CN103145658A - One-step preparation method of composition of sodium (or potassium) 17-hydro-9-dehydroandrographolide-3-sulphate, sodium (or potassium) 17-hydro-9-dehydroandrographolide-19-sulphate and sodium (or potassium) 17-hydro-9-dehydroandrographolide-3,19-disulfate, and use of the composition in drug preparation. - Google Patents

One-step preparation method of composition of sodium (or potassium) 17-hydro-9-dehydroandrographolide-3-sulphate, sodium (or potassium) 17-hydro-9-dehydroandrographolide-19-sulphate and sodium (or potassium) 17-hydro-9-dehydroandrographolide-3,19-disulfate, and use of the composition in drug preparation. Download PDF

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CN103145658A
CN103145658A CN2012101091329A CN201210109132A CN103145658A CN 103145658 A CN103145658 A CN 103145658A CN 2012101091329 A CN2012101091329 A CN 2012101091329A CN 201210109132 A CN201210109132 A CN 201210109132A CN 103145658 A CN103145658 A CN 103145658A
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hydrogen
potassium
rographolide
dehydrogenation rographolide
composition
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吕武清
唐春山
谢宁
杨小玲
刘地发
李志勇
程帆
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JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
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JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
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Abstract

The invention relates to a composition of sodium (or potassium) 17-hydro-9-dehydroandrographolide-3-sulphate, sodium (or potassium) 17-hydro-9-dehydroandrographolide-19-sulphate and sodium (or potassium) 17-hydro-9-dehydroandrographolide-3,19-disulfate, a one-step preparation method of the composition, a use of the composition in preparation of a drug for bringing down a fever, diminishing inflammation and resisting viruses, and a pharmaceutically acceptable preparation prepared from the composition.

Description

17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, a preparation method of 19-di-sulfate sodium (or potassium) composition and prepare pharmaceutical use
Technical field
The present invention relates to a kind of 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, preparation method of 19-di-sulfate sodium (or potassium) composition and prepare pharmaceutical use.
Background technology
The dry aerial parts that Herba Andrographis is acanthaceous plant Herba Andrographis Andrographis paniculata (Burm.f.) Nees, chemical composition and pharmacological evaluation show, the activeconstituents of Herba Andrographis be take diterpene-kind compound that rographolide is representative as main [State Administration of Traditional Chinese Medicine. the 7th of China's book on Chinese herbal medicine. Shanghai: Shanghai science tech publishing house, 1999:439], the structure of rographolide is:
Figure BSA00000701931300011
Molecular formula: C 20h 30o 5, be the crystallization of colourless square square, m.p.230-232 ℃, [α] 0 20-126 ° of (c0.2, H 2o).Flavor is extremely bitter, dissolves in methyl alcohol, ethanol, propyl alcohol, pyridine, is slightly soluble in chloroform, ether, is insoluble in water and sherwood oil.Therefore, the common film-making agent of oral preparations, capsule, dripping pill, soft capsule; Water insoluble because of rographolide, brought difficulty to preparing liquid preparation.
There is no effective especially, as to be suitable for the extraction effective ingredient of actual production preparation method in prior art yet, especially when needs are made into composition by the common use of two kinds of effective 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 a kind of 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, the preparation method of 19-di-sulfate sodium (or potassium) composition.
Said composition is by rographolide, sulfonation obtains, and the preparation method is as follows:
[1] add solvent in reactor, add rographolide to make its dissolving, then add sulphonating agent to carry out sulfonation reaction, conditioned reaction still temperature is at 6～39 ℃, sulfonation reaction 0.5～28 hour;
[2] rographolide solution is in the situation that stirring adopts the mode that slowly drips, sprays or ventilate to add sulphonating agent, and when adopting the mode that slowly drips or spray to add sulphonating agent, add speed control at 1.25ml～4.95ml/min/1g rographolide, when the mode that passes into gas when employing adds sulphonating agent, pass into speed control at 0.010 liter～0.28 liter/min/1g rographolide, by above-mentioned sulfonation reaction thing to saturated salt solution; Perhaps use alkaline solution adjust pH to 7, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-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: 20 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 4～1: 85, 1～12 times of column volume wash-out of water, discard elutriant, with 5%～38 ethanol or 4～15 times of column volume wash-outs of methyl alcohol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) composition, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition, perhaps
Mixture is through the ODS column chromatography for separation, applied sample amount is 1: 4～1: 16 with the ODS ratio, the resin column blade diameter length ratio is 1: 6～1: 77, 1.5～13 times of column volume wash-outs of water, discard elutriant, with 5%～38 ethanol or 3.5～14 times of column volume wash-outs of methyl alcohol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) composition, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition, 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: 82, 1～13 times of column volume wash-out of water, discard elutriant, with 5%～37 ethanol or 3～15 times of column volume wash-outs of methyl alcohol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, the elute soln of 19-di-sulfate sodium (or potassium) composition, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition, perhaps
Mixture successively passes through aforementioned two or more step co-treatment;
Obtain described 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium (or potassium) composition is made by following weight proportion: 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium) 10%～65%, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium) 10%～65%, 17-hydrogen-9-dehydrogenation rographolide-3,19-sodium sulfovinate (or potassium) 10%～60%.
Preferably, the described solvent added in reactor is one or both of aceticanhydride or Glacial acetic acid, and the described dissolution with solvents of 3g～12g for the 1g rographolide is preferred, the described dissolution with solvents of 4g～9g for the 1g rographolide.
Preferably, described solvent is aceticanhydride and Glacial acetic acid, and described aceticanhydride, Glacial acetic acid are made by following weight proportion: aceticanhydride 80%～20%, Glacial acetic acid 20%～80%, and preferred, wherein aceticanhydride 62%, Glacial acetic acid 38%.
Preferably, described sulphonating agent adopts the vitriol oil and Glacial acetic acid, 1g for the 1g rographolide～10g vitriol oil Glacial acetic acid carries out sulfonation, the described vitriol oil and Glacial acetic acid are made by following weight proportion: the vitriol oil 80%～20%, Glacial acetic acid 20%～80%, preferably, 1.5g for the 1g rographolide～5g vitriol oil Glacial acetic acid, and the acid of dense stream is 1: 1 with the Glacial acetic acid part by weight.
Preferably, described sulphonating agent adopts sulphur trioxide, the 1g rographolide passes into the sulphur trioxide that 0.2 liter～5 liters volumetric concentrations are 1%～3% and carries out sulfonation, and preferred, the 1g rographolide passes into the sulphur trioxide that 0.3 liter～3 liters volumetric concentrations are 1.5%～2.5% and carries out sulfonation.
Preferably, described sulphonating agent adopts chlorsulfonic acid, and 0.2g for the 1g rographolide～5g chlorsulfonic acid carries out sulfonation, and preferred, 0.3g for the 1g rographolide～3.5g chlorsulfonic acid carries out sulfonation.
Preferably, described mixture separates through the CG161 macroporous adsorbent resin column chromatography, and applied sample amount is 1: 5.5～1: 10 with CG161 macroporous adsorbent resin ratio; Described mixture is through the ODS column chromatography for separation, and applied sample amount is 1: 6～1: 8 with the ODS ratio; Described mixture is through Sephadex LH-20 column chromatography for separation, and applied sample amount is 1: 5～1: 7 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: 25; 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%～30 ethanol or 4.5～7.5 times of column volume wash-outs of methyl alcohol; Described mixture is through the ODS column chromatography for separation, with 12%～32 ethanol or 4～6.5 times of column volume wash-outs of methyl alcohol; Described mixture is through Sephadex LH-20 column chromatography for separation, with 15%～33 ethanol or 4～8 times of column volume wash-outs of methyl alcohol.
Another object of the present invention has been to provide a kind of 17-hydrogen-9-dehydrogenation rographolide-3, the composite preparation of 19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), it is main active ingredient that said preparation be take the described composition that the described preparation method of claim 1～9 any one obtains.
Another object of the present invention provides the purposes of a kind of claim 1～9 any one described composition that described preparation method obtains for the preparation of analgesic medicine.
Preferably, the refrigeration function of described composition induced by endotoxin pyrogenicity.
Preferably, the refrigeration function of described composition to dry yeast pyrogenicity.
Another object of the present invention has been to provide the purposes of the described composition of the described preparation method's acquisition of a kind of claim 1～9 any one for the preparation of the medicine of anti-inflammatory.
Preferably, the drug effect of described composition to septicemia.
Preferably, described composition p-Xylol induced mice auricle edema anti-inflammatory action.
Preferably, the anti-inflammatory action of rat paw edema due to described composition on Carrageenan.
Another object of the present invention has been to provide the purposes of the described composition of the described preparation method's acquisition of a kind of claim 1～9 any one for the preparation of antiviral drug.
Preferably, described composition is for suppressing neuraminidase.
Preferably, described composition is for suppressing influenza virus.
Preferably, described composition is for suppressing influenza virus FM1.
17-hydrogen provided by the present invention-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, the preparation method of 19-di-sulfate sodium (or potassium) composition is simple and convenient, mild condition, productivity is high, the disposable 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium) of simultaneously preparing easily, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition.
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 sulphonating agent and sulfonation reaction thereof, the processing mode of sulphonating agent and add determining of complex relationship between speed for example, and the determining etc. of suitable numerical range, thereby just finally obtained required reactant.
On above-mentioned working foundation, further adopt again creative purification technique to carry out purifying, 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 rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition, can under multiple different condition, make 17-hydrogen of the present invention-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium) thereby the invention provides, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composite preparation.
The present invention is compared with the prior art and shows: the 17-hydrogen that adopts preparation method of the present invention to make-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium (or potassium) composite preparation, can not change the original chemical attribute of rographolide fully; And 17-hydrogen of the present invention-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium (or potassium) composite preparation compared with prior art has the characteristics such as good water solubility, thermostability is high, hemolytic action is little.Guaranteed to greatest extent the pharmacologically active effect of rographolide pure natural medical.
17-hydrogen provided by the invention-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium (or potassium) composite preparation, in water, solubleness is very good and stability is very high, be applicable to very much practical application, can be applied to various common type, as tablet, capsule, soft capsule, dispersible tablet, oral liquid, particle, chewable tablet, orally disintegrating tablet, dripping pill, slow releasing tablet, controlled release tablet, slow releasing capsule, controlled release capsule.Can certainly make liquid preparation as syrup, injection liquid etc., particularly make injection and overcome the low defect of oral pharmaceutical biological utilisation.
The present invention and prior art are known through the experimental data contrast:
After giving intracellular toxin 1h, blank group Healthy Rabbits body temperature average has risen, and after lasting rising 3h, starts gradually to descend.Compare low dose group 40mgkg with the blank group -1composition 1～2h shows obvious inhibition rabbit temperature rise effect, middle dosage group 80mgkg -1, high dose group 160mgkg -1show the effect of extremely strong inhibition rabbit fervescence in composition 1-2h, also demonstrate the rabbit fervescence effect that suppresses during 4h; In 3 drug study groups with 80mgkg -1the above antipyretic effect of dosage is best.Show 40～160mgkg -1the rabbit fervescence that the composite preparation induced by endotoxin causes all has cooling effect, has 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 control group, have obtained unforeseeable technique effect.
With the control group ratio, due to each dosage group of composite preparation and the equal on Carrageenan of Dexamethasone group, the rat swollen feet has obvious restraining effect, composite preparation obvious restraining effect 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 restraining effect of swelling is had to a certain amount of effect relationship between three dosage groups of composite preparation, obtained unforeseeable technique effect.
Rographolide and composite preparation can significantly improve the survival rate of the septicemia mouse that LPS induces, time, dose-dependently reduce TNF-α in the septicemia mice serum, IL-1 β, ALT, the content of AST, the sick rising that suppresses inflammatory factor mRNA level in liver organization.And the composite preparation onset is fast than rographolide, better effects if.After experimental result shows that rographolide is transformed into sulfonated bodies, it is water-soluble better, and administration is rapid-action, 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 enzymic 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 restraining effect, 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 rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium (or potassium) composition has above-mentioned good purposes.
The accompanying drawing explanation
Fig. 1: 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate hydrogen nuclear magnetic resonance spectrogram.
Fig. 2: 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate carbon-13 nmr spectra figure.
Fig. 3: 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate mass spectrum.
Fig. 4: 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate linear relationship chart.
Fig. 5: 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate hydrogen nuclear magnetic resonance spectrogram
Fig. 6: 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate carbon-13 nmr spectra figure
Fig. 7: 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate linear relationship chart.
Fig. 8: 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium hydrogen nuclear magnetic resonance spectrogram.
Fig. 9: 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium carbon-13 nmr spectra figure.
Figure 10: 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium mass spectrum.
Figure 11: 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium wire sexual intercourse figure.
Embodiment
The invention provides a kind of 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, the composition of 19-di-sulfate sodium (or potassium), the structural formula of wherein said 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium) is as follows:
Figure BSA00000701931300081
R can be sodium or potassium;
The structural formula of described 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium) is as follows:
Figure BSA00000701931300082
R 1can be sodium or potassium;
Wherein said 17-hydrogen-9-dehydrogenation rographolide-3, the molecular formula of 19-sodium sulfovinate (or potassium):
Figure BSA00000701931300083
R 2, R 3can be sodium or potassium;
Embodiment 1
Get creat lactone, add 5.5 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make to dissolve, under agitation, speed with 1g rographolide per minute 1.6ml, slowly drip the sulfuric 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, the aqueous solution is through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, and the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, its sodium salt molecular formula C 20h 29naO 8s, molecular weight: 452.
The reaction formula example:
Figure BSA00000701931300091
17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate physico-chemical property and spectral data:
17-hydrogen-9-dehydrogenation rographolide-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 5.6 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, with the speed of 1g rographolide per minute 2.0ml, slowly drip 4.5 times of equivalent sulfuric acid ice acetic acid, mix, conditioned reaction still temperature, at 18 ℃, is placed and within 100 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 36%NaOH is 7.0 left and right, add 95% ethanol to make to reach more than 82% containing the alcohol amount, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, 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%, collects eluant component, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, the molecular formula of its sodium salt: C 20h 29naO 8s, molecular weight: 452.
The reaction formula example:
Figure BSA00000701931300101
17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate physico-chemical property and spectral data:
17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate: molecular formula C 20h 29naO 8s, white powder; 1h NMR and 13c NMR data.
1NMR(DMSO，600MHz)δ：1.04～1.07(m，1H，H-1α)，1.58～1.75(m，1H，H-1β)，1.58～1.75(m，2H，H-2)，3.18(m，1H，H-3)，1.11(brd，J＝12.6Hz，1H，H-5)，1.43(m，1H，H-6α)，1.58～1.75(m，1H，H-6β)，1.92～2.02(m，2H，H-7)，2.99(dd，J＝17.4，7.8Hz，1H，H-11α)，3.14(dd，J＝17.4，5.6Hz，H-11β)，6.46(ddd，J＝7.8，5.6，1.4Hz，1H，H-12)，4.96(brs，1H，H-14)，4.03(dd，J＝9.8，2.3Hz，1H，H-15α)，4.44(dd，J＝9.8，6.0Hz，1H，H-15β)，1.52(s，3H，H-17)，1.08(s，3H，H-18)，3.28(dd，J＝10.8，6.0Hz，1H，H-19α)，3.84(d，J＝10.9Hz，1H，H-19β)，0.92(s，1H，H-20)，4.96(br?s，3-OH)，5.73(br?s，1H，14-OH)，4.06(br?d，J＝6.0Hz)
13C-NMR(DMSO，150MHz)δ：35.0(C-1)，28.1(C-2)，77.7(C-3)，42.1(C-4)，52.1(C-5)，19.5(C-6)，34.5(C-7)，128.6(C-8)，137.4(C-9)，38.5(C-10)，27.9(C-11)，146.4(C-12)，128.9(C-13)，65.2(C-14)，74.7(C-15)，170.5(C-16)，19.7(C-17)，23.4(C-18)，63.4(C-19)，20.6(C-20)。
Embodiment 3
Get creat lactone, add 5.3 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.2ml, slowly drip the sulfuric acid ice acetic acid of 4.3 times of amount equal proportions, mix, conditioned reaction still temperature is at 15 ℃, place and within 90 minutes, make sulfonation, add the equivalent purified water, pour into again in saturated nacl aqueous solution, again through macroporous adsorbent resin, the ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, the Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium, its sodium salt molecular formula: C 20h 28na 2o 11s 2, molecular weight: 554.
The reaction formula example:
Figure BSA00000701931300111
17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium physico-chemical property and spectral data:
17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium: molecular formula C 20h 28na 2o 11s 2, white powder; ESIMS m/z:531[M-Na] -, HRESIMS:m/z m/z 531.1014[M-Na] -(cald.for C 20h 28o 11s 2na, 531.0971); 1h NMR and 13c NMR data.
1NMR(D 2O，600MHz)δ：1.75～1.83(m，1H，H-1α)，1.17(td，J＝13.6，3.5Hz，1H，H-1β)，1.75～1.83(m，1H，H-2α)，1.94～2.05(m，1H，H-2β)，3.97(dd，J＝12.0，4.5Hz，1H，H-3)，1.28(brd，J＝12.0，1H，H-5)，1.56(m，1H，H-6α)，175～1.83(m，1H，H-6β)，1.75～1.83(m，1H，H-7α)，1.94～2.05(m，1H，H-7β)，3.05(dd，J＝17.7，8.6Hz，1H，H-11α)，3.19(dd，J＝17.7，4.9Hz，H-11β)，6.81(ddd，J＝8.0，5.0，1.4Hz，1H，H-12)，5.13(dd，J＝6.0，1.8z，1H，H-14)，4.23(dd，J?＝10.6，1.8Hz，1H，H-15α)，4.50(dd，J＝10.6，6.0Hz，1H，H-15β)，1.52(s，3H，H-17)，1.12(s，3H，H-18)，3.96(d，J＝10.0Hz，1H，H-19α)，4.20(d，J＝10.0Hz，1H，H-19β)，1.00(s，1H，H-20)
13C-NMR(D 2O，150MHz)δ：32.2(C-1)，22.0(C-2)，84.7(C-3)，39.3(C-4)，49.5(C-5)，17.5(C-6)，31.7(C-7)，128.1(C-8)，133.5(C-9)，36.0(C-10)，26.0(C-11)，148.6(C-12)，124.0(C-13)，63.0(C-14)，73.1(C-15)，171.0(C-16)，16.5(C-17)，19.7(C-18)，67.7(C-19)，16.5(C-20)。
Embodiment 4
Get creat lactone, add 5.5 times of amount aceticanhydrides (60%) and Glacial acetic acid (40) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.1ml, spraying adds 3.5 times of amount sulfuric acid (52%) and Glacial acetic acid (48%), mix, conditioned reaction still temperature is at 17 ℃, place and within 80 minutes, make sulfonation, add the equivalent purified water, pour into again in saturated nacl aqueous solution, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixing solutionss 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: 15, 2 times of column volume wash-outs of water, discard elutriant, with 5 times of column volume wash-outs of 12% ethanol, use again 6 times of column volume wash-outs of 22% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum-drying, must be containing 25.00% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 37.80% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 37.05% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 5
Get creat lactone, add 5.4 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.3ml, slowly drip the sulfuric 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 30%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 84% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures such as 19-di-sulfate potassium, the a small amount of water dissolution of mixture, by the ODS post, the mixture applied sample amount is 1: 4.5 with the ODS ratio, ODS post blade diameter length ratio is 1: 8, 1.8 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition elute soln, reclaim ethanol, vacuum-drying, must be containing 21.00% 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 45.28% 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 65.85% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition.
Embodiment 6
Get creat lactone, add 6.2 times of amount aceticanhydrides (59%) and Glacial acetic acid (41%) to make to dissolve, under agitation, speed with 0.08 liter of 1g rographolide per minute, pass into 1.3 liter of 1.4% sulphur trioxide, conditioned reaction still temperature, at 14 ℃, 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, filter, decompression filtrate recycling ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures 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: 18, 2.5 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 26% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum-drying, must be containing 63.23% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 18.85% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17.46% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 7
Get creat lactone, add 5.6 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, speed with 0.10 liter of 1g rographolide per minute, pass into 1.4 liter of 1.7% sulphur trioxide, conditioned reaction still temperature, at 16 ℃, is placed and within 80 minutes, is made sulfonation.In reactant impouring saturated nacl aqueous solution, obtain 7-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixing solutionss 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: 7 with Sephadex LH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 20, 2.5 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 24% ethanol, HPLC detects, merge 7-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum-drying, must be containing 12.65% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 62.85% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 21.00% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 8
Get creat lactone, add 4.7 times of amount aceticanhydrides (55%) and Glacial acetic acid (45%) to make to dissolve, under agitation, speed with 0.08 liter of 1g rographolide per minute, pass into 1.8 liter of 1.8% sulphur trioxide, conditioned reaction still temperature, at 18 ℃, is placed and within 110 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 32%KOH is 7.0 left and right, add 95% ethanol to make to reach more than 85% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures 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: 6 with Sephadex LH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 15, 2 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 22% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition elute soln, reclaim ethanol, vacuum-drying, must be containing 35.10% 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 35.15% 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 28.85% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition.
Embodiment 9
Get creat lactone, add 5.7 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, speed with 0.07 liter of 1g rographolide per minute, pass into 1.5 liter of 2.3% sulphur trioxide, conditioned reaction still temperature, at 24 ℃, is placed and within 90 minutes, is made sulfonation, add purified water, stir evenly.In reactant impouring saturated potassium chloride solution, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, the mixing solutionss 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: 8 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 12, 3 times of column volume wash-outs of water, discard elutriant, with 4 times of column volume wash-outs of 13% ethanol, use again 4 times of column volume wash-outs of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition elute soln, reclaim ethanol, vacuum-drying, must be containing 26.57% 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 18.85% 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 54.00% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition.
Embodiment 10
Get creat lactone, add 4.9 times of amount aceticanhydrides (59%) and Glacial acetic acid (41%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.0ml, slowly drip 2.2 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 14 ℃, place and within 100 minutes, make sulfonation, add the equivalent purified water, stir evenly, adjusting pH with 30%NaOH 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 rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures 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: 18, 2.5 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum-drying, must be containing 40.00% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 22.65% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 36.90% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 11
Get creat lactone, add aceticanhydride and the Glacial acetic acid of 6.2 times of amount equal proportions to make to dissolve, under agitation, speed with 1g rographolide per minute 1.5ml, slowly drip 2.2 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 22 ℃, place and within 70 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 84% containing the alcohol amount, filter, decompression filtrate recycling ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-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: 6.5 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 16, 3.5 times of column volume wash-outs of water, discard elutriant, with 6 times of column volume wash-outs of 10% ethanol, use again 7 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition elute soln, reclaim ethanol, vacuum-drying, must be containing the 17-hydrogen of 20.26 %-9-dehydrogenation rographolide-3-sulfuric ester potassium, 34.46% 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 44.75% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition.
Embodiment 12
Get creat lactone, add 5.3 times of amount aceticanhydrides (58%) and Glacial acetic acid (42%) to make to dissolve, under agitation, speed with 1g rographolide per minute 1.6ml, slowly drip 2.4 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 26 ℃, place and within 90 minutes, make sulfonation, in reactant impouring saturated potassium chloride solution, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, the mixing solutionss such as 19-di-sulfate potassium, concentrating under reduced pressure, vacuum-drying, obtain mixture, mixture is with passing through Sephadex LH-20 post after a small amount of water dissolution, the mixture applied sample amount is 1: 7.5 with the SephadexLH-20 ratio, the resin column blade diameter length ratio is 1: 20, 3.5 times of column volume wash-outs of water, discard elutriant, with 5 times of column volume wash-outs of 12% ethanol, use again 5 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition elute soln, reclaim ethanol, vacuum-drying, must be containing 42.15% 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 25.64% 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 31.76% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition.
Embodiment 13
Get creat lactone, add 6.2 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, speed with 1g rographolide per minute 1.9ml, slowly drip the sulfuric acid (55%) and Glacial acetic acid (45%) of 4.5 times of amounts, mix, conditioned reaction still temperature, at 23 ℃, is placed and within 110 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 30%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 rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures such as 19-di-sulfate sodium, the a small amount of water dissolution of mixture, by the ODS post, applied sample amount is 1: 6.5 with the ODS ratio, ODS post blade diameter length ratio is 1: 16, 3.2 times of column volume wash-outs of water, discard elutriant, with 7 times of column volume wash-outs of 30% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum-drying, must be containing 62.35% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 20.15% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 16.90% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 14
Get creat lactone, add 5.6 times of amount aceticanhydrides to make to dissolve, under agitation, with the speed of 1g rographolide per minute 2.5ml, the atomization spray adds the sulfuric acid (54%) and Glacial acetic acid (46%) of 4.5 times of amounts, mixes, conditioned reaction still temperature, at 15 ℃, is placed and within 100 minutes, is made sulfonation.In reactant impouring saturated nacl aqueous solution, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixing solutionss 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: 7.5 with the SephadexLH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 22, 4.5 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 22% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium solution, reclaim ethanol, vacuum-drying, must be containing 23.05% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 52.54% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 23.85% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 15
Get creat lactone, add 5.5 times of amount aceticanhydrides (62%) and Glacial acetic acid (38%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.0ml, slowly drip the sulfuric acid (52%) and Glacial acetic acid (48%) of 4.8 times of amounts, mix, conditioned reaction still temperature, at 24 ℃, is placed and within 120 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 84% containing the alcohol amount, filter, filtrate recycling ethanol, concentrating under reduced pressure, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures 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: 5.5 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 18, 3.5 times of column volume wash-outs of water, discard elutriant, with 6 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium part, reclaim ethanol, vacuum-drying, must be containing 33.05% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 32.83% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 33.81%17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 16
Get creat lactone, add 6.2 times of amount aceticanhydrides (63%) and Glacial acetic acid (37%) to make to dissolve, under agitation, speed with 0.09 liter of 1g rographolide per minute, pass into 1.8 liter of 1.5% sulphur trioxide, conditioned reaction still temperature, at 18 ℃, is placed and within 120 minutes, is made sulfonation.In reactant impouring saturated potassium chloride solution, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium etc. mixing solutions, 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: 8.5 with the SephadexLH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 26, 4.5 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, the composition parts such as 19-di-sulfate potassium, reclaim ethanol, vacuum-drying, must be containing 42.85% 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 37.36% 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 19.05% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition.
Embodiment 17
Get creat lactone, add 5.6 times of amount aceticanhydrides (63%) and Glacial acetic acid (37%) to make to dissolve, under agitation, speed with 0.09 liter of 1g rographolide per minute, pass into 1.6 liter of 1.8% sulphur trioxide, conditioned reaction still temperature, at 16 ℃, is placed and within 90 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 32%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 rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures such as 19-di-sulfate sodium, the a small amount of water dissolution of mixture, by the ODS post, applied sample amount is 1: 5.9 with the ODS ratio, ODS post blade diameter length ratio is 1: 19, 3.9 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 26% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium part, reclaim ethanol, vacuum-drying, must be containing 15.25% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 57.07% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 26.85% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 18
Get creat lactone, add 5.8 times of amount aceticanhydrides (58%) and Glacial acetic acid (42%) to make to dissolve, under agitation, speed with 0.09 liter of 1g rographolide per minute, pass into 1.9 liter of 2.0% sulphur trioxide, conditioned reaction still temperature, at 16 ℃, is placed and within 110 minutes, is made sulfonation.Add the equivalent purified water, stir evenly, adjusting pH with 38%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 rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures 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: 6.5 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 16, 4.0 times of column volume wash-outs of water, discard elutriant, with 6 times of column volume wash-outs of 15% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition elute soln, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-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: 8 with Sephadex LH-20 ratio, Sephadex LH-20 post blade diameter length ratio is 1: 25, 3 times of column volume wash-outs of water, discard elutriant, with 6 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition elute soln, reclaim ethanol, vacuum-drying, must be containing 27.62% 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 35.48% 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 39.25% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition.
Embodiment 19
Get creat lactone, add 6 times of amount aceticanhydrides (60%) and Glacial acetic acid (40%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.0ml, slowly drip 3.0 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 16 ℃, place and within 120 minutes, make 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 rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures 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 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 16, 4.5 times of column volume wash-outs of water, discard elutriant, with 6 times of column volume wash-outs of 10% ethanol, use again 6 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum-drying, must be containing 55.06% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17.25% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 26.88% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 20
Get creat lactone, add 5.6 times of amount aceticanhydrides (56%) and Glacial acetic acid (44%) to make to dissolve, under agitation, speed with 1g rographolide per minute 2.3ml, atomization adds 3.4 times of amount chlorsulfonic 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 457%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 rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures 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: 5.5 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 18, 4 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 15% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-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 with the ODS ratio, ODS post blade diameter length ratio is 1: 20, 3 times of column volume wash-outs of water, discard elutriant, with 8 times of column volume wash-outs of 20% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium elute soln, reclaim ethanol, vacuum-drying, must be containing 15.24% 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 46.28% 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 37.65% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate composition of sodium.
Embodiment 21
Get creat lactone, add aceticanhydride and the Glacial acetic acid of 5.5 times of amount equal proportions to make to dissolve, under agitation, speed with 1g rographolide per minute 2.0ml, slowly drip 3.0 times of amount chlorsulfonic acids, mix, conditioned reaction still temperature is at 22 ℃, place and within 100 minutes, make sulfonation, add the equivalent purified water, stir evenly, add the equivalent purified water, stir evenly, adjusting pH with 50%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 rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, the mixtures 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 with the ODS ratio, ODS post blade diameter length ratio is 1: 15, 2 times of column volume wash-outs of water, discard elutriant, with 6 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium mixture elute soln, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-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: 20, 2.5 times of column volume wash-outs of water, discard elutriant, with 6 times of column volume wash-outs of 25% ethanol, HPLC detects, merge 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition elute soln, reclaim ethanol, vacuum-drying, must be containing 46.15% 17-hydrogen-9-dehydrogenation rographolide-3-sulfuric ester potassium, 42.25% 17-hydrogen-9-dehydrogenation rographolide-19-sulfuric ester potassium, 11.00% 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate potassium composition.
Experimental data 1:17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium content is measured
1. instrument and reagent
Instrument: Agilent1100 quarternary low pressure gradient pump series, Chemstation chem workstation, DAD detector; Shimadzu LC-2010A type high performance liquid chromatograph, two channels ultraviolet variable-wavelenght detector; Sartoriuscp211D 100,000/electronic balance.
Chromatographic column: Diamonsil C 18post (250mm * 4.6mm, 5 μ m);
Reagent: acetonitrile is chromatographically pure, and water is ultrapure water prepared by Millipore, and other reagent are analytical pure.
17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate and 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium reference substance, for self-control, is respectively 99.46,99.68% 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 propyl carbinol-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 rographolide-3-sodium sulfovinate is a spot as a result;
Get the sample that embodiment 2 preparation methods obtain, adopt respectively propyl carbinol-Glacial acetic acid-water (4: 0.5: 0.5), chloroform-methanol-water-Glacial acetic acid (6.5: 2.5: 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 rographolide-19-sodium sulfovinate is a spot as a result;
Get the sample that embodiment 3 preparation methods obtain, adopt respectively propyl carbinol-Glacial acetic acid-water (4.5: 1: 1), chloroform-methanol-water-Glacial acetic acid (8: 3: 1: 0.5) carry out the purity of thin layer chromatography inspection, the point sample amount is respectively 5,10,15,20,25 μ g, 17-hydrogen-9-dehydrogenation rographolide-3 as a result, 19-di-sulfate sodium is a spot;
Use high performance liquid chromatography, adopt area normalization method to measure the 17-hydrogen of embodiment 1-9-dehydrogenation rographolide-3-sodium sulfovinate content, select respectively chromatographic column: Diamonsil C 18(250mm * 4.6mm, 5 μ m); Moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-acetonitrile (89: 11) and moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-methyl alcohol (77: 23); Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 25 ℃.Every group of moving phase passes in and out respectively 10 μ l, 20 μ l respectively once, and sample introduction is 4 times altogether, and recording 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate 4 times average content is 99.46%.
Use high performance liquid chromatography, adopt area normalization method to measure the 17-hydrogen of embodiment 2-9-dehydrogenation rographolide-19-sodium sulfovinate content, select respectively chromatographic column: Diamonsil C 18(250mm * 4.6mm, 5 μ m); Moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-acetonitrile (84: 16) and moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-methyl alcohol (76: 24); Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 25 ℃.Every group of moving phase passes in and out respectively 10 μ l, 20 μ l respectively once, and sample introduction is 4 times altogether, and recording 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate 4 times average content is 99.68%.
Use high performance liquid chromatography, adopt area normalization method to measure the 17-hydrogen of embodiment 3-9-dehydrogenation rographolide-3,19-di-sulfate sodium content, select respectively chromatographic column: Diamonsil C 18(250mm * 4.6mm, 5 μ m); Moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-acetonitrile (84: 16) and moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-methyl alcohol (77: 23); Flow velocity: 1ml/min; Detect wavelength: 225nm; Column temperature: 25 ℃.Every group of moving phase passes in and out respectively 10 μ l, 20 μ l respectively once, and sample introduction is 4 times altogether, records 17-hydrogen-9-dehydrogenation rographolide-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 rographolide-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 liquor in contrast, for methodological study.
Precision takes the 17-hydrogen of above-mentioned mark-9-dehydrogenation rographolide-19-sodium sulfovinate reference substance 23.62mg, puts in the 10ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, and obtains, and product mother liquor in contrast, for methodological study.
Precision takes the 17-hydrogen of above-mentioned mark-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium reference substance 24.32mg, put in the 10ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, and obtains, and product mother liquor in contrast, for methodological study.
2.3 the preparation of need testing solution: precision takes No. 15 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 primary 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 rographolide-19-sodium sulfovinate.
4, the investigation of linear relationship
Each accurate 17-hydrogen-9-dehydrogenation rographolide-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 10 μ l injection liquid chromatographies, by chromatographic condition peak area under 3 chromatographic conditions and system suitability item, 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate peak area the integrated value of take respectively is ordinate zou, the reference substance sample size is X-coordinate separately, the drawing standard curve, 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate regression equation is y=2495.26X-128.6, R 2=0.9999,17-hydrogen-9-dehydrogenation rographolide-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 rographolide-3-sodium sulfovinate linear relationship result
Each accurate above-mentioned 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate reference substance mother solution 1ml that draws, put respectively in 100ml, 50ml, 25ml, 10ml, 5ml measuring bottle, thin up becomes following concentration: 0.02362mg/ml, 0.04724mg/ml, 0.09448mg/ml, 0.18896mg/ml, 0.37792mg/ml respectively.Accurate draw solution 10 μ l injection liquid chromatographies, by chromatographic condition peak area under 3 chromatographic conditions and system suitability item, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate peak area the integrated value of take respectively is ordinate zou, the reference substance sample size is X-coordinate separately, the drawing standard curve, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate regression equation is y=2454.7x-105.4, R 2=0.9998,17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate is good linear between 0.2362～3.7792 μ g.The results are shown in Table 2, linear relationship chart is shown in Fig. 7.
Table 2.17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate linear relationship result
Figure BSA00000701931300242
Each accurate above-mentioned 17-hydrogen-9-dehydrogenation rographolide-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 rographolide-3,19-di-sulfate sodium peak area integrated value is ordinate zou, the reference substance sample size is X-coordinate separately, drawing standard curve, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium regression equation is y=2340X-120.3, R 2=0.9999,17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium is good linear between 0.2432～3.8912 μ g.The results are shown in Table 3, linear relationship chart is shown in Figure 11.
Table 3.17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium wire sexual intercourse result
Figure BSA00000701931300251
5. precision test
Draw respectively 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate reference substance solution, 17-hydrogen-9-dehydrogenation rographolide-9-sodium sulfovinate reference substance solution, 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium reference substance solution, repeat sample introduction 6 times, the results are shown in Table 4, the result demonstration, instrument precision is good.
Table 4. Precision test result
Figure BSA00000701931300252
6. stability test
Get sample of the present invention, adding water makes to dissolve, make need testing solution, according to chromatographic condition under 3 chromatographic conditions and system suitability item, measure, sample introduction once at regular intervals, as a result 17-hydrogen in need testing solution-9-dehydrogenation rographolide-3-sodium sulfovinate, peak area is without considerable change in 24 hours for 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (hereinafter to be referred as 19 sodium sulfovinates), its peak area RSD is respectively 0.88% and 0.97%.The results are shown in Table 5.
. table 5 stability test result
Figure BSA00000701931300261
7. replica test
Get No. 38 samples of the embodiment of the present invention, totally 6 parts, accurately weighed, add water and make need testing solution, according to chromatographic condition under 3. chromatographic conditions and system suitability item, measure, 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate average content is that 33.05%, RSD is 0.39%.17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate average content is that 32.83%, RSD is 0.67%, 17-hydrogen-9-dehydrogenation rographolide-3, and 19-di-sulfate sodium average content is that 33.81%, RSD is 0.69%, the results are shown in Table 6.
Table 6. replica test result
Figure BSA00000701931300262
8. recovery test
Adopt the application of sample recovery test, precision takes sample solution of the present invention to the 50ml volumetric flask, totally 6 parts, precision adds 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (lower 3 sodium sulfovinates that are called for short) reference substance solution (0.562mg/ml) 2ml, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (lower 19 sodium sulfovinates that are called for short) reference substance solution (0.656mg/ml) 2ml respectively, 17-hydrogen-9-dehydrogenation rographolide-3,19-sodium sulfovinate (lower abbreviation 3,19 sodium sulfovinates) reference substance solution (0.662mg/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 9.
Table 7.17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate recovery test result
Figure BSA00000701931300271
Table 8.17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate recovery test result
Figure BSA00000701931300281
Below experiment all gets with " composition " sample that embodiment 4 preparation methods obtain.The experimental data that other embodiment obtain is substantially approaching, repeats no more.
Experimental data 2: the impact of composition induced by endotoxin pyrogenicity
Get Japan large ear rabbit, body weight 1.8～2.3kg, male and female have concurrently, and 1d before experiment, choose body temperature between 38.0～39.4 ℃, and body temperature changed the rabbit be no more than 0.4 ℃ and used rabbit as experiment the same day.Experiment same day, get above-mentioned qualified rabbit, measure basal body temperature before modeling, oneself rabbit ear vein bacterial injection intracellular toxin normal saline solution, dosage is 1mL/kg (10EU/mL), observes rabbit body temperature and changes, every 30min record 1 time.Choose the body temperature rise rabbit that surpasses 0.5 ℃ after injection 1h, be divided at random 5 groups, 8 every group.The ear vein injection gives 0.9% sodium chloride solution, composition 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 10.
Medication temperature variation after table 10. endotoxin pyrogenic
Figure BSA00000701931300282
Annotate: with the empty map group, compare ※ P<0.05 ※ ※ P<0.01
As shown in Table 10, after giving intracellular toxin 1h, blank group Healthy Rabbits body temperature average has risen, and after lasting rising 3h, starts gradually to descend.Compare low dose group 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 composition 1-2h, also demonstrate the rabbit fervescence effect that suppresses during 4h; 3 drug study group antipyretic effects are remarkable.Show 40～160mgkg -1the rabbit fervescence that the composition induced by endotoxin causes all has cooling effect.
Experimental data 3: the impact of composition 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, composition low dose group 40mgkg -1, middle dosage group 80mgkg -1, high dose group 160mgkg -1and acetylsalicylic acid 100mgkg -1, measure the rat temperature of 1～6h after administration, per hour 1 time, using the difference of different time points body temperature value and basic value as observation index, the results are shown in Table 11.Table 11 is found out, while giving dry yeast 1h, makes blank group healthy rat body temperature continue the 6h that rises.Compare composition 40,80,160mgkg with the blank group -1show obvious inhibition rat temperature rising effect in 1～4h.
Medication temperature variation ℃ after table 11. dry yeast pyrogenicity,
Figure BSA00000701931300291
Figure BSA00000701931300292
Annotate: with the empty map group, compare ※ P<0.05 ※ ※ P<0.01
Experimental data 4: the impact of composition p-Xylol induced mice auricle edema
Get 50 of mouse, be divided at random 5 groups.Every day is to composition 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 mouse, the outside is dripped and is coated with 0.1ml caused by dimethylbenzene xylene inflammation, and auris dextra is in contrast.Cause scorching latter 2 hours de-cervical vertebras and put to death mouse, and cut mouse two ears along the auricle baseline, with diameter 6mm punch tool, respectively at left and right ear same section, lay auricle, put on electronic balance and weigh and record data.With left and right auricle weight difference value representation swelling.Table 12 demonstration, each medication group of composition, Dexamethasone group mice auricle swelling degree are significantly less than control group.
Table 12. p-Xylol causes the impact of auricle edema
Figure BSA00000701931300293
Figure BSA00000701931300294
Figure BSA00000701931300301
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 composition on Carrageenan
Get 40 of rats, be divided at random 5 groups.Every day is to composition 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 physiological saline 8ml, after 30 minutes, in rat foot claw middle part subcutaneous injection 0.15ml 1% carrageenin solution, and 30min, 1h, 2h, 3h, 4h, 5h measure its left back sufficient pawl thickness as the rat paw edema level index with milscale after injecting.Table 13 result shows, with the control group ratio, due to each dosage group of composition and the equal on Carrageenan of Dexamethasone group, the rat swollen feet has obvious restraining effect, each dosage group composition obvious restraining effect occurs and continues 5 hours at administration 30min, suitable with effects of dexamethasone, three dosage of composition are remarkable to the restraining effect effect of swelling.
Table 13. causes the impact of rat toes swelling on carrageen
Figure BSA00000701931300303
Annotate: with the empty map group, compare ※p<0.05 ※ ※p<0.01
Experimental data 6: the restraining effect of composition to the neuraminic acid enzymic activity
Get composition, add suitable quantity of water and make to dissolve, application neuraminidase inhibitor identification kit is measured composition and is suppressed tiring in Table 22 of neuraminidase (N1).
(1). typical curve is prepared: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases to detect damping fluid; B. every hole adds respectively 0,1,2,5,7.5,10 μ l H5N1 neuraminidases again; C. every hole adds 0～20 μ l Milli-Q water again.
(2). the preparation of sample detection: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases to detect damping fluid; B. every hole adds 10 μ l H5N1 neuraminidases again; C. every hole adds 0～10 μ 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 typical curve is also hatched together;
C. every hole adds 10 μ l neuraminidase fluorogenic substrates;
D. vibration mixes about 1min again;
E.37 after ℃ hatching 20～30min, carry out fluorometric assay.Excitation wavelength is 360nm, and emission wavelength is 440nm.
(4). calculate the inhibition per-cent of sample for the H5N1 neuraminidase according to typical curve, and calculate the IC50 of composition for the H5N1 neuraminidase after doing concentration curve.The inhibiting rate IC50 that composition reaches neuraminidase is 0.20g/L.In Table 14.
Table 14. composition suppresses the activity of neuraminidase
Figure BSA00000701931300311
According to above-mentioned experimental result, can be clear that:
(1). composition can extract effective inhibition neuraminic acid enzyme component;
(2). composition is along with the using dosage size variation, and it suppresses the ability of neuraminic acid enzymic activity, i.e. also corresponding changing of the height of neuraminic acid enzyme inhibition rate, and become positive correlation;
(3). from above-mentioned experiment, composition 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 composition has prevention and treats the good result that the influenza virus sexuality is emitted.
Experimental data 7: the restraining effect of composition infected by influenza infected chicken embryo
Get composition, application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1) identifies that composition suppresses the ability that the FM1 influenza virus is copied and suppresses in the chicken embryo.
(1). FM1 influenza virus liquid is inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, and every embryo 0.2ml, hatch 72h for 37 ℃, observes and calculate half chicken embryo infective dose (EID50).
(2). composition adopts the toxic action of chicken embryo, stroke-physiological saline solution is done to be inoculated in 10d no-special pathogen in age chick embryo allantoic cavity after serial dilution to composition, every embryo 0.2ml, each concentration is inoculated 6 embryos, hatch for 37 ℃, observe chicken embryonic development developmental state, the chicken embryo of usining can be survived the peak concentration of 96h as the TD of medicine.
(3). the restraining effect of composition infected by influenza in the chicken embryo adopts, the influenza virus liquid of 0.1ml and different dilution composition 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, stroke-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 chicken embryo, and its EID50 is 10 -4.92.
(2). after composition is inoculated in the chicken embryo, it grows basically identical with Normal group.96h chicken embryo is all survived.The chicken embryo gives composition stoste and has no chicken embryo death, so can think that TD0 is 2.12g/L.
(4). the restraining effect of composition infected by influenza in the chicken embryo is in Table 15.
The restraining effect of table 15. composition infected by influenza infected chicken embryo
Figure BSA00000701931300321
With the virus control group, compare: * P<0.05
As shown in Table 23, composition has significant restraining effect (P<0.05), ED at 0.0775～0.62g/L infected by influenza 50for 1.0204g ± 0.0066g/L, TI is 62.87 ± 3.42.
Experimental data 8: composition affects the FM1 influenza virus
Get composition, application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1) identifies that composition suppresses the ability of FM1 influenza virus virulence.
(1) .FM1 adopts cell median infective dose (TCID50) micromethod to the toxicity test of dog kidney passage cell (MDCK).
(2). composition adopts the DMEM of serum-free to do to be inoculated in Zhong，Mei hole, the mdck cell hole 100 μ l that form individual layer after serial dilution to composition to the toxicity test of mdck cell, and each extent of dilution repeats 4 holes, establishes the normal cell contrast simultaneously.Culture plate is put to 37 ℃, 5%CO 2in incubator, cultivate, observation of cell pathology every day (CPE), Continuous Observation 3d, with " +～++ ++ " record result, press the Reed-Muench method and calculate medicine median toxic concentration (TD50) and maximal non-toxic concentration (TD0).
(3). composition suppresses the effect of FM1 influenza virus and measures: mdck cell 5 * 10 5/ ml, every hole 100 μ l, in 96 orifice plates, 37 ℃, 5%CO 2cultivate in incubator, suck nutrient solution in hole next day, add 100TCID50 influenza virus liquid, every hole 100 μ l, suck supernatant liquor after 37 ℃ of absorption 1h.With phosphate buffered saline buffer (PBS), wash 2 times, the TD0 of medicine of take is the 1st hole, then with the DMEM liquid of serum-free, composition 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. monolayer cell sex change becomes circle etc., and 3d, calculate 50% of medicine and suppress pathology concentration (IC50) and therapeutic index (TI) continuously.The calculating of TI: TI=TD50/IC50, the TI value is larger, shows that the safety range of medicine is larger.With Kruskal-Walis and Mann-Whitney method of inspection comparison test group and the cytopathic difference of virus control group, to drug dose with to virus infected cell, avoid the inhibiting rate that cytopathy (CPE) occurs to carry out correlation analysis, judge whether amount validity response relation.
(4) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of mdck cell, and its TCID50 is 10 -4.74.(2). the TD0 of composition mdck cell is respectively 1.28g ± 0.064g/L.(3). after composition 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 16.
The IC of table 16. composition to the FM1 influenza virus 50and TI (x ± s) (g/L)
Figure BSA00000701931300331
As shown in Table 16, the IC50 of composition is low, and TI is high.Composition suppresses the cytopathogenic effect of FM1 influenza virus all to be strengthened along with the increase of drug dose.The correlation analysis that drug dose and medicine are carried out the inhibiting rate of CPE shows, the dosage of composition and medicine are to being obvious positive correlation between the CPE inhibiting rate.
Experimental data 9: the impact of composition on spleen index and the lung index of influenza virus infection FM1 strain in Mice Body
Get composition, application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1) is identified the dead provide protection of composition to influenza virus infection FM1 strain in Mice Body.
(1) influenza virus FM1 strain virus is inoculated respectively every group of 10 BALB/C mice, male and female half and half after doing 10 times of doubling dilutions.After the slight anesthesia of ether, give and different dilution virus respectively for every group, every mouse collunarium is inoculated 20 μ l.Observe the dead mouse situation of 10d, calculating LD50 by the Reed-Muench method is 10 -1.68.Therefore determine that experiment modeling concentration used is 10LD50.
(2) the dead provide protection of composition to influenza virus infection FM1 strain in Mice Body: Normal group, influenza virus FM1 strain virus control group, composition 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 physiological saline of same volume simultaneously.4 groups of administration are continued administration, Normal group and influenza virus FM1 strain virus control group administered physiological saline 8 days, and dosage is the same.Day by day observe the animal morbidity and record death toll, observing altogether 14 days, calculating mortality ratio (mortality ratio=every group of death toll/every group of total mice * 100%), the results are shown in Table 17.
(3) impact of composition on influenza virus infection FM1 strain lung index in Mice Body: Normal group, influenza virus FM1 strain virus control group, composition 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 physiological saline of same volume simultaneously.4 groups of administration are continued administration, Normal group and influenza virus FM1 strain virus control group administered physiological saline 8 days, and dosage is the same.Within the 8th day after virus infection, put to death mouse, weigh, get lung and claim the lung weight, calculate lung index (lung index=lung quality/weight * 100%); In addition, get spleen and claim the spleen weight, calculate spleen index (spleen index=spleen quality/weight * 100%), the results are shown in Table 18.
The death protection result of table 17. composition 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 18. composition on spleen index and the lung index of influenza virus infection FM1 strain in Mice Body
Figure BSA00000701931300352
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 17, composition has significant provide protection (p<0.01) at 0.2～0.8g/L influenza virus infected.
As shown in Table 18, composition 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 composition on mouse septicemia
1. composition can significantly improve septicemia mouse survival rate
Rographolide, because of water-soluble extreme difference, carries out gavage (30mg/kg) so adopt 0.9% sodium carboxymethyl cellulose solution to be mixed with suspension, and composition directly is made into settled solution with PBS and carries out tail vein injection (10mg/kg).Abdominal injection 5mg/kg LPS carries out modeling, observes mouse survival number of elements in 60h, calculates survival rate.The model group mouse occurs dead in 16 o'clock after modeling, after 60h, survival rate is 25% (shown in table 19), rographolide administration group survival rate is 50%, composition administration group survival rate is 75%, comparatively speaking, the effect of composition for improved survival rate is slightly better than rographolide group effect, but its dosage is lower.
The septicemia mouse survival rate that table 19. composition is induced LPS
Figure BSA00000701931300353
2. composition significantly suppresses the rising of inflammatory factor in the septicemia mice serum
BALB/C mice abdominal cavity gavage gives rographolide 30mg/kg, tail vein injection composition 0.8,2.4,8mg/kg, abdominal injection 5mg/kg LPS simultaneously, after modeling and administration 2,5,8,4 time points of 12h are respectively put to death 3 mouse, and eye socket is got blood, measure inflammatory factor TNF-α in serum, the content of IL-1 β, shown in table 20.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, composition administration 2h can significantly suppress TNF-α to 3345pg/ml, to the release of IL-1 β being produced significantly and suppresses when the 5h.Rographolide effect 8h just can significantly suppress the rising of TNF-α.Experimental result shows than rographolide, and composition is rapid-action to the restraining effect 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 20. composition time, dose-dependently reduction LPS induce
TNF?α(pg/ml)
IL?1β(pg/ml)
Figure BSA00000701931300362
Figure BSA00000701931300363
N=3, * p<0.05vs model group, #p<0.05vs rographolide group.
3. composition significantly suppresses the hepar damnification of septicemia mouse
Septicemia is a kind of disease that causes multiple organ injury, and liver is one of main organs of its damage.The BALB/C mice gavage gives rographolide 30mg/kg, tail vein injection composition 8mg/kg, and while abdominal injection 5mg/kgLPS, respectively at 2h, 5h, 8h, the 12h eye socket is got blood, measures the content of serum alt, AST.Shown in table 29, after giving LPS, model mice serum alt and AST continue to raise, and give the content of ALT/AST after composition 2h and descend to some extent, with model group ALT/AST, significant difference is arranged to 12h, rographolide is slightly poorer than composition effect to the inhibition of ALT/AST.RT-PCR measures the mouse rna level of each inflammatory factor in liver organization and finds, LPS stimulates lower ifn-γ, il-6, tnf-α, il-β, cox-2mRNA significantly raises, composition and rographolide administration 5h and 8h can significantly suppress ifn-γ, il-6, tnf-α, il-β, the rising of cox-2mRNA.Experimental result shows equally than rographolide, and composition is rapid-action to the restraining effect of septicemia mouse liver injury, and inhibition is more remarkable.
The restraining effect of the septicemia mouse liver injury that table 21. composition is induced LPS
AST(karmen?units)
Figure BSA00000701931300371
ALT(karmen?units)
Figure BSA00000701931300372
N=3, * p<0.05, vs model group.
4, brief summary
Rographolide and composition can significantly improve the survival rate of the septicemia mouse that LPS induces, time, dose-dependently reduce TNF-α in the septicemia mice serum, IL-1 β, ALT, the content of AST, the sick rising that suppresses inflammatory factor mRNA level in liver organization.The composition onset is fast than rographolide, better effects if.After experimental result shows that rographolide is transformed into sulfonated bodies, it is water-soluble better, rapid-action after the composition administration, the improvement effect of mouse septicemia also is enhanced, and dosage is lower.

Claims (21)

1. 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, the preparation method of 19-di-sulfate sodium (or potassium) composition, said composition is by rographolide, sulfonation obtains, and the preparation method is as follows:
[1] add solvent in reactor, add rographolide to make its dissolving, then add sulphonating agent to carry out sulfonation reaction, conditioned reaction still temperature is at 6～39 ℃, sulfonation reaction 0.5～28 hour;
[2] rographolide solution is in the situation that stirring adopts the mode that slowly drips, sprays or ventilate to add sulphonating agent, and when adopting the mode that slowly drips or spray to add sulphonating agent, add speed control at 1.25ml～4.95ml/min/1g rographolide, when the mode that passes into gas when employing adds sulphonating agent, pass into speed control at 0.010 liter～0.28 liter/min/1g rographolide, by above-mentioned sulfonation reaction thing to saturated salt solution; Perhaps use alkaline solution adjust pH to 7, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-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: 20 with CG161 macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 4～1: 85, 1～12 times of column volume wash-out of water, discard elutriant, with 5%～38 ethanol or 4～15 times of column volume wash-outs of methyl alcohol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition elute soln, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition, perhaps
Mixture is through the ODS column chromatography for separation, applied sample amount is 1: 4～1: 16 with the ODS ratio, the resin column blade diameter length ratio is 1: 6～1: 77, 1.5～13 times of column volume wash-outs of water, discard elutriant, with 5%～38 ethanol or 3.5～14 times of column volume wash-outs of methyl alcohol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition elute soln, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition, 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: 82, 1～13 times of column volume wash-out of water, discard elutriant, with 5%～37 ethanol or 3～15 times of column volume wash-outs of methyl alcohol, HPLC detects, merge containing 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition elute soln, reclaim ethanol, vacuum-drying, obtain 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3, 19-di-sulfate sodium (or potassium) composition, perhaps
Mixture successively passes through aforementioned two or more step co-treatment;
Obtain described 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-3,19-di-sulfate sodium (or potassium) composition is made by following weight proportion: 17-hydrogen-9-dehydrogenation rographolide-3-sodium sulfovinate (or potassium) 10%～65%, 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium) 10%～65%, 17-hydrogen-9-dehydrogenation rographolide-3,19-sodium sulfovinate (or potassium) 10%～60%.
2. the preparation method of claim 1, the described solvent added in reactor is one or both of aceticanhydride or Glacial acetic acid, the described dissolution with solvents of 3g～12g for the 1g rographolide is preferred, the described dissolution with solvents of 4g～9g for the 1g rographolide.
3. the preparation method of claim 2, described solvent is aceticanhydride and Glacial acetic acid, described aceticanhydride, Glacial acetic acid are made by following weight proportion: aceticanhydride 80%～20%, Glacial acetic acid 20%～80%, preferred, wherein aceticanhydride 62%, Glacial acetic acid 38%.
4. the preparation method of claim 1, described sulphonating agent adopts the vitriol oil and Glacial acetic acid, 1g for the 1g rographolide～10g vitriol oil Glacial acetic acid carries out sulfonation, the described vitriol oil and Glacial acetic acid are made by following weight proportion: the vitriol oil 80%～20%, Glacial acetic acid 20%～80%, preferably, 1.5g for the 1g rographolide～5g vitriol oil Glacial acetic acid, and the acid of dense stream is 1: 1 with the Glacial acetic acid part by weight.
5. the preparation method of claim 1, described sulphonating agent adopts sulphur trioxide, the 1g rographolide passes into the sulphur trioxide that 0.2 liter～5 liters volumetric concentrations are 1%～3% and carries out sulfonation, preferably, the 1g rographolide passes into the sulphur trioxide that 0.3 liter～3 liters volumetric concentrations are 1.5%～2.5% and carries out sulfonation.
6. the preparation method of claim 1, described sulphonating agent adopts chlorsulfonic acid, and the 0.2g 1g rographolide for～5g chlorsulfonic acid carries out sulfonation, and preferably, 0.3g for the 1g rographolide～3.5g chlorsulfonic acid carries out sulfonation.
7. the preparation method of claim 1, described mixture separates through the CG161 macroporous adsorbent resin column chromatography, and applied sample amount is 1: 5.5～1: 10 with CG161 macroporous adsorbent resin ratio; Described mixture is through the ODS column chromatography for separation, and applied sample amount is 1: 6～1: 8 with the ODS ratio; Described mixture is through Sephadex LH-20 column chromatography for separation, and applied sample amount is 1: 5～1: 7 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: 25; 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%～30 ethanol or 4.5～7.5 times of column volume wash-outs of methyl alcohol; Described mixture is through the ODS column chromatography for separation, with 12%～32 ethanol or 4～6.5 times of column volume wash-outs of methyl alcohol; Described mixture is through Sephadex LH-20 column chromatography for separation, with 15%～33 ethanol or 4～8 times of column volume wash-outs of methyl alcohol.
10. 17-hydrogen-9-dehydrogenation rographolide-3, the composite preparation of 19-sodium sulfovinate (or potassium), 17-hydrogen-9-dehydrogenation rographolide-19-sodium sulfovinate (or potassium), it is main active ingredient that said preparation be take the described composition that the described preparation method of claim 1～9 any one obtains.
11. the described composition 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 composition induced by endotoxin pyrogenicity.
13. as the purposes of claim 11, the refrigeration function of described composition to dry yeast pyrogenicity.
14. the described composition that the described preparation method of claim 1～9 any one obtains is for the preparation of the purposes of the medicine of anti-inflammatory.
15. as the purposes of claim 14, the drug effect of described composition to septicemia.
16. as the purposes of claim 14, described composition p-Xylol induced mice auricle edema anti-inflammatory action.
17. as the purposes of claim 14, the anti-inflammatory action of rat paw edema due to described composition on Carrageenan.
18. the described composition 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 composition is for suppressing neuraminidase.
20., as the purposes of claim 18, described composition is for suppressing influenza virus.
21., as the purposes of claim 18, described composition is for suppressing influenza virus FM1.
CN2012101091329A 2012-04-12 2012-04-12 One-step preparation method of composition of sodium (or potassium) 17-hydro-9-dehydroandrographolide-3-sulphate, sodium (or potassium) 17-hydro-9-dehydroandrographolide-19-sulphate and sodium (or potassium) 17-hydro-9-dehydroandrographolide-3,19-disulfate, and use of the composition in drug preparation. Pending CN103145658A (en)

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CN106810520A (en) * 2015-12-02 2017-06-09 江西青峰药业有限公司 A kind of pharmaceutical composition of andrographolide sulfonate and preparation method thereof
CN106810522A (en) * 2015-12-02 2017-06-09 江西青峰药业有限公司 A kind of method that use 17- hydrogen -9- dehydrogenations andrographolide prepares andrographolide sulfonate composition

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