CN104650024B - The purposes of biological species mouth mountain ketone monomeric compound anti-hepatic fibrosis and hepatic sclerosis - Google Patents

The purposes of biological species mouth mountain ketone monomeric compound anti-hepatic fibrosis and hepatic sclerosis Download PDF

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CN104650024B
CN104650024B CN201410040042.8A CN201410040042A CN104650024B CN 104650024 B CN104650024 B CN 104650024B CN 201410040042 A CN201410040042 A CN 201410040042A CN 104650024 B CN104650024 B CN 104650024B
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hepatic
liver
compound
mountain ketone
zyc
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CN104650024A (en
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蒲小平
郑希元
杨莹帆
赵欣
孙懿
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Yi Yueneng
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Peking University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
    • C07D311/86—Oxygen atoms, e.g. xanthones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/78—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
    • C07C49/753—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups
    • C07C49/755—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups a keto group being part of a condensed ring system with two or three rings, at least one ring being a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms

Abstract

The invention discloses the purposes of biological species mouth mountain ketone monomeric compound anti-hepatic fibrosis and hepatic sclerosis.The present invention discloses the 6 kinds of biological species mouth mountain ketone monomeric compounds for separating and obtaining from Swertia punicea Hemsl first.The present invention further discloses the separation method of 6 kinds of biological species mouth mountain ketone monomeric compounds.Vitro Experimental Results show that 3,4,8 trimethoxy mouth mountain one monomers compound of separated 1,7 dihydroxy has anti-CCl4The hepatocellular injury pharmacological activity of induction；Results of animal shows, 1,7 dihydroxy 3,4,8 trimethoxy mouth mountain ketone have exact therapeutic effect for the hepatic fibrosis rats that DMN is induced, degeneration of liver cells, necrosis, hepatic entorrhagia, liver Diffuse fibrous hyperblastosis degree for Hepatic Fibrosis etc. are significantly improved, and show that the compound has application potential on prevention or the clinical medicine for the treatment of liver fibrosis or hepatic sclerosis is prepared.

Description

The purposes of biological species mouth mountain ketone monomeric compound anti-hepatic fibrosis and hepatic sclerosis
Technical field
The present invention relates to the new pharmacological use of biological mouth xanthones compounds, more particularly to from Swertia punicea Hemsl（Swertia punicea Hemsl.）The mouth mountain ketone monomeric compound and its preventing the new drug of liver fibrosis or hepatic sclerosis that middle separation obtains Purposes is managed, belongs to separation and its application field of biological mouth mountain ketone monomeric compound.
Background technology
Liver fibrosis is that the common etiology of chronic liver disease changes, and is that a kind of compensatory repair is reacted, and various Chronic Livers Only stage which must be passed by of the dirty disease to cirrhosis progress.Liver fiber can occur for the chronic liver disease overwhelming majority caused by the various causes of disease Change, wherein 25%~40% finally develops into hepatic sclerosis even liver cancer.In China, the incidence of virus hepatitis is very high, hepatitis The incidence of hepatic sclerosis is also in rising trend afterwards, seriously endangers people's health.
Liver fibrosis refers to caused by various virulence factors connective tissue paraplasm in liver, causes diffusivity cell in liver The pathologic process of epimatrix excessive deposition.The lighter is known as liver fibrosis, and severe one reconstructs lobuli hepatis structure, pseudolobuli and tubercle, As hepatic sclerosis.Hepatic stellate cells（hepatic stellate cell,HSC）Leading work is played in liver fibrosis occurrence and development With in the liver perisinusoidal space (Disse gaps), its form is irregular, and cell space is rounded or irregular shape, often stretches out several stars Shape cell, which is dashed forward, surrounds hepatic sinusoid.Under the effect of various pathogenic factors, resting stage HSC is activated and breeds, is converted into flesh into fibre Tie up sample mother cell, synthesis and extracellular matrix secretion（extracellular matrix,ECM）Up-regulation, simultaneously synthesizing and release A large amount of matrix metalloproteinase inhibitors（tissue inhibitor of metalloproteinase,TIMPs）, between making The proteinase activities such as matter clostridiopetidase A reduce, and ECM degradeds are reduced, and ultimately result in ECM accumulations and liver fibrosis or even hepatic sclerosis occurs.
ROS plays a significant role into final conclusion with lipid peroxidation in hepatocellular injury.ROS can be acted on directly or indirectly In liver cell, change the structure of its cell membrane and subcellular organelle make its denaturation, necrosis cause hepatocellular injury, necrosis, apoptosis and Liver tissues inflammatory is reacted, and produces substantial amounts of inflammatory mediator and cell factor, further activates Kupffer cells；Kupffer cells The cell factor of secretion, inflammatory mediator are remake for liver cell, further aggravate its damage.Oxidativestress damage is as important machine One of system, participates in the deposition of collagen component in HSC activation and liver, there is close relationship between liver fibrosis.
Recently research have indicated that liver fibrosis is a kind of reversible pathologic stage, as long as being carried out rationally in liver fibrosis stage Treatment, can reverse the process of liver fibrosis, so as to achieve the purpose that to alleviate or even cure liver fibrosis.In basic research side Face, occurrence and development and correlative factor in relation to liver fibrosis wait to illustrate；In clinicing aspect, liver fibrosis and hepatic sclerosis are treated Medicine is less, and the medicine that can reach satisfactory curative effect is then less, therefore tries to explore liver fibrosis pathogenesis, seeks Effective remedy measures and preferable clinical treatment medicine are of great significance to reducing pathogenesis of cirrhosis rate and the death rate.
The content of the invention
An object of the present invention is to provide from Swertia punicea Hemsl（Swertia punicea Hemsl.）In it is isolated Mouth mountain ketone monomeric compound；
The second object of the present invention is to provide a kind of from Swertia punicea Hemsl（Swertia punicea Hemsl.）Middle separation The method of the mouth mountain ketone monomeric compound；
The third object of the present invention is that the mouth mountain ketone monomeric compound is hard applied to prevention liver fibrosis or liver Change.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
The present invention is from Swertia punicea Hemsl（Swertia punicea Hemsl.）Middle separation obtains 6 kinds of biological species mouth mountain ketones Monomeric compound, the biological species mouth mountain ketone monomeric compound are selected from：- 3,4 methoxyl group mouth mountain ketone of 1,7- hydroxyls（ZYC- 1）, 1,5- dihydroxy -3- methoxyl group mouths mountain ketone（ZYC-2）, 1- hydroxy-3-methoxy -8- primrose glycosyl mouths mountain ketone（ZYC-3）、 Swertianolin（ZYC-4）, 7-O- [α-L- rhamnopyranosyls-(1 → 2)-β-D- xylopyranosyls] -1,8- dihydroxy -3- first Epoxide mouth mountain ketone（ZYC-5）, 1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone（ZYC-6）.
Present invention additionally comprises corresponding all pharmaceutically acceptable salt, hydrate or the prodrugs of above-claimed cpd, it is It is applied to prepare in the medicine for preventing liver fibrosis and hepatic sclerosis as unique bulk pharmaceutical chemicals.
The salt of the sour addition of biological species mouth xanthones compounds is also included in the present invention in the present invention.
The salt of the sour addition of the biological species mouth xanthones compounds is preferably the pharmaceutically appropriate acid of acceptable（Example Such as hydrochloric acid, acetic acid, sulfuric acid）Nontoxic salt is formed, in addition to pharmaceutically acceptable salt, other salt are also included within the present invention Among.
Present invention also offers the side that a kind of separation from Swertia punicea Hemsl obtains 6 kinds of mouth mountains ketone monomeric compound Method, this method comprise the following steps：
（1）Extracted and concentrated with ethanol after Swertia punicea Hemsl is crushed, obtain the ethanol extract of Swertia punicea Hemsl；（2）Will After the ethanol extract of Swertia punicea Hemsl adds distilled water to complete suspend, successively with petroleum ether, ethyl acetate, water-saturated n-butanol Extraction；（3）Solvent is recovered under reduced pressure in extract, respectively each extraction position；（4）N-butanol portion is carried out using column chromatography Separation, respectively obtains 6 kinds of mouth mountain ketone monomeric compounds.
Wherein, step（4）Described in column chromatography include：Polyamide column chromatography, HP-20 column chromatographys, Sephadex-LH20 Gel column chromatography and Semi-PHPLC etc..
It is further preferred that step（4）It is middle that 6 kinds of mouth mountain ketone lists are respectively obtained from extraction position using following separation method Body compound：N-butanol portion is taken to be separated through HP-20 resins, alcohol-water gradient elution（5:95→100:0）, obtain F1, F2, Tetra- elution positions of F3 and F4；F3 positions are separated using polyamide, alcohol-water gradient elution（25:75→95:5）, obtain Five elution positions of F3A, F3B, F3C, F3D and F3E；F3B is purified using Sephadex LH-20, and methanol elution, prepares through half Efficient liquid phase separates（Mobile phase：Acetonitrile/water=20:80）, obtain compound 1- hydroxy-3-methoxy -8- primrose glycosyl mouths mountain ketone （ZYC-3）And swertianolin（ZYC-4）；F4 positions are purified using Sephadex LH-20, and methanol elution, produces 52 cuts； Wherein cut 17-21 prepares efficient liquid phase separation through half（Mobile phase：Methanol/water=60:40）, obtain 7-O- [α-L- rhamnopyranosyloxyhies Glycosyl-(1 → 2)-β-D- xylopyranosyls] -1,8- dihydroxy -3- methoxyl group mouths mountain ketone（ZYC-5）With 1,7- dihydroxy -3,4, 8- trimethoxy mouths mountain ketone（ZYC-6）；Cut 17-21 prepares efficient liquid phase separation through half（Mobile phase：Acetonitrile/water=30:70）, Obtain -3,4 methoxyl group mouth mountain ketone of compound 1,7- hydroxyls（ZYC-1）, 1,5- dihydroxy -3- methoxyl group mouths mountain ketone（ZYC-2）.
Use CCl450mM acts on HepG2 cell 4h, can cause significant hepatocellular injury, show in cells and supernatant AST and LDH contents significantly raise；The present invention is tested by pharmacology pharmacodynamic and found, 1,7- dihydroxy -3,4,8- trimethoxy mouths Mountain ketone（ZYC-6）Preact HepG2Cell 24h, can significantly reduce CCl4Inducing hepatocyte damages the content of AST and LDH, with Model group more has significant difference（Respectively*P<0.05 He**P<0.01）, and curative effect is bicyclic better than positive control drug Alcohol；And ZYC-1~ZYC-5 this 5 compound preact HepG2Cell 24h, the content and mould of AST and LDH in culture supernatant Difference that type group compares that there are no significant；Test result indicates that separated other 5 kinds of mouth mountains ketone singulation from Swertia punicea Hemsl Compound does not possess anti-CCl4The hepatocellular injury pharmacological activity of induction, only 1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone With anti-CCl4The hepatocellular injury pharmacological activity of induction, shows that it possesses the medicine potentiality for preparing liver protecting.
N-nitrosodimethylamine (Dimethylvinphos, DMN) intraperitoneal injection induced rat liver fibrosis is classical way, The liver fibrosis that the method modeling is formed is stablized relatively, and the modeling time is short, and pathological development shows as hepatic injury, liver fibrosis, liver Hardening, liver reduce, spleen increase；Change to mankind's hepatic sclerosis early stage and Collagen fiber deposition is similar, can be used as and screen anti-liver The animal model of fibrosis medicine.The present invention is tested by substantial amounts of pharmacology pharmacodynamic and found, 1,7- dihydroxy -3,4,8- front threes Epoxide mouth mountain ketone has obvious therapeutic effect for the hepatic fibrosis rats that DMN is induced, show can effectively adjust weight, Liver body ratio, spleen body ratio and liver and spleen ratio, significantly reduce glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST) and total bile acid (TBIL) Level, significantly improves albumin (ALB) level, and it is horizontal while significantly reduce the third two to significantly improve superoxide dismutase (SOD) Aldehyde (MDA) is horizontal, makes degeneration of liver cells, the necrosis of rat liver, hepatic entorrhagia, liver Diffuse fibrous hyperblastosis degree subtracts Gently there is different degrees of improvement.
Therefore, pharmacodynamic experiment of the invention the result shows that, 1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone has anti- The liver fibrosis pharmacological activity of DMN inductions, has and is prepared into prevention liver fibrosis and the medicinal application of hepatic sclerosis in the latent of clinic Power.
Correspondingly, invention further provides a kind of pharmaceutical composition for preventing liver fibrosis or hepatic sclerosis, the medicine Compositions include prevention or the upper a effective amount of 1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone for the treatment of and pharmaceutically acceptable Carrier；By a effective amount of 1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone and pharmaceutically acceptable carrier or diluent After cooperation, any one suitable pharmaceutical composition is prepared into by the formulation method of this area routine；The usual medicine group Compound is suitable for being administered orally and drug administration by injection, also is adapted for other medications；The pharmaceutical composition can be tablet, capsule The liquid forms such as agent, pulvis, granule, lozenge, suppository or oral liquid.According to different medications, medicine of the present invention Composition can contain the biological species mouth mountain ketone monomeric compound 1 of 0.1%-99% weight, preferably 10-60% weight, 7- dihydroxies Base -3,4,8- trimethoxy mouths mountain ketone.
Brief description of the drawings
Fig. 1 biological species mouths mountain one monomers compound ZYC-1~ZYC-6 separation process figures.
Fig. 2 biological species mouths mountain one monomers compound 1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone (1,7- Dihydroxy-3,4,8-trimethoxyxanthone, ZYC-6) chemical structural formula.
Fig. 3 rat models liver tissues inflammatory changes（Hematoxylin eosin staining, i.e. H＆E dye）；A：Normal rats；B： Model group rats；C：ZYC-6 group rats；D：Positive drug group rat.
Fig. 4 rat model hepatic tissue Collagen fiber deposition dynamic changes（Sirius red stains, i.e. Sirius Red dye）； A：Normal rats；B：Model group rats；C：ZYC-6 group rats；D：Positive drug group rat.
Fig. 5 rat model hepatic tissue Collagen fiber deposition dynamic changes（Horse pine dyeing, i.e. Masson dyeing）；A：Normal group Rat；B：Model group rats；C：ZYC-6 group rats；D：Positive drug group rat.
Embodiment
The present invention is further described with reference to specific embodiment, the advantages and features of the present invention will be with describing It is and apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.Art technology Personnel should be understood that without departing from the spirit and scope of the invention can be to the details and form of technical solution of the present invention Modify or replace, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment 1 separates biological mouth xanthones compounds and its identification from Swertia punicea Hemsl
1st, reagent and medicine
Acetonitrile（HPLC grade, Merck KGaA, Germany/HPLC grade, Fisher, Germany）；H3PO4 （HPLC grade, TEDIA Co., Inc., USA）；Methanol（HPLC grade, Merck KGaA, Germany/HPLC Grade, Fisher, Germany）；Pure water（Wahaha Pure Water Co., Ltd, Hangzhou）；Acetonitrile, methanol（Chromatographically pure, Tianjin City Xihua special reagent factory；Tianjin great Mao chemical reagent factories；Beijing Yili Fine Chemicals Co., Ltd.）；Chloroform, methanol, Ethyl acetate, ethanol（Analyze pure, Beijing Chemical Plant）.
Thin-layer chromatography：Silica GF254, Haiyang Chemical Plant, Qingdao；Polyamide column chromatography, Jiangsu Changfeng Chemical Co., Ltd.； HP-20 resins, mitsubishi chemical industry company produce；Sephadex LH-20, GE Healthcare Products.
2nd, instrument
Agilent1100/1200HPLC systems, equipped with binary pump, autosampler, column oven, DAD detectors （Agilent Technologies, USA）.Data acquisition and issuance uses Agilent Chemstation9.01.
Half prepares HPLC：Alltech426 binary pumps, Alltech UVIS2000 UV detector, Agilent Zorbax SB C18 chromatographic columns (250 × 9mm, 5 μm)；Nuclear Magnetic Resonance：Bruker DRX400/500 nuclear magnetic resonance chemical analysers；Mass spectrograph： Bruker APEX IV FT mass spectrographs（HRESI-MS）, ABI Q-STAR LC-MS instrument（ESI-MS）.
3rd, medicinal material
Swertia punicea Hemsl：The drying herb of gentianaceae plant Swertia punicea Hemsl Swertia punicea Hemsl., the place of production Dali, numbering ZYC001 are identified that medicinal material sample deposits in Peking University's pharmacy by College of Pharmacy, Beijing Univ teacher Liu Guangxue Natural pharmacology system of institute Specimen Room.
4th, extract with separating
The Swertia punicea Hemsl medicinal material collected is further dried, is weighed, amounts to 10kg medicinal materials, it is thick all to crush Powder, the medicinal material after crushing is extracted with ethanol to be concentrated, and obtains the ethanol extract of Swertia punicea Hemsl.By ethanol extract plus distilled water extremely After suspending completely, extracted successively with petroleum ether, ethyl acetate, water-saturated n-butanol.Solvent is recovered under reduced pressure in extract, obtains each extraction Position.Take n-butanol portion（300g）Separated through HP-20 resins, alcohol-water gradient elution（5:95→100:0）, obtain four Elute position（F1-F4）.F3 positions are separated using polyamide, alcohol-water gradient elution（25:75→95:5）, obtain five A elution position（F3A-F3E）.F3B is purified using Sephadex LH-20, methanol elution, and efficient liquid phase separation is prepared through half （Mobile phase：Acetonitrile/water=20：80）, obtain compound 3 and 4.F4 positions are purified using Sephadex LH-20, methanol elution, production Raw 52 cuts.Wherein cut 17-21 prepares efficient liquid phase separation through half（Mobile phase：Methanol/water=60:40）, obtain compound 5, 6.Cut 17-21 prepares efficient liquid phase separation through half（Mobile phase：Acetonitrile/water=30:70）, obtain compound 1 and 2.Specifically separated Journey is shown in Fig. 1.
5th, Structural Identification
From the isolated 6 mouth mountains ketone monomeric compound of n-butanol portion, wherein compound 1, compound 2, compound 3 With compound 5 for first from Swertia plant it is isolated, 6 compound structure appraising datums are as follows：
（1）Compound 1（ZYC-1）：- 3,4 methoxyl group mouth mountain ketone (1,7-dihydroxy-3,4- of 1,7- hydroxyls Dimethoxy-xanthone identification)
Pale yellow powder, dissolves in methanol, acetonitrile.ESI-MS m/z(%)：287.1[M-H]-(100)；1H-NMR (400MHz,DMSO-d6)δppm：6.59(1H,H-4),7.57(1H,d,8.8Hz,H-5),7.32(1H,dd,2.8Hz, 8.8Hz,H-6),7.43(1H,d,2.8Hz,H-8),3.94(3H,OCH3),3.80(3H,OCH3),12.77(1H,OH-1), 10.05(1H,OH-7)；13C-NMR(100MHz,DMSO-d6)δppm：158.07(C-1),94.76(C-2),159.62(C-3), 128.08(C-4),148.83(C-4a),149.14(C-4b),120.12(C-5),124.80(C-6),154.02(C-7), 107.97(C-8),119.25(C-8a),180.33(C-9),102.21(C-9a),60.90(OCH3),56.53(OCH3)
（2）Compound 2（ZYC-2）：1,5- dihydroxy -3- methoxyl group mouths mountain ketone (1,5-dihydroxy-3-methoxy- Xanthone identification)
Yellow needles, dissolve in methanol, acetonitrile.ESI-MS m/z(%)：257.1[M-H]-(100)；1H-NMR (400MHz,DMSO-d6)δppm：6.41(1H,H-2),6.65(1H,H-4),7.34(1H,m,H-6),7.27(1H,m,H-7), 7.57(1H,m,H-8),3.90(3H,OCH3),12.88(1H,OH-1),10.56(1H,OH-5)；13C-NMR(100MHz, DMSO-d6)δppm：162.56(C-1),97.14(C-2),166.55(C-3),92.76(C-4),157.18(C-4a), 144.97(C-4b),146.30(C-5),120.73(C-6),124.32(C-7),114.51(C-8),120.95(C-8a), 180.53(C-9),103.04(C-9a),56.20(OCH3)。
（3）Compound 3（ZYC-3）：1- hydroxy-3-methoxy -7- primrose glycosyl mouths mountain ketone (1-hydroxy-3- Methoxy-7-O-primeverosylxanthone identification)
Colorless needles, dissolve in methanol, acetonitrile.ESI-MS m/z(%)：551.1[M-H]-(100)；1H-NMR(400MHz, DMSO-d6)δppm：7.67(1H,d,J=2.4Hz,H-8),7.65(1H,dd,J=2.4Hz,10.0Hz,H-6),7.61(1H,d, J=10.0Hz,H-5),6.64(1H,d,J=2Hz,H-4),6.41((1H,d,J=2Hz,H-2),4.91(1H,glu-1H),4.17 (1H,rha-1H),3.89(3H,OCH3),12.77(1H,OH-1)；13C-NMR(100MHz,DMSO-d6)δppm：162.96(C- 1),97.50(C-2),167.05(C-3),93.18(C-4),157.82(C-4a),151.24(C-4b),119.75(C-5), 126.10(C-6),154.32(C-7),111.06(C-8),120.75(C-8a),180.36(C-9),103.31(C-9a), 56.66(OCH3),97.50(C-1’),73.82(C-2’),76.31(C-3’),70.01(C-4’),76.96(C-5’),68.75 (C-6’),103.31(C-1”),73.67(C-2”),76.68(C-3”),70.01(C-4”),66.08(C-5”)。
（4）Compound 4（ZYC-4）：The identification of swertianolin (swertianolin)
Yellow amorphous powder, dissolves in methanol, acetonitrile.ESI-MS m/z(%)：435.2[M-H]-(100)；SDS1H- NMR(400MHz,DMSO-d6)δppm：6.38(1H,d,2Hz,H-2),6.58(1H,d,2Hz,H-5),7.26(1H,d, 8.8Hz, H-6), 7.12 (1H, d, 8.8Hz, H-7), 4.81 (1H, d, J=8Hz, 1 '-H), 3.15-3.74 (m, sugar on proton), 3.89(3H,OCH3),13.10(1H,OH-1),10.07(1H,OH-5)；13C-NMR(100MHz,DMSO-d6)δppm：162.73 (C-1),97.21(C-2),166.29(C-3),92.21(C-4),156.43(C-4a),145.03(C-4b),140.98(C- 5),121.10(C-6),112.31(C-7),149.44(C-8),111.90(C-8a),181.08(C-9),103.07(C-9a), 61.01(OCH3),103.551(C-1’),73.52(C-2’),76.09(C-3’),69.72(C-4’),77.44(C-5’), 60.82(C-6’),56.12(OCH3)。
（5）Compound 5（ZYC-5）：7-O- [α-L- rhamnopyranosyls-(1 → 2)-β-D- xylopyranosyls] -1,8- two Hydroxy-3-methoxy mouth mountain ketone (7-O- [α-L-rhamnopyranosyl- (1 → 2)-β-D-xylopyranosyl] -1,8- Dihydroxy-3-methoxy-xanthone identification)
Yellow powder, dissolves in methanol, acetonitrile.ESI-MS m/z(%)：551.1[M-H]-(100)；1H-NMR(400MHz, DMSO-d6)δppm：7.61(1H,d,J=9.0Hz,H-6),7.02(1H,d,J=9.0Hz,H-5),6.66(1H,brs,H-4), 6.45(1H,brs,H-2),5.22(1H,xyl-1H),5.15(1H,rha-1H),3.89(3H,OCH3),11.85(2H,OH-1, OH-8)；13C-NMR(100MHz,DMSO-d6)δppm：161.82(C-1),97.40(C-2),167.15(C-3),92.92(C- 4),157.58(C-4a),150.25(C-4b),105.79(C-5),126.19(C-6),139.09(C-7),150.25(C-8), 107.47(C-8a),184.12(C-9),101.80(C-9a),56.25(OCH3),100.43(C-1’),77.16(C-2’), 76.34(C-3’),70.50(C-4’),65.58(C-5’),99.80(C-1”),69.52(C-2”),70.41(C-3”),71.91 (C-4”),68.40(C-5”),17.78(C-6”),56.12(3-OCH3)。
（6）Compound 6（ZYC-6）：1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone (1,7-dihydroxy-3,4, Identification 8-trimethoxyxanthone)
Yellow powder, dissolves in methanol, acetonitrile.ESI-MS m/z(%)：317.2[M-H]-(100)；1H-NMR(400MHz, DMSO-d6)δppm：6.52(1H,H-2),7.28(1H,d,8.8Hz,H-5),7.38(1H,d,8.8Hz,H-6),3.92(3H, OCH3),3.81(3H,OCH3),3.77(3H,OCH3),13.17(1H,OH-1),9.68(1H,OH-7)；13C-NMR(100MHz, DMSO-d6)δppm：158.38(C-1),94.66(C-2),159.25(C-3),127.52(C-4),148.25(C-4a), 149.37(C-4b),113.41(C-5),124.50(C-6),147.10(C-7),145.34(C-8),114.64(C-8a), 180.80(C-9),102.68(C-9a),61.01(OCH3),60.93(OCH3),56.46(OCH3)。
Test example 1 separated mouth mountain one monomers Compound ira vitro toxicity and CCl from Swertia punicea Hemsl4External model liver protection Active evaluation test
First, test material
1st, test compound：In embodiment 1 from Swertia punicea Hemsl separated 6 kinds of mouth mountains ketone monomeric compound：1,7- The methoxyl group mouth of hydroxyl -3,4 mountain ketone（ZYC-1）, 1,5- dihydroxy -3- methoxyl group mouths mountain ketone（ZYC-2）, 1- hydroxy-3-methoxies- 7- primrose glycosyl mouths mountain ketone（ZYC-3）, swertianolin（ZYC-4）, 7-O- [α-L- rhamnopyranosyls-(1 → 2)-β-D- pyrans Xylosyl] -1,8- dihydroxy -3- methoxyl group mouths mountain ketone（ZYC-5）With 1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone （ZYC-6）；Compound is the sterile 6mM storing solutions prepared.
2nd, positive control drug：Bicyclic alcohols, the present of consonance pharmaceutical factory, DMSO are prepared, 4 DEG C of preservations.
3rd, cell line：Human liver cancer HepG2Cell, in the DMEM nutrient solutions containing 10% hyclone（100U/ containing penicillin Ml, 100 μ g/ml of streptomysin）Middle growth, condition of culture are 37 DEG C, 5%CO2, saturated humidity.With containing 0.25% trypsase and 0.02%EDTA liquid had digestive transfer cultures.
2nd, test method
（1）Mouth mountain one monomers Compound ira vitro toxicity assessment
Using MTT methods：HepG2Cell inoculation is in 96 porocyte culture plates, after cultivating 24h, adds 10 μM of final concentration Test compound, while solvent control group is set, each drug concentration sets 3 parallel holes.After drug effect cell 48h, training is discarded Nutrient solution, 100 μ l of MTT (0.5mg/ml) liquid are added per hole, are continued to cultivate 4h, are discarded MTT liquid, DMSO150 μ l are added per hole, mix Oscillator vibrates, and absorbance is measured at microplate reader 570nm wavelength.
Cell survival rate（%）=（Cell OD average values/solvent control cell OD average values are administered）×100%.
（2）Mouth mountain one monomers compound is to CCl4Cause the Protective Effect of in vitro liver cell damage
With glutamic-oxalacetic transaminease（AST）, lactic dehydrogenase（LDH）Content is evaluation index.HepG2Cell inoculation is thin in 96 holes Merged in born of the same parents' culture plate to cell length to 60-70%, add non-toxic concn testing compound, positive control drug and solvent, effect is thin Born of the same parents 24h, adds damage agent CCl4（50 μM of final concentration）, then collective effect cell 4h, 150 μ L of cells and supernatant are taken, are centrifuged, entirely Automatic biochemistry analyzer（Toshiba）Detect AST, LDH contents.###P ＜ 0.001, compared with blank control group；* P ＜ 0.05, * * P ＜ 0.01, with CCl4Model group compares.
3rd, result of the test
1st, 6 test compounds act on HepG under 10 μM of concentration2Cell 48h, the equal unrestraint effect of cell growth（In detail Carefully it the results are shown in Table 1）, therefore select 10 μM of concentration to be used for follow-up study.
Separated mouth mountain one monomers Compound ira vitro toxicity assessment result of the test in 1 Swertia punicea Hemsl of table
2、CCl450mM acts on HepG2 cell 4h, causes significant hepatocellular injury, shows in cells and supernatant AST and LDH contents significantly raise（Respectively##P<0.01 He###P<0.001）.Positive control drug bicyclic alcohols are to CCl4Caused liver Cellular damage significantly inhibits effect, can effectively reduce the content of AST and LDH in culture supernatant（It is*P<0.05）.ZYC-6 Preact HepG2 cell 24h, can significantly reduce the content of AST and LDH in culture supernatant, more have with model group significantly Sex differernce（Respectively*P<0.05 He**P<0.01）, and curative effect is better than positive control drug bicyclic alcohols（Bicyclol）.And ZYC-1 This 5 compound preact HepG2 cell 24h of~ZYC-5, in culture supernatant AST and LDH with model group relatively without significantly Sex differernce（Table 2）.
Separated mouth mountain one monomers compound is to CCl in 2 Swertia punicea Hemsl of table4（50mM）Cause in vitro liver cell damage Protective effect result of the test
Measurement data is represented with means ± SD.Compared with normal group of blank, statistical significant difference is：###P ＜ 0.001,##P ＜ 0.01.With CCl4Model group compares, and statistical significant difference is：**P ＜ 0.01,*P ＜ 0.05.
The liver fiber of the test example 21,7- dihydroxy -3,4,8- trimethoxy mouths anti-N-nitrosodimethylamines of mountain ketone (DMN) induction Change rat test
First, test material
1st, test compound：1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone（ZYC-6）, through HPLC authenticating compounds Purity ＞ 98%.
2nd, positive control drug：Recombinant human interferon alpha 2 b parenteral solution (IFN-α 2b), has purchased from Tianjin Holley up to bioengineering Limit company.
3rd, experimental animal：Wistar male rats 34, SPF grades, weight 135-165g, purchased from the experiment of Beijing dimension tonneau China Zoo technical Co., Ltd, credit number：SCXK（Capital）2012-0001, Beijing's Quality of Experimental Animals quality certification number： 11400700026788.All rat feedings are in Department Of Medicine, Peking University's Experimental Animal Center, full diet, free water.
4th, experiment reagent：N-nitrosodimethylamine（dimethylnitrosamine,DMN）Purchased from Sigma companies；Serum liver Functional reagent box, including glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST), albumin (ALB) and total bilirubin (TBIL) are equal Bioengineering Research Institute is built up purchased from Nanjing.
2nd, test method
1st, prepared by animal model：Solution with normal saline dilution DMN into concentration for 0.5 (volume ratio), by 2ml/kg (i.e. 10 μ g/kg) dosage row rats by intraperitoneal injection, one time a day, weekly for three days on end, totally 4 weeks, the 1st week injection 2/3 amount（7μg/ kg）.
2nd, animal packet and treatment
Wistar rats 34, are divided into 4 groups according to random digits table.10 rats of Normal group, use physiological saline It is injected intraperitoneally one time a day with the volume of 2ml/k g rat weights, weekly for three days on end, totally 4 weeks, the 1st week 2/3 amount of injection （7μg/kg）.10 rats of positive drug IFN-α 2b treatment groups, with positive drug IFN-α 2b solution according to 8 since modeling the 3rd week ×105IU/kg rat body weights carry out intramuscular injection, 1 time a day, continuous 6 days weekly, totally 4 weeks.
10 rats of 4 rats of ZYC-6 treatment groups and model group, are equally respectively with concentration the 3rd week since modeling The ZYC-6 solution and distilled water of 0.5mg/ml carries out gavage according to 10ml/kg rat body weights, 1 time a day, continuous 6 days weekly, and altogether 4 weeks.
3rd, Indexs measure
After treatment end, 2ml/kg dosage intraperitoneal injection of anesthesia rats, the blood sampling of rat eye socket are pressed with 2% yellow Jackets.Adopt The whole blood of collection be statically placed in 4 DEG C of refrigerators 2 it is small when, 4000rpm centrifugation 10min, collect serum, carry out serum liver function, SOD in serum with And MDA detections；After disconnected neck is put to death, abdominal cavity is opened, wins liver；1.0cm × 0.8cm × 0.3cm size hepatic tissues are taken, with 10% Formalin is fixed, and dehydration of alcohol step by step after 24h, dimethylbenzene is transparent, 60 DEG C of paraffin embedding, for common pathology.Hepatic tissue passes through After 40g/L formalins are fixed, paraffin embedding, 5 μm of slice thick, makes H＆E dyeing, Sirius Red dyeing and Masson dyeing Pathological section, in light microscope（H＆E and Masson dyeing, Bar=200 μM）And petrographic microscope（Sirius Red are dyed； Bar=50μM）Lower observation liver tissues inflammatory and fibrosis.
4th, statistical analysis
Statistical analysis is carried out using SPSS19.0 software kits.Measurement data is represented with means ± SD, using variance analysis Tournament method.Compared with normal group, statistical significant difference is：###P ＜ 0.001,##P ＜ 0.01,#P ＜ 0.05. Compared with model group, statistical significant difference is：***P ＜ 0.001,**P ＜ 0.01,*P ＜ 0.05.
3rd, result of the test
1st, general animal sign
Compared with normal group, weight and liver and spleen ratio conspicuousness decline model group（Respectively P<0.05 and P<0.001）, spleen Body rises than conspicuousness（P<0.001）；Liver body declines than, but no difference of science of statistics.After positive drug is treated, weight and liver Spleen rises than conspicuousness（It is P<0.05）, spleen body is than conspicuousness decline（P<0.05）；Liver body declines than, but without statistics Difference.After medicine ZYC-6 treatments, weight and liver and spleen ratio conspicuousness rise（It is P<0.05）, spleen body is than conspicuousness decline（P< 0.001）；Liver body declines than, but no difference of science of statistics.Detailed results are shown in Table 3.
The change of 3 rat body weight of table and liver and spleen weight
Measurement data is represented with means ± SD.Compared with normal group, statistical significant difference is：###P ＜ 0.001,#P ＜ 0.05.Compared with model group, statistical significant difference is：***P ＜ 0.001,*P ＜ 0.05.
2nd, serum liver function and oxidative stress index
Compared with normal group, Serum ALT, AST and the horizontal conspicuousnesses of TBIL rise model group（Respectively P<0.001,P< 0.05 and P<0.001）, the horizontal conspicuousness declines of serum ALB（P<0.001）.After positive drug is treated, Serum ALT and TBIL water Flat conspicuousness declines（Respectively P<0.001 and P<0.01）；At the same time serum AST levels declined, serum ALB levels on Rise, but equal no difference of science of statistics.
After medicine ZYC-6 treatments, Serum ALT, AST and TBIL are horizontal to be declined in conspicuousness（Respectively P<0.01,P< 0.05 and P<0.001）, the horizontal conspicuousness risings of serum ALB（P<0.01）.Serology AST indexs, medicine ZYC-6 groups with it is normal Group, which is compared, significant difference（P<0.001）, this result is without substantive significance.Detailed results are shown in Table 4.
The influence of 4 rat blood serum liver function of table and oxidative stress index of correlation
Measurement data is represented with means ± SD.Compared with normal group, statistical significant difference is：###P ＜ 0.001,#P ＜ 0.05.Compared with model group, statistical significant difference is：***P ＜ 0.001,**P ＜ 0.01,*P ＜ 0.05。
3rd, pathological examination
H＆E coloration results are shown：Normal rat lobuli hepatis structure is clear, and hepatic cell cords is by central vein around in radiation Shape arranges, it is seen that a little sinus parietal cell, the visible very small amount fibrous connective tissue in portal area, liver cell form are complete（Fig. 3 A）.Mould Extensive hemorrhagic focus in visible hepatic tissue during type exposed rats 4 weeks, liver cell is largely downright bad, and is taken by roomy fibrosis interval Generation, massive inflammatory cells infiltrated in necrosis region, sinus hepaticus torsional deformation（Fig. 3 B）.Through medicine ZYC-6（Fig. 3 C）Or positive drug IFN-α 2b（Fig. 3 D）After treatment, degeneration of liver cells, the necrosis of rat liver, hepatic entorrhagia have different degrees of improvement (Fig. 3).
Sirius Red are dyed and Masson coloration results (Fig. 4 and Fig. 5) display：Only in portal area in normal rat liver A small amount of collagenous fibres are seen with central vein wall（Fig. 4 A, Fig. 5 A）.Rat model hepatic fibrosis tissue diffusivity hyperplasia is serious, greatly Majority forms thicker complete interval, is stretched into lobuli hepatis tissue, and segmentation wrapping hepatic tissue, forms roomy fine and close header Area-central vein fibrous septum and pseudolobuli（Fig. 4 B, Fig. 5 B）（Arrow is signified at collagen deposition in Fig. 5 B）.Through medicine ZYC- 6（Fig. 4 C, Fig. 5 C）Or positive drug IFN-α 2b（Fig. 4 D, Fig. 5 D）After treatment, rat liver Diffuse fibrous hyperblastosis degree subtracts Gently.

Claims (1)

1.1,7- dihydroxy -3,4,8- trimethoxy mouths mountain ketone is in prevention or treatment liver fibrosis or hepatic sclerosis medicine is prepared Purposes.
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