CN102532156B - Swerilactones H-K, 1-4 and medicinal composition and application thereof - Google Patents
Swerilactones H-K, 1-4 and medicinal composition and application thereof Download PDFInfo
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Abstract
The invention discloses a compound shown as a structural formula (I), i.e., swerilactones H-K, 1-4, a medicinal composition consisting of an effective amount of the compound 1-4 and a medicinal carrier or an excipient, preparation methods of a medicinal compound, i.e., swerilactones H-K (1-4) and the medicinal composition thereof, an application of the swerilactones H-K, 1-4 to treatment of human diseases, and particularly an application of the swerilactones H-K, 1-4 to preparation of a medicament for resisting virus B hepatitis and a medicament for treating virus B hepatitis.
Description
Technical field:
The invention belongs to technical field of pharmaceuticals, specifically, relate to swerilactone H-K, contain it and be the pharmaceutical composition of the treatment hepatitis B of effective constituent, its preparation method, and the application in pharmacy, the particularly application in the anti-hepatitis B medicine of preparation.
Background technology:
Hepatitis B is hepatitis B virus (hepatitis B virus, HBV) cause, take the liver inflammatory pathology as leading and cause a kind of communicable disease of multiple organ injury, wherein chronic viral hepatitis is one of Etiological that causes liver cirrhosis, liver cancer.According to the World Health Organization (WHO) statistics, the whole world has 2,000,000,000 people of surpassing to infect or infecting HBV, and wherein approximately 400,000,000 people are HBV chronic infection person, the chronic hepatitis, liver cirrhosis and the primary hepatitis that have every year more than 100 ten thousand populations to die from hepatitis B to cause.China belongs to hepatitis B infected high Endemic Area, and chronic viral hepatitis B cause of disease carrier surpasses 1.2 hundred million, and Chronic Hepatitis B is 2800-3000 ten thousand examples approximately.Therefore the hepatitis B infection is serious world's public health problem, and the expense for the treatment of chronic hepatitis is very expensive, and every year, due to illness virus hepatitis caused direct economic loss to reach 30,000,000,000～50,000,000,000 Renminbi.
The course of infection of hepatitis B virus related generally to for 5 steps: in conjunction with, invade, transcribe, translation, gene replication and packing.At first virion is attached to liver cell by acceptor, sloughs adventitia after the intrusion endochylema, forms core particle.Core particle moves to liver cell nuclear, and HBV DNA core particle shells and goes out, and forms cccDNA (covalently closed circularDNA, cccDNA) in nucleus.CccDNA is the template of hepatitis B replication, is that liver cell continues infected key substance for a long time.CccDNA has 2 kinds of functions, and the various members of the synthetic virion of the first are as HBsAg (surface antigen, consist of adventitia), HBcAg (hepatitis virus c antigens, cAg, consist of inner membrance), HBeAg (being secreted in blood); It two is synthetic HBV DNA of future generation.Most of new synthetic HBV DNA is used for the packing of reovirion, and part reenters liver cell nuclear, becomes another source of cccDNA.At first the new synthetic HBV DNA of part is assembled into core particle with HBcAg, and then is assembled into complete HBV particle with HBsAg, discharges to the extracellular.Superfluous HBsAg is released into alone blood sometimes, forms microspheric form particle or cast particle.
Present multiple therapeutic modality comprises Hepatitis B virus vaccine, immunomodulator and anti-HBV medicine nucleoside analog etc., has been applied to treat HBV and has infected, and obtained certain curative effect.Hepatitis B virus vaccine can effectively prevent HBV to infect, yet invalid to the patient who infects HBV.Immunomodulator as alpha-interferon (IFN) etc., can show anti-HBV effect by improving body immunity, yet itself there is no antiviral activity.And Interferon, rabbit has obvious restraining factors at the treatment chronic hepatitis B: at first, it never obtains real success, unless the substantive immune response to HBV has appearred in patient before treatment.And side effect is obvious, before interferon-induced HBeAg serum conversion, unexpected one property crossed of hepatitis activity increases the weight of, show as ALT (alanine aminotransferase, ALT, alanine aminotransferase) raise, can cause patient with liver cirrhosis generation liver failure, occur once in a while dead.Nucleoside medicine is present internationally recognized curative effect anti-hepatic-B virus medicine preferably, is all nucleoside analog as lamivudine (lamivudine), adefovir ester (adefovir dipivoxiil), Entecavir (enticavir), Telbivudine (tibivudine), the tenofovir (tenofovir) that has gone on the market at present.During these medicines are tested, HBV virus there is remarkable restraining effect in external or body.Its mechanism of action is that medicine enters and becomes the triphosphoric acid compound in cell, by the competition of substrate, polysaccharase or the ThermoScript II of virus is suppressed, and finally suppresses synthetic, the viral propagation of viral DNA.The anti-HBV of nucleoside analog has evident in efficacy, easy administration, characteristics such as bioavailability is high, patient tolerability is good.Yet also have obvious weak point, as single in action target spot, long-term taking causes the virus strain variation, produces resistance, can not thoroughly eradicate HBV, easily recurrence after drug withdrawal.Present anti-HBV medicine far can not satisfy the needs that treatment HBV infects, so the Antihepatitis medicament that the development structure novelty has a novel mechanism has very large market outlook.
China's traditional Chinese medicine aboundresources, medicinal history is long, and shows to have determined curative effect through the human body long-term taking, low toxicity or the advantage such as nontoxic.Mile Swertia Herb is the peculiar anti-hepatitis Chinese medicine in Yunnan, at the acute icterohepatitis that is used for the treatment of for a long time among the people, has good curative effect, yet its anti-HBV active substance is still unclear.
So far, in prior art without the report of compound swerilactone H-K (shown in formula I), there is no it as the report of the pharmaceutical composition of effective constituent yet, there is no swerilactone H-K and pharmaceutical composition thereof the application report in preparation or treatment hepatitis B medicine yet.
Summary of the invention:
The object of the present invention is to provide the swerilactone H-K (1-4) shown in the new formula with pharmaceutical use (I) of a class, contain the swerilactone H-K (I) for the treatment of hepatitis B significant quantity and the treatment hepatitis B pharmaceutical composition of pharmaceutical carrier or vehicle, the preparation method of swerilactone H-K and pharmaceutical composition thereof, the application in the anti-hepatitis B medicine of preparation of this compounds or its pharmaceutical composition.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Swerilactone H-K (1-4) shown in structural formula (I),
Pharmaceutical composition wherein contains formula (I) swerilactone H-K (1-4) any and the carrier that pharmaceutically can give and accept for the treatment of significant quantity.
Described pharmaceutical composition is the pharmaceutical composition for the treatment of hepatitis B.
Any application in the medicine of preparation treatment human diseases or illness of formula (I) swerilactone H-K (1-4).
Above-mentioned application, described disease is hepatitis B.
Contain the application of pharmaceutical composition in the medicine of preparation treatment human diseases or illness of formula (I) swerilactone H-K (1-4) any and the carrier that pharmaceutically can give and accept for the treatment of significant quantity.
Above-mentioned application, described disease is hepatitis B.
the method of preparation formula of the present invention (I) swerilactone H-K (1-4) is, get Mile Swertia Herb (Swertia mileensis) herb, dry, pulverize, use successively 90% and 50% alcohol reflux 3 times, each 2-3 hour, merge alcohol extract, filter, decompression recycling ethanol is to distinguishing the flavor of without alcohol, this extracting solution is suspended in the aqueous solution, use successively sherwood oil, ethyl acetate and n-butanol extraction, ethyl acetate extraction part is adsorbed on silica gel with the chloroform/methanol dissolving, room temperature is placed and is volatilized, grind, through silica gel column chromatography, with chloroform/methanol (100: 1 → 80: 20) wash-out, be divided into ten parts, first part's silica gel column chromatography wherein, chloroform/acetone gradient elution with 95: 5 → 80: 20, then further pass through silica gel column chromatography, with chloroform/methanol (95: 5) wash-out, and obtain compound swerilactone H-K (1-4) through chloroform/methanol (1: 1) recrystallization.
The method that preparation contains the pharmaceutical composition of swerilactone H-K (1-4) is take compound swerilactone H-K (1-4) as raw material, adds pharmaceutically acceptable carrier or vehicle.
When the compounds of this invention is used as medicine, can directly use, perhaps use with the form of pharmaceutical composition.This pharmaceutical composition contains 0.1-99%, is preferably the compounds of this invention of 0.5-90%, and all the other are acceptable on pharmacology, pharmaceutically acceptable carrier and/or the vehicle of and inertia nontoxic to humans and animals.
Described pharmaceutical carrier or vehicle are one or more solids, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention can be through injection (quiet notes, intramuscular injection) and oral two kinds of form administrations.
Embodiment:
In order to understand better essence of the present invention, the below will illustrate with test example of the present invention the pharmacological action result of formula of the present invention (I) swerilactone H-K (1-4), but not limit the present invention with this test example.
Test example 1:
Swerilactone H-K (I) is to the drug toxicity of Hep G2.2.15 cell and the restraining effect of HBV DNA replication dna:
1 materials and methods
1.1 material: swerilactone H-K (I); Lamivudine (lamivudine) [GlaxoSmithKline PLC pharmacy (Suzhou) company limited, the accurate word H20030581 of traditional Chinese medicines]; Hep G2.2.15 cell (Guangzhou air hospital); DMEM in high glucose (GIBCO); G418 (GIBICO); Foetal calf serum (Tianjin blood grinds institute); L-glutaminate (AMRESCO); Penicillin, Streptomycin sulphate [Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., Shiyao Group].
1.2 Hep G2.2.15 cell is cultivated with DMEM in high glucose liquid, nutrient solution adds 10% foetal calf serum, 0.03%L-glutamine, 100mg/L G418,10
5The blue or green enzyme element of IU/L, 100mg/L Streptomycin sulphate, 5%NaCO
3Transfer pH to 6.8-7.0.
1.3 instrument: microplate reader Bio-RAD 680 (U.S.); CO
2Incubator Thermo Forma 3310 (U.S.); Inverted biologic microscope XD-101 type (Nanjing); Quantitative real time PCR Instrument Mastercycler ep realplex type (German eppendorf company); Low-temperature and high-speed whizzer Centrifuge 5415D type (German eppendorf company).
1.4 experimentation: Hep G2.2.15 cell is with having added the 0.03%L-glutamine, 100mg/L G418, the blue or green enzyme element of 100IU, the DMEM in high glucose of 100IU Streptomycin sulphate (GIBICO) culture medium culturing.Be inoculated on cell plate with the cell single cell suspension after trysinization, use instead after 48h and contain 2% foetal calf serum, the pastille substratum of 380 μ g/mL, every kind of medicine is four drug levels with four times of dilutions of substratum, the cell control group and the acellular blank group that do not add medicine are set simultaneously, and do the positive drug contrast with lamivudine.Measure medicine to the toxicity of cell with mtt assay; Detect HBV DNA carrying capacity with fluorescence quantitative PCR method.
1.5 drug cell toxicity test.The cytotoxicity of the mtt assay detection of drugs of setting up according to Mosmann (Y.Nakajima, Y.Saton, M.Katsumata, K.Tsujiyama, Y.Ida, and J.Shoji, Phytochemistry 1994,36,119-127).Concrete grammar is: according to 5 * 10
5Individual every hole is inoculated in and changes supernatant after the cell cultures 48h of 48 orifice plates is the pastille substratum.After continue cultivating 72h, adding concentration by 0.3mL every hole is the MTT of 0.4mg/mL, in 37 ℃ of 5%CO
2Abandon supernatant after hatching 4h, add the 0.3mL dimethyl sulfoxide (DMSO) in every hole, hatch 10min in 37 ℃, measuring the absorbance of solution under 490nm on microplate reader.Calculate medicine to the destruction percentage of cell according to result:
η
Destroy=(A
The cell control group-A
The test sample group)/(A
The cell control group-A
Blank group) * 100.
1.6 the impact of medicine on HBV DNA carrying capacity.Concrete grammar is: Hep G2.2.15 cell is by 5 * 10
5Individual every hole is inoculated in 24 porocyte plates, in 5%CO
2, be replaced by the pastille substratum after cultivation 48h in 37 ℃ of incubators, and change cell conditioned medium every 48h with the pastille nutrient solution.Cell continued to cultivate after 8 days, used blood/cell/tissue genome DNA extracting reagent kit (TIANamp Gemomic DNA Kit, TIANGEN, China) to extract DNA.Method detection by quantitative HBV DNA carrying capacity with quantitative fluorescent PCR.1 μ L DNA sample is with 20 μ L2 * SYBR Green PCR Master Mix (Applied Biosystems, USA) and the special primer of 2 HBV as the pcr amplification system: front primer (5 ' GGA ACC TCT ATG TAT CCC TCC 3 '), rear primer (5 ' TCC GTC CGA AGG TTT GGT AC 3 '). amplification and detection Mastercycler Ep Realplex System quantitative PCR instrument (Eppendorf on probation, Masteraycler Eprealplex, German) 95 ℃ of preheatings 2 minutes, and by 95 ℃ 20 seconds, 58 ℃ 15 seconds, 72 ℃ of conditions of 20 seconds repeat the reaction of 40 circulations.Calculate the inhibition percentage of medicine according to result:
η
Inhibition=(A
The cell control group-A
The test sample group)/(A
The cell control group-A
Blank group) * 100.
2. result: CC
50Be half cell-lethal concentration, according to destroying percentage η
DestroyCalculate.IC
50For half virus inhibition concentration, according to inhibition percentage η
InhibitionCalculate.Net result is with selectivity index (SI=CC
50/ IC
50) estimate.
CC
50, IC
50And the calculation formula of SI:
A=log (the η＞50% time
Destroy/ η
InhibitionDrug level)
B=log (the η＜50% time
Destroy/ η
InhibitionDrug level)
C＝|A-B|
CC
50η during=Anti log[(50-＜50%
Destroy) * C/ (the η＞50% time
Destroyη in the time of-＜50%
Destroy)]+B
IC
50η during=Antilog[(50-＜50%
Inhibition) * C/ (the η＞50% time
Inhibitionη in the time of-＜50%
Inhibition)]+B
SI＝CC
50/IC
50
Concrete outcome sees Table 1:
Table 1 swerilactone H-K (1-4) is (I) in inhibition and the cytotoxicity (unit μ M) of Hep G2.2.15 cell to the HBV DNA replication dna
3, conclusion:
The experimental result demonstration, swerilactone H-K (1-4) has significant restraining effect external to the HBV DNA replication dna, its IC
50Value is between 1.53-5.34 μ M, and the obvious cytotoxicity of nothing.
Come by the following examples further to illustrate preparation method of the present invention and ingredients, being situated between does not limit the present invention with this.
Embodiment 1:
Swerilactone H-K (1-4) preparation (I):
The extraction of swerilactone H-K (1-4) separates:
Gather the Mile Swertia Herb herb, its formal name used at school dries in the shade under room temperature through being accredited as Swertia mileensis T.N.Ho et W.L.Shi, pulverize, get 5.0kg dry powder, use successively 90% and 50% alcohol reflux 3 times, each 2 hours, merge ethanol extract, decompression recycling ethanol is to distinguishing the flavor of without alcohol.This extracting solution is suspended in the aqueous solution, uses successively sherwood oil, ethyl acetate and n-butanol extraction.Ethyl acetate extraction part is adsorbed on silica gel with the chloroform/methanol dissolving, and room temperature is placed and volatilized solvent, grinds, and through silica gel column chromatography, with chloroform/methanol (100: 1 → 80: 20) wash-out, is divided into ten parts.Wherein first part continues to use silica gel column chromatography, chloroform/acetone gradient elution with 95: 5 → 80: 20, then further pass through the silicagel column purifying, with chloroform/methanol (95: 5) wash-out, and through chloroform/methanol (1: 1) recrystallization, obtain respectively Herba Swertiae bimaculatae lactone (swerilactone) H (1,15.0mg), I (2,19.0mg), J (3,20.0mg) and K (4,11.0mg).The structure of above compound is passed through
1H，
13C NMR, IR, UV, mass spectrum, and X-ray crystalline diffraction data are determined.
The structured data of swerilactone H-K (1-4)
Fusing point is measured with the XRC-1 type micro-meldometer that tech factory of Sichuan University produces, and thermometer is not revised; Optically-active is measured by Jascomodel 1020 polarimeters (Horiba, Tokyo, Japan); Infrared spectra (IR) adopts the KBr pressed disc method, by Bio-Rad FTS-135 type infrared spectrometer; UV spectrum is measured by UV-2401A type ultraviolet spectrometer; Nuclear magnetic resonance spectrum (
1H-,
13C-NMR, DEPT) measure with DRX-500 type NMR spectrometer with superconducting magnet with Brucker AM-400 type and DRX-500 type NMR spectrometer with superconducting magnet mensuration, two dimensional NMR spectrum, with pyridine-d
5As solvent, mark in TMS (tetramethylsilane) does; Mass spectrum (MS) is measured with VGAutospec-3000 type mass spectrograph; High resolution mass spectrum is measured with API Qstar Pulsar mass spectrograph; X-ray crystalline diffraction data are used respectively D/max-3B (Rigaku company, Japan) and CAD4/PC (Enraf Noius ﹠amp; Enraf Noius company, Holland) x-ray diffractometer mensuration; Thin-layer chromatography silica gel, column chromatography silica gel (200-300 order) are available from Qingdao Makall Group Co., Ltd.;
Swerilactone H (1)
Molecular formula: C
30H
32O
11
Molecular weight: 568.56
Proterties: colourless prismatic crystal
mp：268-269℃
Optically-active
(c=2.60mg cm
-3, pyridine)
IR(KBr)v
max：3456，1726，1701，1681，1642，1403，1278，1240，1107，1062，1041，1008，964，792cm
-1。
UV/Vis(CHCl
3/MeOH＝1∶1，v/v)λ
max(ε)：277(8989)nm。
ESIMS(-)m/z：603[M
-+Cl]，567[M-H]
-，341，297。
HRESIMS (-) m/z: experimental value 567.1876, calculated value 567.1866 (C
30H
31O
11, [M-H]
-).
1H?NMR(C
5D
5N)δ：6.14(1H，br?s，H-7)，5.86(1H，br?s，H-11)，5.79(1H，brs，H-25)，5.23(1H，d，J＝2.6Hz，H-17)，5.10(1H，m，H-23)，4.96(1H，m，H-20a)，4.91(1H，d，J＝14.6Hz，H-20b)，4.61(1H，d，J＝?3.7Hz，H-13)，4.43(1H，m，H-3a)，4.42(1H，m，H-27a)，4.27(1H，m，H-3b)，3.91(1H，q，J＝6.2Hz，H-19)，3.89(1H，m，H-27b)，3.53(3H，s，H-33)，2.54(1H，m，H-16a)，2.52(1H，m，H-15)，2.29(1H，m，H-4a)，2.27(1H，m，H-28a)，2.16(1H，m，H-4b)，2.15(1H，m，H-14)，2.12(1H，m，H-28b)，1.47(3H，d，J＝6.8Hz，H-32)，1.46(1H，m，H-16b)，0.99(3H，d，J＝6.2Hz，H-31)。
13C?NMR(C
5D
5N)δ：172.4(s，C-30)，164.2(s，C-22)，161.0(s，C-1)，156.0(s，C-5)，141.0(s，C-9)，135.9(s，C-24)，132.4(s，C-8)，131.2(s，C-18)，130.1(s，C-29)，127.6(d，C-7)，126.4(s，C-10)，89.4(d，C-25)，76.3(d，C-19)，71.4(d，C-23)，68.5(t，C-20)，66.7(t，C-3)，66.2(d，C-13)，64.6(d，C-17)，63.3(d，C-11)，57.9(t，C-27)，54.3(s，C-12)，52.5(q，C-33)，47.7(s，C-6)，41.6(d，C-14)，36.1(d，C-15)，31.9(t，C-16)，27.2(t，C-4)，25.6(t，C-28)，18.2(q，C-31)，17.6(q，C-32)。
X-ray crystalline diffraction data: oblique system, spacer are p2
1 
α=90.00, β=96.91 (3), γ=90.00,
Z=4, d=1.427g/cm
3, crystalline size 0.10 * 0.10 * 0.05nm
3, R
1=0.0374, wR
2=0.0710.
Swerilactone I (2)
Molecular formula: C
29H
28O
10
Molecular weight: 536.53
Proterties: colourless prismatic crystal
mp：204-205℃
Optically-active
(c=1.20mg cm
-3, CHCl
3/ MeOH=1: 1, v/v)
IR(KBr)v
max：3437，2925，1720，1672，1640，1462，1405，1376，1287，1247，1128，1100，1073，933，874cm
-1。
UV/Vis(CHCl
3/MeOH＝1∶1，v/v)λ
max(ε)：226(10896)，222(11110)，212(10977)nm。
ESIMS(+)m/z：559[M+Na]
+。
HRESIMS (+) m/z: experimental value 559.1578, calculated value 559.1580 (C
29H
28O
10Na, [M+Na]
+).
1H?NMR(C
5D
5N)δ：10.34(1H，s，H-25)，6.25(1H，s，H-7)，5.42(1H，s，H-11)，5.25(1H，s，H-17)，4.95(1H，d，J＝14.0Hz，H-20a)，4.89(1H，d，J＝14.0Hz，H-20b)，4.85(1H，m，H-23)，4.82(1H，m，H-27a)，4.64(1H，d，J＝3.2Hz，H-13)，4.48(1H，m，H-27b)，4.45(1H，m，H-3a)，4.40(1H，m，H-3b)，4.23(1H，d，J＝12.7Hz，H-14)，4.17(1H，m，H-19)，3.85(1H，m，H-28a)，3.67(1H，m，H-28b)，3.23(1H，m，H-15)，2.66(1H，m，H-4a)，2.48(1H，m，H-4b)，1.93(1H，m，H-16)，1.38(3H，d，J＝6.6Hz，H-32)，0.80(3H，d，J＝6.2Hz，H-31)。
13C?NMR(C
5D
5N)δ：189.5(d，C-25)，168.1(s，C-30)，163.7(s，C-22)，161.6(s，C-1)，159.0(s，C-5)，154.4(s，C-29)，144.3(s，C-9)，135.0(s，C-24)，131.3(s，C-8)，130.0(s，C-18)，128.4(d，C-7)，124.1(s，C-10)，71.3(d，C-19)，71.0(d，C-11)，68.9(d，C-23)，68.6(t，C-20)，67.6(t，C-27)，67.4(d，C-13)，66.8(t，C-3)，64.9(d，C-17)，53.5(s，C-12)，46.5(s，C-6)，37.4(d，C-14)，33.7(d，C-15)，30.8(t，C-16)，25.3(t，C-4)，24.8(t，C-28)，19.6(q，C-31)，18.8(q，C-32)。
X-ray crystalline diffraction data: oblique system, spacer are p2
1 
α=90, β=98.960 (3), γ=90,
Z=4, d=1.545g/cm
3, crystalline size 0.26 * 0.14 * 0.10nm
3, R
1=0.1069 (2391), wR
2=0.3491 (7831).
Swerilactone J (3)
Molecular formula: C
29H
28O
10
Molecular weight: 536.53
Proterties: colourless prismatic crystal
mp：266-267℃
Optically-active
(c=2.52mg cm
-3, CHCl
3/ MeOH=1: 1, v/v)
IR(KBr)v
max：3434，1718，1690，1636，1460，1407，1266，1220，1182，1129，1108，1036，1023，1003，948，881cm
-1。
UV/Vis(CHCl
3/MeOH＝1∶1，v/v)λ
max(ε)：281(18499)，211(11673)，193(11756)nm。
ESIMS(+)m/z：1095[2M
++Na]，559[M+Na]
+。
HRESIMS(+)m/z：found：559.1581[M
++Na]，calcd?for?559.1580(C
29H
28O
10Na，[M+Na]
+)。
1H?NMR(C
5D
5N)δ：6.12(1H，s，H-7)，5.99(1H，br?s，H-28)，0.98(1H，d，J＝6.1Hz，H-31)，5.80(1H，s，H-11)，5.68(1H，br?s，H-25)，5.23?(1H，d，J＝14.6Hz，H-20a)，5.13(1H，d，J＝14.6Hz，H-20b)，5.04(1H，dd，J＝14.3，1.9Hz，H-27a)，4.92(1H，dd，J＝16.4，2.0Hz，H-27b)，4.50(1H，br?s，H-13)，4.44(1H，m，H-23)，4.33(1H，m，H-3a)，4.21(1H，m，H-3b)，3.93(1H，q，J＝6.3Hz，H-19)，2.66(1H，dd，J＝13.0，3.4Hz，H-14)，2.54(1H，br?s，H-24)，2.51(1H，m，H-15)，2.38(1H，m，H-16a)，5.14(1H，m，H-17)，2.31(1H，m，H-4a)，2.25(1H，m，H-4b)，1.35(1H，d，J＝6.6Hz，H-32)，1.19(1H，dd，J＝13.0，5.0Hz，H-16b)。
13C?NMR(C
5D
5N)δ：166.9(s，C-30)，164.0(s，C-22)，161.1(s，C-1)，156.2(s，C-5)，145.4(s，C-9)，132.6(s，C-8)，131.0(s，C-18)，129.4(s，C-29)，127.1(d，C-7)，123.5(s，C-10)，118.4(d，C-28)，95.6(d，C-25)，76.5(d，C-19)，73.0(d，C-23)，69.7(t，C-27)，68.7(t，C-20)，68.1(d，C-13)，66.7(t，C-3)，65.8(d，C-11)，64.7(d，C-17)，49.2(d，C-24)，48.0(s，C-6)，44.9(s，C-12)，42.9(d，C-14)，36.4(d，C-15)，31.0(t，C-16)，27.1(t，C-4)，18.4(q，C-31)，17.1(q，C-32)。
X-ray crystalline diffraction data: triclinic(crystalline)system, spacer are p2
1 
α=70.965 (3), β=84.606 (3), γ=79.944 (3),
Z=2, d=1.442g/cm
3, crystalline size 0.18 * 0.16 * 0.08nm
3, R
1=0.0925, wR
2=0.2123.
Swerilactone K (4)
Molecular formula: C
29H
26O
9
Molecular weight: 518.51
Proterties: colourless prismatic crystal
mp：250-251℃
Optically-active
(c=1.35mg cm
-3, CHCl
3/ MeOH=1: 1, v/v)
IR(KBr)v
max：3456，2958，2927，1713，1683，1645，1596，1468，1405，1281，1131，1103，1072，942cm
-1。
UV/Vis(CHCl
3/MeOH＝1∶1，v/v)λ
max(ε)：275(6337)，263(6122)，256(6058)，226(5641)，223(5666)，212(5505)，206(5407)nm。
ESIMS(+)m/z：541[M+Na]
+。
HRESIMS (+) m/z: experimental value 541.1476, calculated value 541.1474 (C
29H
26O
9Na, [M+Na]
+).
1H?NMR(C
5D
5N)δ：8.20(1H，d，J＝7.6Hz，H-26)，7.88(1H，d，J＝7.6Hz，H-24)，7.26(1H，dd，J＝7.6，7.6Hz，H-25)，6.26(1H，s，H-7)，6.40(1H，br?s，H-11)，5.74(1H，d，J＝9.8Hz，H-13)，5.28(1H，d，J＝14.1Hz，H-20a)，5.17(1H，d，J＝14.1Hz，H-20b)，4.88(1H，d，J＝3.5Hz，H-17)，4.49(1H，m，H-3)，4.46(1H，m，H-30b)，4.26(1H，m，H-30a)，3.39(1H，q，J＝6.1Hz，H-19)，3.25(1H，m，H-29)，2.95(1H，dd，J＝11.9，9.8Hz，H-14)，2.59(1H，m，H-4a)，2.39(1H，m，H-4b)，2.27(1H，m，H-15)，1.91(1H，m，H-16a)，1.01(1H，d，J＝6.1Hz，H-31)，0.65(1H，dd，J＝12.9，5.2Hz，H-16b)。
13C?NMR(C
5D
5N)δ：165.0(s，C-32)，162.7(s，C-22)，160.8(s，C-1)，155.9(s，C-5)，146.0(s，C-9)，138.9(s，C-23)，138.6(s，C-28)，133.2(d，C-24)，131.5(s，C-18)，131.0(d，C-26)，131.0(s，C-8)，127.8(d，C-25)，127.6(d，C-7)，126.6(s，C-27)，125.1(s，C-10)，88.5(d，C-11)，76.5(d，C-19)，69.0(t，C-20)，66.8(t，C-3)，66.8(t，C-30)，66.6(d，C-13)，64.2(d，?C-17)，47.5(s，C-6)，27.1(t，C-4)，42.8(d，C-14)，38.7(d，C-15)，32.8(t，C-16)，24.8(t，C-29)，18.3(q，C-31)。
X-ray crystalline diffraction data: oblique system, spacer are p2
1 
α=90.00, β=106.921 (3), γ=90.00,
Z=4, d=1.447g/cm
3, crystalline size 0.23 * 0.15 * 0.10nm
3, R
1=0.0656 (1877), wR
2=0.2035 (5631).
Embodiment 2:
Method by embodiment 1 first makes swerilactone H-K (1-4), with after a small amount of DMSO dissolving, injects routinely water respectively, the essence filter, and injection liquid is made in the embedding sterilization.
Embodiment 3:
Method by embodiment 1 first makes swerilactone H-K (1-4), with after a small amount of DMSO dissolving, it is dissolved in sterile water for injection respectively, be stirred to dissolve, filter with aseptic suction funnel, more aseptic essence filter, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing gets powder injection.
Embodiment 4:
With separate of the swerilactone H-K (1-4) that obtains, be that the ratio of 9: 1 adds vehicle in itself and vehicle weight ratio respectively, make pulvis.
Embodiment 5:
Method by embodiment 1 first makes swerilactone H-K (1-4), is that the ratio of 5: 1 adds vehicle, pelletizing press sheet in itself and vehicle weight ratio respectively.
Embodiment 6:
Method by embodiment 1 first makes swerilactone H-K (1-4), and the oral liquid method for making is made oral liquid routinely respectively.
Embodiment 7:
Method by embodiment 1 first makes swerilactone H-K (1-4), is that the ratio of 5: 1 adds vehicle in itself and vehicle weight ratio respectively, makes capsule.
Embodiment 8:
Method by embodiment 1 first makes swerilactone H-K (1-4), is that the ratio of 3: 1 adds vehicle in itself and vehicle weight ratio respectively, makes capsule.
Claims (6)
1. the swerilactone H (1) shown in structural formula (I), I (2), J (3), K (4),
（I）。
2. pharmaceutical composition wherein contains the described formula of the claim 1 for the treatment of significant quantity (I) swerilactone H (1)-K (4) any and pharmaceutically acceptable carrier.
3. any application in the medicine of preparation treatment hepatitis B of the described formula of claim 1 (I) swerilactone H (1)-K (4).
4. the application of pharmaceutical composition claimed in claim 2 in the medicine of preparation treatment hepatitis B.
5. prepare the method for the described formula of claim 1 (I) swerilactone H (1)-K (4), it is characterized in that getting the Mile Swertia Herb herb, drying, pulverize, use successively 90% and 50% alcohol reflux 3 times, each 2-3 hour, merge ethanol extract, filter, be evaporated to without the alcohol flavor; This extracting solution is suspended in the aqueous solution, use successively sherwood oil, ethyl acetate and n-butanol extraction, ethyl acetate extraction part is adsorbed on silica gel with the chloroform-methanol dissolving, room temperature is placed and is volatilized solvent, grind, through silica gel column chromatography, with the chloroform/methanol wash-out of 100: 1 → 80: 20, obtain ten parts; Wherein first part continues to use silica gel column chromatography, chloroform/acetone gradient elution with 95: 5 → 80: 20, then further pass through silica gel column chromatography, with the chloroform/methanol wash-out of 95: 5, and obtain compound swerilactone H (1)-K (4) through 1:1 chloroform/methanol recrystallization.
6. the method for preparing pharmaceutical composition claimed in claim 2, compound swerilactone H (1)-K (4) adds respectively pharmaceutically acceptable carrier or vehicle.
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| CN104497004A (en) * | 2014-12-30 | 2015-04-08 | 云南中医学院 | Method for preparing swertia lactone I-K |
| CN104478893B (en) * | 2014-12-30 | 2017-04-05 | 云南中医学院 | A kind of Herba Swertiae bimaculatae lactone compound and preparation method thereof, preparation and application |
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| CN1709412A (en) * | 2005-06-10 | 2005-12-21 | 北京正大绿洲医药科技有限公司 | Liver-benefiting dropping pill prepared from total neterosides of swertia mileensis, and its preparing method |
| WO2010008521A1 (en) * | 2008-07-14 | 2010-01-21 | Cropsolution, Inc. | Modulators of acetyl-coenzyme a carboxylase and methods of use thereof |
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| CN1709412A (en) * | 2005-06-10 | 2005-12-21 | 北京正大绿洲医药科技有限公司 | Liver-benefiting dropping pill prepared from total neterosides of swertia mileensis, and its preparing method |
| WO2010008521A1 (en) * | 2008-07-14 | 2010-01-21 | Cropsolution, Inc. | Modulators of acetyl-coenzyme a carboxylase and methods of use thereof |
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| Title |
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| JP平1-230593A 1989.09.14 |
| 獐牙菜属药用植物的研究及应用概况;陈家春等;《中西医结合肝病杂志》;19981231;第8卷;第223-226页 * |
| 陈家春等.獐牙菜属药用植物的研究及应用概况.《中西医结合肝病杂志》.1998,第8卷第223-226页. |
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