CN103156840A - Once preparation method of composite of 17-hydrogen-9-dehydro-andrographolidume-3-sodium (or potassium) disulfate and 17-hydrogen-9-dehydro-andrographolidume-3, 19-sodium (or potassium) disulfate, and purposes of prepared medicine - Google Patents

Abstract

The invention relates to a composite of 17-hydrogen-9-dehydro-andrographolidume-3-sodium (or potassium) disulfate and 17-hydrogen-9-dehydro-andrographolidume-3, 19-sodium (or potassium) disulfate, and discloses components of the composite, and a once preparation method of the components. The composite can be used for preparing medicine having antipyretic, anti-inflammatory and antiviral purposes. The composite is made into dosage forms which can be accepted pharmaceutically.

Description

17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, a preparation method of 19-di-sulfate sodium (or potassium) compositions and prepare medicinal usage

Technical field

The present invention relates to 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, a preparation method of 19-di-sulfate sodium (or potassium) compositions and prepare medicinal usage.

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 active component of Herba Andrographis be take diterpene-kind compound that andrographolide 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 andrographolide 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 methanol, ethanol, propanol, pyridine, is slightly soluble in chloroform, ether, is insoluble in water and petroleum ether.Therefore, the common film-making agent of oral formulations, capsule, drop pill, soft capsule; Water insoluble because of andrographolide, brought difficulty to preparing liquid preparation.

There is no effective especially, as to be suitable for the extraction active ingredient of actual production preparation method in prior art yet, especially when needs are made into compositions by the common use of two kinds of active ingredients, can only extract respectively, and then be mixed in proportion, complex manufacturing, production cost is high, has seriously restricted production efficiency.

Summary of the invention

The invention provides 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the preparation method of 19-di-sulfate sodium (or potassium) compositions.

Said composition is by andrographolide, sulfonation obtains, and preparation method is as follows:

[1] add solvent in reactor, add andrographolide to make its dissolving, then add sulfonating agent to carry out sulfonating reaction, conditioned reaction still temperature is at 5.5～39.5 ℃, sulfonating reaction 0.5～28.5 hour;

[2] andrographolide solution is in the situation that stirring adopts the mode that slowly drips, sprays or ventilate to add sulfonating agent, and when adopting the mode that slowly drips or spray to add sulfonating agent, add speed controlling at 1.15ml～4.8ml/min/1g andrographolide, when the mode that passes into gas when employing adds sulfonating agent, pass into speed controlling at 0.011 liter～0.28 liter/min/1g andrographolide, by above-mentioned sulfonating reaction thing to saturated salt solution; Perhaps use aqueous slkali adjust pH to 7, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture of 19-di-sulfate sodium (or potassium);

[3] mixture separates through the CG161 macroporous adsorbent resin column chromatography, applied sample amount is 1: 3～1: 18 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 5～1: 78, 1～10 times of column volume eluting of water, discard eluent, with 5%～35 ethanol or 3～9 times of column volume eluting of methanol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) compositions, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium (or potassium) compositions, perhaps

Mixture is through the ODS column chromatography for separation, applied sample amount is 1: 4.5～1: 21 with the ODS ratio, the resin column blade diameter length ratio is 1: 6～1: 78, 1～10.5 times of column volume eluting of water, discard eluent, with 8%～38 ethanol or 3～10 times of column volume eluting of methanol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) compositions, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium (or potassium) compositions, perhaps

Mixture is through Sephadex LH-20 column chromatography for separation, applied sample amount is 1: 4～1: 19 with Sephadex LH-20 ratio, the resin column blade diameter length ratio is 1: 6～1: 85, 1～10 times of column volume eluting of water, discard eluent, with 5%～35 ethanol or 2～10 times of column volume eluting of methanol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) compositions, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium (or potassium) compositions, perhaps

Mixture successively passes through aforementioned two or more step co-treatment;

Obtain described 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) compositions, the two weight content is: 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium) 94%～6%, 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) 6%～94%.

Preferably, the described solvent added in reactor is one or both of acetic anhydride or glacial acetic acid, and the described dissolution with solvents of 3g～12g for the 1g andrographolide is preferred, the described dissolution with solvents of 4g～9g for the 1g andrographolide.

Preferably, described solvent is acetic anhydride and glacial acetic acid, and described acetic anhydride, glacial acetic acid are made by following weight proportion: acetic anhydride 80%～20%, glacial acetic acid 20%～80%, and preferred, wherein acetic anhydride 62%, glacial acetic acid 38%.

Preferably, described sulfonating agent adopts concentrated sulphuric acid and glacial acetic acid, 1g for the 1g andrographolide～10g concentrated sulphuric acid glacial acetic acid carries out sulfonation, described concentrated sulphuric acid and glacial acetic acid are made by following weight proportion: concentrated sulphuric acid 80%～20%, glacial acetic acid 20%～80%, preferably, 1.5g for the 1g andrographolide～5g concentrated sulphuric acid glacial acetic acid, and the acid of dense stream is 1: 1 with the glacial acetic acid part by weight.

Preferably, described sulfonating agent adopts sulfur trioxide, the 1g andrographolide passes into the sulfur trioxide that 0.2 liter～5 liters volumetric concentrations are 1%～3% and carries out sulfonation, and preferred, the 1g andrographolide passes into the sulfur trioxide that 0.3 liter～3 liters volumetric concentrations are 1.5%～2.5% and carries out sulfonation.

Preferably, described sulfonating agent adopts chlorosulfonic acid, and 0.2g for the 1g andrographolide～5g chlorosulfonic acid carries out sulfonation, and preferred, 0.3g for the 1g andrographolide～3.5g chlorosulfonic acid carries out sulfonation.

Preferably, described mixture separates through the CG161 macroporous adsorbent resin column chromatography, and applied sample amount is 1: 5～1: 7.5 with CG161 macroporous adsorbent resin ratio; Described mixture is through the ODS column chromatography for separation, and applied sample amount is 1: 5～1: 7 with the ODS ratio; Described mixture is through Sephadex LH-20 column chromatography for separation, and applied sample amount is 1: 5～1: 6.5 with Sephadex LH-20 ratio.

Preferably, described mixture separates through the CG161 macroporous adsorbent resin column chromatography, and the resin column blade diameter length ratio is 1: 8～1: 23; Described mixture is through the ODS column chromatography for separation, and the resin column blade diameter length ratio is 1: 9～1: 26; Described mixture is through Sephadex LH-20 column chromatography for separation, and the resin column blade diameter length ratio is 1: 10～1: 28.

Preferably, described mixture separates through the CG161 macroporous adsorbent resin column chromatography, with 10%～20 ethanol or 3～6 times of column volume eluting of methanol; Described mixture is through the ODS column chromatography for separation, with 12%～25 ethanol or 3～7 times of column volume eluting of methanol; Described mixture is through Sephadex LH-20 column chromatography for separation, with 15%～28 ethanol or 4～8 times of column volume eluting of methanol.

Another object of the present invention has been to provide a kind of 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the composite preparation of 19-di-sulfate sodium (or potassium), it is main active that said preparation be take the described compositions that the described preparation method of claim 1～9 any one obtains.

Another object of the present invention has been to provide the purposes of the described compositions of described preparation method acquisition for the preparation of analgesic medicine.

Preferably, the refrigeration function of described compositions induced by endotoxin pyrogenicity.

Preferably, the refrigeration function of described compositions to dry yeast pyrogenicity.

Another object of the present invention has been to provide the purposes of the described compositions of described preparation method acquisition for the preparation of the medicine of antiinflammatory.

Preferably, the drug effect of described compositions to septicemia.

Preferably, described compositions xylol induced mice auricle edema antiinflammatory action.

Preferably, the antiinflammatory action of rat paw edema due to described compositions on Carrageenan.

Another object of the present invention has been to provide the purposes of the described compositions of described preparation method acquisition for the preparation of antiviral drug.

Preferably, described compositions is for suppressing neuraminidase.

Preferably, described compositions is for suppressing influenza virus.

Preferably, described compositions is for suppressing influenza virus FM1.

17-hydrogen provided by the present invention-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the preparation method of 19-di-sulfate sodium (or potassium) compositions is simple and convenient, mild condition, productivity ratio are high, disposable 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) compositions simultaneously prepared easily.

Simultaneously, 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 sulfonating agent and sulfonating reaction thereof, the processing mode of sulfonating 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 above-mentioned working foundation, further adopt again creative purification technique to carry out purification, adopted respectively the CG161 macroporous adsorbent resin column chromatography to separate, the ODS column chromatography for separation, Sephadex LH-20 column chromatography for separation, for above-mentioned chromatograph packing material different qualities, applied sample amount and chromatograph packing material ratio under different chromatographic conditions have been optimized, the blade diameter length ratio of chromatographic column, the concentration range of eluting solvent and the consumption of eluting solvent, under the high performance liquid chromatography monitoring, be enriched to 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium (or potassium) compositions, can under multiple different condition, make 17-hydrogen of the present invention-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium) thereby the invention provides, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium (or potassium) composite preparation.

The present invention is compared with the prior art and shows: adopt the 17-hydrogen that preparation method of the present invention makes-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) composite preparation, can not change the original chemical attribute of andrographolide fully; And 17-hydrogen of the present invention-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) composite preparation compared with prior art has the characteristics such as good water solubility, heat stability is high, haemolysis is little.Guaranteed to greatest extent the pharmacologically active effect of andrographolide pure natural medical.

17-hydrogen provided by the invention-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) composite preparation, in water, dissolubility 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, granule, chewable tablet, oral cavity disintegration tablet, drop pill, slow releasing tablet, controlled release tablet, slow releasing capsule, controlled release capsule.Can certainly make liquid preparation as syrup, injection etc., particularly make injection and overcome the low defect of oral drugs biological utilisation.

The present invention and prior art are known through the experimental data contrast:

After giving endotoxin 1h, blank group Healthy Rabbits body temperature average has risen, and after lasting rising 3h, starts gradually to descend.Compare low dose group 40mgkg with the blank group ^-1, middle dosage group 80mgkg ^-1, high dose group 160mgkg ^-1show the effect of extremely strong inhibition rabbit fervescence in compositions 1～2h, also demonstrate the rabbit fervescence effect that suppresses during 4h; Antipyretic effect presents dose-effect relationship.Show 40～160mgkg ^-1the rabbit fervescence that the compositions induced by endotoxin causes all has cooling effect.Obtained unforeseeable technique effect.

While giving dry yeast 1h, make blank group healthy rat body temperature continue the 6h that rises.Compare composite preparation 80,160mgkg with the blank group ^-1show obvious inhibition rat temperature rising effect in 1～4h, obtained unforeseeable technique effect.

Each medication group of composite preparation, Dexamethasone group mice auricle swelling degree are significantly less than matched group, have obtained unforeseeable technique effect.

With the matched group ratio, due to each dosage group of composite preparation and the equal on Carrageenan of Dexamethasone group, the rat swollen feet has obvious inhibitory action, composite preparation obvious inhibitory action occurs and continues 5 hours in administration 30min when 160mg/kg, 80mg/kg effect and effects of dexamethasone are suitable, at same time point, the inhibitory action of swelling is had to a certain amount of effect relationship between three dosage groups of composite preparation, obtained unforeseeable technique effect.

Andrographolide and composite preparation can significantly improve the survival rate of the septicemia mice that LPS induces, time, dose dependent 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 the composite preparation onset is fast than andrographolide, better effects if.After experimental result shows that andrographolide is transformed into sulfonated bodies, its water solublity is better, and administration is rapid-action, the improvement effect of mouse septicemia also is enhanced, and composite preparation dosage is lower, has obtained unforeseeable technique effect.

Composite preparation can extract effective inhibition neuraminic acid enzyme component; Composite preparation is along with the using dosage size variation, and it suppresses the ability of neuraminic acid enzymatic activity, i.e. also corresponding changing of the height of neuraminic acid enzyme inhibition rate, and become positive correlation; Composite preparation 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 that in advance composite preparation has prevention and treats the good result that the influenza virus sexuality is emitted, and has obtained unforeseeable technique effect.

Known through the experimental data contrast, the composite preparation infected by influenza has significant inhibitory action, has obtained unforeseeable technique effect.

Medical science and study of pharmacy personnel can't be in advance under the prerequisites of not doing related experiment, learn in advance 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) compositions has above-mentioned good purposes.

The accompanying drawing explanation

Fig. 1: 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate hydrogen nuclear magnetic resonance spectrogram.

Fig. 2: 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate carbon-13 nmr spectra figure.

Fig. 3: 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate mass spectrum.

Fig. 4: 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate linear relationship chart.

Fig. 5: 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium hydrogen nuclear magnetic resonance spectrogram.

Fig. 6: 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium carbon-13 nmr spectra figure.

Fig. 7: 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium mass spectrum.

Fig. 8: 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium wire sexual relationship figure.

The specific embodiment

The invention provides 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the compositions of 19-di-sulfate sodium (or potassium), the molecular formula of wherein said 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium):

R can be sodium or potassium;

Wherein said 17-hydrogen-9-dehydrogenation andrographolide-3, the molecular formula of 19-di-sulfate sodium (or potassium):

R ₁, R ₂can be sodium or potassium;

Embodiment 1

Get creat lactone, add 5.5 times of amount acetic anhydride (62%) and glacial acetic acid (38%) to make to dissolve, under agitation, speed with 1g andrographolide 1.6ml per minute, slowly drip the sulphuric acid (52%) and glacial acetic acid (48%) of 4.5 times of amounts, mix, 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 82% containing the alcohol amount, decompression recycling ethanol, 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 eluent, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, its sodium salt molecular formula C ₂₀h ₂₉naO ₈s, molecular weight: 452.

The reaction equation example:

17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate physicochemical property and spectral data:

17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate: molecular formula C ₂₀h ₂₉naO ₈s, white powder; ESIMS m/z:429[M-Na] ^-, HRESIMS:m/z 429.1590[M-Na] ^-(cald.for C ₂₀h ₂₉o ₈s, 429.1583); ¹h NMR and ¹³the CNMR data.

¹NMR(DMSO，600MHz)δ：1.01(overlapping，1H，H-1α)，1.74(m，1H，H-1β)，1.55～1.66(m，1H，H-2α)，2.04(m，1H，H-2β)，3.70(dd，J＝11.9，4.2Hz，1H，H-3)，1.10(d，J＝12.3Hz，1H，H-5)，1.55～1.66(m，1H，H-6α)，1.71(dd，J＝13.0，5.4Hz，1H，H-6β)，1.90～1.98(m，2H，H-7)，3.02(dd，J＝＝17.4，8.2Hz，1H，H-11α)，3.13(dd，J＝17.4，4.8Hz，H-11β)，6.46(dd，J＝8.2，4.8Hz，1H，H-12)，4.97(brt，J＝＝6.0Hz，1H，H-14)，4.02(dd，J＝9.6，1.7Hz，1H，H-15α)，4.45(dd，J＝9.6，6.0Hz，1H，H-15β)，1.53(s，3H，H-17)，1.04(s，3H，H-18)，3.46(dd，J＝9.6，3.2Hz，1H，H-19α)，3.57(dd，J＝9.6，3.2Hz，1H，H-19β)，0.98(s，1H，H-20)，5.73(d，J＝6.0Hz，14-OH)，3.55(brs，1H，19-OH)

¹³C-NMR(DMSO，150MHz)δ：34.5(C-1)，24.3(C-2)，83.3(C-3)，42.2(C-4)，52.2(C-5)，19.8(C-6)，34.0(C-7)，128.0(C-8)，136.7(C-9)，38.1(C-10)，27.4(C-11)，145.8(C-12)，128.3(C-13)，64.6(C-14)，74.1(C-15)，170.1(C-16)，19.5(C-17)，22.8(C-18)，62.5(C-19)，19.5(C-20)。

Embodiment 2

Get creat lactone, add 5.3 times of amount acetic anhydride (62%) and glacial acetic acid (38%) to make to dissolve, under agitation, speed with 1g andrographolide 2.2ml per minute, 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 eluent, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium, its sodium salt molecular formula: C ₂₀h ₂₈na ₂o ₁₁s ₂, molecular weight: 554.

The reaction equation example:

17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium physicochemical property and spectral data:

17-hydrogen-9-dehydrogenation andrographolide-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.forC 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 3

Get creat lactone, add 6.0 times of amount acetic anhydride (58%) and glacial acetic acid (42%) to make to dissolve, under agitation, speed with 0.07 liter per minute of 1g andrographolide, pass into 1.3 liter of 1.4% sulfur trioxide, 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 45%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 85% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate potassium, the a small amount of water dissolution of mixture, by Sephadex LH-20 post, the mixture applied sample amount is 1: 5.5 with Sephadex LH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 22, 2.5 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions elute soln, reclaim ethanol, vacuum drying, must be containing 65.55% 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 33.58% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions.

Embodiment 4

Get creat lactone, add 4.2 times of amount acetic anhydride to make to dissolve, under agitation, with the speed of 1g andrographolide 2.0ml per minute, the atomization spray adds the sulphuric 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 potassium chloride solution, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixed solutions such as 19-di-sulfate potassium, concentrating under reduced pressure, vacuum drying, obtain mixture, the a small amount of water dissolution of mixture, by Sephadex LH-20 post, the mixture applied sample amount is 1: 6.5 with Sephadex LH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 30, 3 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions elute soln, reclaim ethanol, vacuum drying, must be containing 66.35% 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 33.12% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions.

Embodiment 5

Get creat lactone, add 6 times of amount acetic anhydride (62%) and glacial acetic acid (38) to make to dissolve, under agitation, speed with 1g andrographolide 2.0ml per minute, spraying adds 3.3 times of amount sulphuric acid (52%) and glacial acetic acid (48%), mix, conditioned reaction still temperature is at 16 ℃, place and within 90 minutes, make sulfonation, add the equivalent purified water, pour into again in saturated nacl aqueous solution, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixed solutions such as 19-di-sulfate sodium, concentrating under reduced pressure, vacuum drying, obtain mixture, mixture is with passing through the CG161 macroporous adsorptive resins after a small amount of water dissolution, the mixture applied sample amount is 1: 5.5 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 22, 2.5 times of column volume eluting of water, discard eluent, with 4 times of column volume eluting of 10% ethanol, use again 6 times of column volume eluting of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 18.34% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 80.92% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 6

Get creat lactone, add 5.4 times of amount acetic anhydride (61%) and glacial acetic acid (39%) to make to dissolve, under agitation, speed with 0.09 liter per minute of 1g andrographolide, pass into 1.5 liter of 1.6% sulfur trioxide, conditioned reaction still temperature, at 14 ℃, is placed and within 100 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 40%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 82% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate sodium, the a small amount of water dissolution of mixture, by the ODS post, applied sample amount is 1: 5 with the ODS ratio, ODS post blade diameter length ratio is 1: 17, 3 times of column volume eluting of water, discard eluent, with 6 times of column volume eluting of 30% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 74.98% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 24.52% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 7

Get creat lactone, add 4.4 times of amount acetic anhydride (56%) and glacial acetic acid (44%) to make to dissolve, under agitation, speed with 0.08 liter per minute of 1g andrographolide, pass into 1.6 liter of 2.0% sulfur trioxide, conditioned reaction still temperature, at 20 ℃, is placed and within 100 minutes, is made sulfonation.In reactant impouring saturated nacl aqueous solution, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixed solutions such as 19-di-sulfate sodium, concentrating under reduced pressure, vacuum drying, obtain mixture, the a small amount of water dissolution of mixture, by Sephadex LH-20 post, the mixture applied sample amount is 1: 6 with Sephadex LH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 20, 2 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 85.06% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 14.57% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 8

Get creat lactone, add 5.9 times of amount acetic anhydride (60%) and glacial acetic acid (40%) to make to dissolve, under agitation, speed with 0.07 liter per minute of 1g andrographolide, pass into 1.6 liter of 1.8% sulfur trioxide, conditioned reaction still temperature is at 22 ℃, place and within 80 minutes, make sulfonation, add the equivalent purified water, stir evenly, adjusting pH with 35%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 83% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium mixture, mixture is with passing through the CG161 macroporous adsorptive resins after a small amount of water dissolution, the mixture applied sample amount is 1: 6 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 17, 2.5 times of column volume eluting of water, discard eluent, with 6 times of column volume eluting of 10% ethanol, use again 5 times of column volume eluting of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions elute soln, reclaim ethanol, vacuum drying, must be containing 33.62% 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 65.77% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions.

Embodiment 9

Get creat lactone, add 4.8 times of amount acetic anhydride (58%) and glacial acetic acid (42%) to make to dissolve, under agitation, with the speed of 1g andrographolide 2.2ml per minute, slowly drip 2.3 times of amount chlorosulfonic acids, mix, conditioned reaction still temperature is at 19 ℃, place and make sulfonation in 110 minutes, add purified water, stir evenly.In reactant impouring saturated potassium chloride solution, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixed solutions such as 19-di-sulfate potassium, concentrating under reduced pressure, vacuum drying, obtain mixture, mixture is with passing through the CG161 macroporous adsorptive resins after a small amount of water dissolution, the mixture applied sample amount is 1: 6.5 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 26, 2 times of column volume eluting of water, discard eluent, with 5 times of column volume eluting of 10% ethanol, use again 4 times of column volume eluting of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions elute soln, reclaim ethanol, vacuum drying, must be containing 18.53% 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 81.00% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions.

Embodiment 10

Get creat lactone, add 5.4 times of amount acetic anhydride (57%) and glacial acetic acid (43%) to make to dissolve, under agitation, speed with 1g andrographolide 1.9ml per minute, atomization adds 2.2 times of amount chlorosulfonic acids, mix, mix, conditioned reaction still temperature is at 16 ℃, place and within 80 minutes, make sulfonation, add the equivalent purified water, stir evenly, adjusting pH with 42%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 82% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate sodium, the a small amount of water dissolution of mixture, by the ODS post, applied sample amount is 1: 7 with the ODS ratio, ODS post blade diameter length ratio is 1: 23, 2.0 times of column volume eluting of water, discard eluent, with 6 times of column volume eluting of 26% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 68.24% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 31.17% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 11

Get creat lactone, add 4.5 times of amount acetic anhydride (62%) and glacial acetic acid (38%) to make to dissolve, under agitation, speed with 0.09 liter per minute of 1g andrographolide, pass into 1.4 liter of 2.2% sulfur 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 36%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 85% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate potassium, the a small amount of water dissolution of mixture, by the ODS post, applied sample amount is 1: 5.6 with the ODS ratio, ODS post blade diameter length ratio is 1: 17, 2.5 times of column volume eluting of water, discard eluent, with 6 times of column volume eluting of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium mixture elute soln, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium mixture.The a small amount of water dissolution of mixture, again by Sephadex LH-20 post, the mixture applied sample amount is 1: 6 with Sephadex LH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 24, 2.5 times of column volume eluting of water, discard eluent, with 6 times of column volume eluting of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions elute soln, reclaim ethanol, vacuum drying, must be containing 59.85% 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 39.48% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions.

Embodiment 12

Get creat lactone, add 4.9 times of amount acetic anhydride (56%) and glacial acetic acid (44%) to make to dissolve, under agitation, speed with 1g andrographolide 1.8ml per minute, slowly drip 2.5 times of amount chlorosulfonic 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 35%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 83% containing the alcohol amount, filter, filtrate recycling ethanol, concentrating under reduced pressure, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate sodium, mixture is with passing through the CG161 macroporous adsorptive resins after a small amount of water dissolution, the mixture applied sample amount is 1: 9 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 25, 2.5 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 25.95% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 73.42% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 13

Get creat lactone, add 5.4 times of amount acetic anhydride (60%) and glacial acetic acid (40%) to make to dissolve, under agitation, speed with 1g andrographolide 2.3ml per minute, slowly drip the sulphuric acid (57%) and glacial acetic acid (43%) of 4.8 times of amounts, mix, conditioned reaction still temperature, at 15 ℃, 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, filter, decompression filtrate recycling ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate sodium, the a small amount of water dissolution of mixture, by the ODS post, the mixture applied sample amount is 1: 6.5 with the ODS ratio, ODS post blade diameter length ratio is 1: 18, 3 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 48.87% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 51.08% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 14

Get creat lactone, add 5.4 times of amount acetic anhydride (60%) and glacial acetic acid (40%) to make to dissolve, under agitation, speed with 1g andrographolide 1.8ml per minute, the atomization spray adds the sulphuric 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 40%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 82% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate sodium, the a small amount of water dissolution of mixture, by the ODS post, applied sample amount is 1: 6 with the ODS ratio, ODS post blade diameter length ratio is 1: 24, 2 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 24% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 74.85% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 24.38% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 15

Get creat lactone, add 5.5 times of amount acetic anhydride (59%) and glacial acetic acid (41%) to make to dissolve, under agitation, speed with 1g andrographolide 2.6ml per minute, slowly drip the sulphuric 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 45%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 84% containing the alcohol amount, filter, filtrate recycling ethanol, concentrating under reduced pressure, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate potassium, mixture is with passing through the CG161 macroporous adsorptive resins after a small amount of water dissolution, the mixture applied sample amount is 1: 5.5 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 28, 3 times of column volume eluting of water, discard eluent, with 6 times of column volume eluting of 15% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions elute soln, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium mixture.The a small amount of water dissolution of mixture, again by Sephadex LH-20 post, the mixture applied sample amount is 1: 6 with the SephadexLH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 26, 2 times of column volume eluting of water, discard eluent, with 7 times of column volume eluting of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions elute soln, reclaim ethanol, vacuum drying, must be containing 36.57% 17-hydrogen-9-dehydrogenation andrographolide-3-sulfuric ester potassium, 62.75% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate potassium compositions.

Embodiment 16

Get creat lactone, add 6.0 times of amount acetic anhydride (60%) and glacial acetic acid (40%) to make to dissolve, under agitation, speed with 0.075 liter per minute of 1g andrographolide, pass into 1.4 liter of 1.6% sulfur 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 32%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 82% containing the alcohol amount, filter, filtrate recycling ethanol, concentrating under reduced pressure, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate sodium, mixture is with passing through the CG161 macroporous adsorptive resins after a small amount of water dissolution, the mixture applied sample amount is 1: 7.5 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 26, 2 times of column volume eluting of water, discard eluent, with 6 times of column volume eluting of 10% ethanol, use again 6 times of column volume eluting of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 26.43% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 72.91% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 17

Get creat lactone, add 5 times of amount acetic anhydride (56%) and glacial acetic acid (44%) to make to dissolve, under agitation, speed with 0.085 liter per minute of 1g andrographolide, pass into 1.6 liter of 1.8% sulfur trioxide, conditioned reaction still temperature, at 18 ℃, is placed and within 90 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 40%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 82% containing the alcohol amount, filter, filtrate recycling ethanol, concentrating under reduced pressure, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture such as 19-di-sulfate sodium, mixture is with passing through the CG161 macroporous adsorptive resins after a small amount of water dissolution, the mixture applied sample amount is 1: 5.4 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 18, 2.5 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 15% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium mixture.The a small amount of water dissolution of mixture, by the ODS post, applied sample amount is 1: 6.5 with the ODS ratio, ODS post blade diameter length ratio is 1: 25, 2 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 85.12% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 14.25% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Embodiment 18

Get creat lactone, add acetic anhydride and the glacial acetic acid of 5.8 times of amount equal proportions to make to dissolve, under agitation, speed with 1g andrographolide 1.6ml per minute, slowly drip 2.4 times of amount chlorosulfonic acids, mix, conditioned reaction still temperature is at 20 ℃, place and within 60 minutes, make sulfonation, in reactant impouring saturated nacl aqueous solution, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, the mixed solutions such as 19-di-sulfate sodium, concentrating under reduced pressure, vacuum drying, obtain mixture, the a small amount of water dissolution of mixture, by the SephadexLH-20 post, the mixture applied sample amount is 1: 8 with Sephadex LH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 27, 2.5 times of column volume eluting of water, discard eluent, with 8 times of column volume eluting of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum drying, must be containing 32.46% 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate, 66.95% 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate composition of sodium.

Experimental data 1:17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate and 17-hydrogen-9-dehydrogenation andrographolide-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, dual pathways 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 ultra-pure water prepared by Millipore, and other reagent are analytical pure.

17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate and 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium reference substance, for self-control, is respectively 99.46 and 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 n-butyl alcohol-glacial acetic acid-water (5: 1.5: 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 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate is a speckle as a result;

Get the sample that embodiment 2 preparation methoies obtain, adopt respectively n-butyl alcohol-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, 17-hydrogen-9-dehydrogenation andrographolide-3 as a result, 19-di-sulfate sodium is a speckle;

Use high performance liquid chromatography, adopt area normalization method to measure 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate content, select respectively chromatographic column: Diamonsil C ₁₈(250mm * 4.6mm, 5 μ m); Mobile phase: phosphate buffer (potassium dihydrogen phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-acetonitrile (89: 11) and mobile phase: phosphate buffer (potassium dihydrogen phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-methanol (77: 23); Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 25 ℃.Every group of mobile phase passes in and out respectively 10 μ l, 20 μ l respectively once, and sample introduction is 4 times altogether, and recording 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate 4 times average content is 99.46%.

Use high performance liquid chromatography, adopt area normalization method to measure 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium content, select respectively chromatographic column: Diamonsil C ₁₈(250mm * 4.6mm, 5 μ m); Mobile phase: phosphate buffer (potassium dihydrogen phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-acetonitrile (84: 16) and mobile phase: phosphate buffer (potassium dihydrogen phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-methanol (77: 23); Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 25 ℃.Every group of mobile phase passes in and out respectively 10 μ l, 20 μ l respectively once, and sample introduction is 4 times altogether, records 17-hydrogen-9-dehydrogenation andrographolide-3, and 4 average contents of 19-di-sulfate sodium are 99.06%.

2.2 reference substance solution preparation: precision takes the 17-hydrogen of above-mentioned mark-9-dehydrogenation andrographolide-3-sodium sulfovinate reference substance 22.40mg, puts in the 10ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, and obtains, and product mother solution in contrast, for methodological study.

Precision takes the 17-hydrogen of above-mentioned mark-9-dehydrogenation andrographolide-3, and 19-di-sulfate sodium reference substance 24.32mg, put in the 10ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, and obtains, and product mother solution in contrast, for methodological study.

2.3 the preparation of need testing solution: precision takes No. 13 sample 100mg of embodiment, puts in the 500ml measuring bottle, is diluted with water to scale, shakes up, and obtains;

3. chromatographic condition and system suitability

Take acetonitrile as mobile phase A, and the potassium phosphate buffer (adding potassium dihydrogen phosphate 1.361g and heptane-1-sodium sulfonate 1.0g in every 1000ml water) of take is Mobile phase B, and according to the form below, regulation is carried out gradient elution; Column temperature is 25 ℃; The detection wavelength is 225nm.Number of theoretical plate calculates and should be not less than 10000 by 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate.

4, the investigation of linear relationship

Each accurate 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate reference substance mother solution 1ml that draws, put respectively in 100ml, 50ml, 25ml, 10ml, 5ml measuring bottle, thin up becomes following concentration: 0.0224mg/ml, 0.0448mg/ml, 0.0896mg/ml, 0.1792mg/ml, 0.3584mg/ml respectively.Accurate draw solution 10l injection liquid chromatography, by chromatographic condition peak area under 3 chromatographic conditions and system suitability item, 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate peak area the integrated value of take respectively is vertical coordinate, the reference substance sample size is abscissa separately, the drawing standard curve, 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate regression equation is y=2495.26X-128.6, R ²=0.9999,17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate is good linear between 0.224～3.584 μ g.The results are shown in Table 1, linear relationship chart is shown in Fig. 4.

Table 1.17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate linear relationship result

Each accurate above-mentioned 17-hydrogen-9-dehydrogenation andrographolide-3 of drawing, 19-di-sulfate sodium reference substance mother solution 1ml, 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 andrographolide-3,19-di-sulfate sodium peak area integrated value is vertical coordinate, the reference substance sample size is abscissa separately, drawing standard curve, 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium regression equation is y=2340X-120.3, R ²=0.9999,17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium is good linear between 0.2432～3.8912 μ g.The results are shown in Table 2, linear relationship chart is shown in Fig. 8.

Table 2.17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium wire sexual relationship result

5. precision test

Get 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate reference substance solution, repeat sample introduction 6 times, 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate peak area RSD is 0.91% as a result, the results are shown in Table 3.

Table 3. Precision test result

Get 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium reference substance solution, repeat sample introduction 6 times, 17-hydrogen-9-dehydrogenation andrographolide-3 as a result, and 19-di-sulfate sodium peak area RSD is 0.19%, in Table 4.

Table 4. 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 andrographolide-3-sodium sulfovinate (hereinafter to be referred as 3 sodium sulfovinates) and 17-hydrogen-9-dehydrogenation andrographolide-3 as a result, 19-di-sulfate sodium is (hereinafter to be referred as 3,19 sodium sulfovinates) in 24 hours, peak area is without significant change, and its peak area RSD is respectively 0.48% and 1.33%.The results are shown in Table 5.

Table 5. stability test result

7. replica test

Precision takes sample of the present invention, and totally 6 parts, add water and make need testing solution, according to chromatographic condition under 3 chromatographic conditions and system suitability item, to measure, 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate average content is that 48.87%, RSD is 0.34%.17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium average content is that 51.08%, RSD is 1.33%, the results are shown in Table 6.

Table 6. replica test result

8. recovery test

Adopt the application of sample recovery test, precision measures sample solution of the present invention to the 50ml volumetric flask, totally 6 parts, precision adds 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate reference substance solution (0.928mg/ml) 2ml and 17-hydrogen-9-dehydrogenation andrographolide-3 respectively, 19-di-sulfate sodium reference substance solution (0.986mg/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 7, table 8.

Table 7.17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate recovery test result

Table 8.17-hydrogen-9-dehydrogenation andrographolide-3,1-di-sulfate sodium recovery test result

Below experiment all gets with " compositions " sample that embodiment 13 preparation methoies obtain.The experimental data that other embodiment obtain is substantially approaching, repeats no more.

Experimental data 2: the impact of compositions 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 endotoxin 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, compositions low dose group 40mgkg respectively ^-1, middle dosage group 80mgkg ^-1, high dose group 160mgkg ^-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 9.

Variations in temperature after table 9. compositions induced by endotoxin pyrogenicity

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

As shown in Table 9, after giving endotoxin 1h, blank group Healthy Rabbits body temperature average has risen, and after lasting rising 3h, starts gradually to descend.Compare low dose group 40mgkg with the blank group ^-1, middle dosage group 80mgkg ^-1, high dose group 160mgkg ^-1show the effect of extremely strong inhibition rabbit fervescence in compositions 1～2h, also demonstrate the rabbit fervescence effect that suppresses during 4h; Antipyretic effect presents dose-effect relationship.Show 40～160mgkg ^-1the rabbit fervescence that the compositions induced by endotoxin causes all has cooling effect.

Experimental data 3: the impact of compositions 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, compositions low dose group 40mgkg ^-1, middle dosage group 80mgkg ^-1, high dose group 160mgkg ^-1and aspirin 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 10.Table 10 is found out, while giving dry yeast 1h, makes blank group healthy rat body temperature continue the 6h that rises.Compare compositions 80,160mgkg with the blank group ^-1show obvious inhibition rat temperature rising effect in 1～4h.

Medication variations in temperature ℃ after table 10. dry yeast pyrogenicity,

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

Experimental data 4: the impact of compositions xylol induced mice auricle edema

Get 50 of mices, be divided at random 5 groups.Every day is to compositions low dose group 40mgkg ^-1, middle dosage group 80mgkg ^-1, high dose group 160mgkg ^-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 mice, 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 mice, and cut Mus two ears along the auricle baseline, with diameter 6mm card punch, 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 11 demonstration, each medication group of compositions, Dexamethasone group mice auricle swelling degree are significantly less than matched group.

Table 11. xylol causes the impact of auricle edema

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

Experimental data 5: the impact of rat paw edema due to the compositions on Carrageenan

Get 40 of rats, be divided at random 5 groups.Every day is to compositions low dose group 40mgkg ^-1, middle dosage group 80mgkg ^-1, high dose group 160mgkg ^-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 normal 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 micrometer after injecting.Table 12 result shows, with the matched group ratio, due to each dosage group of compositions and the equal on Carrageenan of Dexamethasone group, the rat swollen feet has obvious inhibitory action, compositions obvious inhibitory action occurs and continues 5 hours in administration 30min when 160mg/g, 80mg/kg effect and effects of dexamethasone are suitable, between three dosage groups of compositions, at same time point, the inhibitory action of swelling are had to a certain amount of effect relationship.

Table 12. causes the impact of rat toes swelling on chondrus ocellatus Holmes

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

Experimental data 6: the inhibitory action of compositions to the neuraminic acid enzymatic activity

Get compositions, add suitable quantity of water and make to dissolve, application neuraminidase inhibitor identification kit is measured compositions and is suppressed tiring in Table 13 of neuraminidase (N1).

(1). standard curve is prepared: a. every hole in 96 hole luciferase targets adds the 70l neuraminidase to detect buffer; 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 buffer; B. every hole adds 10 μ l H5N1 neuraminidases again; C. every hole adds 0～10 μ l composition 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 standard 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 fluoremetry.Excitation wavelength is 360nm, and emission wavelength is 440nm.

(4). calculate the inhibition percentage ratio of sample for the H5N1 neuraminidase according to standard curve, and calculate the IC50 of compositions for the H5N1 neuraminidase after doing concentration curve.The suppression ratio IC50 that compositions reaches neuraminidase is 0.274g/L.In Table 13.

Table 13. compositions suppresses the activity of neuraminidase

According to above-mentioned experimental result, can be clear that:

(1). compositions can extract effective inhibition neuraminic acid enzyme component;

(2). compositions is along with the using dosage size variation, and it suppresses the ability of neuraminic acid enzymatic activity, i.e. also corresponding changing of the height of neuraminic acid enzyme inhibition rate, and become positive correlation;

(3). from above-mentioned experiment, compositions 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 is copied,

Propagation, thus infection, the growth of influenza virus to cell reduced, 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 that in advance compositions has prevention and treats the good result that the influenza virus sexuality is emitted.

Experimental data 7: the compositions infected by influenza infects the inhibitory action of Embryo Gallus domesticus

Get compositions, application influenza virus A-prime Mus lung adapted strain (FM1) (H1N1) identifies that compositions suppresses the ability that the FM1 influenza virus is copied and suppresses in Embryo Gallus domesticus.

(1). FM1 influenza virus liquid is inoculated in to 10d no-special pathogen in age chick embryo allantois intracavity, and every embryo 0.2ml, hatch 72h for 37 ℃, observes and calculate half Embryo Gallus domesticus infective dose (EID50).

(2). compositions adopts the toxic action of Embryo Gallus domesticus, physiological saline solution is done to be inoculated in 10d no-special pathogen in age chick embryo allantois intracavity after serial dilution to compositions, every embryo 0.2ml, each concentration is inoculated 6 embryos, hatch for 37 ℃, observe Embryo Gallus domesticus growth promoter situation, the Embryo Gallus domesticus of usining can be survived the Cmax of 96h as the TD of medicine.

(3). the inhibitory action of compositions infected by influenza in Embryo Gallus domesticus adopts, the influenza virus liquid of 0.1ml and different dilution compositions mixing, 37 ℃ of effect 2h, be inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, inoculates 6 embryos for every group, hatches 72h for 37 ℃.The virus attack amount is 50EID50, establishes virus control, physiological saline solution normal control simultaneously, the median effective dose (ED50) of calculation composition to viral inhibition.

(1) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of Embryo Gallus domesticus, and its EID50 is 10 ^-4.78.

(2). after compositions is inoculated in Embryo Gallus domesticus, its growth promoter and Normal group are basically identical.The 96h Embryo Gallus domesticus is all survived.Embryo Gallus domesticus gives compositions stock solution and has no chicken embryo death, so can think that TD0 is 2.32g/L.

(3). the inhibitory action of compositions infected by influenza in Embryo Gallus domesticus is in Table 14.

Table 14. compositions infected by influenza infects the inhibitory action of Embryo Gallus domesticus

With the virus control group, compare: * P<0.05

As shown in Table 14, compositions has significant inhibitory action (P<0.05), ED at 0.0725～0.58g/L infected by influenza ₅₀for 0.1024g ± 0.0105g/L, TI is 63.42 ± 3.28.

Experimental data 8: compositions affects the FM1 influenza virus

Get compositions, application influenza virus A-prime Mus lung adapted strain (FM1) (H1N1) identifies that compositions suppresses the ability of FM1 influenza virus virulence.

(1) .FM1 adopts cell median infective dose (TCID50) micromethod to the toxicity test of Testis et Pentis Canis passage cell (MDCK).

(2). compositions adopts the DMEM of serum-free to do to be inoculated in Zhong，Mei hole, the mdck cell hole 100l that forms monolayer after serial dilution to compositions to the toxicity test of mdck cell, and each dilution factor repeats 4 holes, establishes the normal cell contrast simultaneously.Culture plate is put to 37 ℃, 5%CO ₂in incubator, cultivate, observation of cell pathological changes 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). compositions 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 culture fluid in hole next day, add 100TCID50 influenza virus liquid, every hole 100 μ l, suck supernatant after 37 ℃ of absorption 1h.With phosphate buffer (PBS), wash 2 times, the TD0 of medicine of take is the 1st hole, then with the DMEM liquid of serum-free, compositions is made to serial dilution, adds respectively above-mentioned the infection in viral cell, establishes 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. cell monolayer degeneration becomes circle etc., and 3d, calculate 50% of medicine and suppress pathological changes 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 comparative test group and the cytopathic difference of virus control group, to drug dose with to virus infected cell, avoid the suppression ratio 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 ^-4.68.(2). the TD0 of compositions mdck cell is respectively 1.24g ± 0.027g/L.(3). after compositions is made to 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 15.

The IC of table 15. compositions to the FM1 influenza virus ₅₀and TI (x ± s) (g/L)

As shown in Table 15, the IC50 of compositions is low, and TI is high.Compositions suppresses the cytopathogenic effect of FM1 influenza virus and all strengthens along with the increase of drug dose.The correlation analysis that drug dose and medicine are carried out the suppression ratio of CPE shows, the dosage of compositions and medicine are to being obvious positive correlation between the CPE suppression ratio.

Experimental data 9: the impact of compositions on spleen index and the lung index of influenza virus infection FM1 strain in Mice Body

Get compositions, application influenza virus A-prime Mus lung adapted strain (FM1) (H1N1) is identified the dead protective effect of compositions 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 mice collunarium is inoculated 20 μ l.Observe the dead mouse situation of 10d, calculating LD50 by the Reed-Muench method is 10 ^-1.88.Therefore determine that experiment modeling concentration used is 10LD50.

(2) the dead protective effect of compositions to influenza virus infection FM1 strain in Mice Body: Normal group, influenza virus FM1 strain virus matched group, compositions 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/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 normal 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 rate (mortality rate=every group of death toll/every group of total mice * 100%), the results are shown in Table 16.

(3) impact of compositions on influenza virus infection FM1 strain lung index in Mice Body: Normal group, influenza virus FM1 strain virus matched group, compositions 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/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 normal 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 viral infection, put to death mice, 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 17.

The death protection result of table 16. compositions to influenza virus infection FM1 strain in Mice Body

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

The impact of table 17. compositions 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

As shown in Table 16, compositions has significant protective effect (p<0.01) at 0.2～0.8g/L influenza virus infected.

As shown in Table 17, compositions has significant effect (p<0.01) at the lung index of 0.4～0.8g/L influenza virus infected.

Experimental data 10: the impact of compositions on mouse septicemia

1. compositions can significantly be improved septicemia mouse survival rate

Andrographolide, because of the water solublity extreme difference, carries out gavage (30mg/kg) so adopt 0.9% sodium carboxymethyl cellulose solution to be mixed with suspension, and compositions directly is made into settled solution with PBS and carries out tail vein injection (10mg/kg).Lumbar injection 5mg/kg LPS carries out modeling, observes mouse survival number of elements in 60h, calculates survival rate.The model group mice occurs dead in 16 o'clock after modeling, after 60h, survival rate is 25% (shown in table 18), andrographolide administration group survival rate is 50%, compositions administration group survival rate is 72.5%, comparatively speaking, the effect of composition for improved survival rate is slightly better than andrographolide group effect, but its dosage is lower.

The septicemia mouse survival rate that table 18. compositions is induced LPS

2. compositions significantly suppresses the rising of inflammatory factor in the septicemia mice serum

BALB/C mice abdominal cavity gavage gives andrographolide 30mg/kg, tail vein injection compositions 0.8,2.4,8mg/kg, lumbar injection 5mg/kg LPS simultaneously, after modeling and administration 2,5,8,12h4 time point respectively put to death 3 mices, and eye socket is got blood, measures inflammatory factor TNF-α in serum, the content of IL-1 β, shown in table 19.After lps injection 2h, TNF-α in the model group mice serum, the content of IL-1 β reach peak value, is respectively 4815pg/ml and 391pg/ml, compositions administration 2h can significantly suppress TNF-α to 3513pg/ml, to the release of IL-1 β being produced significantly and suppresses when the 5h.Andrographolide effect 8h just can significantly suppress the rising of TNF-α.Experimental result shows than andrographolide, and compositions is rapid-action to the inhibitory action of inflammatory factor in the septicemia mice serum, and inhibition is more remarkable, and dosage is lower.

Inflammatory factor in the septicemia mice serum that table 19. compositions time, dose dependent reduction LPS induce

TNF?α(pg/ml)

IL?1β(pg/ml)

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

3. compositions significantly suppresses the hepar damnification of septicemia mice

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 andrographolide 30mg/kg, tail vein injection compositions 8mg/kg, and while lumbar 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 20, after giving LPS, model mice serum alt and AST continue to raise, and give the content of ALT/AST after compositions 2h and descend to some extent, with model group ALT/AST, significant difference is arranged to 12h, andrographolide is slightly poorer than composition effect to the inhibition of ALT/AST.RT-PCR measures the mice 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, compositions and andrographolide 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 andrographolide, and compositions is rapid-action to the inhibitory action of septicemia mouse liver injury, and inhibition is more remarkable.

The inhibitory action of the septicemia mouse liver injury that table 20. compositions is induced LPS

AST(karmen?units)

ALT(karmen?units)

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

4, brief summary

Andrographolide and compositions can significantly be improved the survival rate of the septicemia mice that LPS induces, time, dose dependent 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.The compositions onset is fast than andrographolide, better effects if.After experimental result shows that andrographolide is transformed into sulfonated bodies, its water solublity is better, rapid-action after the compositions administration, the improvement effect of mouse septicemia also is enhanced, and dosage is lower.

Claims (21)

1. 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the preparation method of 19-di-sulfate sodium (or potassium) compositions, said composition is by andrographolide, sulfonation obtains, and preparation method is as follows:

[1] add solvent in reactor, add andrographolide to make its dissolving, then add sulfonating agent to carry out sulfonating reaction, conditioned reaction still temperature is at 5.5～39.5 ℃, sulfonating reaction 0.5～28.5 hour;

[2] andrographolide solution is in the situation that stirring adopts the mode that slowly drips, sprays or ventilate to add sulfonating agent, and when adopting the mode that slowly drips or spray to add sulfonating agent, add speed controlling at 1.15ml～4.8ml/min/1g andrographolide, when the mode that passes into gas when employing adds sulfonating agent, pass into speed controlling at 0.011 liter～0.28 liter/min/1g andrographolide, by above-mentioned sulfonating reaction thing to saturated salt solution; Perhaps use aqueous slkali adjust pH to 7, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the mixture of 19-di-sulfate sodium (or potassium);

[3] mixture separates through the CG161 macroporous adsorbent resin column chromatography, applied sample amount is 1: 3～1: 18 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 5～1: 78, 1～10 times of column volume eluting of water, discard eluent, with 5%～35 ethanol or 3～9 times of column volume eluting of methanol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) compositions, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium (or potassium) compositions, perhaps

Mixture is through the ODS column chromatography for separation, applied sample amount is 1: 4.5～1: 21 with the ODS ratio, the resin column blade diameter length ratio is 1: 6～1: 78, 1～10.5 times of column volume eluting of water, discard eluent, with 8%～38 ethanol or 3～10 times of column volume eluting of methanol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) compositions, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium (or potassium) compositions, perhaps

Mixture is through Sephadex LH-20 column chromatography for separation, applied sample amount is 1: 4～1: 19 with Sephadex LH-20 ratio, the resin column blade diameter length ratio is 1: 6～1: 85, 1～10 times of column volume eluting of water, discard eluent, with 5%～35 ethanol or 2～10 times of column volume eluting of methanol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) compositions, reclaim ethanol, vacuum drying, obtain 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3, 19-di-sulfate sodium (or potassium) compositions, perhaps

Mixture successively passes through aforementioned two or more step co-treatment;

Obtain described 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) compositions, the two weight content is: 17-hydrogen-9-dehydrogenation andrographolide-3-sodium sulfovinate (or potassium) 94%～6%, 17-hydrogen-9-dehydrogenation andrographolide-3,19-di-sulfate sodium (or potassium) 6%～94%.

2. the preparation method of claim 1, the described solvent added in reactor is one or both of acetic anhydride or glacial acetic acid, the described dissolution with solvents of 3g～12g for the 1g andrographolide is preferred, the described dissolution with solvents of 4g～9g for the 1g andrographolide.

3. the preparation method of claim 2, described solvent is acetic anhydride and glacial acetic acid, described acetic anhydride, glacial acetic acid are made by following weight proportion: acetic anhydride 80%～20%, glacial acetic acid 20%～80%, preferred, wherein acetic anhydride 62%, glacial acetic acid 38%.

4. the preparation method of claim 1, described sulfonating agent adopts concentrated sulphuric acid and glacial acetic acid, 1g for the 1g andrographolide～10g concentrated sulphuric acid glacial acetic acid carries out sulfonation, described concentrated sulphuric acid and glacial acetic acid are made by following weight proportion: concentrated sulphuric acid 80%～20%, glacial acetic acid 20%～80%, preferably, 1.5g for the 1g andrographolide～5g concentrated sulphuric acid glacial acetic acid, and the acid of dense stream is 1: 1 with the glacial acetic acid part by weight.

5. the preparation method of claim 1, described sulfonating agent adopts sulfur trioxide, the 1g andrographolide passes into the sulfur trioxide that 0.2 liter～5 liters volumetric concentrations are 1%～3% and carries out sulfonation, preferably, the 1g andrographolide passes into the sulfur trioxide that 0.3 liter～3 liters volumetric concentrations are 1.5%～2.5% and carries out sulfonation.

6. the preparation method of claim 1, described sulfonating agent adopts chlorosulfonic acid, and the 0.2g 1g andrographolide for～5g chlorosulfonic acid carries out sulfonation, and preferably, 0.3g for the 1g andrographolide～3.5g chlorosulfonic acid carries out sulfonation.

7. the preparation method of claim 1, described mixture separates through the CG161 macroporous adsorbent resin column chromatography, and applied sample amount is 1: 5～1: 7.5 with CG161 macroporous adsorbent resin ratio; Described mixture is through the ODS column chromatography for separation, and applied sample amount is 1: 5～1: 7 with the ODS ratio; Described mixture is through Sephadex LH-20 column chromatography for separation, and applied sample amount is 1: 5～1: 6.5 with Sephadex LH-20 ratio.

8. the preparation method of claim 1, described mixture separates through the CG161 macroporous adsorbent resin column chromatography, and the resin column blade diameter length ratio is 1: 8～1: 23; Described mixture is through the ODS column chromatography for separation, and the resin column blade diameter length ratio is 1: 9～1: 26; Described mixture is through Sephadex LH-20 column chromatography for separation, and the resin column blade diameter length ratio is 1: 10～1: 28.

9. the preparation method of claim 1, described mixture separates through the CG161 macroporous adsorbent resin column chromatography, with 10%～20 ethanol or 3～6 times of column volume eluting of methanol; Described mixture is through the ODS column chromatography for separation, with 12%～25 ethanol or 3～7 times of column volume eluting of methanol; Described mixture is through Sephadex LH-20 column chromatography for separation, with 15%～28 ethanol or 4～8 times of column volume eluting of methanol.

10. 17-hydrogen-9-dehydrogenation andrographolide-3, the composite preparation of 19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation andrographolide-19-sodium sulfovinate (or potassium), it is main active that said preparation be take the described compositions that the described preparation method of claim 1～9 any one obtains.

11. the described compositions that the described preparation method of claim 1～9 any one obtains is for the preparation of the purposes of analgesic medicine.

12. as the purposes of claim 11, the refrigeration function of described compositions induced by endotoxin pyrogenicity.

13. as the purposes of claim 11, the refrigeration function of described compositions to dry yeast pyrogenicity.

14. the described compositions that the described preparation method of claim 1～9 any one obtains is for the preparation of the purposes of the medicine of antiinflammatory.

15. as the purposes of claim 14, the drug effect of described compositions to septicemia.

16. as the purposes of claim 14, described compositions xylol induced mice auricle edema antiinflammatory action.

17. as the purposes of claim 14, the antiinflammatory action of rat paw edema due to described compositions on Carrageenan.

18. the described compositions that the described preparation method of claim 1～9 any one obtains is for the preparation of the purposes of antiviral drug.

19., as the purposes of claim 18, described compositions is for suppressing neuraminidase.

20., as the purposes of claim 18, described compositions is for suppressing influenza virus.

21., as the purposes of claim 18, described compositions is for suppressing influenza virus FM1.

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