CN103159717A - 17-hydrogen-9-dehydro-andrographolidume-3, 19-disulfate, preparation method and purposes of medicine prepared by the same - Google Patents

17-hydrogen-9-dehydro-andrographolidume-3, 19-disulfate, preparation method and purposes of medicine prepared by the same Download PDF

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CN103159717A
CN103159717A CN201210109518XA CN201210109518A CN103159717A CN 103159717 A CN103159717 A CN 103159717A CN 201210109518X A CN201210109518X A CN 201210109518XA CN 201210109518 A CN201210109518 A CN 201210109518A CN 103159717 A CN103159717 A CN 103159717A
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hydrogen
rographolide
salt
sulfate
dehydrogenation rographolide
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CN103159717B (en
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吕武清
谢宁
唐春山
杨小玲
刘地发
程帆
李志勇
刘荛琦
廖祝元
刘艳红
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JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
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Abstract

The invention discloses 17-hydrogen-9-dehydro-andrographolidume-3, 19-disulfate or salt thereof, a preparation method thereof, and antipyretic, anti-inflammatory and antiviral purposes of the 17-hydrogen-9-dehydro-andrographolidume-3, 19-disulfate or the salt thereof. The 17-hydrogen-9-dehydro-andrographolidume-3, 19-disulfate or the salt thereof formed by the preparation method does not change chemical properties of andrographolidume, the 17-hydrogen-9-dehydro-andrographolidume-3, 19-disulfate or the salt thereof has the advantages of being good in water-solubility, high in heat stability, small in hemoclasis, and the like, and pharmacoactive effects of andrographolidume pure natural medicine are guaranteed to the greatest extent. In addition, medicine and pharmaceutical research workers cannot know the good purposes of 17-hydrogen-9-dehydro-andrographolidume-3, 19-sodium disulfate in advance on the premise that related experiments are not conducted.

Description

17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate compound, preparation method and prepare pharmaceutical use
Technical field
The present invention relates to a kind of 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate compound, preparation method and prepare pharmaceutical use.
Background technology
The dry aerial parts that Herba Andrographis is acanthaceous plant Herba Andrographis Andrographis paniculata (Burm.f.) Nees, chemical composition and pharmacological evaluation show, the activeconstituents of Herba Andrographis be take diterpene-kind compound that rographolide is representative as main [State Administration of Traditional Chinese Medicine. the 7th of China's book on Chinese herbal medicine. Shanghai: Shanghai science tech publishing house, 1999:439], the structure of rographolide is:
Figure BSA00000702067400011
Molecular formula: C 20h 30o 5, be the crystallization of colourless square square, m.p.230-232 ℃, [α] 0 20-126 ° of (c0.2, H 2o).Flavor is extremely bitter, dissolves in methyl alcohol, ethanol, propyl alcohol, pyridine, is slightly soluble in chloroform, ether, is insoluble in water and sherwood oil.Therefore, the common film-making agent of oral preparations, capsule, dripping pill, soft capsule; Water insoluble because of rographolide, brought difficulty to preparing liquid preparation, at present, existing several different methods is used to transform rographolide and becomes various derivatives, to improve the water-soluble of rographolide.Be prepared into the injection liquid of various soluble derivatives after general extraction rographolide, as with S-WAT add sulfuric acid or with sodium bisulfite generation addition reaction, the water-soluble sulfonate made, rographolidum Natrii Bisulfis (LIANBIZHI ZHUSHEYE), the succinic acid half-ester monopotassium salt, rographolide is through esterification, dehydration, become salt refining to form 14-deshydroxy-11, 12-bis-dehydrogenation rographolides-3, 19-disuccinic acid half ester k-na salt (' Tanhuning ' injection) or 14-deshydroxy-11, 12-bis-dehydrogenation rographolides-3, 19-disuccinic acid half ester monopotassium salt, but above-mentioned salt solubleness in water, stability is not good especially.
Summary of the invention
One object of the present invention is to provide a kind of general formula (I) represented compound or its salt, its chemistry 17-hydrogen by name-9-dehydrogenation rographolide-3, and 1-di-sulfate or its salt,
Figure BSA00000702067400021
R 1, R 2can be H, sodium, potassium or NH 4.
Another object of the present invention is to provide the preparation method of the compound or its salt of a kind of general formula (I), comprises the following steps:
[1] add solvent in reactor, add rographolide to make its dissolving, then add sulphonating agent to carry out sulfonation reaction, conditioned reaction still temperature is at 3.5～38.5 ℃, sulfonation reaction 0.5～27.5 hour;
[2] rographolide solution is in the situation that stirring adopts the mode that slowly drips, sprays or ventilate to add sulphonating agent, and when adopting the mode that slowly drips or spray to add sulphonating agent, add speed control at 1.4ml～4.8ml/min/1g rographolide, when the mode that passes into gas when employing adds sulphonating agent, pass into speed control at 0.015 liter～0.21 liter/min/1g rographolide, obtain 17-hydrogen-9-dehydrogenation rographolide-3, the reactant of 19-di-sulfate and the mixture of unreacted reactant after sulfonation reaction;
[3] mixture is through solvent extraction, crystallization, obtains final 17-hydrogen-9-dehydrogenation rographolide-3, the 19-di-sulfate; Perhaps
Mixture to saturated salt solution, crystallization, obtain final 17-hydrogen-9-dehydrogenation rographolide-3,1-di-sulfate or its salt; Perhaps
Alkaline solution adjust pH to 7 for mixture, through macroporous adsorbent resin, ODS or Sephadex LH-20 column chromatography for separation, take water: ethanol or methyl alcohol are elutriant, gradient elution, collect eluant component, crystallization, final 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt; Perhaps
Mixture successively passes through aforementioned two or more step co-treatment, obtains final 17-hydrogen-9-dehydrogenation
Rographolide-3,19-di-sulfate or its salt.
Preferably, the described solvent added in reactor is one or both of aceticanhydride or Glacial acetic acid, and the described dissolution with solvents of 3g～12g for the 1g rographolide is preferred, the described dissolution with solvents of 4g～9g for the 1g rographolide.
Preferably, described solvent is aceticanhydride and Glacial acetic acid, and described aceticanhydride, Glacial acetic acid are made by following weight proportion: aceticanhydride 80%～20%, Glacial acetic acid 20%～80%, and preferred, wherein aceticanhydride 62%, Glacial acetic acid 38%.
Preferably, described sulphonating agent adopts the vitriol oil and Glacial acetic acid, 1g for the 1g rographolide～10g vitriol oil Glacial acetic acid carries out sulfonation, the described vitriol oil and Glacial acetic acid are made by following weight proportion: the vitriol oil 80%～20%, Glacial acetic acid 20%～80%, preferably, 1.5g for the 1g rographolide～5g vitriol oil Glacial acetic acid, and the acid of dense stream is 1: 1 with the Glacial acetic acid part by weight.
Preferably, described sulphonating agent adopts sulphur trioxide, the 1g rographolide passes into the sulphur trioxide that 0.2 liter～5 liters volumetric concentrations are 1%～3% and carries out sulfonation, and preferred, the 1g rographolide passes into the sulphur trioxide that 0.3 liter～3 liters volumetric concentrations are 1.5%～2.5% and carries out sulfonation.
Preferably, described sulphonating agent adopts chlorsulfonic acid, and 0.2g for the 1g rographolide～5g chlorsulfonic acid carries out sulfonation, and preferred, 0.3g for the 1g rographolide～3.5g chlorsulfonic acid carries out sulfonation.
Preferably, described saturated salt solution adopts sodium-chlor or saturated potassium chloride solution, and described alkaline solution adopts 5%～50% sodium hydroxide or potassium hydroxide or 25% following ammonia soln.
Preferably, wherein said temperature of reaction kettle is at 6～24 ℃, and the described sulfonation reaction time should be controlled at 0.5～3 hour.
Preferably, wherein said sulphonating agent is vitriol oil Glacial acetic acid or chlorsulfonic acid, and adopts the mode that slowly drips, sprays to add, and adds speed control at 2.2ml～3.8ml/min/1g rographolide.
Preferably, wherein said sulphonating agent is sulphur trioxide, and adopts the mode that passes into gas to add, and passes into speed control at 0.06 liter～0.14 liter/min/1g rographolide.
Preferably, in wherein said gradient elution, the ratio of water is 80～20%, and the ratio of ethanol or methyl alcohol is 20～80%.
A further object of the present invention is to provide the measuring method of the compound or its salt of a kind of general formula (I), and it adopts the described 17-hydrogen of high effective liquid chromatography for measuring-9-dehydrogenation rographolide-3,19-di-sulfate or its salt, and condition determination is as follows:
Take octadecylsilane chemically bonded silica as weighting agent;
The detection wavelength is 225nm;
Column temperature is 25 ℃;
Number of theoretical plate is pressed 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate or its salt calculate and is not less than 10000;
The preparation of reference substance solution is by described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are made reference substance by purity test and content mark, and dry in the Vanadium Pentoxide in FLAKES vacuum drier, accurately weighed appropriate, add water and make the solution of desired concn, obtain;
The preparation precision of need testing solution takes sample 5mg, puts in the 50ml measuring bottle, is diluted with water to scale, shakes up, and obtains;
Wash-out be take acetonitrile as mobile phase A, take potassium phosphate buffer as Mobile phase B, and described potassium phosphate buffer is to add potassium primary phosphate 1.361g and heptane-1-sodium sulfonate 1.0g in every 1000ml water, by following condition, carries out gradient elution, moves 70 minutes;
In the time of 0～10 minute, the acetonitrile ratio is 8.0%, and the potassium phosphate buffer ratio is 92.0%;
In the time of 10～60 minutes, the acetonitrile ratio rises to 35.0% by 8.0%, and the potassium phosphate buffer ratio drops to 65.0% by 92.0%;
In the time of 60～62 minutes, the acetonitrile ratio rises to 70.0% by 35.0%, and the potassium phosphate buffer ratio drops to 30.0% by 65.0%;
In the time of 62～70 minutes, keep acetonitrile: potassium phosphate buffer was with 70.0%: 30.0% ratio wash-out;
Under these conditions, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt approximately have a chromatographic peak in 5 minutes in retention time.
Assay method is accurate reference substance solution and the need testing solution drawn respectively, and the injection liquid chromatography, measure, and obtains.
Another object of the present invention is to provide a kind of 17-hydrogen-9-dehydrogenation rographolide-3, the preparation of 19-di-sulfate or its salt, said preparation is 17-hydrogen claimed in claim 1-9-dehydrogenation rographolide-3, and 19-di-sulfate or its salt are made with acceptable carrier pharmaceutically.
Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate or its salt are for the preparation of the purposes of analgesic medicine.
Preferably, described 17-hydrogen-9-dehydrogenation rographolide-3, the refrigeration function of 19-di-sulfate or its salt pair endotoxin pyrogenic.
Preferably, described 17-hydrogen-9-dehydrogenation rographolide-3, the refrigeration function of 19-di-sulfate or its salt pair dry yeast pyrogenicity.
Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate or its salt are for the preparation of the purposes of the medicine of anti-inflammatory.
Preferably, described 17-hydrogen-9-dehydrogenation rographolide-3, the drug effect of 19-di-sulfate or its salt pair septicemia.
Preferably, described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt p-Xylol induced mice auricle edema anti-inflammatory action.
Preferably, described 17-hydrogen-9-dehydrogenation rographolide-3, the anti-inflammatory action of rat paw edema due to 19-di-sulfate or its salt on Carrageenan.
Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate or its salt are for the preparation of the purposes of antiviral drug.
Preferably, described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for suppressing neuraminidase.
Preferably, described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for suppressing influenza virus.
Preferably, described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for suppressing influenza virus FM1.
17-hydrogen provided by the invention-9-dehydrogenation rographolide-3,19-di-sulfate or its salt, in water, solubleness is very good and stability is very high, be applicable to very much practical application, can be applied to various common type, as tablet, capsule, soft capsule, dispersible tablet, oral liquid, particle, chewable tablet, orally disintegrating tablet, dripping pill, slow releasing tablet, controlled release tablet, slow releasing capsule, controlled release capsule.Can certainly make liquid preparation as syrup, injection liquid etc., particularly make injection and overcome the low defect of oral pharmaceutical biological utilisation.
On the other hand, 17-hydrogen provided by the present invention-9-dehydrogenation rographolide-3, the preparation method of 19-di-sulfate or its salt is simple and convenient, mild condition, productivity are high, can prepare easily 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt.
What is more important, the present invention is by thousands of up to a hundred performing creative labours, finally determined suitable solvent, and the important technical parameter of creative sulphonating agent and sulfonation reaction thereof, the processing mode of sulphonating agent and add determining of complex relationship between speed for example, and the determining etc. of suitable numerical range, thereby just finally obtained required reactant.On this basis, more further adopt creative purification technique to carry out purifying, thereby the invention provides to prepare, form 17-hydrogen of the present invention-9-dehydrogenation rographolide-3,19-di-sulfate or its salt under multiple different condition.
The present invention is compared with the prior art and shows: the 17-hydrogen that adopts preparation method of the present invention to form-9-dehydrogenation rographolide-3, and 19-di-sulfate or its salt, can not change the original chemical attribute of rographolide fully; And 17-hydrogen of the present invention-9-dehydrogenation rographolide-3,19-di-sulfate or its salt compared with prior art have the characteristics such as good water solubility, thermostability is high, hemolytic action is little.Guaranteed to greatest extent the pharmacologically active effect of rographolide pure natural medical.
Known through the experimental data contrast, after giving intracellular toxin 1h, blank group Healthy Rabbits body temperature average has risen, and after lasting rising 3h, starts gradually to descend.Compare low dose group 50mgkg with the blank group -117-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium 1～2h shows obvious inhibition rabbit temperature rise effect, middle dosage group 100mgkg -1, high dose group 200mgkg -117-hydrogen-9-dehydrogenation rographolide-3, show the effect of extremely strong inhibition rabbit fervescence in 19-di-sulfate sodium 1～2h, also demonstrate the rabbit fervescence effect that suppresses during 4h; In 3 drug study groups with 100mgkg -1the above antipyretic effect of dosage is best.Show 50～200mgkg -117-hydrogen-9-dehydrogenation rographolide-3, the rabbit fervescence that 19-di-sulfate sodium induced by endotoxin causes all has cooling effect, has obtained unforeseeable technique effect.
Known through the experimental data contrast, while giving dry yeast 1h, make blank group healthy rat body temperature continue the 6h that rises.Compare 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium 100,200mgkg with the blank group -1show obvious inhibition rat temperature rising effect in 1～4h, obtained unforeseeable technique effect.
Known through the experimental data contrast, rographolide and 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium can significantly improve the survival rate of the septicemia mouse that LPS induces, time, dose-dependently reduce TNF-α in the septicemia mice serum, IL-1 β, ALT, the content of AST, the sick rising that suppresses inflammatory factor mRNA level in liver organization.And 17-hydrogen-9-dehydrogenation rographolide-3, the onset of 19-di-sulfate sodium is fast than rographolide, better effects if.After experimental result shows that rographolide is transformed into sulfonated bodies, it is water-soluble better, and administration is rapid-action, and the improvement effect of mouse septicemia also is enhanced, and has obtained unforeseeable technique effect.
Known through the experimental data contrast, 17-hydrogen-9-dehydrogenation rographolide-3, each medication group of 19-di-sulfate sodium, Dexamethasone group mice auricle swelling degree are significantly less than control group, have obtained unforeseeable technique effect.
Known through the experimental data contrast, with the control group ratio, 17-hydrogen-9-dehydrogenation rographolide-3, due to each dosage group of 19-di-sulfate sodium and the equal on Carrageenan of Dexamethasone group, the rat swollen feet has obvious restraining effect, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium obvious restraining effect occurs and continues 5 hours in administration 30min when 200mg/kg, 100mg/kg effect and effects of dexamethasone are suitable, 17-hydrogen-9-dehydrogenation rographolide-3, at same time point, the restraining effect of swelling is had to a certain amount of effect relationship between three dosage groups of 19-di-sulfate sodium, obtained unforeseeable technique effect.
Known through the experimental data contrast, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium can extract effective inhibition neuraminic acid enzyme component; And 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium is along with the using dosage size variation, and it suppresses the ability of neuraminic acid enzymic activity, i.e. also corresponding changing of the height of neuraminic acid enzyme inhibition rate, and become positive correlation.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium can be by suppressing influenza virus surface neuraminidase, and then suppress that influenza virus enters the cell the inside, the influenza virus that suppresses to have entered the cell the inside copies, breeds, thereby reduced infection, the growth of influenza virus to cell, and prevention and treatment influenza and complication thereof, obtained unforeseeable technique effect.
Known through the experimental data contrast, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium has significant restraining effect (P<0.05), ED at 0.075～0.6g/L infected by influenza 50for 0.0918g ± 0.0052g/L, TI is 59.88 ± 1.96.17-hydrogen-9-dehydrogenation rographolide-3, the IC50 of 19-di-sulfate sodium is low, and TI is high.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium suppresses the cytopathogenic effect of FM1 influenza virus all to be strengthened along with the increase of drug dose.The correlation analysis that drug dose and medicine are carried out the inhibiting rate of CPE shows, 17-hydrogen-9-dehydrogenation rographolide-3, and the dosage of 19-di-sulfate sodium and medicine are to being obvious positive correlation between the CPE inhibiting rate.
Medical science and study of pharmacy personnel can't learn in advance 17-hydrogen-9-dehydrogenation rographolide-3 in advance under the prerequisite of not doing related experiment, and 19-di-sulfate sodium has above-mentioned good purposes.
The accompanying drawing explanation
Fig. 1: 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium hydrogen nuclear magnetic resonance spectrogram.
Fig. 2: 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium carbon-13 nmr spectra figure.
Fig. 3: 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium mass spectrum.
Fig. 4: 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium wire sexual intercourse figure.
Embodiment
Embodiment 1
Get creat lactone, add 5.3 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.2ml, slowly drip the sulfuric acid ice acetic acid of 4.3 times of amount equal proportions, mix, conditioned reaction still temperature is at 15 ℃, place and within 90 minutes, make sulfonation, add the equivalent purified water, pour into again in saturated nacl aqueous solution, again through macroporous adsorbent resin, the ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium, its sodium salt molecular formula: C 20h 28na 2o 11s 2, molecular weight: 554.
The reaction formula example:
Figure BSA00000702067400081
17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium physico-chemical property and spectral data:
17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium: molecular formula C 20h 28na 2o 11s 2, white powder; ESIMS m/z:531[M-Na] -, HRESIMS:m/z m/z 531.1014[M-Na] -(cald.for C 20h 28o 11s 2na, 531.0971); 1h NMR and 13c NMR data.
1NMR(D 2O，600MHz)δ：1.75～1.83(m，1H，H-1α)，1.17(td，J＝13.6，3.5Hz，1H，H-1β)，1.75～1.83(m，1H，H-2α)，1.94～2.05(m，1H，H-2β)，3.97(dd，J＝12.0，4.5Hz，1H，H-3)，1.28(brd，J＝12.0，1H，H-5)，1.56(m，1H，H-6α)，175～1.83(m，1H，H-6β)，1.75～1.83(m，1H，H-7α)，1.94～2.05(m，1H，H-7β)，3.05(dd，J＝17.7，8.6Hz，1H，H-11α)，3.19(dd，J＝17.7，4.9Hz，H-11β)，6.81(ddd，J＝8.0，5.0，1.4Hz，1H，H-12)，5.13(dd，J＝6.0，1.8z，1H，H-14)，4.23(dd，J＝10.6，1.8Hz，1H，H-15α)，4.50(dd，J＝10.6，6.0Hz，1H，H-15β)，1.52(s，3H，H-17)，1.12(s，3H，H-18)，3.96(d，J＝10.0Hz，1H，H-19α)，4.20(d，J＝10.0Hz，1H，H-19β)，1.00(s，1H，H-20)
13C-NMR(D 2O，150MHz)δ：32.2(C-1)，22.0(C-2)，84.7(C-3)，39.3(C-4)，49.5(C-5)，17.5(C-6)，31.7(C-7)，128.1(C-8)，133.5(C-9)，36.0(C-10)，26.0(C-11)，148.6(C-12)，124.0(C-13)，63.0(C-14)，73.1(C-15)，171.0(C-16)，16.5(C-17)，19.7(C-18)，67.7(C-19)，16.5(C-20)。
Embodiment 2
Get creat lactone, add 4.2 times of amount aceticanhydrides to make to dissolve, under agitation, with the speed of 1g rographolide per minute 2.0ml, the atomization spray adds the sulfuric acid (52%) and Glacial acetic acid (48%) of 3.7 times of amounts, mixes, conditioned reaction still temperature, at 20 ℃, is placed and within 80 minutes, is made sulfonation.In reactant impouring saturated nacl aqueous solution, throw out is respectively with saturated nacl aqueous solution, water washing, and solid substance refluxes with chloroform, insolubles adds dehydrated alcohol to be made to dissolve, and removes alcohol insoluble solids, reclaims ethanol, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium.
Embodiment 3
Get creat lactone, add 5.4 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, speed with 1g rographolide per minute 1.8ml, the atomization spray adds the sulfuric acid (53%) and Glacial acetic acid (47%) of 4.3 times of amounts, mix, conditioned reaction still temperature, at 17 ℃, is placed and within 100 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 46%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 85% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium.
Embodiment 4
Get creat lactone, add 4.6 times of amount aceticanhydrides (53%) and Glacial acetic acid (47%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.3ml, slowly drip the sulfuric acid (51%) and Glacial acetic acid (49%) of 3.7 times of amounts, mix, conditioned reaction still temperature, at 16 ℃, is placed and within 110 minutes, is made sulfonation.Add 95% ethanol to make to reach more than 85% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, Sephadex LH-20 column chromatography for separation, with water: the methyl alcohol gradient elution, the ratio of water is 80～20%, the ratio of methyl alcohol is 20～80%, collect eluant component, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3, the 19-di-sulfate.
Embodiment 5
Get creat lactone, add 5.5 times of amount aceticanhydrides (59%) and Glacial acetic acid (41%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.6ml, slowly drip the sulfuric acid (52%) and Glacial acetic acid (48%) of 3.7 times of amounts, mix, conditioned reaction still temperature, at 19 ℃, is placed and within 100 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 32%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 85% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate potassium.
Embodiment 6
Get creat lactone, add 5.6 times of amount aceticanhydrides (54%) and Glacial acetic acid (46%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.5ml, slowly drip the sulfuric acid (53%) and Glacial acetic acid (47%) of 3.8 times of amounts, mix, conditioned reaction still temperature, at 20 ℃, is placed and within 90 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, use 23%NH 4it is 7.0 left and right that OH adjusts pH, add 95% ethanol to make to reach more than 85% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, Sephadex LH-20 column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate ammonium.
Embodiment 7
Get creat lactone, add 6.0 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, speed with 0.075 liter of 1g rographolide per minute, pass into 1.4 liter of 1.6% sulphur trioxide, conditioned reaction still temperature, at 16 ℃, is placed and within 90 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 33%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 85% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, ODS column chromatography for separation, with water: the methyl alcohol gradient elution, the ratio of water is 80～20%, and the ratio of methyl alcohol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium.
Embodiment 8
Get creat lactone, add 5 times of amount aceticanhydrides (56%) and Glacial acetic acid (44%) to make to dissolve, under agitation, speed with 0.085 liter of 1g rographolide per minute, pass into 1.6 liter of 1.8% sulphur trioxide, conditioned reaction still temperature, at 18 ℃, is placed and within 100 minutes, is made sulfonation.In reactant impouring saturated nacl aqueous solution, throw out is respectively with saturated nacl aqueous solution, water washing, and solid substance refluxes with chloroform, insolubles adds dehydrated alcohol to be made to dissolve, and removes alcohol insoluble solids, reclaims ethanol, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium.
Embodiment 9
Get creat lactone, add 5.6 times of amount aceticanhydrides (58%) and Glacial acetic acid (42%) to make to dissolve, under agitation, speed with 0.11 liter of 1g rographolide per minute, pass into 2.2 liter of 1.4% sulphur trioxide, conditioned reaction still temperature, at 17 ℃, is placed and within 110 minutes, is made sulfonation.Add 95% ethanol to make to reach more than 80% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, Sephadex LH-20 column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3, the 19-di-sulfate.
Embodiment 10
Get creat lactone, add 4.5 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make to dissolve, under agitation, speed with 0.09 liter of 1g rographolide per minute, pass into 1.4 liter of 2.2% sulphur trioxide, conditioned reaction still temperature, at 18 ℃, is placed and within 120 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 32%KOH is 7.0 left and right, then through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate potassium.
Embodiment 11
Get creat lactone, add 5.8 times of amount aceticanhydrides (61%) and Glacial acetic acid (39%) to make to dissolve, under agitation, speed with 0.095 liter of 1g rographolide per minute, pass into 1.4 liter of 2.0% sulphur trioxide, conditioned reaction still temperature, at 16 ℃, is placed and within 90 minutes, is made sulfonation, add purified water, stir evenly.In reactant impouring saturated potassium chloride solution, throw out is respectively with saturated potassium chloride solution, water washing, and solid substance refluxes with chloroform, insolubles adds dehydrated alcohol to be made to dissolve, and removes alcohol insoluble solids, reclaims ethanol, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate potassium.
Embodiment 12
Get creat lactone, add 5.3 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, speed with 0.065 liter of 1g rographolide per minute, pass into 1.6 liter of 1.5% sulphur trioxide, conditioned reaction still temperature, at 20 ℃, is placed and within 70 minutes, is made sulfonation, add the equivalent purified water, stir evenly.Use 22%NH 4it is 7.0 left and right that OH adjusts pH, add 95% ethanol to make to reach more than 80% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate ammonium.
Embodiment 13
Get creat lactone, add 5.3 times of amount aceticanhydrides (58%) and Glacial acetic acid (42%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.1ml, slowly drip 2.6 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 15 ℃, place and within 100 minutes, make sulfonation, add the equivalent purified water, stir evenly, adjusting pH with 34%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 82% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, the ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium.
Embodiment 14
Get creat lactone, add 6.2 times of amount aceticanhydrides (58%) and Glacial acetic acid (42%) to make to dissolve, under agitation, speed with 1g rographolide per minute 1.8ml, atomization adds 2.2 times of amount chlorsulfonic acids, mix, mix, conditioned reaction still temperature is at 16 ℃, place and within 110 minutes, make sulfonation, in reactant impouring saturated nacl aqueous solution, throw out is respectively with saturated nacl aqueous solution, water washing, solid substance refluxes with chloroform, insolubles adds dehydrated alcohol to be made to dissolve, remove alcohol insoluble solids, reclaim ethanol, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium.
Embodiment 15
Get creat lactone, add 5.4 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.0ml, slowly drip 2.3 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 15 ℃, place and within 70 minutes, make sulfonation, add the equivalent purified water, stir evenly, adding 95% ethanol makes to reach more than 85% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, the ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3, the 19-di-sulfate.
Embodiment 16
Get creat lactone, add aceticanhydride and the Glacial acetic acid of 5.6 times of amount equal proportions to make to dissolve, under agitation, speed with 1g rographolide per minute 2.1ml, slowly drip 2.8 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 20 ℃, place and within 90 minutes, make sulfonation, add the equivalent purified water, stir evenly, adjusting pH with 32%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 85% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, the ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium.
Embodiment 17
Get creat lactone, add 4.3 times of amount aceticanhydrides (56%) and Glacial acetic acid (44%) to make to dissolve, under agitation, with the speed of 1g rographolide per minute 2.3ml, slowly drip 2.6 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature, at 21 ℃, is placed and is made sulfonation in 100 minutes, adds the equivalent purified water, stir evenly, use 18%NH 4it is 7.0 left and right that OH adjusts pH, add 95% ethanol to make to reach more than 85% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate ammonium.
Embodiment 18
Get creat lactone, add 5.6 times of amount aceticanhydrides (55%) and Glacial acetic acid (45%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.1ml, slowly drip 2.4 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 22 ℃, place and within 90 minutes, make sulfonation, in reactant impouring saturated potassium chloride solution, throw out is respectively with saturated potassium chloride solution, water washing, and solid substance refluxes with chloroform, insolubles adds dehydrated alcohol to be made to dissolve, remove alcohol insoluble solids, reclaim ethanol, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate potassium.
Below test with 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium is all got the sample that embodiment 1 preparation method obtains.
Experimental data 1:17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium content is measured
1. instrument and reagent
Instrument: Agilent1100 quarternary low pressure gradient pump series, Chemstation chem workstation, DAD detector; Shimadzu LC-2010A type high performance liquid chromatograph, two channels ultraviolet variable-wavelenght detector; Sartorius cp211D 100,000/electronic balance.
Chromatographic column: Diamonsil C 18post (250mm * 4.6mm, 5 μ m);
Reagent: acetonitrile is chromatographically pure, and water is ultrapure water prepared by Millipore, and other reagent are analytical pure.
17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium reference substance, for self-control, is 99.06% through purity mark content.
2. the preparation of reference substance solution and need testing solution
2.1 reference substance purity test and content mark: get the sample that embodiment 1 preparation method obtains, adopt respectively propyl carbinol-Glacial acetic acid-water (4.5: 1: 1), chloroform-methanol-water-Glacial acetic acid (8: 3: 1: 0.5) carry out the purity of thin layer chromatography inspection, the point sample amount is respectively 5,10,15,20,25 μ g, and result is a spot;
Use high performance liquid chromatography, adopt area normalization method, select respectively chromatographic column: Diamonsil C 18(250mm * 4.6mm, 5 μ m); Moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-acetonitrile (84: 16) and moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-methyl alcohol (77: 23); Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 25 ℃.Every group of moving phase passes in and out respectively 10 μ l, 20 μ l respectively once, and sample introduction is 4 times altogether, and recording 4 average contents is 99.06%.
2.2 reference substance solution preparation: precision takes the 17-hydrogen of above-mentioned mark-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium reference substance 24.32mg, put in the 10ml measuring bottle, be dissolved in water and be diluted to scale, shaking up, obtaining, product mother liquor in contrast, for methodological study.
2.3 the preparation of need testing solution: the sample 50mg that precision takes the present embodiment, put in the 500ml measuring bottle, be diluted with water to scale, shake up, obtain;
3. chromatographic condition and system suitability
Take octadecylsilane chemically bonded silica as weighting agent; The detection wavelength is 225nm; Column temperature is 25 ℃; Number of theoretical plate is pressed 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate or its salt calculate and is not less than 10000;
Take acetonitrile as mobile phase A, take potassium phosphate buffer as Mobile phase B, described potassium phosphate buffer is to add potassium primary phosphate 1.361g and heptane-1-sodium sulfonate 1.0g in every 1000ml water, by following condition, carries out gradient elution, moves 70 minutes:
In the time of 0～10 minute, the acetonitrile ratio is 8.0%, and the potassium phosphate buffer ratio is 92.0%;
In the time of 10～60 minutes, the acetonitrile ratio rises to 35.0% by 8.0%, and the potassium phosphate buffer ratio drops to 65.0% by 92.0%;
In the time of 60～62 minutes, the acetonitrile ratio rises to 70.0% by 35.0%, and the potassium phosphate buffer ratio drops to 30.0% by 65.0%;
In the time of 62～70 minutes, keep acetonitrile: potassium phosphate buffer was with 70.0%: 30.0% ratio wash-out; Under these conditions, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt approximately have a chromatographic peak in 5 minutes in retention time.
4, the investigation of linear relationship
The above-mentioned reference substance mother solution 1ml of each accurate absorption, put respectively in 100ml, 50ml, 25ml, 10ml, 5ml measuring bottle, thin up becomes following concentration: 0.02432mg/ml, 0.0864mg/ml, 0.09728mg/ml, 0.19456mg/ml, 0.38912mg/ml respectively.Accurate draw solution 10 μ l injection liquid chromatographies, by chromatographic condition peak area under 3 chromatographic conditions and system suitability item, respectively with 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium peak area integrated value is ordinate zou, the reference substance sample size is X-coordinate separately, drawing standard curve, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium regression equation is y=2340X-120.3, R 2=0.9999,17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium is good linear between 0.2432～3.8912 μ g.The results are shown in Table 1, Fig. 4.
Table 1.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium wire sexual intercourse result
Figure BSA00000702067400151
5. precision test
Get 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium reference substance solution, repeat sample introduction 6 times, 17-hydrogen-9-dehydrogenation rographolide-3 as a result, and 19-di-sulfate sodium peak area RSD is 0.19%, the results are shown in Table 2.
Table 2 reference substance Precision test result
6. stability test
Get need testing solution, according to chromatographic condition under 3 chromatographic conditions and system suitability item, measure, sample introduction once at regular intervals, 17-hydrogen in need testing solution-9-dehydrogenation rographolide-3 as a result, peak area is without considerable change in 24 hours for 19-di-sulfate sodium, and its peak area RSD is respectively 0.30%.The results are shown in Table 3.
. table 3 stability test result
Figure BSA00000702067400161
7. replica test
Get sample of the present invention, add water and make 6 parts of need testing solutions, measure according to chromatographic condition under 3 chromatographic conditions and system suitability item, 17-hydrogen in sample-9-dehydrogenation rographolide-3,19-di-sulfate sodium average content is 98.89%, RSD=0.28%.The results are shown in Table 4.
Table 4.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium replica test result
Figure BSA00000702067400162
8. recovery test
Adopt the application of sample recovery test, accurate absorption sample solution of the present invention is to the 50ml volumetric flask, totally 6 parts, precision adds 17-hydrogen-9-dehydrogenation rographolide-3 respectively, 19-di-sulfate sodium is (in table with 3,19 sodium sulfovinates mean) reference substance solution (0.964mg/ml) 2ml, be diluted with water to scale, shake up.According to method under 3 chromatographic conditions and system suitability item, measure, calculate recovery rate, the results are shown in Table 5.
Table 5 recovery test result
Figure BSA00000702067400171
Experimental data 2:17-hydrogen-9-dehydrogenation rographolide-3, the impact of 19-di-sulfate sodium induced by endotoxin pyrogenicity
Get Japan large ear rabbit, body weight 1.8-2.3kg, male and female have concurrently, and 1d before experiment, choose body temperature between 38.0～39.4 ℃, and body temperature changed the rabbit be no more than 0.4 ℃ and used rabbit as experiment the same day.Experiment same day, get above-mentioned qualified rabbit, measure basal body temperature before modeling, oneself rabbit ear vein bacterial injection intracellular toxin normal saline solution, dosage is 1mL/kg (10EU/mL), observes rabbit body temperature and changes, every 30min record 1 time.Choose the body temperature rise rabbit that surpasses 0.5 ℃ after injection 1h, be divided at random 5 groups, 8 every group.The ear vein injection gives 0.9% sodium chloride solution, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium low dose group 50mgkg respectively -1, middle dosage group 100mgkg -1, high dose group 200mgkg -1and injection Aspirin-arginine 100mgkg -1, respectively at after administration 1,2,3,4,5,6h measures each rabbit body temperature.The body temperature average before administration of take is radix, calculates each minute rabbit temperature changing value.In Table 6.
Medication temperature variation ℃ after table 6. endotoxin pyrogenic,
Figure BSA00000702067400172
Annotate: with the empty map group, compare ※p<0.05 ※ ※p<0.01
As shown in Table 6, after giving intracellular toxin 1h, blank group Healthy Rabbits body temperature average has risen, and after lasting rising 3h, starts gradually to descend.Compare low dose group 50mgkg with the blank group -117-hydrogen-9-dehydrogenation rographolide-3,1-di-sulfate sodium 1-2h shows obvious inhibition rabbit temperature rise effect, middle dosage group 100mgkg -1, high dose group 200mgkg -117-hydrogen-9-dehydrogenation rographolide-3, show the effect of extremely strong inhibition rabbit fervescence in 19-di-sulfate sodium 1-2h, also demonstrate the rabbit fervescence effect that suppresses during 4h; In 3 drug study groups with 100mgkg -1the above antipyretic effect of dosage is best.Show 50～200mgkg -117-hydrogen-9-dehydrogenation rographolide-3, the rabbit fervescence that 19-di-sulfate sodium induced by endotoxin causes all has cooling effect.
Experimental data 3:17-hydrogen-9-dehydrogenation rographolide-3, the impact of 19-di-sulfate sodium on rat fever due to dry yeast
Experiment is surveyed body temperature 3d in advance with rat, and experiment measured value on the same day is the rat basal body temperature, and screening body temperature changes the animal that is no more than 0.3 ℃, is divided at random 5 groups, 8 every group.Subcutaneous injection 20% dry yeast suspension 5mL/kg, the pyrogenicity pneumoretroperitoneum is injected 0.9% sodium chloride solution, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium low dose group 50mgkg -1, middle dosage group 100mgkg -1, high dose group 200mgkg -1and acetylsalicylic acid 100mgkg -1, measure the rat temperature of 1～6h after administration, per hour 1 time, using the difference of different time points body temperature value and basic value as observation index, the results are shown in Table 7.
Medication temperature variation ℃ after table 7. dry yeast pyrogenicity,
Figure BSA00000702067400181
Figure BSA00000702067400182
Annotate: with the empty map group, compare ※ P<0.05 ※ ※ P<0.01
Table 7 is found out, while giving dry yeast 1h, makes blank group healthy rat body temperature continue the 6h that rises.Compare 17-hydrogen-9-dehydrogenation rographolide-3,1-di-sulfate sodium 100,200mgkg with the blank group -1show obvious inhibition rat temperature rising effect in 1～4h.
Experimental data 4:17-hydrogen-9-dehydrogenation rographolide-3, the impact of 19-di-sulfate sodium p-Xylol induced mice auricle edema
Get 50 of mouse, be divided at random 5 groups.Every day is to 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium low dose group 50mgkg -1, middle dosage group 100mgkg -1, high dose group 200mgkg -12 times, for three days on end, dexamethasone sodium phosphate injection is administration before causing inflammation only.1h after the last administration, in every left auricle of mouse, the outside is dripped and is coated with 0.1ml caused by dimethylbenzene xylene inflammation, and auris dextra is in contrast.Cause scorching latter 2 hours de-cervical vertebras and put to death mouse, and cut mouse two ears along the auricle baseline, with diameter 6mm punch tool, respectively at left and right ear same section, lay auricle, put on electronic balance and weigh and record data.With left and right auricle weight difference value representation swelling.
Table 8. p-Xylol causes the impact of auricle edema
Figure BSA00000702067400191
Figure BSA00000702067400192
Annotate: with the empty map group, compare ※p<0.05 ※ ※p<0.01
Table 8 shows, 17-hydrogen-9-dehydrogenation rographolide-3, and each medication group of 19-di-sulfate sodium, Dexamethasone group mice auricle swelling degree are significantly less than control group.
Experimental data 5:17-hydrogen-9-dehydrogenation rographolide-3, the impact of rat paw edema due to 19-di-sulfate sodium on Carrageenan
Get 40 of rats, be divided at random 5 groups.Every day is to 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium low dose group 50mgkg -1, middle dosage group 100mgkg -1, high dose group 200mgkg -12 times, for three days on end, dexamethasone sodium phosphate injection is administration before causing inflammation only.After the last administration, every rat oral gavage gives physiological saline 8ml, after 30 minutes, in rat foot claw middle part subcutaneous injection 0.15ml 1% carrageenin solution, and 30min, 1h, 2h, 3h, 4h, 5h measure its left back sufficient pawl thickness as the rat paw edema level index with milscale after injecting.
Table 9 causes the impact of rat toes swelling on carrageen
Figure BSA00000702067400194
Annotate: with the empty map group, compare ※ P<0.05 ※ ※ P<0.01
Table 9 result shows, with the control group ratio, 17-hydrogen-9-dehydrogenation rographolide-3, due to each dosage group of 19-di-sulfate sodium and the equal on Carrageenan of Dexamethasone group, the rat swollen feet has obvious restraining effect, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium obvious restraining effect occurs and continues 5 hours in administration 30min when 200mg/kg, 100mg/kg effect and effects of dexamethasone are suitable, 17-hydrogen-9-dehydrogenation rographolide-3, have a certain amount of effect relationship at same time point to the restraining effect of swelling between three dosage groups of 19-di-sulfate sodium.
Experimental data 6:17-hydrogen-9-dehydrogenation rographolide-3, the restraining effect of 19-di-sulfate sodium to the neuraminic acid enzymic activity
Get 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium, add suitable quantity of water and make to dissolve, application neuraminidase inhibitor identification kit is measured 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium suppresses tiring in Table 10 of neuraminidase (N1).
(1). typical curve is prepared: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases to detect damping fluid; B. every hole adds respectively 0,1,2,5,7.5,10 μ l H5N1 neuraminidases again; C. every hole adds 0～20 μ l Milli-Q water again.
(2). the preparation of sample detection: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases to detect damping fluid; B. every hole adds 10 μ l H5N1 neuraminidases again; C. every hole adds 0～10 μ l17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium sample again; D. every hole adds 0～10 μ l Milli-Q water again.
(3). detecting step:
A. vibration mixes about 1min;
B.37 ℃ hatch 2min inhibitor and H5N1 neuraminidase are fully interacted, the sample of doing typical curve is also hatched together;
C. every hole adds 10 μ l neuraminidase fluorogenic substrates;
D. vibration mixes about 1min again;
E.37 after ℃ hatching 20～30min, carry out fluorometric assay.Excitation wavelength is 360nm, and emission wavelength is 440nm.
(4). calculate the inhibition per-cent of sample for the H5N1 neuraminidase according to typical curve, and calculate 17-hydrogen-9-dehydrogenation rographolide-3 after doing concentration curve, 19-di-sulfate sodium is for the IC50 of H5N1 neuraminidase.17-hydrogen-9-dehydrogenation rographolide-3, the inhibiting rate IC50 that 19-di-sulfate sodium reaches neuraminidase is 0.22g/L.In Table 10.
Table 10.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium suppresses the activity of neuraminidase
Figure BSA00000702067400201
Figure BSA00000702067400211
According to above-mentioned experimental result, can be clear that:
(1) .17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium can extract effective inhibition neuraminic acid enzyme component;
(2) .17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium is along with the using dosage size variation, and it suppresses the ability of neuraminic acid enzymic activity, i.e. also corresponding changing of the height of neuraminic acid enzyme inhibition rate, and become positive correlation;
(3). from above-mentioned experiment, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium can be by suppressing influenza virus surface neuraminidase, and then suppress that influenza virus enters the cell the inside, the influenza virus that suppresses to have entered the cell the inside copies, breeds, thereby reduced infection, the growth of influenza virus to cell, and prevention and treatment influenza and complication thereof.
Medical science and study of pharmacy personnel can't not do the inhibition influenza infection, copy in advance, or under the prerequisite of the experiment of inhibition neuraminidase, learn in advance 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium has prevention and treats the good result that the influenza virus sexuality is emitted.
Experimental data 7:17-hydrogen-9-dehydrogenation rographolide-3, the restraining effect of 19-di-sulfate sodium infected by influenza infected chicken embryo
Get 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium, application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1) is identified 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium suppresses the ability that the FM1 influenza virus is copied and suppresses in the chicken embryo.
(1). FM1 influenza virus liquid is inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, and every embryo 0.2ml, hatch 72h for 37 ℃, observes and calculate half chicken embryo infective dose (EID50).
(2) .17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium adopts the toxic action of chicken embryo, stroke-physiological saline solution is to 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium is done to be inoculated in 10d no-special pathogen in age chick embryo allantoic cavity after serial dilution, every embryo 0.2ml, each concentration is inoculated 6 embryos, hatches for 37 ℃, observe chicken embryonic development developmental state, the chicken embryo of usining can be survived the peak concentration of 96h as the TD of medicine.
(3) .17-hydrogen-9-dehydrogenation rographolide-3, the restraining effect of 19-di-sulfate sodium infected by influenza in the chicken embryo adopts, 0.1ml influenza virus liquid and different dilution 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium mixes, 37 ℃ of effect 2h, be inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, inoculate 6 embryos for every group, hatch 72h for 37 ℃.The virus attack amount is 50EID50, establishes virus control, stroke-physiological saline solution normal control simultaneously, calculates 17-hydrogen-9-dehydrogenation rographolide-3, the median effective dose (ED50) of 19-di-sulfate sodium to viral inhibition.
(1) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of chicken embryo, and its EID50 is 10 -4.76.
(2) .17-hydrogen-9-dehydrogenation rographolide-3, after 19-di-sulfate sodium is inoculated in the chicken embryo, it grows basically identical with Normal group.96h chicken embryo is all survived.The chicken embryo gives 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium stoste has no chicken embryo death, so can think that TD0 is 2.08g/L.
(3) .17-hydrogen-9-dehydrogenation rographolide-3, the restraining effect of 19-di-sulfate sodium infected by influenza in the chicken embryo is in Table 11.
Table 11.17-hydrogen-9-dehydrogenation rographolide-3, the restraining effect of 19-di-sulfate sodium infected by influenza infected chicken embryo
Figure BSA00000702067400221
With the virus control group, compare: *p<0.05
As shown in Table 11,17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium has significant restraining effect (P<0.05), ED at 0.0575～0.46g/L infected by influenza 50for 0.0826g ± 0.0026g/L, TI is 61.34 ± 2.27.
Experimental data 8:17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium affects the FM1 influenza virus
Get 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium, application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1) is identified 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium suppresses the ability of FM1 influenza virus virulence.
(1) .FM1 adopts cell median infective dose (TCID50) micromethod to the toxicity test of dog kidney passage cell (MDCK).
(2) .17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium adopts the DMEM of serum-free to 17-hydrogen-9-dehydrogenation rographolide-3 to the toxicity test of mdck cell, 19-di-sulfate sodium is done to be inoculated in the mdck cell hole that forms individual layer after serial dilution, every hole 100 μ l, each extent of dilution repeats 4 holes, establishes the normal cell contrast simultaneously.Culture plate is put to 37 ℃, 5%CO 2in incubator, cultivate, observation of cell pathology every day (CPE), Continuous Observation 3d, with " +～++ ++ " record result, press the Reed-Muench method and calculate medicine median toxic concentration (TD50) and maximal non-toxic concentration (TD0).
(3) .17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium suppresses the effect of FM1 influenza virus and measures: mdck cell 5 * 10 5/ ml, every hole 100 μ l, in 96 orifice plates, 37 ℃, 5%CO 2cultivate in incubator, suck nutrient solution in hole next day, add 100TCID50 influenza virus liquid, every hole 100 μ l, suck supernatant liquor after 37 ℃ of absorption 1h.With phosphate buffered saline buffer (PBS), wash 2 times, the TD0 of medicine of take is the 1st hole, use again the DMEM liquid of serum-free to 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium is made serial dilution, add respectively above-mentioned the infection in viral cell, establish virus control and Normal group, 37 ℃, 5%CO simultaneously 2cultivate in incubator, observe the mdck cell characteristics of lesion that influenza virus produces every day, i.e. monolayer cell sex change becomes circle etc., and 3d, calculate 50% of medicine and suppress pathology concentration (IC50) and therapeutic index (TI) continuously.The calculating of TI: TI=TD50/IC50, the TI value is larger, shows that the safety range of medicine is larger.With Kruskal-Walis and Mann-Whitney method of inspection comparison test group and the cytopathic difference of virus control group, to drug dose with to virus infected cell, avoid the inhibiting rate that cytopathy (CPE) occurs to carry out correlation analysis, judge whether amount validity response relation.
(4) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of mdck cell, and its TCID50 is 10 -4.64.(2) .17-hydrogen-9-dehydrogenation rographolide-3, the TD0 of 19-di-sulfate sodium mdck cell is respectively 1.24g ± 0.042g/L.(3). by 17-hydrogen-9-dehydrogenation rographolide-3, after 19-di-sulfate sodium is made serial dilution, the 100TCID50 influenza virus is carried out to inhibition test, calculate median effective dose IC50 and the TI value size of medicine, the results are shown in Table 12.
Table 12.17-hydrogen-9-dehydrogenation rographolide-3, the IC of 19-di-sulfate sodium to the FM1 influenza virus 50and TI (x ± s) (g/L)
Figure BSA00000702067400241
As shown in Table 12,17-hydrogen-9-dehydrogenation rographolide-3, the IC50 of 19-di-sulfate sodium is low, and TI is high.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium suppresses the cytopathogenic effect of FM1 influenza virus all to be strengthened along with the increase of drug dose.The correlation analysis that drug dose and medicine are carried out the inhibiting rate of CPE shows, 17-hydrogen-9-dehydrogenation rographolide-3, and the dosage of 19-di-sulfate sodium and medicine are to being obvious positive correlation between the CPE inhibiting rate.
Experimental data 9:17-hydrogen-9-dehydrogenation rographolide-3, the impact of 19-di-sulfate sodium on spleen index and the lung index of influenza virus infection FM1 strain in Mice Body
Get 17-hydrogen-9-dehydrogenation rographolide-3; 19-di-sulfate sodium; application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1) is identified 17-hydrogen-9-dehydrogenation rographolide-3, the dead provide protection of 19-di-sulfate sodium to influenza virus infection FM1 strain in Mice Body.
(1) influenza virus FM1 strain virus is inoculated respectively every group of 10 BALB/C mice, male and female half and half after doing 10 times of doubling dilutions.After the slight anesthesia of ether, give and different dilution virus respectively for every group, every mouse collunarium is inoculated 20 μ l.Observe the dead mouse situation of 10d, calculating LD50 by the Reed-Muench method is 10 -1.56.Therefore determine that experiment modeling concentration used is 10LD50.
(2) 17-hydrogen-9-dehydrogenation rographolide-3; the dead provide protection of 19-di-sulfate sodium to influenza virus infection FM1 strain in Mice Body: Normal group, influenza virus FM1 strain virus control group, 17-hydrogen-9-dehydrogenation rographolide-3; 19-di-sulfate sodium 0.125g/L, 0.25g/L, 0.5g/L, 1.0g/L dosage group philosophy gavage, the gavage capacity only is 0.4ml/.After 3d, except Normal group, each group is only used 10LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ under the slight anesthesia of ether.Normal group gives the physiological saline of same volume simultaneously.4 groups of administration are continued administration, Normal group and influenza virus FM1 strain virus control group administered physiological saline 8 days, and dosage is the same.Day by day observe the animal morbidity and record death toll, observing altogether 14 days, calculating mortality ratio (mortality ratio=every group of death toll/every group of total mice * 100%), the results are shown in Table 13.
(3) 17-hydrogen-9-dehydrogenation rographolide-3, the impact of 19-di-sulfate sodium on influenza virus infection FM1 strain lung index in Mice Body: Normal group, influenza virus FM1 strain virus control group, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium 0.125g/L, 0.25g/L, 0.5g/L, 1.0g/L dosage group philosophy gavage, the gavage capacity only is 0.4ml/.After 3 days, except Normal group, each group is only used 1.0LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ under the slight anesthesia of ether.Normal group gives the physiological saline of same volume simultaneously.4 groups of administration are continued administration, Normal group and influenza virus FM1 strain virus control group administered physiological saline 8 days, and dosage is the same.Within the 8th day after virus infection, put to death mouse, weigh, get lung and claim the lung weight, calculate lung index (lung index=lung quality/weight * 100%); In addition, get spleen and claim the spleen weight, calculate spleen index (spleen index=spleen quality/weight * 100%), the results are shown in Table 14.
Table 13.17-hydrogen-9-dehydrogenation rographolide-3, the death protection result of 19-di-sulfate sodium to influenza virus infection FM1 strain in Mice Body
Annotate: ※ ※ P<0.01VS influenza virus model group ※ P<0.05VS influenza virus model group
Table 14.17-hydrogen-9-dehydrogenation rographolide-3, the impact of 19-di-sulfate sodium on spleen index and the lung index of influenza virus infection FM1 strain in Mice Body
Figure BSA00000702067400252
Annotate: #P<0.05VS Normal group is annotated: ※ ※ p<0.001 VS influenza virus model group ※ p<0.05VS influenza virus model group
(1). as shown in Table 13,17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium has significant provide protection (p<0.01) at 0.25～1.0g/L influenza virus infected.
(2). as shown in Table 14,17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium has significant effect (p<0.01) at the lung index of 0.5～1.0g/L influenza virus infected.
Experimental data 10:17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium affects 1.17-hydrogen-9-dehydrogenation rographolide-3 to mouse septicemia, and 19-di-sulfate sodium significantly improves septicemia mouse survival rate
Rographolide, because of water-soluble extreme difference, carries out gavage (30mg/kg) so adopt 0.9% sodium carboxymethyl cellulose solution to be mixed with suspension, and sulfonated bodies directly is made into settled solution with PBS and carries out tail vein injection (10mg/kg).Abdominal injection 5mg/kg LPS carries out modeling, observes mouse survival number of elements in 60h, calculates survival rate.The model group mouse occurs dead in 16 o'clock after modeling, after 60h, survival rate is 25% (as shown in Table 15), rographolide administration group survival rate is 50%, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium administration group survival rate is 67.5%, comparatively speaking, and 17-hydrogen-9-dehydrogenation rographolide-3, the effect of improving survival rate of 19-di-sulfate sodium (being called for short in following form: wear di-sulfate sodium) is slightly better than rographolide group effect, but its dosage is lower.
Table 15.17-hydrogen-9-dehydrogenation rographolide-3, the septicemia mouse survival rate that 19-di-sulfate sodium is induced LPS
2.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium significantly suppresses the rising of inflammatory factor in the septicemia mice serum
BALB/C mice abdominal cavity gavage gives rographolide 30mg/kg, tail vein injection 17-hydrogen-9-dehydrogenation rographolide-3,19-di- sulfate sodium 1,3,10mg/kg, abdominal injection 5mg/kg LPS simultaneously, after modeling and administration 2,5,8,12h4 time point respectively put to death 3 mouse, eye socket is got blood, measure inflammatory factor TNF-α in serum, the content of IL-1 β, shown in table 16.After lps injection 2h, TNF-α in the model group mice serum, the content of IL-1 β reach peak value, be respectively 4815pg/ml and 391pg/ml, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium administration 2h can significantly suppress TNF-α to 3741pg/ml, to the release of IL-1 β being produced significantly and suppresses when the 5h.Rographolide effect 8h just can significantly suppress the rising of TNF-α.Experimental result shows than rographolide, and sulfonated bodies is rapid-action to the restraining effect of inflammatory factor in the septicemia mice serum, and inhibition is more remarkable.
Table 16.17-hydrogen-9-dehydrogenation rographolide-3, inflammatory factor in the septicemia mice serum that 19-di-sulfate sodium time, dose-dependently reduction LPS induce
TNF?α(pg/ml)
Figure BSA00000702067400271
IL?1β(pg/ml)
Figure BSA00000702067400272
Figure BSA00000702067400273
N=3, *p<0.05vs model group, #p<0.05vs rographolide group.
3.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium significantly suppresses the hepar damnification of septicemia mouse
Septicemia is a kind of disease that causes multiple organ injury, and liver is one of main organs of its damage.The BALB/C mice gavage gives rographolide 30mg/kg, tail vein injection 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium 10mg/kg, while abdominal injection 5mg/kg LPS, respectively at 2h, 5h, 8h, the 12h eye socket is got blood, measures the content of serum alt, AST.Shown in table 17, after giving LPS, model mice serum alt and AST continue to raise, give 17-hydrogen-9-dehydrogenation rographolide-3, after 19-di-sulfate sodium 2h, the content of ALT/AST descend to some extent, with model group ALT/AST, significant difference is arranged to 12h, rographolide is to the inhibition of ALT/AST than 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium effect is slightly poor.RT-PCR measures the mouse rna level of each inflammatory factor in liver organization and finds, LPS stimulates lower ifn-γ, il-6, tnf-α, il-β, cox-2mRNA significantly raises, 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium and rographolide administration 5h and 8h can significantly suppress ifn-γ, il-6, tnf-α, il-β, the rising of cox-2mRNA.Experimental result shows equally than rographolide, and sulfonated bodies is rapid-action to the restraining effect of septicemia mouse liver injury, and inhibition is more remarkable.
Table 17.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium is induced LPS
The restraining effect of septicemia mouse liver injury
AST(karmen?units)
Figure BSA00000702067400281
ALT(karmen?units)
Figure BSA00000702067400282
N=3, *p<0.05, the vs model group.
4, brief summary
Rographolide and 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium can significantly improve the survival rate of the septicemia mouse that LPS induces, time, dose-dependently reduce TNF-α in the septicemia mice serum, IL-1 β, ALT, the content of AST, the sick rising that suppresses inflammatory factor mRNA level in liver organization.17-hydrogen-9-dehydrogenation rographolide-3, the onset of 19-di-sulfate sodium is fast than rographolide, better effects if.After experimental result shows that rographolide is transformed into sulfonated bodies, it is water-soluble better, and administration is rapid-action, and the improvement effect of mouse septicemia also is enhanced.

Claims (25)

1. the represented compound or its salt of a general formula (I), its chemistry 17-hydrogen by name-9-dehydrogenation rographolide-3,19-di-sulfate or its salt,
R 1, R 2can be H, sodium, potassium or NH 4.
2. the preparation method of the compound or its salt of claim 1 comprises the following steps:
(1) add solvent in reactor, add rographolide to make its dissolving, then add sulphonating agent to carry out sulfonation reaction, conditioned reaction still temperature is at 3.5～38.5 ℃, sulfonation reaction 0.5～27.5 hour;
(2) rographolide solution is in the situation that stirring adopts the mode that slowly drips, sprays or ventilate to add sulphonating agent, and when adopting the mode that slowly drips or spray to add sulphonating agent, add speed control at 1.4ml～4.8ml/min/1g rographolide, when the mode that passes into gas when employing adds sulphonating agent, pass into speed control at 0.015 liter～0.21 liter/min/1g rographolide, obtain 17-hydrogen-9-dehydrogenation rographolide-3, the reactant of 19-di-sulfate and the mixture of unreacted reactant after sulfonation reaction;
(3) mixture is through solvent extraction, crystallization, obtains final 17-hydrogen-9-dehydrogenation rographolide-3, the 19-di-sulfate; Perhaps
Mixture to saturated salt solution, crystallization, obtain final 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt; Perhaps
Alkaline solution adjust pH to 7 for mixture, through macroporous adsorbent resin, ODS or Sephadex LH-20 column chromatography for separation, take water: ethanol or methyl alcohol are elutriant, gradient elution, collect eluant component, crystallization, final 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt; Perhaps
Mixture successively passes through aforementioned two or more step co-treatment, obtains final 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt.
3. the preparation method of claim 2, the described solvent added in reactor is one or both of aceticanhydride or Glacial acetic acid, the described dissolution with solvents of 3g～12g for the 1g rographolide is preferred, the described dissolution with solvents of 4g～9g for the 1g rographolide.
4. claim 2 or 3 preparation method, described solvent is aceticanhydride and Glacial acetic acid, described aceticanhydride, Glacial acetic acid are made by following weight proportion: aceticanhydride 80%～20%, Glacial acetic acid 20%～80%, preferred, wherein aceticanhydride 62%, Glacial acetic acid 38%.
5. the preparation method of claim 2, described sulphonating agent adopts the vitriol oil and Glacial acetic acid, 1g for the 1g rographolide～10g vitriol oil Glacial acetic acid carries out sulfonation, the described vitriol oil and Glacial acetic acid are made by following weight proportion: the vitriol oil 80%～20%, Glacial acetic acid 20%～80%, preferably, 1.5g for the 1g rographolide～5g vitriol oil Glacial acetic acid, and the acid of dense stream is 1: 1 with the Glacial acetic acid part by weight.
6. the preparation method of claim 2, described sulphonating agent adopts sulphur trioxide, the 1g rographolide passes into the sulphur trioxide that 0.2 liter～5 liters volumetric concentrations are 1%～3% and carries out sulfonation, preferably, the 1g rographolide passes into the sulphur trioxide that 0.3 liter～3 liters volumetric concentrations are 1.5%～2.5% and carries out sulfonation.
7. the preparation method of claim 2, described sulphonating agent adopts chlorsulfonic acid, and the 0.2g 1g rographolide for～5g chlorsulfonic acid carries out sulfonation, and preferably, 0.3g for the 1g rographolide～3.5g chlorsulfonic acid carries out sulfonation.
8. the preparation method of claim 2, described saturated salt solution adopts sodium-chlor or saturated potassium chloride solution, and described alkaline solution adopts 5%～50% sodium hydroxide or potassium hydroxide or 25% following ammonia soln.
9. the preparation method of claim 2～8 any one, wherein said temperature of reaction kettle is at 6～24 ℃, and the described sulfonation reaction time should be controlled at 1～2.5 hour.
10. the preparation method of claim 2, wherein said sulphonating agent is vitriol oil Glacial acetic acid or chlorsulfonic acid, and adopts the mode that slowly drips, sprays to add, and adds speed control at 2.2ml～3.8ml/min/1g rographolide.
11. the preparation method of claim 2, wherein said sulphonating agent is sulphur trioxide, and adopts the mode that passes into gas to add, and passes into speed control at 0.06 liter～0.14 liter/min/1g rographolide.
12. the preparation method of claim 2～8 any one, in wherein said gradient elution, the ratio of water is 80～20%, and the ratio of ethanol or methyl alcohol is 20～80%.
13. the measuring method of the compound or its salt of claim 1, it adopts the described 17-hydrogen of high effective liquid chromatography for measuring-9-dehydrogenation rographolide-3,19-di-sulfate or its salt, and condition determination is as follows:
Take octadecylsilane chemically bonded silica as weighting agent;
The detection wavelength is 225nm;
Column temperature is 25 ℃;
Number of theoretical plate is pressed 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate or its salt calculate and is not less than 10000;
The preparation of reference substance solution is by described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are made reference substance by purity test and content mark, and dry in the Vanadium Pentoxide in FLAKES vacuum drier, accurately weighed appropriate, add water and make the solution of desired concn, obtain;
The preparation precision of need testing solution takes sample 5mg, puts in the 50ml measuring bottle, is diluted with water to scale, shakes up, and obtains;
Wash-out be take acetonitrile as mobile phase A, take potassium phosphate buffer as Mobile phase B, and described potassium phosphate buffer is to add potassium primary phosphate 1.361g and heptane-1-sodium sulfonate 1.0g in every 1000ml water, by following condition, carries out gradient elution, moves 70 minutes;
In the time of 0～10 minute, the acetonitrile ratio is 8.0%, and the potassium phosphate buffer ratio is 92.0%;
In the time of 10～60 minutes, the acetonitrile ratio rises to 35.0% by 8.0%, and the potassium phosphate buffer ratio drops to 65.0% by 92.0%;
In the time of 60～62 minutes, the acetonitrile ratio rises to 70.0% by 35.0%, and the potassium phosphate buffer ratio drops to 30.0% by 65.0%;
In the time of 62～70 minutes, keep acetonitrile: potassium phosphate buffer was with 70.0%: 30.0% ratio wash-out;
Under these conditions, 17-hydrogen-9-dehydrogenation rographolide-3,19-sulfuric ester or its salt approximately have a chromatographic peak in 5 minutes in retention time,
Assay method is accurate reference substance solution and the need testing solution drawn respectively, and the injection liquid chromatography, measure, and obtains.
14. 17-hydrogen-9-dehydrogenation rographolide-3, the preparation of 19-di-sulfate or its salt, said preparation is 17-hydrogen claimed in claim 1-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are made with acceptable carrier pharmaceutically.
15. 17-hydrogen claimed in claim 1-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for the preparation of the purposes of analgesic medicine.
16. as the purposes of claim 15, described 17-hydrogen-9-dehydrogenation rographolide-3, the refrigeration function of 19-di-sulfate or its salt pair endotoxin pyrogenic.
17. as the purposes of claim 15, described 17-hydrogen-9-dehydrogenation rographolide-3, the refrigeration function of 19-di-sulfate or its salt pair dry yeast pyrogenicity.
18. 17-hydrogen claimed in claim 1-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for the preparation of the purposes of the medicine of anti-inflammatory.
19. as the purposes of claim 18, described 17-hydrogen-9-dehydrogenation rographolide-3, the drug effect of 19-di-sulfate or its salt pair septicemia.
20. as the purposes of claim 18, described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt p-Xylol induced mice auricle edema anti-inflammatory action.
21. as the purposes of claim 18, described 17-hydrogen-9-dehydrogenation rographolide-3, the anti-inflammatory action of rat paw edema due to 19-di-sulfate or its salt on Carrageenan.
22. 17-hydrogen claimed in claim 1-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for the preparation of the purposes of antiviral drug.
23. as the purposes of claim 22, described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for suppressing neuraminidase.
24. as the purposes of claim 22, described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for suppressing influenza virus.
25. as the purposes of claim 22, described 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate or its salt are for suppressing influenza virus FM1.
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