liters to 5 liters of sulfur trioxide with a volume concentration of 1% to 3% for sulfonation; and when the sulfonating agent is added slowly or by spraying, the addition rate is controlled at 1.25ml to 4.65ml /min/1g andrographolide, when the sulfonating agent is added by feeding gas, the feeding speed is controlled at 0.026 liters to 0.27 liters/min/1g andrographolide, after the sulfonation reaction, the chemical name is 17-hydrogen -A mixture of reactants and unreacted substances of 9-dehydroandrographolide; (3) The mixture is subjected to solvent extraction and crystallization to obtain the final chemical name of 17-hydrogen-9-dehydroandrographolide; or The mixture is put into a saturated saline solution and crystallized to obtain the final chemical name of 17-hydrogen-9-dehydroandrographolide or its salt; or The mixture was adjusted to pH 7 with alkaline solution, separated by macroporous adsorption resin, ODS or SephadexLH-20 column chromatography, using water: ethanol or methanol as eluent, gradient elution, collected eluent components, crystallized, to obtain the final chemical name of 17-hydro-9-dehydroandrographolide or a salt thereof; or The mixture is jointly treated by the aforementioned two or more steps to obtain the final chemical name of 17-hydrogen-9-dehydroandrographolide or its salt. 2. the preparation method of claim 1, the described solvent that adds in reactor is one or both of acetic anhydride or glacial acetic acid, and 1g andrographolide dissolves with 3g～12g described solvent. 3. The preparation method according to claim 2, wherein 1 g of andrographolide is dissolved with 4 g to 9 g of said solvent. 4. the preparation method of claim 1, described solvent is acetic anhydride and glacial acetic acid, and described acetic anhydride, glacial acetic acid are made by following weight ratio: acetic anhydride 80%～20%, glacial acetic acid 20%～80% . 5. the preparation method of claim 4, wherein acetic anhydride 62%, glacial acetic acid 38%. 6. The preparation method of claim 1, wherein 1 g of andrographolide uses 1.5 g to 5 g of concentrated sulfuric acid glacial acetic acid, and the weight ratio of concentrated flowing acid to glacial acetic acid is 1:1. 7. The preparation method of claim 1, wherein 1 g of andrographolide is passed through 0.3 liters to 3 liters of sulfur trioxide with a volume concentration of 1.5% to 2.5% for sulfonation. 8. The preparation method of claim 1, wherein 1 g of andrographolide is sulfonated with 0.3 g to 3.5 g of chlorosulfonic acid. 9. the preparation method of claim 1, described saturated saline solution adopts sodium chloride or potassium chloride saturated solution, and described alkali solution adopts 5%～50% sodium hydroxide or potassium hydroxide or ammoniacal solution below 25% . 10. The preparation method of claim 1, wherein the temperature of the reaction kettle is 9-25° C., and the sulfonation reaction time should be controlled within 1-3 hours. 11. The preparation method according to claim 1, wherein said sulfonating agent is concentrated sulfuric acid glacial acetic acid or chlorosulfonic acid, and is added by slowly dripping and spraying, and the adding speed is controlled at 1.8ml～3.6ml/min/ 1 g of andrographolide. 12. The preparation method according to claim 1, wherein the sulfonating agent is sulfur trioxide, which is added by feeding gas, and the feeding speed is controlled at 0.05 liters to 0.12 liters/min/1g andrographolide. 13. The preparation method of claim 1, wherein in the gradient elution, the proportion of water is 80-20%, and the proportion of ethanol or methanol is 20-80%. 14. the preparation method of claim 1, it adopts high performance liquid chromatography to measure described chemical by name 17-hydrogen-9-dehydroandrographolide or its salt, and assay condition is as follows: Filler with octadecylsilane bonded silica gel; The detection wavelength is 225nm; The column temperature is 25°C; The number of theoretical plates is not less than 10,000 based on the chemical name of 17-hydro-9-dehydroandrographolide or its salts; Preparation of reference substance solution The chemical name 17-hydrogen-9-dehydroandrographolide or its salt is made into reference substance through purity inspection and content standardization, and dried in a phosphorus pentoxide vacuum desiccator. Accurately weigh an appropriate amount, add water to make a solution of the required concentration, and obtain; Preparation of the test solution Precisely weigh 5mg of the sample, put it in a 50ml measuring bottle, add water to dilute to the mark, shake well, and get final product; For elution, acetonitrile is used as mobile phase A, and potassium dihydrogen phosphate buffer is used as mobile phase B. The potassium dihydrogen phosphate buffer is 1.361 g of potassium dihydrogen phosphate and 1.0 g of sodium heptane-1-sulfonate per 1000 ml of water. g, carry out gradient elution according to the following conditions, and run for 70 minutes; From 0 to 10 minutes, the proportion of acetonitrile is 8.0%, and the proportion of potassium dihydrogen phosphate buffer is 92.0%; From 10 to 60 minutes, the proportion of acetonitrile increased from 8.0% to 35.0%, and the proportion of potassium dihydrogen phosphate buffer decreased from 92.0% to 65.0%; At 60-62 minutes, the proportion of acetonitrile increased from 35.0% to 70.0%, and the proportion of potassium dihydrogen phosphate buffer decreased from 65.0% to 30.0%; At 62 to 70 minutes, keep acetonitrile: potassium dihydrogen phosphate buffer solution at a ratio of 70.0%: 30.0% for elution; Under the above-mentioned conditions, 17-hydrogen-9-dehydroandrographolide or its salt has a chromatographic peak at about 53 minutes of retention time, Determination method Precisely draw the reference substance solution and the test solution respectively, inject it into the liquid chromatograph, measure it, and get it. 15. The preparation method according to any one of claims 1 to 14, wherein the chemical name is 17-hydro-9-dehydroandrographolide or its salt for the preparation of antipyretic drugs. 16. The preparation method as claimed in claim 15, wherein the chemical name is 17-hydrogen-9-dehydroandrographolide or its salt on the antipyretic effect of endotoxin-induced pyrexia. 17. The preparation method according to claim 15, said chemical name is 17-hydrogen-9-dehydroandrographolide or its salt on the antipyretic effect of dry yeast. 18. The preparation method according to any one of claims 1 to 14, wherein the chemical name is 17-hydro-9-dehydroandrographolide or its salt for the preparation of anti-inflammatory drugs. 19. The preparation method according to claim 18, said chemical name is 17-hydrogen-9-dehydroandrographolide or its salt on sepsis. 20. The preparation method according to claim 18, said chemical name is 17-hydrogen-9-dehydroandrographolide or its salt anti-inflammatory effect on mouse auricle swelling caused by p-xylene. 21. The preparation method according to claim 18, said chemical name is 17-hydrogen-9-dehydroandrographolide or its salt on the anti-inflammatory effect of carrageenan-induced paw swelling in rats. 22. The preparation method according to any one of claims 1 to 14, wherein the chemical name is 17-hydro-9-dehydroandrographolide or its salt for the preparation of antiviral drugs. 23. The preparation method according to claim 22, said chemical name is 17-hydrogen-9-dehydroandrographolide or a salt thereof for inhibiting neuraminidase. 24. The preparation method according to claim 22, said chemical name is 17-hydrogen-9-dehydroandrographolide or a salt thereof for inhibiting influenza virus. 25. The preparation method according to claim 22, said chemical name is 17-hydrogen-9-dehydroandrographolide or a salt thereof for inhibiting influenza virus FM1.