CN104370871B - The mouth diphenylene ketone oxide class separated from Swertia punicea Hemsl. and the application of suppression hepatitis B virus - Google Patents
The mouth diphenylene ketone oxide class separated from Swertia punicea Hemsl. and the application of suppression hepatitis B virus Download PDFInfo
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- CN104370871B CN104370871B CN201310349568.XA CN201310349568A CN104370871B CN 104370871 B CN104370871 B CN 104370871B CN 201310349568 A CN201310349568 A CN 201310349568A CN 104370871 B CN104370871 B CN 104370871B
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- diphenylene ketone
- ketone oxide
- mouth diphenylene
- mouth
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- 230000011506 response to oxidative stress Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000000814 tuberculostatic agent Substances 0.000 description 1
Classifications
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
- C07C2603/24—Anthracenes; Hydrogenated anthracenes
Abstract
The invention discloses the mouth diphenylene ketone oxide class separated from Swertia punicea Hemsl. and the application of suppression hepatitis B virus thereof.Effect on hepatitics B virus in vitro screening test shows, it is the most notable that the mouth diphenylene ketone oxide class monomeric compound separated shows suppression ratio difference between inhibitory action in various degree, and different compound to the expression of HBsAg or HBeAg in HepG2.2.15 cell.At test compound HepG2On the basis of hepatotoxicity is evaluated, pass through HepG2The protective effect test of hepatocyte injury finds, the mouth diphenylene ketone oxide class monomeric compound separated all has certain protection or repair for hepatocyte, and wherein, Bellis perennis leaf Radix Gentianae element is the most notable for the repair of hepatocellular protection or hepatocyte injury.The present invention further discloses them at suppression HBsAg or/and HBeAg expresses, protects the application in hepatocyte, repair medicine hepatocyte injury and in preparation purposes in biomarker or diagnostic reagent.
Description
Technical field
The present invention relates to biological mouth xanthones compounds, particularly relate to from Swertia punicea Hemsl. (Swertia
Punicea Hemsl.) the middle mouth diphenylene ketone oxide class monomeric compound separated, the invention further relates to them in system
Standby anti-hepatitis virus HBsAg or HBeAg expresses, protects hepatocyte or repair damage hepatocyte or system
It is ready for use on the purposes in biomarker or diagnostic reagent, belongs to biological mouth xanthones compounds and application thereof
Field.
Background technology
Liver is the central metabolites organ of human body, and hepatitis is the inflammation of liver, as a kind of frequently-occurring propagation
Disease, our healthy and daily life of hepatitis the most serious threat, hepatitis kind is a lot,
Different reasons causes different hepatitis.Hepatitis is divided according to the cause of disease, can be divided into viral hepatitis, medicine
Physical property hepatitis, alcoholic hepatitis, toxic hepatitis etc..
China is a hepatitis big country, and viral hepatitis morbidity numerical digit occupies first of cura legitima infectious disease,
Only chronic hepatitis B virus infecton just reaches 1.2 hundred million.Chronic hepatitis b disease journey is delayed, as can not get
Treat timely, it will develop into liver cirrhosis even hepatocarcinoma, serious harm human health.
Simultaneously during drug use, because of medicine itself and/or its metabolite or due to special body constitution pair
The hypersensitivity of medicine or toleration reduce also can cause hepar damnification, i.e. drug induced hepatic injury, clinically
Can behave as various acute and chronic hepatitis, can recover voluntarily after the lighter's drug withdrawal, severe one may threat to life, need
Active treatment, rescue.Meanwhile, drug-induced hepatopathy accounts for the 20%-50% in Non-viral liver disease,
The 15%-30% of fulminant hepatic failure.Multi-medicament can cause drug induced hepatic injury, such as antineoplastic
Treat medicine, antitubercular agent, antipyretic analgesic, immunosuppressant, hypoglycemic medicine, antibacterium, antifungal
And antiviral agents etc..
Acetaminophen (APAP) is one of global most widely used ntipyretic analgesic medicine, although its
Recommend therapeutic dose safe and effective, but treatment window is narrower.The application of APAP has become western countries and has occurred
The main cause of drug induced hepatic injury, in the U.S., Britain and Australia, APAP is to cause Acute Hepatic
The main cause (in the U.S., the acute hepatic failure more than 50% is by caused by APAP) of nonfunction.
Clinicist should watch out for APAP potential danger, especially long-term alcohol, the patient that has liver underlying diseases
In, more should be careful.
The drug induced hepatic injury that APAP causes is a complicated process.It is now recognized that work as excessive use
After APAP, under Cytochrome P450 (CYP) acts on, APAP is converted into N-acetyl-p-benzoquinone
Imines (NAPQI), causes glutathione depletion and the covalent bond with protein, then causes freedom
Base (including oxygen-derived free radicals and nitrogen free radical) increases.Oxidative stress increase may be relevant with calcium ion, continues
And enabling signal transduction pathway and the conversion of mitochondrial membrane permeability.Mitochondrial membrane permeability conversion cause into
The response to oxidative stress of one step, mitochondrial membrane potential declines, and adenosine triphyosphate (ATP) synthesizes
Obstacle, ATP exhausts, then causes necrocytosis.It is thin that glutathion can block the liver that APAP caused
Born of the same parents are downright bad.
China is resourceful Chinese medicine big country, and the much Chinese medicine of report and effective ingredient thereof all have liver in recent years
Cytoprotection, studies these Chinese medicines and effective ingredient thereof, develops and improves the new drug of hepatitis and will have
Wide application prospect.
Swertia punicea Hemsl. (Swertia punicea Hemsl.) is that Gentianaceae Swertia annual herb is planted
Thing, has another name called Rheum alexandrae Batal. lotus, Radix Berberidis (Hunan), Radix Gentianae, grass Radix Gentianae, Sang Di (Tibetan medicine), Bush
All mend this (Yi nationality, distributed over Yunnan, Sichuan and Guizhou), be distributed in Yunnan, Sichuan, Guizhou, West of Hubei Province, Hunan.It is born in hillside grass
In ground, river shoal, sylvan life, shrubbery, height above sea level 400-3800 rice.Swertia Punicea with all herbal medicine, this medicine
Bitter in the mouth is cold in nature, has liver heat removing function of gallbladder promoting, heat-clearing and toxic substances removing, effect of damp eliminating stomach invigorating.Although treating hepatitis at present
Medicine more, but the medicine that can reach satisfactory curative effect is less.Guarantor in " Chinese Pharmacopoeia "
Liver medicine Herba Swertiae Mileensis resource is the most exhausted, it is difficult to meets and produces needs.Separate from Swertia punicea Hemsl. and obtain
Have definite protect the liver, the monomeric compound of hepatoprotective pharmacologically active, this will for prevention or the treatment of hepatitis
Significant.
Summary of the invention
An object of the present invention is to provide a class from Swertia punicea Hemsl. (Swertia punicea Hemsl.)
Definite anti-hepatitis virus HBsAg and/or HBeAg of having of middle separation expresses, and protects the liver, hepatinica
The mouth diphenylene ketone oxide class monomeric compound of reason activity；
The two of the purpose of the present invention are to provide a kind of from Swertia punicea Hemsl. (Swertia punicea Hemsl.)
The method of middle separation described mouth diphenylene ketone oxide class monomeric compound；
The three of the purpose of the present invention are to be applied to suppress B-mode liver by described mouth diphenylene ketone oxide class monomeric compound
Scorching virus HBsAg and/or HBeAg expresses, protects hepatocyte or repair damage hepatocyte；
The four of the purpose of the present invention be to provide a kind of prevention or treatment hepatitis B or protect the liver, the medicine of hepatoprotective
Compositions.
The five of the purpose of the present invention are to provide and a kind of can be used for preparing the little of biomarker or diagnostic reagent
Molecule conjugate, described biomarker includes quanta point biological fluorescent probe and other fluorescence or non-fluorescence
Probe conjugate.
The above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention separates 7 kinds of biological species of acquisition from Swertia punicea Hemsl. (Swertia punicea Hemsl.)
Mouth diphenylene ketone oxide class monomeric compound, described biological species mouth xanthones compounds is selected from: 1,7-hydroxyl-3,4 first
Epoxide mouth diphenylene ketone oxide, 1,5-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide, 1-hydroxy-3-methoxy-8-primrose glycosyl mouth
Diphenylene ketone oxide, swertianolin, 7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-
Dihydroxy-3-methoxyl group mouth diphenylene ketone oxide, 1,7-dihydroxy-3,4,8-trimethoxy mouth diphenylene ketone oxide and Bellis perennis leaf Radix Gentianae
Element.
The present invention is found by the test of substantial amounts of pharmacology pharmacodynamic, is separated and obtain from Swertia punicea Hemsl.
7 kinds of mouth diphenylene ketone oxide class monomeric compound compounds express tool for HepG2.2.15 cell HBsAg and HBeAg
Having inhibitory action, the hepatocyte injury causing APAP can improve cell survival rate, above-mentioned experimental result
Show, from Swertia punicea Hemsl., separated the 7 kinds of biological species mouth xanthones compounds obtained have certain
Anti-hepatitis virus HBsAg and/or HBeAg expresses, the pharmacologically active of hepatoprotective, clinically can be by it
It is prepared as the medicine of anti-hepatitis virus or hepatoprotective.
Meanwhile, the present invention is further discovered that separating the different biological species mouth mountain obtained from Swertia punicea Hemsl.
Ketone monomeric compound is expressed at suppression HBsAg and/or HBeAg, protection hepatocyte, reparation damage
The difference of pole significance is there is in the activity of the pharmacological effects such as hepatocyte.
The present invention carries out effect on hepatitics B virus in vitro sieve by separating the 7 kinds of mouth diphenylene ketone oxide class monomeric compounds obtained
Choosing test, result of the test shows, these 7 kinds of mouth diphenylene ketone oxide class monomeric compounds are in HepG2.2.15 cell
The expression of HBsAg and/or HBeAg all shows inhibitory action in various degree, and different compound it
Between suppression ratio significant difference:
In 7 kinds of mouth diphenylene ketone oxide class monomeric compounds, 1,7-hydroxyl-3,4 methoxyl group mouth diphenylene ketone oxide (compound 1),
Swertianolin (compound 4) and 7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]
-1,8-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide (compound 5) table to HepG2.2.15 cell HBsAg
Reaching and present the strongest inhibitory action, its suppression ratio is respectively 31.74%, 25.37% and 37.66%,
These three mouth diphenylene ketone oxide class monomeric compound exceeds the most far away remaining four kinds of mouth diphenylene ketone oxide class monomeric compound
Suppression ratio, significantly exceeds positive control drug oleanolic acid and presses down HepG2.2.15 cell HBsAg
(suppression ratio of HepG2.2.15 cell HBsAg is rate processed by positive control drug oleanolic acid
22.58%).
Compared to other 6 kinds of mouth diphenylene ketone oxide class monomeric compounds and positive control drug, 7-O-[α-L-pyrans
Rhamanopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide (compound 5)
Expression to HepG2.2.15 cell HBeAg presents the most significantly inhibitory action, its suppression ratio
Reach 31.72%, exceeded the most far away suppression ratio (its of remaining 6 kinds of mouth diphenylene ketone oxide class monomeric compound
The suppression ratio of HepG2.2.15 cell HBeAg is only up to by 6 kinds of mouth diphenylene ketone oxide class monomeric compounds of Yuing
7.77%) positive control drug oleanolic acid, is significantly exceeded to HepG2.2.15 cell HBeAg's
(suppression ratio of HepG2.2.15 cell HBeAg is only suppression ratio by positive control drug oleanolic acid
8.27%).
Comprehensive effect on hepatitics B virus in vitro screening test result is visible, separates 7 obtained from Swertia punicea Hemsl.
Kind of mouth diphenylene ketone oxide class monomeric compound shows pressing down in various degree for the expression of HBsAg and/or HBeAg
Make use, only 7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy
-3-methoxyl group mouth diphenylene ketone oxide all presents for the expression of HBsAg and HBeAg in HepG2.2.15 cell
Go out inhibitory action strongly, significantly exceed other 6 kinds of mouth diphenylene ketone oxide class monomeric compounds for
The suppression ratio of HepG2.2.15 cell HBsAg and HBeAg, significantly exceed positive control drug for
The suppression ratio of HepG2.2.15 cell HBsAg and HBeAg.
The present invention, on the basis of evaluating toxicity of compound, have rated further and separates from Swertia punicea Hemsl.
The 7 kinds of mouth diphenylene ketone oxide class monomeric compounds the obtained protective effect to hepatocyte injury, result of the test finds, 7
Plant mouth diphenylene ketone oxide class monomeric compound and hepatocyte is all had to certain protection or repair, wherein, chemical combination
Thing 7(is i.e.: Bellis perennis leaf Radix Gentianae element) for hepatocellular protection or hepatocyte injury repair the most
Significantly (*P < 0.05).
In the present invention, the salt of the sour addition of biological species mouth xanthones compounds is also included in the present invention.
The salt of the sour addition of described biological species mouth xanthones compounds the most pharmaceutically acceptable is with suitable
When acid (such as hydrochloric acid, acetic acid, sulphuric acid) form nontoxic salt, except pharmaceutically acceptable salt with
Outward, other salt is also included in the present invention.
Heretofore described mouth diphenylene ketone oxide class monomeric compound can be by the method disclosed in existing document
Prepare, it is possible to the method isolated from Swertia punicea Hemsl. provided according to this specification.
As reference, the invention provides a kind of separation from Swertia punicea Hemsl. and obtain 7 kinds of mouth diphenylene ketone oxide class lists
The method of body compound, the method comprises the following steps:
(1) concentrate with ethanol extraction after Swertia punicea Hemsl. being pulverized, obtain the ethanol of Swertia punicea Hemsl.
Extract；(2) ethanol extraction of Swertia punicea Hemsl. is added distilled water to completely suspend after, successively
With petroleum ether, ethyl acetate, water-saturated n-butanol extraction；(3) extract decompression and solvent recovery,
Obtain each extraction position；(4) utilize column chromatography that n-butanol portion is separated, respectively obtain
7 kinds of mouth diphenylene ketone oxide class monomeric compounds.
Wherein, the column chromatography described in step (4) includes: polyamide column chromatography, HP-20 column chromatography,
Sephadex-LH20 gel column chromatography and Semi-PHPLC etc..
Another object of the present invention is to provide a kind of suppression hepatitis B virus HBsAg or HBeAg
The pharmaceutical composition expressed, this pharmaceutical composition is by the biological species mouth diphenylene ketone oxide class preventing or treating effective dose
Compound or its pharmaceutically useful salt form with pharmaceutically acceptable carrier combination；
The present invention also provides for a kind of pharmaceutical composition protected hepatocyte or repair hepatocyte injury, this medicine
Compositions is by biological species mouth xanthones compounds or its pharmaceutically useful salt and the medicine preventing or treating effective dose
On, acceptable carrier cooperation forms；Wherein, described hepatocyte injury is caused by acetaminophen
Hepatocyte injury.
By the biological species mouth xanthones compounds of pharmaceutically acceptable consumption and pharmaceutically acceptable carrier or
After diluent coordinates, the formulation method conventional by this area is prepared into any one suitable medicine group
Compound.Generally said composition is suitable for oral administration and drug administration by injection, also is adapted for other medication.
Said composition can be the liquid such as tablet, capsule, powder, granule, lozenge, suppository, or oral liquid
Body preparation form.According to different medications, pharmaceutical composition of the present invention can contain 0.1%-99%
Weight, the biological species mouth diphenylene ketone oxide class monomeric compound of preferably 10-60% weight.
It is even that the mouth diphenylene ketone oxide class monomeric compound that the present invention separates from Swertia punicea Hemsl. is alternatively arranged as little molecule
Connection thing is used for preparing biomarker or diagnostic reagent, and described biomarker includes quanta point biological fluorescence
Probe and other fluorescence or non-fluorescence probe conjugate.
Described biological species mouth xanthones compounds is selected from 1,7-hydroxyl-3,4 methoxyl group mouth diphenylene ketone oxide, 1,5-bis-
Hydroxy-3-methoxy mouth diphenylene ketone oxide, 1-hydroxy-3-methoxy-7-primrose glycosyl mouth diphenylene ketone oxide, swertianolin,
7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyl group mouth
In diphenylene ketone oxide, 1,7-dihydroxy-3,4,8-trimethoxy mouth diphenylene ketone oxide or Bellis perennis leaf Radix Gentianae element any one or
Multiple；It is preferably: 1,7-hydroxyl-3,4 methoxyl group mouth diphenylene ketone oxide, 1-hydroxy-3-methoxy-7-primrose glycosyl
Mouth diphenylene ketone oxide, swertianolin, 7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-
Dihydroxy-3-methoxyl group mouth diphenylene ketone oxide or Bellis perennis leaf Radix Gentianae element；Most preferably: 7-O-[α-L-rhamnopyranosyloxyhy
Glycosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide.
Accompanying drawing explanation
Fig. 1 compound 1-7 separation process figure.
The suppression that HepG2.2.15 cell HBsAg and/or HBeAg is expressed by Fig. 2 compound 1-7 is made
By result；###P ＜ 0.001,##P ＜ 0.01,#P ＜ 0.05, compares with cell controls group.
Fig. 3 compound 1-7 is to HepG2Study of cytotoxicity.
Fig. 4 compound 1-7 causes HepG to APAP2The protective effect of cell injury；###P ＜ 0.001,
Compare with blank group；* P ＜ 0.05 compares with model group.
Detailed description of the invention
Further describing the present invention, advantages of the present invention and feature below in conjunction with specific embodiments will be with
Description and apparent.But these embodiments are only exemplary, the scope of the present invention is not constituted
Any restriction.It will be understood by those skilled in the art that under without departing from the spirit and scope of the present invention
The details of technical solution of the present invention and form can be modified or replace, but these amendments and replacement are all
Fall within the scope of protection of the present invention.
Embodiment 1 separates biological mouth xanthones compounds and qualification thereof from Swertia punicea Hemsl.
1, 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)；(Wahaha Pure Water has pure water
Limit company, Hangzhou)；Acetonitrile, methanol (chromatographically pure, Xihua, Tianjin special reagent factory；Tianjin great Mao
Chemical reagent factory；Beijing Yili Fine Chemicals Co., Ltd.)；Chloroform, methanol, ethyl acetate, ethanol
(analytical pure, Beijing Chemical Plant).
Thin layer chromatography: silica GF254, Haiyang Chemical Plant, Qingdao；Polyamide column chromatography, Changfeng, Jiangsu
Chemical Co., Ltd.；HP-20 resin, mitsubishi chemical industry company produces；Sephadex LH-20, GE
Healthcare Products.
2, instrument
Agilent1100/1200HPLC system, is furnished with binary pump, automatic sampler, column oven, DAD
Detector (Agilent Technologies, USA).Data acquisition and issuance uses Agilent
Chemstation9.01。
Half preparation HPLC:Alltech426 binary pump, Alltech UVIS2000 UV-detector,
Agilent Zorbax SB C18 chromatographic column (250 × 9mm, 5 μm)；Nuclear magnetic resonance analyser: Bruker DRX
400/500 nuclear magnetic resonance chemical analyser；Mass spectrograph: Bruker APEX IV FT mass spectrograph (HRESI-MS),
ABI Q-STAR LC-MS instrument (ESI-MS).
3, medical material
Swertia punicea Hemsl.: being dried of gentianaceae plant Swertia punicea Hemsl. Swertia punicea Hemsl. is complete
Grass, Dali, the place of production, numbering ZYC001, College of Pharmacy, Beijing Univ teacher Liu Guangxue identify, medicine
Material specimen deposits in natural pharmacology system of College of Pharmacy, Beijing Univ Specimen Room.
4, extract and separate
The Swertia punicea Hemsl. medical material collected is dried further, weighs, altogether 10kg medical material, all
Pulverizing is coarse powder, and the ethanol extraction of the medical material after pulverizing concentrates, and obtains the ethanol extraction of Swertia punicea Hemsl..
Ethanol extraction is added distilled water to completely suspend after, successively with petroleum ether, ethyl acetate, water saturation just
Butanol, before immunoassay.Extract decompression and solvent recovery, obtains each extraction position.Take n-butanol portion (300g) warp
HP-20 resin separates, and alcohol-water gradient elution (5:95 → 100:0) obtains four eluting positions
(F1-F4).F3 position uses polyamide to separate, alcohol-water gradient elution (25:75 → 95:5),
Obtain five eluting positions (F3A-F3E).F3B uses Sephadex LH-20 purification, and methanol is washed
De-, it is separated (flowing phase: acetonitrile/water=20:80) through partly preparing high-efficient liquid, obtains compound 3 He
4.F4 position uses Sephadex LH-20 purification, methanol-eluted fractions, produces 52 fractions.Wherein
Fraction 17-21 is separated (flowing phase: methanol/water=60:40) through partly preparing high-efficient liquid, obtains chemical combination
Thing 5,6 and 7.Fraction 17-21 through partly prepare high-efficient liquid be separated (flowing phase: acetonitrile/water=30:
70) compound 1 and 2, is obtained.Figure-1 is shown in concrete separation process.
5, Structural Identification
From 7 mouth xanthones compounds of n-butanol portion isolated, wherein compound 1, compound 2,
Compound 3 and compound 5 are isolated from Swertia plant first.Compound structure identifies number
According to as follows:
(1) compound 1:1,7-hydroxyl-3,4 methoxyl group mouth diphenylene ketone oxide (1,7-dihydroxy-3,4-dimethoxy-
Xanthone) qualification
Pale yellow powder, dissolves in methanol, acetonitrile.ESI-MS m/z (%): 287.1 [M-H]-(100)；1H-NMR(400MHz,DMSO-d6) δ ppm: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) δ ppm: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).With polishing wax
Data and document (Yang XD, Xu LZ, Yang SL.Xanthones from the stems of
Report be consistent Securidaca inappendiculata.Phytochemistry.200158 (8): 1245-1249.),
Therefore this compound structure is accredited as 1,7-dihydroxy-3,4-dimethoxy-xanthone.
(2) compound 2:1,5-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide (1,5-dihydroxy-3-methoxy-xanthone)
Qualification
Yellow needles, dissolves in methanol, acetonitrile.ESI-MS m/z (%): 257.1 [M-H]-(100)；1H-NMR(400MHz,DMSO-d6) δ ppm: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) δ ppm: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).Above spectroscopic data and document (Cheng WY, Zhong FF, Zhao YH, Yang
GZ, Chen Yu.Study on the Antioxidant Constituents from the Barks of Garcinia
Xanthochymus.Nat Prod Res Dev2008,20:836-838.) report is consistent, therefore this compound
Structural Identification is 1,5-dihydroxy-3-methoxyxanthone.
(3) compound 3:1-hydroxy-3-methoxy-7-primrose glycosyl mouth diphenylene ketone oxide (1-hydroxy-3-
Methoxy-7-O-primeverosylxanthone) qualification
Colorless needles, dissolves in methanol, acetonitrile.ESI-MS m/z (%): 551.1 [M-H]-(100)；1H-NMR(400MHz,DMSO-d6) δ ppm: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) δ ppm: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”)。
Above spectroscopic data and document (Takaaki Hayashi, Takashi Yamagishi.Two xanthone
Report phase glycosides from gentiana lutea.Phytochemistry.198827 (11): 3696-3699.)
Symbol, therefore this compound structure is accredited as 1-hydroxy-3-methoxy-7-O-primeverosylxanthone.
(4) compound 4: the qualification of swertianolin (swertianolin)
Yellow amorphous powder, dissolves in methanol, acetonitrile.ESI-MS m/z (%): 435.2 [M-H]-(100)；
SDS1H-NMR(400MHz,DMSO-d6) δ ppm: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, proton on sugar), 3.89 (3H, OCH3),13.10(1H,OH-1),10.07(1H,
OH-5)；13C-NMR(100MHz,DMSO-d6) δ ppm: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).Above spectroscopic data and document (Tan P, Liu YL, Hou CY.
Structure of swertiapuniside from Swertia punicea Hemsl.Yao Xue Xue Bao.
Report be consistent, therefore this compound structure be accredited as swertianolin 1992:27 (6): 476-479.).
(5) compound 5:7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy
-3-methoxyl group mouth diphenylene ketone oxide (7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-
Xylopyranosyl]-1,8-dihydroxy-3-methoxy-xanthone) qualification
Yellow powder, dissolves in methanol, acetonitrile.ESI-MS m/z (%): 551.1 [M-H]-(100)；1H-NMR(400MHz,DMSO-d6) δ ppm: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) δ ppm: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).With polishing wax
Data and document (Li, Y.L., et al., 2008.The glycosides from Lomatogoniumrotatum.
Natural Product Research.22 (3), 198-202.) report is consistent, therefore this compound structure is accredited as
7-O-[α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxy
-xanthone。
(6) compound 6:1,7-dihydroxy-3,4,8-trimethoxy mouth diphenylene ketone oxide (1,7-dihydroxy-3,4,8-
Trimethoxyxanthone) qualification
Yellow powder, dissolves in methanol, acetonitrile.ESI-MS m/z (%): 317.2 [M-H]-(100)；1H-NMR(400MHz,DMSO-d6) δ ppm: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) δ ppm:
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).Above spectroscopic data and document
(Edilberto R.Silveira.Maria Jos é C.Alisio Menezes Jr,David G.I.
Kingston,Thomas E.Glass.Pentaoxygenated xanthones from Bredemeyera
Report be consistent floribunda.Phytochemistry.199539 (6): 1433-1436.), therefore this compound knot
Structure is accredited as 1,7-dihydroxy-3,4,8-trimethoxyxanthone.
(7) compound 7: the qualification of Bellis perennis leaf Radix Gentianae element (bellidifolin)
Yellow needles, dissolves in methanol, acetonitrile.ESI-MS m/z (%): 273.1 [M-H]-(100)；1H-NMR(400MHz,DMSO-d6) δ ppm:6.43 (1H, H-2), 6.64 (1H, H-4), 7.27 (1H, d,
8.8Hz,H-6),6.66(1H,d,8.8Hz,H-7),3.91(3H,OCH3),11.93(1H,OH-8),
11.09(1H,OH-1),9.73(1H,OH-5)；13C-NMR(100MHz,DMSO-d6) δ ppm:
161.95(C-1),97.52(C-2),167.10(C-3),93.01(C-4),157.43(C-4a),143.39(C-4b),
137.35(C-5),123.87(C-6),109.55(C-7),151.77(C-8),107.55(C-8a),184.09(C-9),
102.18(C-9a),56.29(OCH3).Above spectroscopic data and document (Tan P, Liu YL, Hou CY.
Structure of swertiapuniside from Swertia punicea Hemsl.Yao Xue Xue Bao.
Report be consistent, therefore this compound structure be accredited as Bellis perennis leaf Radix Gentianae element 1992:27 (6): 476-479.)
(bellidifolin)。
The mouth diphenylene ketone oxide class monomeric compound effect on hepatitics B virus in vitro screening that test example 1 separates from Swertia punicea Hemsl.
Test
One, test material
1, test compound: 7 kinds of mouth diphenylene ketone oxide class monomer chemical combination that embodiment 1 separates from Swertia punicea Hemsl.
Thing: 1,7-hydroxyl-3,4 methoxyl group mouth diphenylene ketone oxide (compound 1), 1,5-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide (is changed
Compound 2), 1-hydroxy-3-methoxy-7-primrose glycosyl mouth diphenylene ketone oxide (compound 3), swertianolin (is changed
Compound 4), 7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-first
Epoxide mouth diphenylene ketone oxide (compound 5), 1,7-dihydroxy-3,4,8-trimethoxy mouth diphenylene ketone oxide (compound 6) and
Bellis perennis leaf Radix Gentianae element (compound 7).Compound is and aseptic has prepared 6mM storing solution.
2, positive control drug: oleanolic acid, purchased from National Institute for Food and Drugs Control.
3, cell strain: HepG2.2.15 cell (people's hepatocarcinoma of hepatitis B virus (HBV) genome transfection
Cell (HepG2) it is that its dyeing interbody fusion has hepatitis B virus full-length genome, can stably express hepatitis B virus
Surface antigen (HBsAg) and e antigen (HBeAg), and the hepatitis B virus granule of infectious can be secreted,
Preclinical medicine institute of Department Of Medicine, Peking University department of microbiology provides), high at the DMEM containing 10% hyclone
Growth in sugar culture fluid (containing penicillin 100U/ml, streptomycin 100 μ g/ml, G-4181 μ l/ml), training
Foster condition is 37 DEG C, 5%CO2, saturated humidity.With containing 0.25% trypsin and 0.02%EDTA liquid
Had digestive transfer culture.
Two, experimental apparatus and reagent
Abbott Laboratories' automatic lmunoassays analyzer (model: ARCHITECT i2000sr)；DMEM height sugar is trained
Nutrient solution 500mL, China's match is silent flies generation that biological company limited product；G-418 (Geneticin), tire cattle
Serum, U.S.'s GIBCO Products；HBsAg and HBeAg chemiluminescence micropartical immunoassay
Box, Ireland diagnosis department of Abbott Laboratories product；Dimethyl sulfoxide, SIGMA Products.
Three, test method
Test sets HBsAg and HBeAg positive controls, negative control group, cell controls group, 10 μMs
Drug level group and positive drug control group (50 μMs).HepG2.2.15 cell is with every hole 6 × 104Individual inoculation
24 porocyte culture plates, every hole 1ml, 37 DEG C, 5%CO2Culture medium 24 hours.Discard culture fluid,
Every hole adds 10 μMs of compound medicinal liquids, every multiple hole of concentration 3,37 DEG C, 5%CO2Cultivate, cultivate continuously
4 days, obtain the 4th day culture supernatant.HBsAg and HBeAg it is separately added in culture supernatant
Chemiluminescence micropartical immunoassay reagent (operating by test kit operation instructions) detects.Antigen
Inhibition percentage (%)=(cell controls group-administration group)/cell controls group × 100%.With cell controls
Group compares, and statistical significant difference is:###P ＜ 0.001,##P ＜ 0.01,#P ＜ 0.05,.
Four, result of the test
Result of the test is shown in Fig. 2.Result of the test shows, positive control drug oleanolic acid (50 μMs) and chemical combination
Thing 1-7(10 μM) inhibitory action is all shown for the expression of HepG2.2.15 cell HBsAg, its
Suppression ratio is respectively 22.58%(P ＜ 0.05), 31.74%(P ＜ 0.05), 12.01%, 3.27%, 25.37%
(P ＜ 0.01), 37.66%(P ＜ 0.001), 7.28% and 8.72%.Positive control drug oleanolic acid
(50 μMs) and compound 1-7(10 μM) expression of 2.2.15 cell HBeAg also shown and presses down
Make use, its suppression ratio is respectively 8.27%(P ＜ 0.05), 0.42%, 7.77%, 7.53%(P ＜ 0.05),
2.65%, 31.72%(P ＜ 0.001), 3.60% and 2.40%.
Mouth diphenylene ketone oxide monomeric compound in vitro toxicity and liver-protecting activity that test example 2 separates from Swertia punicea Hemsl. are commented
Valency is tested
One, test material
1, test compound: 7 kinds of mouth diphenylene ketone oxide class singulation that embodiment 1 separates from Swertia punicea Hemsl.
Compound: 1,7-hydroxyl-3,4 methoxyl group mouth diphenylene ketone oxide (compound 1), 1,5-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide
(compound 2), 1-hydroxy-3-methoxy-7-primrose glycosyl mouth diphenylene ketone oxide (compound 3), swertianolin
(compound 4), 7-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy
-3-methoxyl group mouth diphenylene ketone oxide (compound 5), 1,7-dihydroxy-3,4,8-trimethoxy mouth diphenylene ketone oxide (compound 6)
With Bellis perennis leaf Radix Gentianae element (compound 7).Compound is and aseptic has prepared 6mM storing solution.
2, positive control drug: bicyclol, the present of consonance pharmaceutical factory, DMSO prepares, 4 DEG C of preservations.
3, cell strain: people's hepatocarcinoma HepG2Cell, at the DMEM culture fluid containing 10% hyclone
Growth in (containing penicillin 100U/ml, streptomycin 100 μ g/ml), condition of culture is 37 DEG C, 5%CO2,
Saturated humidity.With containing 0.25% trypsin and 0.02%EDTA liquid had digestive transfer culture.
4, test reagent: DMEM solution, mycillin mixed liquor, trypsinization liquid, Beijing Suo Lai
Precious company limited；Hyclone, U.S.'s GIBCO Products；Dimethyl sulfoxide, SIGMA company
Product；Toshiba's automatic clinical chemistry analyzer.
Two, test method
(1) mouth diphenylene ketone oxide monomeric compound in vitro toxicity is evaluated
Use MTT method: HepG2Cell is inoculated in 96 porocyte culture plates, after cultivating 24h,
Adding 10 μMs of test compounds of final concentration, set solvent control group simultaneously, each drug level sets 3
Parallel hole.After medicine function cells 48h, discarding culture fluid, every hole adds MTT (0.5mg/ml)
Liquid 100 μ l, continues to cultivate 4h, discards MTT liquid, and every hole adds DMSO150 μ l, mixing vibration
Device vibrates, and measures absorbance at microplate reader 570nm wavelength.Cell survival rate (%)=(give
Medicine cell OD meansigma methods/solvent control cell OD meansigma methods) × 100%.
(2) mouth diphenylene ketone oxide monomeric compound liver-protecting activity is evaluated
Use MTT method.HepG2Cell is inoculated in 96 porocyte culture plates, after cultivating 24h,
Add test compound and the acetaminophen (APAP, final concentration 8mM) of 10 μMs, set positive drug simultaneously
Thing matched group (bicyclol, bicyclol), solvent blank matched group and model group.Continue function cells 48h.
Discarding culture fluid, every hole adds MTT (0.5mg/ml) liquid 100 μ l, continues to cultivate 4h, discards MTT liquid,
Every hole adds DMSO150 μ l, and mixing agitator vibrates and measures absorbance at microplate reader 570nm wavelength
Value.Cell survival rate (%)=100 × administration group OD meansigma methods/blank group OD meansigma methods.###P
＜ 0.001, compares with blank group；* P ＜ 0.05 compares with model group.
Three, result of the test
Result of the test is shown in Fig. 3 and Fig. 4 respectively.Result of the test shows: (1) 7 test compound exists
HepG is acted under 10 μMs of concentration2Cell 48h, cell growth equal unrestraint effect, therefore select
This concentration is used for follow-up study；(2) APAP8mM effect HepG2Cell 48h, causes significantly
Cell injury, compare with blank have pole significant difference (###P < 0.001).Compound 7 and positive drug
Bicyclol effect group cell survival rate compare with model group have significance improve (*P < 0.05).
Claims (6)
1. prepare biological species mouth diphenylene ketone oxide class monomeric compound 1,7-dihydroxy-3,4-dimethoxy for one kind
The method of base mouth diphenylene ketone oxide, it is characterised in that comprise the following steps:
(1) Swertia punicea Hemsl. ethanol extraction is concentrated, obtain the ethanol of Swertia punicea Hemsl.
Extract；(2) ethanol extraction of Swertia punicea Hemsl. adds distilled water suspend, use successively
Petroleum ether, ethyl acetate, water-saturated n-butanol extract；(3) extract recovered under reduced pressure is molten
Agent, obtains each extraction position；(4) utilize column chromatography that n-butanol portion is separated,
Obtain.
The most in accordance with the method for claim 1, it is characterised in that described in step (4)
Column chromatography include: polyamide column chromatography, HP-20 column chromatography, Sephadex-LH20 are solidifying
Glue column chromatography and Semi-PHPLC.
3. the medicine that biological species mouth diphenylene ketone oxide class monomeric compound is expressed at preparation suppression HBsAg
In purposes；Wherein, described biological species mouth diphenylene ketone oxide class monomeric compound is selected from: 1,7-dihydroxy
Base-3,4-dimethoxy mouth diphenylene ketone oxide, swertianolin or 7-O-[α-L-rhamnopyranosyl
-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide.
4. the medicine that biological species mouth diphenylene ketone oxide class monomeric compound is expressed at preparation suppression HBeAg
In purposes；Wherein, described biological species mouth diphenylene ketone oxide class monomeric compound is 7-O-[α-L-pyrrole
Mutter rhamanopyranosyl-(1 → 2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyl group mouth diphenylene ketone oxide.
5. biological species mouth diphenylene ketone oxide class monomeric compound is repaiied at preparation protection hepatocyte medicine or preparation
Purposes in multiple hepatocyte injury medicine；Wherein, described biological species mouth diphenylene ketone oxide class monomer chemical combination
Thing is Bellis perennis leaf Radix Gentianae element.
6. according to the purposes described in claim 5, it is characterised in that: described hepatocyte injury
It it is the Drug hepatocyte injury that caused of acetaminophen.
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