CN103145665A

CN103145665A - 17-hydro-9-dehydro-14,17-cyclo-andrographolide-19-sulphate and its preparation method and use in drug preparation

Info

Publication number: CN103145665A
Application number: CN2012101095207A
Authority: CN
Inventors: 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
Original assignee: JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
Current assignee: JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
Priority date: 2012-04-12
Filing date: 2012-04-12
Publication date: 2013-06-12
Anticipated expiration: 2032-04-12
Also published as: CN103145665B

Abstract

The invention discloses 17-hydro-9-dehydro-14,17-cyclo-andrographolide-19-sulphate or its salt, and its preparation method and use in drug preparation. The 17-hydro-9-dehydro-14,17-cyclo-andrographolide-19-sulphate or its salt has effects of bringing down a fever, diminishing inflammation and resisting viruses. The 17-hydro-9-dehydro-14,17-cyclo-andrographolide-19-sulphate or its salt does not change chemical properties of andrographolide and has characteristics of good water-solubility, high thermostability and low hemolysis, and guarantees the pharmacological activity of an andrographolide all-natural drug to the maximum degree. Medical and pharmaceutical researchers cannot know the above uses of the 17-hydro-9-dehydro-14,17-cyclo-andrographolide-19-sulphate in advance without related experiments.

Description

17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester compound, preparation method and preparation pharmaceutical use thereof

Technical field

The present invention relates to a kind of 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester compound, preparation method and preparation pharmaceutical use thereof.

Background technology

Herba Andrographis is the dry aerial parts of acanthaceous plant Herba Andrographis Andrographis paniculata (Burm.f.) Nees, chemical composition and pharmacological evaluation show, the activeconstituents of Herba Andrographis be take rographolide as representative diterpene-kind compound 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:

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 for the preparation 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 that makes, 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-two dehydrogenation rographolide-3, 19-disuccinic acid half ester k-na salt (' Tanhuning ' injection) or 14-deshydroxy-11, 12-two dehydrogenation rographolide-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-14, and 17-ring-rographolide-19-sulfuric ester or its salt,

R can 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 6～39 ℃, sulfonation reaction 1～28.5 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 1.3ml～4.7ml/min/1g rographolide, when the mode that passes into gas when employing adds sulphonating agent, pass into speed control at 0.03 liter～0.22 liter/min/1g rographolide, get 17-hydrogen-9-dehydrogenation-14, the 17-ring-rographolide-reactant of 19-sulfuric ester and the mixture of unreacted reactant after sulfonation reaction;

[3] mixture is through solvent extraction, crystallization, gets final 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester; Perhaps

Mixture to saturated salt solution, crystallization, get final 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt; Perhaps

Mixture alkaline solution adjust pH to 7, through macroporous adsorbent resin, ODS or Sephadex LH-20 column chromatography for separation, take water: ethanol or methyl alcohol are as elutriant, gradient elution, collect eluant component, crystallization gets final 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt; Perhaps

Mixture successively passes through aforementioned two or more step co-treatment, gets final 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt.

Preferably, the described solvent that adds in reactor is one or both of aceticanhydride or Glacial acetic acid, and the 1g rographolide is with the described dissolution with solvents of 3g～12g, and is preferred, the 1g rographolide described dissolution with solvents of 4g～9g.

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, the 1g rographolide carries out sulfonation with 1g～10g vitriol oil Glacial acetic acid, 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, 1g rographolide 1.5g～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, it is that 1%～3% sulphur trioxide carries out sulfonation that the 1g rographolide passes into 0.2 liter～5 liters volumetric concentrations, preferred, and it is that 1.5%～2.5% sulphur trioxide carries out sulfonation that the 1g rographolide passes into 0.3 liter～3 liters volumetric concentrations.

Preferably, described sulphonating agent adopts chlorsulfonic acid, and the 1g rographolide carries out sulfonation with 0.2g～5g chlorsulfonic acid, and preferred, the 1g rographolide carries out sulfonation with 0.3g～3.5g chlorsulfonic acid.

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 7～25 ℃, 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 2ml～4ml/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 0.05 liter～0.15 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), it adopts the described 17-hydrogen of high effective liquid chromatography for measuring-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt, 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-14, and 17-ring-rographolide-19-sulfuric ester or its salt calculate and is not less than 10000;

The preparation of reference substance solution is with described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt are made reference substance by purity test and content mark, and dry in the Vanadium Pentoxide in FLAKES vacuum drier, and is accurately weighed appropriate, add water and make the solution of desired concn, and get final product;

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 get final product;

Wash-out is take acetonitrile as mobile phase A, and 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, carries out gradient elution by following condition, 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-14,17-ring-rographolide-19-sulfuric ester or its salt approximately had a chromatographic peak in 42 minutes in retention time.

Assay method is accurate reference substance solution and the need testing solution drawn respectively, and the injection liquid chromatography is measured, and be get final product.

Another object of the present invention is to provide a kind of 17-hydrogen-9-dehydrogenation-14, the preparation of 17-ring-rographolide-19-sulfuric ester or its salt, said preparation is 17-hydrogen claimed in claim 1-9-dehydrogenation-14, and 17-ring-rographolide-19-sulfuric ester or its salt are made with acceptable carrier pharmaceutically.

Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation-14, and 17-ring-rographolide-19-sulfuric ester or its salt are for the preparation of the purposes of analgesic medicine.

Preferably, described 17-hydrogen-9-dehydrogenation-14, the refrigeration function of 17-ring-rographolide-19-sulfuric ester or its salt pair endotoxin pyrogenic.

Preferably, described 17-hydrogen-9-dehydrogenation-14, the refrigeration function of 17-ring-rographolide-19-sulfuric ester or its salt pair dry yeast pyrogenicity.

Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation-14, and 17-ring-rographolide-19-sulfuric ester or its salt are for the preparation of the purposes of the medicine of anti-inflammatory.

Preferably, described 17-hydrogen-9-dehydrogenation-14, the drug effect of 17-ring-rographolide-19-sulfuric ester or its salt pair septicemia.

Preferably, described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt p-Xylol induced mice auricle edema anti-inflammatory action.

Preferably, described 17-hydrogen-9-dehydrogenation-14, the anti-inflammatory action of rat paw edema due to 17-ring-rographolide-19-sulfuric ester or its salt on Carrageenan.

Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation-14, and 17-ring-rographolide-19-sulfuric ester or its salt are for the preparation of the purposes of antiviral drug.

Preferably, described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt are used for suppressing neuraminidase.

Preferably, described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt are used for suppressing influenza virus.

Preferably, described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt are used for suppressing influenza virus FM1.

17-hydrogen provided by the invention-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt, solubleness is very good and stability is very high in water, be fit 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 such as syrup, injection liquid etc., particularly make injection and overcome the low defective of oral pharmaceutical biological utilisation.

On the other hand, 17-hydrogen provided by the present invention-9-dehydrogenation-14, the preparation method of 17-ring-rographolide-19-sulfuric ester or its salt is simple and convenient, mild condition, productivity are high, can prepare easily 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester 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, form 17-hydrogen of the present invention-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt thereby the invention provides to prepare under multiple different condition.

The present invention is compared with the prior art and shows: adopt the 17-hydrogen that preparation method of the present invention forms-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt can not change the original chemical attribute of rographolide fully; And 17-hydrogen of the present invention-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester 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.

Through the experimental data contrast as can be known, after giving intracellular toxin 1h, blank group Healthy Rabbits body temperature average has risen, and begins gradually after lasting rising 3h to descend.Compare low dose group 50mgkg with the blank group ^-117-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 1～2h shows obvious inhibition rabbit temperature rise effect, middle dosage group 100mgkg ^-1, high dose group 200mgkg ^-117-hydrogen-9-dehydrogenation-14 show the effect of extremely strong inhibition rabbit fervescence in 17-ring-rographolide-19-sodium sulfovinate 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-14, the rabbit fervescence that 17-ring-rographolide-19-sodium sulfovinate induced by endotoxin causes all has cooling effect, has obtained unforeseeable technique effect.

Through the experimental data contrast as can be known, make blank group healthy rat body temperature continue the 6h that rises when giving dry yeast 1h.Compare 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 100,200mgkg with the blank group ^-1Show obvious inhibition rat temperature rising effect in 1～4h, obtained unforeseeable technique effect.

Through the experimental data contrast as can be known, rographolide and 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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-14,17-ring-rographolide-19-sodium sulfovinate onset is fast than rographolide, better effects if.After experimental result showed that rographolide is transformed into sulfonated bodies, it was water-soluble better, and administration is rapid-action, and the improvement effect of mouse septicemia also is enhanced, and had obtained unforeseeable technique effect.

Through the experimental data contrast as can be known, 17-hydrogen-9-dehydrogenation-14, each medication group of 17-ring-rographolide-19-sodium sulfovinate, Dexamethasone group mice auricle swelling degree are significantly less than control group, have obtained unforeseeable technique effect.

through the experimental data contrast as can be known, with the control group ratio, 17-hydrogen-9-dehydrogenation-14, due to each dosage group of 17-ring-rographolide-19-sodium sulfovinate and the equal on Carrageenan of Dexamethasone group, the rat swollen feet has obvious restraining effect, 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate 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-14, at same time point, the restraining effect of swelling there is a certain amount of effect relationship between 17-ring-rographolide-three of 19-sodium sulfovinates dosage group, obtained unforeseeable technique effect.

Through the experimental data contrast as can be known, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate can extract effective inhibition neuraminic acid enzyme component; And 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate are 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-14,17-ring-rographolide-19-sodium sulfovinate can be by suppressing the surperficial neuraminidase of influenza virus, 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.

Through the experimental data contrast as can be known, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate has significant restraining effect (P＜0.05), ED at 0.075～0.6g/L infected by influenza ₅₀Be 0.0918g ± 0.0052g/L, TI is 59.88 ± 1.96.17-hydrogen-9-dehydrogenation-14, the IC50 of 17-ring-rographolide-19-sodium sulfovinate is low, and TI is high.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate suppress the cytopathogenic effect of FM1 influenza virus and all strengthen along with the increase of drug dose.Drug dose and medicine are shown the correlation analysis that the inhibiting rate of CPE carries out, 17-hydrogen-9-dehydrogenation-14, the dosage of 17-ring-rographolide-19-sodium sulfovinate and medicine are to being obvious positive correlation between the CPE inhibiting rate.

Medical science and study of pharmacy personnel can't learn 17-hydrogen-9-dehydrogenation-14 in advance in advance under the prerequisite of not doing related experiment, 17-ring-rographolide-19-sodium sulfovinate has above-mentioned good purposes.

Description of drawings

Fig. 1: 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate hydrogen nuclear magnetic resonance spectrogram.

Fig. 2: 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate carbon-13 nmr spectra figure.

Fig. 3: 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate mass spectrum.

Fig. 4: 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate linear relationship chart.

Embodiment

Embodiment 1

Get creat lactone, add 4.5 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.3ml, slowly drip the sulfuric acid (53%) and Glacial acetic acid (47%) of 4.8 times of amounts, mixing, conditioned reaction still temperature are placed and were made sulfonation in 100 minutes at 16 ℃.Add the equivalent purified water, stir evenly, transferring pH with 35%NaOH is 7.0 left and right, add 95% ethanol to make to contain the alcohol amount to reach more than 83%, 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 are 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization gets 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate, its sodium salt molecular formula C ₂₀H ₂₇NaO ₇S, molecular weight: 434,

The reaction formula example:

17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate physico-chemical property and spectral data:

17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate: faint yellow amorphous, [α] _D ²⁰=-212 (c 1.15, MeOH), and molecular formula: C ₂₀H ₂₇NaO ₇S。ESI-MSm/z?457[M+Na] ⁺，411[M-Na] ^-，891[2M+Na] ⁺，HRESIMS?m/z：411.1483[M-Na] ^-(calcd?411.1472)。 ¹H (400MHz, CD ₃OD) and ¹³C NMR (100MHz, CD ₃OD) data.

¹NMR(CD ₃OD，400MHz)δ：1.99(brd，J＝13.1Hz，1H，H-1α)，1.21(m，1H，H-1β)，1.81(m，1H，H-2α)，1.70(m，1H，H-2β)，3.24(dd，J＝11.5.5.0Hz，1H，H-3)，1.16(m，1H，H-5)，1.63(m，1H，H-6α)，1.88(m，1H，H-6β)，2.14(dd，J＝17.8，6.0Hz，1H，H-7α)，2.07(dd，J＝12.0，6.0Hz，1H，H-7β)，3.07(brd，2H，H-11)，4.74(dd，J＝7.8，4.4Hz，1H，H-12)，2.99(m，1H，H-14)，4.47(t，J＝8.6Hz，1H，H-15α)，3.70(t，J＝8.6Hz，1H，H-15β)，1.93(dd，J＝11.9，2.3Hz，1H，H-17α)，2.35(dd，J＝11.9Hz，1H，H-17β)，1.20(s，3H，H-18)，4.16(s，2H，H-19)，1.02(s，.3H，H-20)

¹³C-NMR(CD ₃OD，100MHz)δ：35.7(C-1)，28.4(C-2)，79.5(C-3)，43.2(C-4)，53.0(C-5)，21.5(C-6)，35.1(C-7)，133.0(C-8)，141.6(C-9)，40.0(C-10)，28.0(C-11)，138.3(C-12)，132.9(C-13)，38.2(C-14)，72.1(C-15)，174.4(C-16)，36.1(C-17)，23.4(C-18)，70.8(C-19)，19.4(C-20)。

Embodiment 2

get creat lactone, add 4.6 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.5ml, slowly drip the sulfuric acid ice acetic acid of 3.6 times of amount equal proportions, mixing, conditioned reaction still temperature is at 17 ℃, place and made sulfonation in 90 minutes, 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, get 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate.

Embodiment 3

Get creat lactone, add 4.2 times of amount aceticanhydrides (52%) and Glacial acetic acid (48%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.7ml, slowly drip the sulfuric acid (58%) and Glacial acetic acid (42%) of 3.5 times of amounts, mixing, conditioned reaction still temperature are placed and were made sulfonation in 100 minutes at 12 ℃.Add 95% ethanol to make to contain the alcohol amount to reach more than 82%, 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 are 80～20%, and the ratio of ethanol is 20～80%, collect eluant component, merge same composition, crystallization gets 8-table-Isorographolide-19-sulfuric ester.

Embodiment 4

Get creat lactone, add 4.7 times of amount aceticanhydrides to make dissolving, under agitation, with the speed of 1g rographolide per minute 2.2ml, the atomizing spray adds the sulfuric acid (53%) and Glacial acetic acid (47%) of 3.3 times of amounts, mixing, conditioned reaction still temperature is placed and was made sulfonation in 120 minutes at 20 ℃.In reactant impouring saturated nacl aqueous solution, with saturated nacl aqueous solution, water washing, solid substance refluxes with chloroform throw out respectively, insolubles adds dehydrated alcohol makes dissolving, removes alcohol insoluble solids, reclaims ethanol, crystallization gets 8-table-Isorographolide-19-sodium sulfovinate.

Embodiment 5

Get creat lactone, add 5.2 times of amount aceticanhydrides (65%) and Glacial acetic acid (35%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.5ml, slowly drip the sulfuric acid (51%) and Glacial acetic acid (49%) of 3.4 times of amounts, mixing, conditioned reaction still temperature are placed and were made sulfonation in 110 minutes at 17 ℃.Add the equivalent purified water, stir evenly, transferring pH with 32%KOH is 7.0 left and right, add 95% ethanol to make to contain the alcohol amount to reach more than 84%, decompression recycling ethanol, the aqueous solution be through macroporous adsorbent resin, ODS 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%, and the Fractional Collections elutriant merges same composition, crystallization gets 8-table-Isorographolide-19-sulfuric ester potassium.

Embodiment 6

Get creat lactone, add 5.5 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.0ml, slowly drip the sulfuric acid (57%) and Glacial acetic acid (43%) of 4.8 times of amounts, mixing, conditioned reaction still temperature are placed and were made sulfonation in 90 minutes at 14 ℃.Add the equivalent purified water, stir evenly, use 22%NH ₄It is 7.0 left and right that OH transfers pH, add 95% ethanol to make to contain the alcohol amount to reach more than 82%, decompression recycling ethanol, the aqueous solution be through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, and the Fractional Collections elutriant merges same composition, crystallization gets 8-table-Isorographolide-19-sulfuric ester ammonium.

Embodiment 7

Get creat lactone, add 6.2 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make dissolving, under agitation, speed with 0.09 liter of 1g rographolide per minute, pass into 1.5 liter of 1.4% sulphur trioxide, conditioned reaction still temperature is placed and was made sulfonation in 90 minutes at 20 ℃.Add the equivalent purified water, stir evenly, transferring pH with 33%NaOH is 7.0 left and right, add 95% ethanol to make to contain the alcohol amount to reach more than 85%, decompression recycling ethanol, the aqueous solution be through macroporous adsorbent resin, ODS 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%, and the Fractional Collections elutriant merges same composition, crystallization gets 8-table-Isorographolide-19-sodium sulfovinate.

Embodiment 8

Get creat lactone, add 5.2 times of amount aceticanhydrides (64%) and Glacial acetic acid (36%) to make dissolving, under agitation, speed with 0.07 liter of 1g rographolide per minute, pass into 1.9 liter of 1.4% sulphur trioxide, conditioned reaction still temperature is placed and was made sulfonation in 110 minutes at 19 ℃.In reactant impouring saturated nacl aqueous solution, with saturated nacl aqueous solution, water washing, solid substance refluxes with chloroform throw out respectively, insolubles adds dehydrated alcohol makes dissolving, removes alcohol insoluble solids, reclaims ethanol, crystallization gets 8-table-Isorographolide-19-sodium sulfovinate.

Embodiment 9

Get creat lactone, add 5.3 times of amount aceticanhydrides (56%) and Glacial acetic acid (44%) to make dissolving, under agitation, speed with 0.11 liter of 1g rographolide per minute, pass into 1.8 liter of 1.6% sulphur trioxide, conditioned reaction still temperature is placed and was made sulfonation in 100 minutes at 11 ℃.Add 95% ethanol to make to contain the alcohol amount to reach more than 80%, 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 are 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization gets 8-table-Isorographolide-19-sulfuric ester.

Embodiment 10

Get creat lactone, add 5.5 times of amount aceticanhydrides (68%) and Glacial acetic acid (32%) to make dissolving, under agitation, speed with 0.09 liter of 1g rographolide per minute, pass into 1.3 liter of 2.2% sulphur trioxide, conditioned reaction still temperature is placed and was made sulfonation in 120 minutes at 18 ℃.Add the equivalent purified water, stir evenly, transferring pH with 35%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%, the ratio of ethanol is 20～80%, and the Fractional Collections elutriant merges same composition, crystallization gets 8-table-Isorographolide-19-sulfuric ester potassium.

Embodiment 11

Get creat lactone, add 5.4 times of amount aceticanhydrides (61%) and Glacial acetic acid (39%) to make dissolving, under agitation, speed with 0.06 liter of 1g rographolide per minute, pass into 0.9 liter of 2.5% sulphur trioxide, conditioned reaction still temperature is placed and was made sulfonation in 80 minutes at 19 ℃, add purified water, stir evenly.In reactant impouring saturated potassium chloride solution, with saturated potassium chloride solution, water washing, solid substance refluxes with chloroform throw out respectively, insolubles adds dehydrated alcohol makes dissolving, removes alcohol insoluble solids, reclaims ethanol, crystallization gets 8-table-Isorographolide-19-sulfuric ester potassium.

Embodiment 12

Get creat lactone, add 5.4 times of amount aceticanhydrides (66%) and Glacial acetic acid (34%) to make dissolving, under agitation, speed with 0.07 liter of 1g rographolide per minute, pass into 1.3 liter of 1.5% sulphur trioxide, conditioned reaction still temperature is placed and was made sulfonation in 70 minutes at 20 ℃, add the equivalent purified water, stir evenly.Use 21%NH ₄It is 7.0 left and right that OH transfers pH, add 95% ethanol to make to contain the alcohol amount to reach more than 80%, decompression recycling ethanol, the aqueous solution be through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, and the Fractional Collections elutriant merges same composition, crystallization gets 8-table-Isorographolide-19-sulfuric ester ammonium.

Embodiment 13

Get creat lactone, add 5.6 times of amount aceticanhydrides (59%) and Glacial acetic acid (41%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.1ml, atomizing adds 1.9 times of amount chlorsulfonic acids, mixing, mixing, conditioned reaction still temperature is at 15 ℃, place and made sulfonation in 100 minutes, 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 makes dissolving, removes alcohol insoluble solids, reclaims ethanol, crystallization gets 8-table-Isorographolide-19-sodium sulfovinate.

Embodiment 14

get creat lactone, add 5.3 times of amount aceticanhydrides (56%) and Glacial acetic acid (44%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.3ml, slowly drip 2.6 times of amount chlorsulfonic acids, mixing, conditioned reaction still temperature is at 16 ℃, place and made sulfonation in 100 minutes, add the equivalent purified water, stir evenly, transferring pH with 28%NaOH is 7.0 left and right, add 95% ethanol to make to contain the alcohol amount to reach more than 83%, 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, get 8-table-Isorographolide-19-sodium sulfovinate.

Embodiment 15

get creat lactone, add 5.0 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.3ml, slowly drip 2.6 times of amount chlorsulfonic acids, mixing, conditioned reaction still temperature is at 18 ℃, place and made sulfonation in 80 minutes, add the equivalent purified water, stir evenly, adding 95% ethanol makes and contains alcohol amount and reach more than 85%, 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, get 8-table-Isorographolide-19-sulfuric ester.

Embodiment 16

get creat lactone, add aceticanhydride and the Glacial acetic acid of 5.3 times of amount equal proportions to make dissolving, under agitation, speed with 1g rographolide per minute 2.1ml, slowly drip 2.2 times of amount chlorsulfonic acids, mixing, conditioned reaction still temperature is at 21 ℃, place and made sulfonation in 90 minutes, add the equivalent purified water, stir evenly, transferring pH with 34%KOH is 7.0 left and right, add 95% ethanol to make to contain the alcohol amount to reach more than 84%, 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, get 8-table-Isorographolide-19-sulfuric ester potassium.

Embodiment 17

Get creat lactone, add 4.8 times of amount aceticanhydrides (59%) and Glacial acetic acid (421%) to make dissolving, under agitation, with the speed of 1g rographolide per minute 2.7ml, slowly drip 2.6 times of amount chlorsulfonic acids, mixing, conditioned reaction still temperature is placed and was made sulfonation in 80 minutes at 21 ℃, adds the equivalent purified water, stir evenly, use 21%NH ₄It is 7.0 left and right that OH transfers pH, add 95% ethanol to make to contain the alcohol amount to reach more than 85%, decompression recycling ethanol, the aqueous solution be through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, and the Fractional Collections elutriant merges same composition, crystallization gets 8-table-Isorographolide-19-sulfuric ester ammonium.

Embodiment 18

Get creat lactone, add 4.9 times of amount aceticanhydrides (58%) and Glacial acetic acid (42%) to make dissolving, under agitation, speed with 1g rographolide per minute 2.4ml, slowly drip 2.7 times of amount chlorsulfonic acids, mixing, conditioned reaction still temperature are placed and were made sulfonation in 80 minutes at 21 ℃, in reactant impouring saturated potassium chloride solution, with saturated potassium chloride solution, water washing, solid substance refluxes with chloroform throw out respectively, and insolubles adds dehydrated alcohol makes dissolving, remove alcohol insoluble solids, reclaim ethanol, crystallization gets 8-table-Isorographolide-19-sulfuric ester potassium.

Below test with 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate is all got the sample that embodiment 1 preparation method obtains.

Experimental data 1:17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate assay

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; Sartoriuscp211D 100,000/electronic balance.

Chromatographic column: Diamonsil C ₁₈Post (250mm * 4.6mm, 5 μ m);

Reagent: acetonitrile is chromatographically pure, and water is the ultrapure water of Millipore preparation, and other reagent are analytical pure.

17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate reference substance for self-control, is 99.27% 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, adopted respectively propyl carbinol-Glacial acetic acid-water (4: 0.5: 1), chloroform-methanol-water-Glacial acetic acid (7.5: 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 (80: 20) and moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-methyl alcohol (72: 28); 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 99.27%.

2.2 the reference substance solution preparation: precision takes 17-hydrogen-9-dehydrogenation-14, and 17-ring-rographolide-19-sodium sulfovinate reference substance 23.88mg puts in the 10ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, and get final product, in contrast the product mother liquor.

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, and get final product;

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-14, and 17-ring-rographolide-19-sulfuric ester 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, carries out gradient elution by following condition, moves 70 minutes:

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-14,17-ring-rographolide-19-sulfuric ester or its salt approximately had a chromatographic peak in 42 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-14, 17-ring-rographolide-19-sodium sulfovinate peak area integrated value is ordinate zou, the reference substance sample size is X-coordinate separately, the drawing standard curve, 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate regression equation is y=2187.0X-62.0, R2=1, 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate is good linear between 0.2388～3.8208 μ g.The results are shown in Table 1, Fig. 4.

Table 1.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate linear relationship result

5. precision test

Get 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate reference substance solution repeats sample introduction 6 times, 17-hydrogen-9-dehydrogenation-14 as a result, and 17-ring-rographolide-19-sodium sulfovinate peak area RSD is 0.29%, the results are shown in Table 2.

Table 2 reference substance Precision test result

6. stability test

Get need testing solution, measure according to chromatographic condition under 3 chromatographic conditions and system suitability item, sample introduction once at regular intervals, 17-hydrogen-9-dehydrogenation-14 in need testing solution as a result, peak area is without considerable change in 24 hours for 17-ring-rographolide-19-sodium sulfovinate, and its peak area RSD is respectively 0.50%.The results are shown in Table 3.

Table 3 stability test result

7. replica test

Get sample of the present invention, add water and make 6 parts of need testing solutions, measure 17-hydrogen-9-dehydrogenation-14 in sample according to chromatographic condition under 3 chromatographic conditions and system suitability item, 17-ring-rographolide-19-sodium sulfovinate average content is 98.79%, RSD=0.41%.The results are shown in Table 4.

Table 4.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate replica test result

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-14 respectively, 17-ring-rographolide-19-sodium sulfovinate (representing with 19 sodium sulfovinates in table) reference substance solution (0.942mg/ml) 2ml, be diluted with water to scale, shake up.Measure according to method under 3 chromatographic conditions and system suitability item, calculate recovery rate the results are shown in Table 5.

Table 5 recovery test result

Experimental data 2:17-hydrogen-9-dehydrogenation-14, the impact of 17-ring-rographolide-19-sodium sulfovinate induced by endotoxin pyrogenicity

Get Japan large ear rabbit, body weight 1.8-2.3kg, male and female have concurrently, and 1d before experiment chooses 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-14,17-ring-rographolide-19-sodium sulfovinate 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 as radix, is calculated each minute rabbit temperature changing value before the administration.See Table 6.

Medication temperature variation ℃ after table 6. endotoxin pyrogenic,

Annotate: compare with the empty map group ^※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 begins gradually after lasting rising 3h to descend.Compare low dose group 50mgkg with the blank group ^-117-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 1-2h shows obvious inhibition rabbit temperature rise effect, middle dosage group 100mgkg ^-1, high dose group 200mgkg ^-117-hydrogen-9-dehydrogenation-14 show the effect of extremely strong inhibition rabbit fervescence in 17-ring-rographolide-19-sodium sulfovinate 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-14, the rabbit fervescence that 17-ring-rographolide-19-sodium sulfovinate induced by endotoxin causes all has cooling effect.

Experimental data 3:17-hydrogen-9-dehydrogenation-14, the impact of 17-ring-rographolide-19-sodium sulfovinate 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 the variation of screening body temperature is no more than the animal of 0.3 ℃, is divided at random 5 groups, 8 every group.Subcutaneous injection 20% dry yeast suspension 5mL/kg, pyrogenicity pneumoretroperitoneum injection 0.9% sodium chloride solution, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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, as observation index, the results are shown in Table 7 with the difference of different time points body temperature value and basic value.

Medication temperature variation ℃ after table 7. dry yeast pyrogenicity,

Annotate: compare with the empty map group ^※P＜0.05 ^{※ ※}P＜0.01

Table 7 is found out, makes blank group healthy rat body temperature continue the 6h that rises when giving dry yeast 1h.Compare 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 100,200mgkg with the blank group ^-1Show obvious inhibition rat temperature rising effect in 1～4h.

Experimental data 4:17-hydrogen-9-dehydrogenation-14, the impact of 17-ring-rographolide-19-sodium sulfovinate p-Xylol induced mice auricle edema

Get 50 of mouse, be divided at random 5 groups.Every day is to 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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 rear 2 hours and take off cervical vertebra execution mouse, and cut mouse two ears along the auricle baseline, lay auricle with diameter 6mm punch tool respectively at left and right ear same section, put and weigh on electronic balance and record data.With left and right auricle weight difference value representation swelling.

Table 8. p-Xylol causes the impact of auricle edema

Annotate: compare with the empty map group ^※P＜0.05 ^{※ ※}P＜0.01

Table 8 shows, 17-hydrogen-9-dehydrogenation-14, and each medication group of 17-ring-rographolide-19-sodium sulfovinate, Dexamethasone group mice auricle swelling degree are significantly less than control group.

The impact of rat paw edema due to experimental data 5:17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate on Carrageenan

Get 40 of rats, be divided at random 5 groups.Every day is to 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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

Annotate: compare ※ P＜0.05 ※ ※ P＜0.01 with the empty map group

table 9 result shows, with the control group ratio, 17-hydrogen-9-dehydrogenation-14, due to each dosage group of 17-ring-rographolide-19-sodium sulfovinate and the equal on Carrageenan of Dexamethasone group, the rat swollen feet has obvious restraining effect, 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate 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-14, at same time point, the restraining effect of swelling there is a certain amount of effect relationship between 17-ring-rographolide-three of 19-sodium sulfovinates dosage group.

Experimental data 6:17-hydrogen-9-dehydrogenation-14, the restraining effect of 17-ring-rographolide-19-sodium sulfovinate to the neuraminic acid enzymic activity

Get 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate, add suitable quantity of water and make dissolving, use the neuraminidase inhibitor identification kit and measure 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate suppresses tiring of neuraminidase (N1) and sees Table 10.

(1). typical curve is prepared: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases to detect damping fluids; 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 fluids; B. every hole adds 10 μ l H5N1 neuraminidases again; C. every hole adds 0～10 μ l17-hydrogen-9-dehydrogenation-14 again, 17-ring-rographolide-19-sodium sulfovinate sample; D. every hole adds 0～10 μ l Milli-Q water again.

(3). detecting step:

A. vibrate approximately 1min of mixing;

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. vibrate again approximately 1min of mixing;

E.37 carry out fluorometric assay after ℃ hatching 20～30min.Excitation wavelength is 360nm, and emission wavelength is 440nm.

(4). calculate sample for H5N1 neuraminic acid enzymeinhibition per-cent according to typical curve, and calculate 17-hydrogen-9-dehydrogenation-14 after doing concentration curve, 17-ring-rographolide-19-sodium sulfovinate is for the IC50 of H5N1 neuraminidase.The inhibiting rate IC50 that 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate reach neuraminidase is 0.28g/L.See Table 10.

Table 10.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate suppresses the activity of neuraminidase

Can be clear that according to above-mentioned experimental result:

(1) .17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate can extract effective inhibition neuraminic acid enzyme component;

(2) .17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate are 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). by above-mentioned experiment as seen, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate can be by suppressing the surperficial neuraminidase of influenza virus, 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-14,17-ring-rographolide-19-sodium sulfovinate has prevention and treats the good result that the influenza virus sexuality is emitted.

Experimental data 11:17-hydrogen-9-dehydrogenation-14, the restraining effect of 17-ring-rographolide-19-sodium sulfovinate infected by influenza infected chicken embryo

Get 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate, use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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 is hatched 72h for 37 ℃, observes and calculate half chicken embryo infective dose (EID50).

(2) .17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate adopts the toxic action of chicken embryo, stroke-physiological saline solution is to 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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 inoculation 6 embryo is hatched for 37 ℃, observe chicken embryonic development developmental state, can survive the peak concentration of 96h as the TD of medicine with the chicken embryo.

(3) .17-hydrogen-9-dehydrogenation-14, the restraining effect of 17-ring-rographolide-19-sodium sulfovinate infected by influenza in the chicken embryo adopts, 0.1ml influenza virus liquid and different dilution 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate mixes, 37 ℃ of effect 2h, be inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, every winding kind 6 embryos are hatched 72h for 37 ℃.The virus attack amount is 50EID50, establishes simultaneously virus control, stroke-physiological saline solution normal control, calculates 17-hydrogen-9-dehydrogenation-14, the median effective dose (ED50) of 17-ring-rographolide-19-sodium sulfovinate 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 ^-4.82

(2) .17-hydrogen-9-dehydrogenation-14, after 17-ring-rographolide-19-sodium sulfovinate was inoculated in the chicken embryo, it grew basically identical with Normal group.96h chicken embryo is all survived.The chicken embryo gives 17-hydrogen-9-dehydrogenation-14, and 17-ring-rographolide-19-sodium sulfovinate stoste has no chicken embryo death, so can think that TD0 is 2.60g/L.(3) .17-hydrogen-9-dehydrogenation-14, the restraining effect of 17-ring-rographolide-19-sodium sulfovinate infected by influenza in the chicken embryo sees Table 11.

Table 11.17-hydrogen-9-dehydrogenation-14, the restraining effect of 17-ring-rographolide-19-sodium sulfovinate infected by influenza infected chicken embryo

Compare with the virus control group: * P＜0.05

As shown in Table 11,17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate has significant restraining effect (P＜0.05), ED at 0.09375～0.75g/L infected by influenza ₅₀Be 0.1012g ± 0.0098g/L, TI is 61.08 ± 3.28.

Experimental data 8:17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate affects the FM1 influenza virus

Get 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate, use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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-14,17-ring-rographolide-19-sodium sulfovinate adopts the DMEM of serum-free to 17-hydrogen-9-dehydrogenation-14 to the toxicity test of mdck cell, 17-ring-rographolide-19-sodium sulfovinate is done to be inoculated in after serial dilution in the mdck cell hole that forms individual layer, every hole 100 μ l, each extent of dilution repeats 4 holes, establishes simultaneously the normal cell contrast.Culture plate is put 37 ℃, 5%CO ₂Cultivate in incubator, 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-14,17-ring-rographolide-19-sodium sulfovinate suppress the effect of FM1 influenza virus and measure: 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 the hole next day, add 100TCID50 influenza virus liquid, every hole 100 μ l suck supernatant liquor after 37 ℃ of absorption 1h.Wash 2 times with phosphate buffered saline buffer (PBS), take the TD0 of medicine as the 1st hole, use again the DMEM liquid of serum-free to 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate is made serial dilution, add respectively above-mentioned the infection in viral cell, establish simultaneously virus control and Normal group, 37 ℃, 5%CO 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, drug dose and inhibiting rate that virus infected cell is avoided cytopathy (CPE) occurs are carried 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 ^-4.92(2) .17-hydrogen-9-dehydrogenation-14, the TD0 of 17-ring-rographolide-19-sodium sulfovinate mdck cell is respectively 1.19g ± 0.048g/L.(3). with 17-hydrogen-9-dehydrogenation-14, after 17-ring-rographolide-the 19-sodium sulfovinate is made serial dilution, the 100TCID50 influenza virus is carried out 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-14, the IC of 17-ring-rographolide-19-sodium sulfovinate to the FM1 influenza virus ₅₀(g/L) and TI (x ± s)

As shown in Table 12,17-hydrogen-9-dehydrogenation-14, the IC50 of 17-ring-rographolide-19-sodium sulfovinate is low, and TI is high.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate suppress the cytopathogenic effect of FM1 influenza virus and all strengthen along with the increase of drug dose.Drug dose and medicine are shown the correlation analysis that the inhibiting rate of CPE carries out, 17-hydrogen-9-dehydrogenation-14, the dosage of 17-ring-rographolide-19-sodium sulfovinate and medicine are to being obvious positive correlation between the CPE inhibiting rate.

Experimental data 9:17-hydrogen-9-dehydrogenation-14, the impact of 17-ring-rographolide-19-sodium sulfovinate on spleen index and the lung index of influenza virus infection FM1 strain in Mice Body

Get 17-hydrogen-9-dehydrogenation-14; 17-ring-rographolide-19-sodium sulfovinate; use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify 17-hydrogen-9-dehydrogenation-14, the dead provide protection of 17-ring-rographolide-19-sodium sulfovinate 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 viruses respectively every mouse collunarium inoculation 20 μ l for every group.Observe the dead mouse situation of 10d, calculating LD50 by the Reed-Muench method is 10 ^-1.38Therefore determine that experiment modeling concentration used is 10LD50.

(2) 17-hydrogen-9-dehydrogenation-14; the dead provide protection of 17-ring-rographolide-19-sodium sulfovinate to influenza virus infection FM1 strain in Mice Body: Normal group, influenza virus FM1 strain virus control group, 17-hydrogen-9-dehydrogenation-14; 17-ring-rographolide-19-sodium sulfovinate 0.125g/L, 0.25g/L, 0.5g/L, 1.0g/L dosage group philosophy gavage, gavage capacity only are 0.4ml/.After 3d, each group is only used 10LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ under the slight anesthesia of ether except Normal group.Normal group gives the physiological saline with 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-14, the impact of 17-ring-rographolide-19-sodium sulfovinate on influenza virus infection FM1 strain lung index in Mice Body: Normal group, influenza virus FM1 strain virus control group, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 0.125g/L, 0.25g/L, 0.5g/L, 1.0g/L dosage group philosophy gavage, gavage capacity only are 0.4ml/.After 3 days, each group is only used 1.0LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ under the slight anesthesia of ether except Normal group.Normal group gives the physiological saline with 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.Put to death mouse on the 8th day after virus infection, weigh, get lung and claim lung heavy, calculate lung index (lung index=lung quality/weight * 100%); In addition, get spleen and claim spleen heavy, calculate spleen index (spleen index=spleen quality/weight * 100%), the results are shown in Table 14.

Table 13.17-hydrogen-9-dehydrogenation-14, the death protection result of 17-ring-rographolide-19-sodium sulfovinate 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-14, the impact of 17-ring-rographolide-19-sodium sulfovinate on spleen index and the lung index of influenza virus infection FM1 strain in Mice Body

Annotate: #P＜0.05VS Normal group is annotated: ※ ※ p＜0.001VS influenza virus model group ※ p＜0.05VS influenza virus model group

(1). as shown in Table 13,17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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-14,17-ring-rographolide-19-sodium sulfovinate 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-14, the impact of 17-ring-rographolide-19-sodium sulfovinate on mouse septicemia

1.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate significantly improves septicemia mouse survival rate

Rographolide carries out gavage (30mg/kg) because of water-soluble extreme difference 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 appearance in 16 o'clock after modeling of model group mouse is dead, after 60h, survival rate is 25% (as shown in Table 15), rographolide administration group survival rate is 50%, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate administration group survival rate is 65%, comparatively speaking, 17-hydrogen-9-dehydrogenation-14, (be called for short in following form: the effect of the improving survival rate 19-sodium sulfovinate) is slightly better than rographolide group effect, but its dosage is lower for 17-ring-rographolide-19-sodium sulfovinate.

Table 15.17-hydrogen-9-dehydrogenation-14, the septicemia mouse survival rate that 17-ring-rographolide-19-sodium sulfovinate is induced LPS

2.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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-14,17-ring-rographolide-19-sodium sulfovinate 1,3,10mg/kg, abdominal injection 5mg/kg LPS simultaneously, after modeling and administration 2,4 time points of 5,8,12h are 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 namely reaches peak value, be respectively 4815pg/ml and 391pg/ml, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate administration 2h can significantly suppress TNF-α to 3672pg/ml, suppresses the release of IL-1 β being produced significantly 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-14, inflammatory factor in the septicemia mice serum that 17-ring-rographolide-19-sodium sulfovinate time, dose-dependently reduction LPS induce

TNFα(pg/ml)

IL?1β(pg/ml)

N=3, * p＜0.05vs model group, #p＜0.05vs rographolide group.

3.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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-14,17-ring-rographolide-19-sodium sulfovinate 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-14, after 17-ring-rographolide-19-sodium sulfovinate 2h, the content of ALT/AST namely descends to some extent, with model group ALT/AST, significant difference is arranged to 12h, than 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate effect is slightly poor to the inhibition of ALT/AST for rographolide.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-14, and 17-ring-rographolide-19-sodium sulfovinate 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.

The restraining effect of the septicemia mouse liver injury that induce LPS table 17.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate

AST(karmen?units)

ALT(karmen?units)

N=3, * p＜0.05, vs model group.

4, brief summary

Rographolide and 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 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-14,17-ring-rographolide-19-sodium sulfovinate onset is fast than rographolide, better effects if.After experimental result showed that rographolide is transformed into sulfonated bodies, it was water-soluble better, and administration is rapid-action, and the improvement effect of mouse septicemia also is enhanced.

Claims

1. the represented compound or its salt of a general formula (I), its chemistry 17-hydrogen by name-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt,

R can 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 6～39 ℃, sulfonation reaction 1～28.5 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 1.3ml～4.7ml/min/1g rographolide, when the mode that passes into gas when employing adds sulphonating agent, pass into speed control at 0.03 liter～0.22 liter/min/1g rographolide, get 17-hydrogen-9-dehydrogenation-14, the 17-ring-rographolide-reactant of 19-sulfuric ester and the mixture of unreacted reactant after sulfonation reaction;

(3) mixture is through solvent extraction, crystallization, gets final 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester; Perhaps

Mixture alkaline solution adjust pH to 7, through macroporous adsorbent resin, ODS or Sephadex LH-20 column chromatography for separation, take water: ethanol or methyl alcohol are as elutriant, gradient elution, collect eluant component, crystallization gets final 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt; Perhaps mixture successively passes through aforementioned two or more step co-treatment, gets final 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt.

3. the preparation method of claim 2, the described solvent that adds in reactor is one or both of aceticanhydride or Glacial acetic acid, and the 1g rographolide is with the described dissolution with solvents of 3g～12g, and is preferred, and the 1g rographolide is with the described dissolution with solvents of 4g～9g.

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, the 1g rographolide carries out sulfonation with 1g～10g vitriol oil Glacial acetic acid, 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, 1g rographolide 1.5g～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, it is that 1%～3% sulphur trioxide carries out sulfonation that the 1g rographolide passes into 0.2 liter～5 liters volumetric concentrations, preferably, to pass into 0.3 liter～3 liters volumetric concentrations be that 1.5%～2.5% sulphur trioxide carries out sulfonation to the 1g rographolide.

7. the preparation method of claim 2, described sulphonating agent adopts chlorsulfonic acid, and the 1g rographolide carries out sulfonation with 0.2g～5g chlorsulfonic acid, and preferably, the 1g rographolide carries out sulfonation with 0.3g～3.5g chlorsulfonic acid.

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 7～25 ℃, and the described sulfonation reaction time should be controlled at 0.5～2.5 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 1.8ml～3.5ml/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 0.05 liter～0.15 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-14,17-ring-rographolide-19-sulfuric ester 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 ℃;

Under these conditions, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt approximately had a chromatographic peak in 42 minutes in retention time,

14. 17-hydrogen-9-dehydrogenation-14, the preparation of 17-ring-rographolide-19-sulfuric ester or its salt, said preparation is 17-hydrogen claimed in claim 1-9-dehydrogenation-14, and 17-ring-rographolide-19-sulfuric ester or its salt are made with acceptable carrier pharmaceutically.

15. 17-hydrogen claimed in claim 1-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester 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-14, the refrigeration function of 17-ring-rographolide-19-sulfuric ester or its salt pair endotoxin pyrogenic.

17. as the purposes of claim 15, described 17-hydrogen-9-dehydrogenation-14, the refrigeration function of 17-ring-rographolide-19-sulfuric ester or its salt pair dry yeast pyrogenicity.

18. 17-hydrogen claimed in claim 1-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester 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-14, the drug effect of 17-ring-rographolide-19-sulfuric ester or its salt pair septicemia.

20. as the purposes of claim 18, described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt p-Xylol induced mice auricle edema anti-inflammatory action.

21. as the purposes of claim 18, described 17-hydrogen-9-dehydrogenation-14, the anti-inflammatory action of rat paw edema due to 17-ring-rographolide-19-sulfuric ester or its salt on Carrageenan.

22. 17-hydrogen claimed in claim 1-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester 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-14,17-ring-rographolide-19-sulfuric ester or its salt are used for suppressing neuraminidase.

24. as the purposes of claim 22, described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt are used for suppressing influenza virus.

25. as the purposes of claim 22, described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt are used for suppressing influenza virus FM1.