CN103145665B - 17-hydro-9-dehydro-14,17-cyclo-andrographolide-19-sulphate and its preparation method and use in drug preparation - Google Patents

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

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CN103145665B
CN103145665B CN201210109520.7A CN201210109520A CN103145665B CN 103145665 B CN103145665 B CN 103145665B CN 201210109520 A CN201210109520 A CN 201210109520A CN 103145665 B CN103145665 B CN 103145665B
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rographolide
dehydrogenation
hydrogen
rings
salt
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CN103145665A (en
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吕武清
谢宁
唐春山
杨小玲
刘地发
程帆
李志勇
刘荛琦
廖祝元
刘艳红
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JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
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Abstract

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

Description

17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester compound, preparation method and prepare pharmaceutical use
Technical field
The present invention relates to a kind of 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester compound, preparation method and prepare pharmaceutical use.
Background technology
Herba Andrographis is the dry aerial parts of acanthaceous plant Herba Andrographis Andrographis paniculata (Burm.f.) Nees, chemical composition and pharmacological evaluation show, the activeconstituents of Herba Andrographis be with rographolide be representative diterpene-kind compound for master [State Administration of Traditional Chinese Medicine. China book on Chinese herbal medicine the 7th. Shanghai: Shanghai science tech publishing house, 1999:439], the structure of rographolide is:
Molecular formula: C 20h 30o 5, be the crystallization of colourless square square, m.p.230-232 DEG C, [α] 0 20-126 ° of (c0.2, H 2o).Taste 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 usual film-making agent of oral preparations, capsule, dripping pill, soft capsule; Because rographolide is water insoluble, bring difficulty to preparing liquid preparation, at present, existing multiple method is used to transform rographolide and becomes various derivative, to improve the water-soluble of rographolide.The injection liquid of various soluble derivative is prepared into after general extraction rographolide, as with S-WAT add sulfuric acid or with sodium bisulfite generation addition reaction, obtained water-soluble sulfonate, rographolidum Natrii Bisulfis (LIANBIZHI ZHUSHEYE), succinic acid half-ester monopotassium salt, rographolide is through esterification, dehydration, salt refining is become to form 14-deshydroxy-11, 12-bis-dehydrogenation rographolide-3, 19-disuccinic acid half ester k-na salt (' Tanhuning ' injection) or 14-deshydroxy-11, 12-bis-dehydrogenation rographolide-3, 19-disuccinic acid half ester monopotassium salt, but above-mentioned salt solubleness in water, stability is not good especially.
Summary of the invention
One object of the present invention is to provide the compound or its salt represented by a kind of general formula (I), its chemistry 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester by name or its salt,
R can be H, sodium, potassium or NH 4.
Another object of the present invention is the preparation method of the compound or its salt providing a kind of general formula (I), comprises the following steps:
[1] add solvent in a kettle., add rographolide and make it dissolve, then add sulphonating agent and carry out sulfonation reaction, regulate temperature of reaction kettle at 6 ~ 39 DEG C, sulfonation reaction 1 ~ 28.5 hour;
[2] rographolide solution adopts the mode slowly dripping, spray or ventilate to add sulphonating agent when stirring, and when adopting the mode slowly dripping or spray to add sulphonating agent, add speed control at 1.3ml ~ 4.7ml/min/1g rographolide, when adopting the mode passing into gas to add sulphonating agent, pass into speed control at 0.03 liter ~ 0.22 liter/min/1g rographolide, 17-hydrogen-9-dehydrogenation-14, the 17-rings-reactant of rographolide-19-sulfuric ester and the mixture of unreacted reactant is obtained after sulfonation reaction;
[3] mixture is through solvent extraction, crystallization, obtains 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester finally; Or
Mixture in saturated salt solution, crystallization, obtain 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester finally or its salt; Or
Mixture alkaline solution adjust pH to 7, through macroporous adsorbent resin, ODS or Sephadex LH-20 column chromatography for separation, with water: ethanol or methyl alcohol are elutriant, gradient elution, collect eluant component, crystallization, obtains 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester finally or its salt; Or
Mixture, successively through two or more step co-treatment aforementioned, obtains 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester finally or its salt.
Preferably, the described solvent added in a kettle. is one or both of aceticanhydride or Glacial acetic acid, 1g rographolide dissolution with solvents described in 3g ~ 12g, preferably, and 1g rographolide dissolution with solvents described in 4g ~ 9g.
Preferably, described solvent is aceticanhydride and Glacial acetic acid, and described aceticanhydride, Glacial acetic acid are made up of following weight proportion: aceticanhydride 80% ~ 20%, Glacial acetic acid 20% ~ 80%, preferably, and wherein aceticanhydride 62%, Glacial acetic acid 38%.
Preferably, described sulphonating agent adopts the vitriol oil and Glacial acetic acid, 1g rographolide 1g ~ 10g vitriol oil Glacial acetic acid carries out sulfonation, the described vitriol oil and Glacial acetic acid are made up of following weight proportion: the vitriol oil 80% ~ 20%, Glacial acetic acid 20% ~ 80%, preferably, 1g rographolide 1.5g ~ 5g vitriol oil Glacial acetic acid, and the acid of dense stream is 1: 1 with Glacial acetic acid part by weight.
Preferably, described sulphonating agent adopts sulphur trioxide, 1g rographolide pass into 0.2 liter ~ 5 liters volumetric concentrations be 1% ~ 3% sulphur trioxide carry out sulfonation, preferably, 1g rographolide pass into 0.3 liter ~ 3 liters volumetric concentrations be 1.5% ~ 2.5% sulphur trioxide carry out sulfonation.
Preferably, described sulphonating agent adopts chlorsulfonic acid, and 1g rographolide 0.2g ~ 5g chlorsulfonic acid carries out sulfonation, and preferably, 1g rographolide 0.3g ~ 3.5g chlorsulfonic acid carries out sulfonation.
Preferably, described saturated salt solution adopts sodium-chlor or saturated potassium chloride solution, described alkaline solution adopt 5% ~ 50% sodium hydroxide potassium hydroxide or less than 25% ammonia soln.
Preferably, wherein said temperature of reaction kettle is at 7 ~ 25 DEG C, and the described sulfonation reaction time should control at 0.5 ~ 3 hour.
Preferably, wherein said sulphonating agent is vitriol oil Glacial acetic acid or chlorsulfonic acid, and adopt slowly drip, the mode of spraying adds, and adds speed control at 2ml ~ 4ml/min/1g rographolide.
Preferably, wherein said sulphonating agent is sulphur trioxide, and adopts the mode passing into gas to add, and passes into speed control at 0.05 liter ~ 0.15 liter/min/1g rographolide.
Preferably, in wherein said gradient elution, the ratio of water is 80 ~ 20%, and the ratio of ethanol or methyl alcohol is 20 ~ 80%.
Another object of the present invention is the measuring method of the compound or its salt providing a kind of general formula (I), it adopts the 17-hydrogen-9-dehydrogenation-14 described in high effective liquid chromatography for measuring, 17-ring-rographolide-19-sulfuric ester or its salt, condition determination is as follows:
Take octadecylsilane chemically bonded silica as weighting agent;
Determined wavelength is 225nm;
Column temperature is 25 DEG C;
Number of theoretical plate presses 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or the calculating of its salt is not less than 10000;
The preparation of reference substance solution is by described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt make reference substance by purity test and content mark, and dry, in right amount accurately weighed in Vanadium Pentoxide in FLAKES vacuum drier, add water and make the solution of desired concn, to obtain final product;
The preparation precision of need testing solution takes sample 5mg, puts in 50ml measuring bottle, is diluted with water to scale, shake up, to obtain final product;
Wash-out is mobile phase A with acetonitrile, take potassium phosphate buffer as Mobile phase B, and described potassium phosphate buffer is add potassium primary phosphate 1.361g and heptane-1-sodium sulfonate 1.0g in every 1000ml water, carries out gradient elution, run 70 minutes by following condition;
When 0 ~ 10 minute, acetonitrile ratio is 8.0%, and potassium phosphate buffer ratio is 92.0%;
When 10 ~ 60 minutes, acetonitrile ratio rises to 35.0% by 8.0%, and potassium phosphate buffer ratio drops to 65.0% by 92.0%;
When 60 ~ 62 minutes, acetonitrile ratio rises to 70.0% by 35.0%, and potassium phosphate buffer ratio drops to 30.0% by 65.0%;
When 62 ~ 70 minutes, keep acetonitrile: potassium phosphate buffer is with 70.0%: 30.0% ratio wash-out;
Under these conditions, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt have a chromatographic peak in about 42 minutes in retention time.
Assay method is accurate respectively draws reference substance solution and need testing solution, injection liquid chromatography, measures, to obtain final product.
Another object of the present invention is to provide a kind of 17-hydrogen-9-dehydrogenation-14, the preparation of 17-ring-rographolide-19-sulfuric ester or its salt, to be 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester according to claim 1 or its salt make with pharmaceutically acceptable carrier said preparation.
Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt for the preparation of the purposes of antipyretic medicine.
Preferably, the refrigeration function of described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt pair endotoxin pyrogenic.
Preferably, the refrigeration function of described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt pair dry yeast pyrogenicity.
Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt for the preparation of the purposes of the medicine of anti-inflammatory.
Preferably, the drug effect of described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt pair septicemia.
Preferably, described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt p-Xylol induced mice auricle edema anti-inflammatory action.
Preferably, the anti-inflammatory action of rat paw edema caused by described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt on Carrageenan.
Another object of the present invention is to provide described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt for the preparation of the purposes of antiviral drug.
Preferably, described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt are for suppressing neuraminidase.
Preferably, described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt are for suppressing influenza virus.
Preferably, described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt are for suppressing Influenza B virus.
17-hydrogen-9-dehydrogenation-14 provided by the invention, 17-ring-rographolide-19-sulfuric ester or its salt, in water, solubleness is very good and stability is very high, very be applicable to practical application, various common type can be applied to, 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.Liquid preparation can certainly be made as syrup, injection liquid etc., particularly make injection and overcome the low defect of oral pharmaceutical biological utilisation.
On the other hand, 17-hydrogen-9-dehydrogenation-14 provided by the present invention, the preparation method of 17-ring-rographolide-19-sulfuric ester or its salt is simple and convenient, mild condition, productivity are high, can prepare 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt easily.
What is more important, the present invention is by thousands of up to a hundred performing creative labours, finally determine suitable solvent, and the important technical parameter of creative sulphonating agent and sulfonation reaction thereof, the processing mode of such as sulphonating agent and add the determination of the complex relationship between speed, and the determination etc. of suitable numerical range, thus just finally obtain required reactant.On this basis, then adopt further creative purification technique to carry out purifying, thus the invention provides to prepare in many different conditions and form 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester of the present invention or its salt.
The present invention is compared with the prior art and shows: 17-hydrogen-9-dehydrogenation-14, the 17-rings-rographolide-19-sulfuric ester adopting preparation method of the present invention to be formed or its salt, can not change the original chemical attribute of rographolide completely; And 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester of the present invention or its salt compared with prior art have the features such as good water solubility, thermostability is high, hemolytic action is little.Ensure that the pharmacologically active effect of rographolide pure natural medical to greatest extent.
Known through experimental data contrast, after intracellular toxin 1h, blank group Healthy Rabbits body temperature average rises, and starts gradually after continuing rising 3h to decline.Compare with blank group, low dose group 50mgkg -117-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate 1 ~ 2h shows and obviously suppresses rabbit temperature rise effect, middle dosage group 100mgkg -1, high dose group 200mgkg -1show the effect of extremely strong suppression rabbit fervescence in 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate 1 ~ 2h, also demonstrate during 4h and suppress the effect of rabbit fervescence; With 100mgkg in 3 drug study groups -1the above antipyretic effect of dosage is best.Show 50 ~ 200mgkg -1the rabbit fervescence that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate induced by endotoxin causes all has cooling effect, achieves unforeseeable technique effect.
Known through experimental data contrast, make blank group healthy rat body temperature continue rising 6h when giving dry yeast 1h.Compare with blank group, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate 100,200mgkg -1show in 1 ~ 4h and obviously suppress rat temperature rising effect, achieve unforeseeable technique effect.
Known through experimental data contrast, rographolide and 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate significantly can improve the survival rate of the septicemia mouse of LPS induction, time, dose-dependently reduce TNF-α in septicemia mice serum, IL-1 β, the content of ALT, AST, the sick rising suppressing inflammatory factor mRNA level in-site in liver organization.And 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate onset comparatively rographolide is fast, better effects if.After experimental result display rographolide is transformed into sulfonated bodies, it is better water-soluble, and administration is rapid-action, is also enhanced to the improvement result of mouse septicemia, achieves unforeseeable technique effect.
Known through experimental data contrast, each medication group of 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate, Dexamethasone group mice auricle swelling degree are significantly less than control group, achieve unforeseeable technique effect.
Known through experimental data contrast, with control group ratio, 17-hydrogen-9-dehydrogenation-14, caused by 17-ring-rographolide-19-sodium sulfovinate each dosage group and the equal on Carrageenan of Dexamethasone group, rat swollen feet has obvious restraining effect, 17-hydrogen-9-dehydrogenation-14, obvious restraining effect is there is in administration 30min and continues 5 hours in 17-ring-rographolide-19-sodium sulfovinate when 200mg/kg, 100mg/kg effect is suitable with effects of dexamethasone, 17-hydrogen-9-dehydrogenation-14, a certain amount of effect relationship is had in the restraining effect of same time point to swelling between 17-ring-rographolide-19-sodium sulfovinate three dosage groups, achieve unforeseeable technique effect.
Known through experimental data contrast, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate can extract effective suppression neuraminic acid enzyme component; And 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is along with using dosage size variation, it suppresses the ability of neuraminidase activity, and namely the height of neuraminic acid enzyme inhibition rate is also corresponding changes, and becomes positive correlation.17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate can by suppressing influenza surface neuraminidase, and then suppression influenza virus enters inside cell, suppress the influenza virus entered inside cell to copy, breed, thus decrease influenza virus to the infection of cell, growth, and prevention and therapy influenza and complication thereof, achieve unforeseeable technique effect.
Known through experimental data contrast, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate has significant restraining effect (P < 0.05) at 0.075 ~ 0.6g/L infected by influenza, ED 50for 0.0918g ± 0.0052g/L, TI are 59.88 ± 1.96.The IC50 of 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is low, and TI is high.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate suppresses the cytopathogenic effect of FM1 influenza virus all to strengthen along with the increase of drug dose.Drug dose and medicine are shown the correlation analysis that the inhibiting rate of CPE carries out, the dosage of 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate and medicine between CPE inhibiting rate in obvious positive correlation.
Medical science and study of pharmacy personnel in advance under the prerequisite not doing related experiment, cannot learn that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate has above-mentioned good purposes in advance.
Accompanying drawing explanation
Fig. 1: 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate hydrogen nuclear magnetic resonance spectrogram.
Fig. 2: 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate carbon-13 nmr spectra figure.
Fig. 3: 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate mass spectrum.
Fig. 4: 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate linear relationship chart.
Embodiment
Embodiment 1
Get creat lactone, add 4.5 times amount aceticanhydrides (62%) and make dissolving with Glacial acetic acid (38%), under agitation, with the speed of 1g rographolide per minute 2.3ml, slowly drip the sulfuric acid (53%) of 4.8 times amount and Glacial acetic acid (47%), mixing, regulates temperature of reaction kettle at 16 DEG C, places and make sulfonation in 100 minutes.Add equivalent purified water, stir evenly, adjust pH to be about 7.0 with 35%NaOH, adding 95% ethanol makes alcohol content reach more than 83%, decompression recycling ethanol, and 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%, Fractional Collections elutriant, merge same composition, crystallization, obtains 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate, its sodium salt molecular formula C 20h 27naO 7s, molecular weight: 434,
Reaction formula example:
17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate physico-chemical property and spectral data:
17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate: faint yellow amorphous, [α] d 20=-212 (c 1.15, MeOH), molecular formula: C 20h 27naO 7s.ESI-MSm/z?457[M+Na] +，411[M-Na] -，891[2M+Na] +，HRESIMS?m/z：411.1483[M-Na] -(calcd?411.1472)。 1h (400MHz, CD 3oD) and 13c NMR (100MHz, CD 3oD) data.
1NMR(CD 3OD，400MHz)δ：1.99(brd，J＝13.1Hz，1H，H-1α)，1.21(m，1H，H-1β)，1.81(m，1H，H-2α)，1.70(m，1H，H-2β)，3.24(dd，J＝11.5.5.0Hz，1H，H-3)，1.16(m，1H，H-5)，1.63(m，1H，H-6α)，1.88(m，1H，H-6β)，2.14(dd，J＝17.8，6.0Hz，1H，H-7α)，2.07(dd，J＝12.0，6.0Hz，1H，H-7β)，3.07(brd，2H，H-11)，4.74(dd，J＝7.8，4.4Hz，1H，H-12)，2.99(m，1H，H-14)，4.47(t，J＝8.6Hz，1H，H-15α)，3.70(t，J＝8.6Hz，1H，H-15β)，1.93(dd，J＝11.9，2.3Hz，1H，H-17α)，2.35(dd，J＝11.9Hz，1H，H-17β)，1.20(s，3H，H-18)，4.16(s，2H，H-19)，1.02(s，.3H，H-20)
13C-NMR(CD 3OD，100MHz)δ：35.7(C-1)，28.4(C-2)，79.5(C-3)，43.2(C-4)，53.0(C-5)，21.5(C-6)，35.1(C-7)，133.0(C-8)，141.6(C-9)，40.0(C-10)，28.0(C-11)，138.3(C-12)，132.9(C-13)，38.2(C-14)，72.1(C-15)，174.4(C-16)，36.1(C-17)，23.4(C-18)，70.8(C-19)，19.4(C-20)。
Embodiment 2
Get creat lactone, add 4.6 times amount aceticanhydrides (60%) and make dissolving with Glacial acetic acid (40%), under agitation, with the speed of 1g rographolide per minute 2.5ml, slowly drip the sulfuric acid ice acetic acid of 3.6 times amount equal proportions, mixing, regulate temperature of reaction kettle at 17 DEG C, place and make sulfonation in 90 minutes, add equivalent purified water, pour into again in saturated nacl aqueous solution, again through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merge same composition, crystallization, obtain 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate.
Embodiment 3
Get creat lactone, add 4.2 times amount aceticanhydrides (52%) and make dissolving with Glacial acetic acid (48%), under agitation, with the speed of 1g rographolide per minute 2.7ml, slowly drip the sulfuric acid (58%) of 3.5 times amount and Glacial acetic acid (42%), mixing, regulates temperature of reaction kettle at 12 DEG C, places and make sulfonation in 100 minutes.Adding 95% ethanol makes alcohol content reach more than 82%, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, and the ratio of ethanol is 20 ~ 80%, collect eluant component, merge same composition, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester.
Embodiment 4
Get creat lactone, add 4.7 times amount aceticanhydrides and make dissolving, under agitation, with the speed of 1g rographolide per minute 2.2ml, atomization spray adds the sulfuric acid (53%) of 3.3 times amount and Glacial acetic acid (47%), mixing, regulates temperature of reaction kettle at 20 DEG C, places and make sulfonation in 120 minutes.In reactant impouring saturated nacl aqueous solution, throw out is respectively with saturated nacl aqueous solution, water washing, and solid substance refluxes with chloroform, insolubles adds dehydrated alcohol makes dissolving, and removing alcohol insoluble solids, reclaims ethanol, crystallization, obtains 8-table-Isorographolide-19-sodium sulfovinate.
Embodiment 5
Get creat lactone, add 5.2 times amount aceticanhydrides (65%) and make dissolving with Glacial acetic acid (35%), under agitation, with the speed of 1g rographolide per minute 2.5ml, slowly drip the sulfuric acid (51%) of 3.4 times amount and Glacial acetic acid (49%), mixing, regulates temperature of reaction kettle at 17 DEG C, places and make sulfonation in 110 minutes.Add equivalent purified water, stir evenly, adjust pH to be about 7.0 with 32%KOH, adding 95% ethanol makes alcohol content reach more than 84%, decompression recycling ethanol, the aqueous solution through macroporous adsorbent resin, ODS column chromatography for separation, with water: methanol elution gradient, the ratio of water is 80 ~ 20%, the ratio of methyl alcohol is 20 ~ 80%, Fractional Collections elutriant, merges same composition, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester potassium.
Embodiment 6
Get creat lactone, add 5.5 times amount aceticanhydrides (60%) and make dissolving with Glacial acetic acid (40%), under agitation, with the speed of 1g rographolide per minute 2.0ml, slowly drip the sulfuric acid (57%) of 4.8 times amount and Glacial acetic acid (43%), mixing, regulates temperature of reaction kettle at 14 DEG C, places and make sulfonation in 90 minutes.Add equivalent purified water, stir evenly, use 22%NH 4oH adjusts pH to be about 7.0, adding 95% ethanol makes alcohol content reach more than 82%, decompression recycling ethanol, the aqueous solution through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merges same composition, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester ammonium.
Embodiment 7
Get creat lactone, add 6.2 times amount aceticanhydrides (62%) and make dissolving with Glacial acetic acid (38%), under agitation, with the speed of 1g rographolide per minute 0.09 liter, pass into 1.5 liter of 1.4% sulphur trioxide, regulate temperature of reaction kettle at 20 DEG C, place and make sulfonation in 90 minutes.Add equivalent purified water, stir evenly, adjust pH to be about 7.0 with 33%NaOH, adding 95% ethanol makes alcohol content reach more than 85%, decompression recycling ethanol, the aqueous solution through macroporous adsorbent resin, ODS column chromatography for separation, with water: methanol elution gradient, the ratio of water is 80 ~ 20%, the ratio of methyl alcohol is 20 ~ 80%, Fractional Collections elutriant, merges same composition, crystallization, obtains 8-table-Isorographolide-19-sodium sulfovinate.
Embodiment 8
Get creat lactone, add 5.2 times amount aceticanhydrides (64%) and make dissolving with Glacial acetic acid (36%), under agitation, with the speed of 1g rographolide per minute 0.07 liter, pass into 1.9 liter of 1.4% sulphur trioxide, regulate temperature of reaction kettle at 19 DEG C, place and make sulfonation in 110 minutes.In reactant impouring saturated nacl aqueous solution, throw out is respectively with saturated nacl aqueous solution, water washing, and solid substance refluxes with chloroform, insolubles adds dehydrated alcohol makes dissolving, and removing alcohol insoluble solids, reclaims ethanol, crystallization, obtains 8-table-Isorographolide-19-sodium sulfovinate.
Embodiment 9
Get creat lactone, add 5.3 times amount aceticanhydrides (56%) and make dissolving with Glacial acetic acid (44%), under agitation, with the speed of 1g rographolide per minute 0.11 liter, pass into 1.8 liter of 1.6% sulphur trioxide, regulate temperature of reaction kettle at 11 DEG C, place and make sulfonation in 100 minutes.Adding 95% ethanol makes alcohol content reach more than 80%, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, Sephadex LH-20 column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, and the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merge same composition, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester.
Embodiment 10
Get creat lactone, add 5.5 times amount aceticanhydrides (68%) and make dissolving with Glacial acetic acid (32%), under agitation, with the speed of 1g rographolide per minute 0.09 liter, pass into 1.3 liter of 2.2% sulphur trioxide, regulate temperature of reaction kettle at 18 DEG C, place and make sulfonation in 120 minutes.Add equivalent purified water, stir evenly, pH is adjusted to be about 7.0 with 35%KOH, then through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merges same composition, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester potassium.
Embodiment 11
Get creat lactone, add 5.4 times amount aceticanhydrides (61%) and make dissolving with Glacial acetic acid (39%), under agitation, with the speed of 1g rographolide per minute 0.06 liter, pass into 0.9 liter of 2.5% sulphur trioxide, regulate temperature of reaction kettle at 19 DEG C, place and make sulfonation in 80 minutes, add purified water, stir evenly.In reactant impouring saturated potassium chloride solution, throw out is respectively with saturated potassium chloride solution, water washing, and solid substance refluxes with chloroform, insolubles adds dehydrated alcohol makes dissolving, and removing alcohol insoluble solids, reclaims ethanol, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester potassium.
Embodiment 12
Get creat lactone, add 5.4 times amount aceticanhydrides (66%) and make dissolving with Glacial acetic acid (34%), under agitation, with the speed of 1g rographolide per minute 0.07 liter, pass into 1.3 liter of 1.5% sulphur trioxide, regulate temperature of reaction kettle at 20 DEG C, place and make sulfonation in 70 minutes, add equivalent purified water, stir evenly.Use 21%NH 4oH adjusts pH to be about 7.0, adding 95% ethanol makes alcohol content reach more than 80%, decompression recycling ethanol, the aqueous solution through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merges same composition, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester ammonium.
Embodiment 13
Get creat lactone, add 5.6 times amount aceticanhydrides (59%) and make dissolving with Glacial acetic acid (41%), under agitation, with the speed of 1g rographolide per minute 2.1ml, atomization adds 1.9 times amount chlorsulfonic acids, mixing, mixing, regulate temperature of reaction kettle at 15 DEG C, place and make sulfonation in 100 minutes, in reactant impouring saturated nacl aqueous solution, throw out is respectively with saturated nacl aqueous solution, water washing, solid substance refluxes with chloroform, insolubles adds dehydrated alcohol makes dissolving, removing alcohol insoluble solids, reclaim ethanol, crystallization, obtain 8-table-Isorographolide-19-sodium sulfovinate.
Embodiment 14
Get creat lactone, add 5.3 times amount aceticanhydrides (56%) and make dissolving with Glacial acetic acid (44%), under agitation, with the speed of 1g rographolide per minute 2.3ml, slowly drip 2.6 times amount chlorsulfonic acids, mixing, regulate temperature of reaction kettle at 16 DEG C, place and make sulfonation in 100 minutes, add equivalent purified water, stir evenly, pH is adjusted to be about 7.0 with 28%NaOH, adding 95% ethanol makes alcohol content reach more than 83%, decompression recycling ethanol, the aqueous solution is through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merge same composition, crystallization, obtain 8-table-Isorographolide-19-sodium sulfovinate.
Embodiment 15
Get creat lactone, add 5.0 times amount aceticanhydrides (62%) and make dissolving with Glacial acetic acid (38%), under agitation, with the speed of 1g rographolide per minute 2.3ml, slowly drip 2.6 times amount chlorsulfonic acids, mixing, regulate temperature of reaction kettle at 18 DEG C, place and make sulfonation in 80 minutes, add equivalent purified water, stir evenly, adding 95% ethanol makes alcohol content reach more than 85%, 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%, the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merge same composition, crystallization, obtain 8-table-Isorographolide-19-sulfuric ester.
Embodiment 16
Get creat lactone, the aceticanhydride and the Glacial acetic acid that add 5.3 times amount equal proportions make dissolving, under agitation, with the speed of 1g rographolide per minute 2.1ml, slowly drip 2.2 times amount chlorsulfonic acids, mixing, regulate temperature of reaction kettle at 21 DEG C, place and make sulfonation in 90 minutes, add equivalent purified water, stir evenly, pH is adjusted to be about 7.0 with 34%KOH, adding 95% ethanol makes alcohol content reach more than 84%, 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%, the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merge same composition, crystallization, obtain 8-table-Isorographolide-19-sulfuric ester potassium.
Embodiment 17
Get creat lactone, add 4.8 times amount aceticanhydrides (59%) and make dissolving with Glacial acetic acid (421%), under agitation, with the speed of 1g rographolide per minute 2.7ml, slowly drip 2.6 times amount chlorsulfonic acids, mixing, regulate temperature of reaction kettle at 21 DEG C, place and make sulfonation in 80 minutes, add equivalent purified water, stir evenly, use 21%NH 4oH adjusts pH to be about 7.0, adding 95% ethanol makes alcohol content reach more than 85%, decompression recycling ethanol, the aqueous solution through macroporous adsorbent resin, ODS column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, the ratio of ethanol is 20 ~ 80%, Fractional Collections elutriant, merges same composition, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester ammonium.
Embodiment 18
Get creat lactone, add 4.9 times amount aceticanhydrides (58%) and make dissolving with Glacial acetic acid (42%), under agitation, with the speed of 1g rographolide per minute 2.4ml, slowly drip 2.7 times amount chlorsulfonic acids, mixing, regulates temperature of reaction kettle at 21 DEG C, places and make sulfonation in 80 minutes, in reactant impouring saturated potassium chloride solution, throw out is respectively with saturated potassium chloride solution, water washing, and solid substance refluxes with chloroform, and insolubles adds dehydrated alcohol makes dissolving, removing alcohol insoluble solids, reclaim ethanol, crystallization, obtains 8-table-Isorographolide-19-sulfuric ester potassium.
The below experiment sample that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate equal Example 1 preparation method obtains.
Experimental data 1:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate assay
1. instrument and reagent
Instrument: Agilent1100 quarternary low pressure gradient pump series, Chemstation chem workstation, DAD detector; Shimadzu LC-2010A type high performance liquid chromatograph, two channels ultraviolet variable-wavelenght detector; Sartoriuscp211D 100,000/electronic balance.
Chromatographic column: Diamonsil C 18post (250mm × 4.6mm, 5 μm);
Reagent: acetonitrile is chromatographically pure, water is ultrapure water prepared by Millipore, and other reagent are analytical pure.
17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate reference substance is self-control, is 99.27% through purity mark content.
2. the preparation of reference substance solution and need testing solution
2.1 reference substance purity tests and content mark: the sample that Example 1 preparation method obtains, propyl carbinol-Glacial acetic acid-water (4: 0.5: 1), chloroform-methanol-water-Glacial acetic acid (7.5: 3: 1: 0.5) is adopted to carry out purity of thin layer chromatography inspection respectively, point sample amount is respectively 5,10,15,20,25 μ g, and result is a spot;
With high performance liquid chromatography, adopt area normalization method, select chromatographic column respectively: Diamonsil C 18(250mm × 4.6mm, 5 μm); Moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-acetonitrile (80: 20) and moving phase: phosphate buffered saline buffer (potassium primary phosphate 1.36g/L+ sodium heptanesulfonate 1g/L)-methyl alcohol (72: 28); Flow velocity: 1ml/min; Determined wavelength: 225nm; Column temperature: 25 DEG C.Often organize moving phase and pass in and out 10 μ l, 20 μ l respectively respectively once, sample introduction 4 times altogether, recording 4 average contents is 99.27%.
2.2 reference substance solution preparations: precision takes 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate reference substance 23.88mg, puts in 10ml measuring bottle, is dissolved in water and is diluted to scale, shaking up, to obtain final product, in contrast product mother liquor.
The preparation of 2.3 need testing solutions: precision takes the sample 50mg of the present embodiment, puts in 500ml measuring bottle, is diluted with water to scale, shake up, to obtain final product;
3. chromatographic condition and system suitability
Take octadecylsilane chemically bonded silica as weighting agent; Determined wavelength is 225nm; Column temperature is 25 DEG C; Number of theoretical plate presses 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or the calculating of its salt is not less than 10000;
Be mobile phase A with acetonitrile, take potassium phosphate buffer as Mobile phase B, described potassium phosphate buffer is add potassium primary phosphate 1.361g and heptane-1-sodium sulfonate 1.0g in every 1000ml water, carries out gradient elution, run 70 minutes by following condition:
When 0 ~ 10 minute, acetonitrile ratio is 8.0%, and potassium phosphate buffer ratio is 92.0%;
When 10 ~ 60 minutes, acetonitrile ratio rises to 35.0% by 8.0%, and potassium phosphate buffer ratio drops to 65.0% by 92.0%;
When 60 ~ 62 minutes, acetonitrile ratio rises to 70.0% by 35.0%, and potassium phosphate buffer ratio drops to 30.0% by 65.0%;
When 62 ~ 70 minutes, keep acetonitrile: potassium phosphate buffer is with 70.0%: 30.0% ratio wash-out; Under these conditions, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt have a chromatographic peak in about 42 minutes in retention time.
4, the investigation of linear relationship
Each precision draws above-mentioned reference substance mother solution 1ml, put in 100ml, 50ml, 25ml, 10ml, 5ml measuring bottle respectively, thin up becomes following concentration respectively: 0.02432mg/ml, 0.0864mg/ml, 0.09728mg/ml, 0.19456mg/ml, 0.38912mg/ml.Accurate draw solution 10 μ l injection liquid chromatography, by chromatographic condition peak area under 3 chromatographic conditions and system suitability item, respectively with 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate integrating peak areas value is ordinate zou, respective reference substance sample size is X-coordinate, drawing standard curve, 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate regression equation is y=2187.0X-62.0, R2=1, 17-hydrogen-9-dehydrogenation-14, 17-ring-rographolide-19-sodium sulfovinate is good linear between 0.2388 ~ 3.8208 μ g.The results are shown in Table 1, Fig. 4.
Table 1.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate linear relationship result
5. precision test
Get 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate reference substance solution, repeat sample introduction 6 times, result 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate peak area RSD is 0.29%, the results are shown in Table 2.
Table 2 reference substance Precision test result
6. stability test
Get need testing solution, measure according to chromatographic condition under 3 chromatographic conditions and system suitability item, sample introduction once at regular intervals, 17-hydrogen-9-dehydrogenation-14 in result need testing solution, peak area is without considerable change in 24 hours for 17-ring-rographolide-19-sodium sulfovinate, and its peak area RSD is respectively 0.50%.The results are shown in Table 3.
Table 3 stability test result
7. replica test
Get inventive samples, add water and make need testing solution 6 parts, measure according to chromatographic condition under 3 chromatographic conditions and system suitability item, 17-hydrogen-9-dehydrogenation-14 in sample, 17-ring-rographolide-19-sodium sulfovinate average content is 98.79%, RSD=0.41%.The results are shown in Table 4.
Table 4.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate replica test result
8. recovery test
Adopt application of sample recovery test, accurate inventive samples solution of drawing is in 50ml volumetric flask, totally 6 parts, precision adds 17-hydrogen-9-dehydrogenation-14 respectively, 17-ring-rographolide-19-sodium sulfovinate (representing with 19 sodium sulfovinates in table) reference substance solution (0.942mg/ml) 2ml, be diluted with water to scale, shake up.Measure according to method under 3 chromatographic conditions and system suitability item, calculate the rate of recovery, the results are shown in Table 5.
Table 5 recovery test result
The impact of experimental data 2:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate induced by endotoxin pyrogenicity
Get Japan large ear rabbit, body weight 1.8-2.3kg, male and female have concurrently, and 1d before experiment, chooses body temperature between 38.0 ~ 39.4 DEG C, and the rabbit that the same day, Temperature changing was no more than 0.4 DEG C uses rabbit as experiment.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), and observe rabbit Temperature changing, every 30min records 1 time.The rabbit that after choosing injection 1h, body temperature rise surpasses 0.5 DEG C, is divided into 5 groups at random, often organizes 8.Ear vein injection gives 0.9% sodium chloride solution, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate low dose group 50mgkg respectively -1, middle dosage group 100mgkg -1, high dose group 200mgkg -1and injection Aspirin-arginine 100mgkg -1, respectively at 1,2,3,4,5,6h measuring each rabbit body temperature after administration.With body temperature average before administration for radix, calculate each minute rabbit temperature changing value.In table 6.
Medication temperature variation DEG C after table 6. endotoxin pyrogenic,
Note: compare with empty map group ※p < 0.05 ※ ※p < 0.01
As shown in Table 6, after intracellular toxin 1h, blank group Healthy Rabbits body temperature average rises, and starts gradually after continuing rising 3h to decline.Compare with blank group, low dose group 50mgkg -117-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate 1-2h shows and obviously suppresses rabbit temperature rise effect, middle dosage group 100mgkg -1, high dose group 200mgkg -1show the effect of extremely strong suppression rabbit fervescence in 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate 1 ~ 2h, also demonstrate during 4h and suppress the effect of rabbit fervescence; With 100mgkg in 3 drug study groups -1the above antipyretic effect of dosage is best.Show 50-200mgkg -1the rabbit fervescence that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate induced by endotoxin causes all has cooling effect.
Experimental data 3:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is on the impact of rat fever caused by dry yeast
Experiment surveys body temperature 3d in advance with rat, and experiment measured value on the same day is rat basal body temperature, and screening Temperature changing is no more than the animal of 0.3 DEG C, is divided into 5 groups at random, often organizes 8.Subcutaneous injection 20% dry yeast suspension 5mL/kg, pyrogenicity pneumoretroperitoneum injects 0.9% sodium chloride solution, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate low dose group 50mgkg -1, middle dosage group 100mgkg -1, high dose group 200mgkg -1and acetylsalicylic acid 100mgkg -1, measure the rat temperature of 1 ~ 6h after administration, 1 time per hour, using the difference of different time points body temperature value and basic value as observation index, the results are shown in Table 7.
Medication temperature variation DEG C after table 7. dry yeast pyrogenicity,
Note: compare with empty map group ※p < 0.05 ※ ※p < 0.01
Table 7 is found out, makes blank group healthy rat body temperature continue rising 6h when giving dry yeast 1h.Compare with blank group, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate 100,200mgkg -1show in 1 ~ 4h and obviously suppress rat temperature rising effect.
The impact of experimental data 4:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate p-Xylol induced mice auricle edema
Get mouse 50, be divided into 5 groups at random.Every day is to 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate low dose group 50mgkg -1, middle dosage group 100mgkg -1, high dose group 200mgkg -12 times, for three days on end, dexamethasone sodium phosphate injection is administration before causing inflammation only.1h after last administration, in every left auricle of mouse, outside is dripped and is coated with 0.1ml caused by dimethylbenzene xylene inflammation, and auris dextra in contrast.Cause scorching latter 2 hours de-cervical vertebras and put to death mouse, and cut mouse two ear along auricle baseline, lay auricle with diameter 6mm punch tool respectively at left and right ear same section, put on electronic balance and weigh and record data.Swelling is represented with left and right auricle weight difference.
Table 8. p-Xylol causes the impact of auricle edema
Note: compare with empty map group ※p < 0.05 ※ ※p < 0.01
Table 8 shows, and each medication group of 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate, Dexamethasone group mice auricle swelling degree are significantly less than control group.
The impact of rat paw edema caused by experimental data 5:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate on Carrageenan
Get rat 40, be divided into 5 groups at random.Every day is to 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate low dose group 50mgkg -1, middle dosage group 100mgkg -1, high dose group 200mgkg -12 times, for three days on end, dexamethasone sodium phosphate injection is administration before causing inflammation only.After last administration, every rat oral gavage gives physiological saline 8ml, after 30 minutes in the middle part of rat foot claw subcutaneous injection 0.15ml 1% Carrageenan solution, and in injection after 30min, 1h, 2h, 3h, 4h, 5h milscale measure its left back sufficient pawl thickness as rat paw edema level index.
Table 9 causes the impact of rat toes swelling to carrageen
Note: compare ※ P < 0.05 ※ ※ P < 0.01 with empty map group
Table 9 result shows, with control group ratio, 17-hydrogen-9-dehydrogenation-14, caused by 17-ring-rographolide-19-sodium sulfovinate each dosage group and the equal on Carrageenan of Dexamethasone group, rat swollen feet has obvious restraining effect, 17-hydrogen-9-dehydrogenation-14, obvious restraining effect is there is in administration 30min and continues 5 hours in 17-ring-rographolide-19-sodium sulfovinate when 200mg/kg, 100mg/kg effect is suitable with effects of dexamethasone, 17-hydrogen-9-dehydrogenation-14, a certain amount of effect relationship is had in the restraining effect of same time point to swelling between 17-ring-rographolide-19-sodium sulfovinate three dosage groups.
Experimental data 6:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is to the restraining effect of neuraminidase activity
Get 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate, add suitable quantity of water and make dissolving, application neuraminidase inhibitor identification kit measures 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate and suppresses tiring in table 10 of neuraminidase (N1).
(1). typical curve prepares: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases and detects damping fluid; B. every hole adds 0,1,2,5,7.5,10 μ l H5N1 neuraminidases more respectively; 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 and detects damping fluid; B. every hole adds 10 μ l H5N1 neuraminidases again; C. every hole adds 0 ~ 10 μ l17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate sample again; D. every hole adds 0 ~ 10 μ l Milli-Q water again.
(3). detecting step:
A. vibration mixes about 1min;
B.37 DEG C hatching 2min makes inhibitor and H5N1 neuraminidase fully interact, and the sample 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 fluorometric assay is carried out after DEG C hatching 20 ~ 30min.Excitation wavelength is 360nm, and emission wavelength is 440nm.
(4). calculate the suppression per-cent of sample for H5N1 neuraminidase according to typical curve, and after doing concentration curve, calculate the IC50 of 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate for H5N1 neuraminidase.It is 0.28g/L that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate reaches the inhibiting rate IC50 of neuraminidase.In table 10.
Table 10.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate suppresses the activity of neuraminidase
Can be clear that according to above-mentioned experimental result:
(1) .17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate can extract effective suppression neuraminic acid enzyme component;
(2) .17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is along with using dosage size variation, and it suppresses the ability of neuraminidase activity, and namely the height of neuraminic acid enzyme inhibition rate is also corresponding changes, and becomes positive correlation;
(3). from above-mentioned experiment, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate can by suppressing influenza surface neuraminidase, and then suppression influenza virus enters inside cell, suppress the influenza virus entered inside cell to copy, breed, thus decrease influenza virus to the infection of cell, growth, and prevention and therapy influenza and complication thereof.
Medical science and study of pharmacy personnel cannot not do suppression influenza infection, copy in advance, or under the prerequisite of the experiment of suppression neuraminidase, learn that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate has the sexy good result emitted of prevention and therapy influenza virus in advance.
The restraining effect of experimental data 11:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate infected by influenza infected chicken embryo
Get 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate, the ability that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate suppression FM1 influenza virus is copied and suppresses in chicken embryo is identified in application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1).
(1). be inoculated in by FM1 influenza virus liquid in 10d no-special pathogen in age chick embryo allantoic cavity, every embryo 0.2ml, hatches 72h for 37 DEG C, observes and calculates half egg infectious amount (EID50).
(2) .17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate adopts the toxic action of chicken embryo, stroke-physiological saline solution is to 17-hydrogen-9-dehydrogenation-14, be inoculated in 10d no-special pathogen in age chick embryo allantoic cavity after 17-ring-rographolide-19-sodium sulfovinate makes serial dilution, every embryo 0.2ml, each concentration inoculates 6 embryos, hatches for 37 DEG C, observe chicken embryonic development developmental state, the peak concentration of 96h can be survived as the TD of medicine using chicken embryo.
(3) .17-hydrogen-9-dehydrogenation-14, the restraining effect of 17-ring-rographolide-19-sodium sulfovinate infected by influenza in chicken embryo adopts, influenza virus liquid and the difference dilution 17-hydrogen-9-dehydrogenation-14 of 0.1ml, 17-ring-rographolide-19-sodium sulfovinate mixing, 37 DEG C of effect 2h, be inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, often organize inoculation 6 embryo, hatch 72h for 37 DEG C.Virus attack amount is 50EID50, establishes virus control, stroke-physiological saline solution normal control simultaneously, calculates 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate to the median effective dose (ED50) of viral inhibition.
(1) .FM1 influenza virus calculates through Reed-Muench method the virulence of chicken embryo, and its EID50 is 10 -4.82.
(2), after .17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is inoculated in chicken embryo, it grows basically identical with Normal group.96h chicken embryo is all survived.Chicken embryo gives 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate stoste and has no chicken embryo death, so can think that TD0 is 2.60g/L.(3) restraining effect of .17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate infected by influenza in chicken embryo is in table 11.
The restraining effect of table 11.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate infected by influenza infected chicken embryo
Compare with virus control group: * P < 0.05
As shown in Table 11,17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate has significant restraining effect (P < 0.05) at 0.09375 ~ 0.75g/L infected by influenza, ED 50for 0.1012g ± 0.0098g/L, TI are 61.08 ± 3.28.
Experimental data 8:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate affects FM1 influenza virus
Get 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate, application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1) identifies that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate suppresses the ability of FM1 influenza virus virulence.
(1) .FM1 adopts cell median infective dose (TCID50) micromethod to the toxicity test of dog kidney passage cell (MDCK).
(2) .17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate adopts the DMEM of serum-free to 17-hydrogen-9-dehydrogenation-14 to the toxicity test of mdck cell, being inoculated in after 17-ring-rographolide-19-sodium sulfovinate makes serial dilution is formed in the mdck cell hole of individual layer, every hole 100 μ l, each extent of dilution repeats 4 holes, establishes normal cell controls simultaneously.Culture plate is put 37 DEG C, 5%CO 2cultivate in incubator, observation of cell pathology every day (CPE), Continuous Observation 3d, with "+~ ++++" record result, calculate medicine median toxic concentration (TD50) and maximal non-toxic concentration (TD0) by Reed-Muench method.
(3) .17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate suppresses the effect of FM1 influenza virus to measure: mdck cell 5 × 10 5/ ml, every hole 100 μ l, respectively in 96 orifice plates, 37 DEG C, 5%CO 2cultivate in incubator, suck nutrient solution in hole next day, add 100TCID50 influenza virus liquid, every hole 100 μ l, after 37 DEG C of absorption 1h, suck supernatant liquor.2 times are washed with phosphate buffered saline buffer (PBS), be the 1st hole with the TD0 of medicine, use the DMEM liquid of serum-free to 17-hydrogen-9-dehydrogenation-14 again, 17-ring-rographolide-19-sodium sulfovinate makes serial dilution, add respectively above-mentioned infected virus cell in, establish virus control and Normal group, 37 DEG C, 5%CO simultaneously 2cultivate in incubator, observe the mdck cell characteristics of lesion that influenza virus produces every day, namely monolayer cell sex change becomes circle etc., continuous 3d, and calculate medicine 50% suppresses pathology concentration (IC50) and therapeutic index (TI).The calculating of TI: TI=TD50/IC50, 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, avoid the inhibiting rate that cytopathy (CPE) occurs to carry out correlation analysis to drug dose with to virus infected cell, judge whether amount validity response relation.
(4) .FM1 influenza virus calculates through Reed-Muench method the virulence of mdck cell, and its TCID50 is 10 -4.92.(2) TD0 of .17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate mdck cell is respectively 1.19g ± 0.048g/L.(3). after-14,17-rings-rographolide-19-sodium sulfovinate makes serial dilution by 17-hydrogen-9-dehydrogenation, inhibition test is carried out to 100TCID50 influenza virus, calculate median effective dose IC50 and the TI value size of medicine, the results are shown in Table 12.
Table 12.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is to the IC of FM1 influenza virus 50and TI (x ± s) (g/L)
As shown in Table 12, the IC50 of 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is low, and TI is high.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate suppresses the cytopathogenic effect of FM1 influenza virus all to strengthen along with the increase of drug dose.Drug dose and medicine are shown the correlation analysis that the inhibiting rate of CPE carries out, the dosage of 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate and medicine between CPE inhibiting rate in obvious positive correlation.
Experimental data 9:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is on the spleen index of influenza virus infection FM1 strain in Mice Body and the impact of Lung Exponent
Get 17-hydrogen-9-dehydrogenation-14; 17-ring-rographolide-19-sodium sulfovinate; application influenza virus A-prime mouse lung adapted strain (FM1) (H1N1) identifies that 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is to the dead provide protection of influenza virus infection FM1 strain in Mice Body.
(1) Influenza B virus strain virus is inoculated respectively after doing 10 times of doubling dilutions and is often organized 10 BALB/C mice, male and female half and half.After ether light anesthesia, often organize respectively give from different dilution virus, every mouse collunarium inoculates 20 μ l.Observe the dead mouse situation of 10d, calculating LD50 by Reed-Muench method is 10 -1.38.Therefore determine that experiment modeling concentration used is 10LD50.
(2) 17-hydrogen-9-dehydrogenation-14; 17-ring-rographolide-19-sodium sulfovinate is to the dead provide protection of influenza virus infection FM1 strain in Mice Body: Normal group, Influenza B virus strain virus control group, 17-hydrogen-9-dehydrogenation-14; 17-ring-rographolide-19-sodium sulfovinate 0.125g/L, 0.25g/L, 0.5g/L, 1.0g/L dosage groups etc. are gavage respectively, and gavage capacity is only 0.4ml/.After 3d, except Normal group, under ether light anesthesia, only use 10LD50 Influenza B virus strain collunarium infecting mouse 20 μ l/ for each group.Normal group gives the physiological saline of same volume simultaneously.4 groups of administration continue administrations, and Normal group and Influenza B virus strain virus control group administered physiological saline 8 days, dosage is the same.Day by day observe animal morbidity and record death toll, observe 14 days altogether, calculate mortality ratio (mortality ratio=often organize death toll/often organize total mice × 100%), the results are shown in Table 13.
(3) 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate is on the impact of influenza virus infection FM1 strain Lung Exponent in Mice Body: Normal group, Influenza B virus strain virus control group, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate 0.125g/L, 0.25g/L, 0.5g/L, 1.0g/L dosage groups etc. are gavage respectively, and gavage capacity is only 0.4ml/.After 3 days, except Normal group, under ether light anesthesia, only use 1.0LD50 Influenza B virus strain collunarium infecting mouse 20 μ l/ for each group.Normal group gives the physiological saline of same volume simultaneously.4 groups of administration continue administrations, and Normal group and Influenza B virus strain virus control group administered physiological saline 8 days, dosage is the same.Within the 8th day after virus infection, put to death mouse, weigh, get lung and claim lung weight, calculate Lung Exponent (Lung Exponent=lung quality/weight × 100%); In addition, get spleen and claim spleen weight, calculate spleen index (spleen index=spleen quality/weight × 100%), the results are shown in Table 14.
Table 13.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is to the death protection result of influenza virus infection FM1 strain in Mice Body
Note: ※ ※ P < 0.01VS influenza model group ※ P < 0.05VS influenza model group
Table 14.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is on the spleen index of influenza virus infection FM1 strain in Mice Body and the impact of Lung Exponent
Note: #P < 0.05VS Normal group note: ※ ※ p < 0.001VS influenza model group ※ p < 0.05VS influenza model group
(1). as shown in Table 13,17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate has significant provide protection (p < 0.01) at 0.25 ~ 1.0g/L influenza virus infected.
(2). as shown in Table 14,17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate has significant effect (p < 0.01) at the lung index of 0.5 ~ 1.0g/L influenza virus infected.
Experimental data 10:17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is on the impact of mouse septicemia
1.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate significantly improves septicemia mouse survival rate
Rographolide is because of water-soluble extreme difference, so adopt 0.9% sodium carboxymethyl cellulose solution to be mixed with suspension carry out gavage (30mg/kg), sulfonated bodies is directly 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.Model group mouse after modeling 16 time occur dead, after 60h, survival rate is 25% (as shown in Table 15), rographolide administration group survival rate is 50%, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate administration group survival rate is 65%, comparatively speaking, 17-hydrogen-9-dehydrogenation-14, the effect of the improvement survival rate of 17-ring-rographolide-19-sodium sulfovinate (being called for short in following form: 19-sodium sulfovinate) is slightly better than rographolide group effect, but its dosage is lower.
The septicemia mouse survival rate that table 15.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is induced LPS
2.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate significantly suppresses the rising of inflammatory factor in septicemia mice serum
BALB/C mice abdominal cavity gavage gives rographolide 30mg/kg, tail vein injection 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate 1,3,10mg/kg, simultaneously abdominal injection 5mg/kg LPS, after modeling and administration 2,5,8,12h, 4 time points respectively put to death 3 mouse, eye socket gets blood, measure the content of inflammatory factor TNF-α, IL-1 β in serum, shown in table 16.After lps injection 2h, TNF-α in model group mice serum, namely the content of IL-1 β reach peak value, be respectively 4815pg/ml and 391pg/ml, 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate administration 2h can significantly suppress TNF-α to 3672pg/ml, significantly suppresses producing the release of IL-1 β when 5h.Rographolide effect 8h just significantly can suppress the rising of TNF-α.Experimental result shows compared to rographolide, and sulfonated bodies is rapid-action to the restraining effect of inflammatory factor in septicemia mice serum, and inhibition is more remarkable.
Table 16.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate time, dose-dependently reduce inflammatory factor in the septicemia mice serum of LPS induction
TNFα(pg/ml)
IL?1β(pg/ml)
N=3, * p < 0.05vs model group, #p < 0.05vs rographolide group.
3.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate significantly suppresses the hepar damnification of septicemia mouse
Septicemia is a kind of disease causing multiple organ injury, and liver is one of main organs of its damage.BALB/C mice gavage gives rographolide 30mg/kg, tail vein injection 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate 10mg/kg, abdominal injection 5mg/kg LPS simultaneously, respectively at 2h, 5h, 8h, 12h eye socket gets blood, measures the content of serum alt, AST.Shown in table 17, after giving LPS, model mice serum alt and AST continue to raise, give 17-hydrogen-9-dehydrogenation-14, after 17-ring-rographolide-19-sodium sulfovinate 2h, namely the content of ALT/AST decline to some extent, to 12h, have significant difference with model group ALT/AST, rographolide is slightly poorer than 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate effect to the inhibition of ALT/AST.The mouse rna level that RT-PCR measures each inflammatory factor in liver organization finds, LPS stimulates lower ifn-γ, il-6, tnf-α, il-β, cox-2mRNA significantly raises, and 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate and rographolide administration 5h and 8h can significantly suppress ifn-γ, il-6, tnf-α, il-β, the rising of cox-2mRNA.Experimental result shows equally compared to rographolide, and the restraining effect of sulfonated bodies to septicemia mouse liver injury is rapid-action, and inhibition is more remarkable.
Table 17.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate is to the restraining effect of the septicemia mouse liver injury that LPS induces
AST(karmen?units)
ALT(karmen?units)
N=3, * p < 0.05, vs model group.
4, brief summary
Rographolide and 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sodium sulfovinate significantly can improve the survival rate of the septicemia mouse of LPS induction, time, dose-dependently reduce TNF-α in septicemia mice serum, IL-1 β, ALT, the content of AST, the sick rising suppressing inflammatory factor mRNA level in-site in liver organization.17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sodium sulfovinate onset comparatively rographolide is fast, better effects if.After experimental result display rographolide is transformed into sulfonated bodies, it is better water-soluble, and administration is rapid-action, is also enhanced to the improvement result of mouse septicemia.

Claims (30)

1. the compound or its salt represented by general formula (I), its chemistry 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester by name or its salt,
R can be H, sodium, potassium or NH 4.
2. the preparation method of the compound or its salt of claim 1, comprises the following steps:
(1) solvent is added in a kettle., adding rographolide makes it dissolve, then adds sulphonating agent and carry out sulfonation reaction, regulates temperature of reaction kettle at 6 ~ 39 DEG C, sulfonation reaction 1 ~ 28.5 hour, the described solvent added in a kettle. is one or both of aceticanhydride or Glacial acetic acid;
(2) rographolide solution adopts the mode slowly dripping, spray or ventilate to add sulphonating agent when stirring, and when adopting the mode slowly dripping or spray to add sulphonating agent, add speed control at 1.3ml ~ 4.7ml/min/1g rographolide, when adopting the mode passing into gas to add sulphonating agent, pass into speed control at 0.03 liter ~ 0.22 liter/min/1g rographolide, 17-hydrogen-9-dehydrogenation-14, the 17-rings-reactant of rographolide-19-sulfuric ester and the mixture of unreacted reactant is obtained after sulfonation reaction;
(3) mixture is through solvent extraction, crystallization, obtains 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester finally; Or
Mixture in saturated salt solution, crystallization, obtain 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester finally or its salt; Or
Mixture alkaline solution adjust pH to 7, through macroporous adsorbent resin, ODS or Sephadex LH-20 column chromatography for separation, with water: ethanol or methyl alcohol are elutriant, gradient elution, collect eluant component, crystallization, obtains 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester finally or its salt; Or
Mixture, successively through two or more step co-treatment aforementioned, obtains 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester finally or its salt.
3. the preparation method of claim 2,1g rographolide dissolution with solvents described in 3g ~ 12g.
4. the preparation method of claim 3,1g rographolide dissolution with solvents described in 4g ~ 9g.
5. the preparation method of claim 2, described solvent is aceticanhydride and Glacial acetic acid, and described aceticanhydride, Glacial acetic acid are made up of following weight proportion: aceticanhydride 80% ~ 20%, Glacial acetic acid 20% ~ 80%.
6. the preparation method of claim 5, wherein aceticanhydride 62%, Glacial acetic acid 38%.
7. the preparation method of claim 2, described sulphonating agent adopts the vitriol oil and Glacial acetic acid, 1g rographolide 1g ~ 10g vitriol oil Glacial acetic acid carries out sulfonation, and the described vitriol oil and Glacial acetic acid are made up of following weight proportion: the vitriol oil 80% ~ 20%, Glacial acetic acid 20% ~ 80%.
8. the preparation method of claim 7,1g rographolide 1.5g ~ 5g vitriol oil Glacial acetic acid, and the acid of dense stream is 1: 1 with Glacial acetic acid part by weight.
9. the preparation method of claim 2, described sulphonating agent adopts sulphur trioxide, 1g rographolide pass into 0.2 liter ~ 5 liters volumetric concentrations be 1% ~ 3% sulphur trioxide carry out sulfonation.
10. the preparation method of claim 9,1g rographolide pass into 0.3 liter ~ 3 liters volumetric concentrations be 1.5% ~ 2.5% sulphur trioxide carry out sulfonation.
The preparation method of 11. claims 2, described sulphonating agent adopts chlorsulfonic acid, and 1g rographolide 0.2g ~ 5g chlorsulfonic acid carries out sulfonation.
The preparation method of 12. claims 11,1g rographolide 0.3g ~ 3.5g chlorsulfonic acid carries out sulfonation.
The preparation method of 13. claims 2, described saturated salt solution adopts sodium-chlor or saturated potassium chloride solution, described alkaline solution adopt 5% ~ 50% sodium hydroxide potassium hydroxide or less than 25% ammonia soln.
The preparation method of 14. any one of claim 2 ~ 13, wherein said temperature of reaction kettle is at 7 ~ 25 DEG C, and the described sulfonation reaction time should control at 0.5 ~ 2.5 hour.
The preparation method of 15. claims 2, wherein said sulphonating agent is vitriol oil Glacial acetic acid or chlorsulfonic acid, and adopts the mode slowly dripping or spray to add, and adds speed control at 1.8ml ~ 3.5ml/min/1g rographolide.
The preparation method of 16. claims 2, wherein said sulphonating agent is sulphur trioxide, and adopts the mode passing into gas to add, and passes into speed control at 0.05 liter ~ 0.15 liter/min/1g rographolide.
The preparation method of 17. any one of claim 2 ~ 13, in wherein said gradient elution, the ratio of water is 80 ~ 20%, and the ratio of ethanol or methyl alcohol is 20 ~ 80%.
The measuring method of the compound or its salt of 18. claims 1, it adopts 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester described in high effective liquid chromatography for measuring or its salt, and condition determination is as follows:
Take octadecylsilane chemically bonded silica as weighting agent;
Determined wavelength is 225nm;
Column temperature is 25 DEG C;
Number of theoretical plate presses 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or the calculating of its salt is not less than 10000;
The preparation of reference substance solution is by described 17-hydrogen-9-dehydrogenation-14,17-ring-rographolide-19-sulfuric ester or its salt make reference substance by purity test and content mark, and dry, in right amount accurately weighed in Vanadium Pentoxide in FLAKES vacuum drier, add water and make the solution of desired concn, to obtain final product;
The preparation precision of need testing solution takes sample 5mg, puts in 50ml measuring bottle, is diluted with water to scale, shake up, to obtain final product;
Wash-out is mobile phase A with acetonitrile, take potassium phosphate buffer as Mobile phase B, and described potassium phosphate buffer is add potassium primary phosphate 1.361g and heptane-1-sodium sulfonate 1.0g in every 1000ml water, carries out gradient elution, run 70 minutes by following condition;
When 0 ~ 10 minute, acetonitrile ratio is 8.0%, and potassium phosphate buffer ratio is 92.0%;
When 10 ~ 60 minutes, acetonitrile ratio rises to 35.0% by 8.0%, and potassium phosphate buffer ratio drops to 65.0% by 92.0%;
When 60 ~ 62 minutes, acetonitrile ratio rises to 70.0% by 35.0%, and potassium phosphate buffer ratio drops to 30.0% by 65.0%;
When 62 ~ 70 minutes, keep acetonitrile: potassium phosphate buffer is with 70.0%:30.0% ratio wash-out;
Under these conditions, 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt have a chromatographic peak in 42 minutes in retention time,
Assay method is accurate respectively draws reference substance solution and need testing solution, injection liquid chromatography, measures, to obtain final product.
19. 1 kinds of 17-hydrogen-9-dehydrogenations-14, the preparation of 17-ring-rographolide-19-sulfuric ester or its salt, to be 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester according to claim 1 or its salt make with pharmaceutically acceptable carrier said preparation.
20. 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric esters according to claim 1 or its salt are for the preparation of the purposes of antipyretic medicine.
21. as the purposes of claim 20, the refrigeration function of described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt pair endotoxin pyrogenic.
22. as the purposes of claim 20, the refrigeration function of described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt pair dry yeast pyrogenicity.
23. 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric esters according to claim 1 or its salt are for the preparation of the purposes of the medicine of anti-inflammatory.
24. as the purposes of claim 23, the drug effect of described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt pair septicemia.
25. as the purposes of claim 23, described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt p-Xylol induced mice auricle edema anti-inflammatory action.
26. as the purposes of claim 23, the anti-inflammatory action of rat paw edema caused by described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt on Carrageenan.
27. 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric esters according to claim 1 or its salt are for the preparation of the purposes of antiviral drug.
28. as the purposes of claim 27, and described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt are for suppressing neuraminidase.
29. as the purposes of claim 27, and described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt are for suppressing influenza virus.
30. as the purposes of claim 27, and described 17-hydrogen-9-dehydrogenation-14,17-rings-rographolide-19-sulfuric ester or its salt are for suppressing Influenza B virus.
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