CN104496974A - Novel diterpene lactone compound in andrographis paniculata, and preparation method and application thereof - Google Patents

Novel diterpene lactone compound in andrographis paniculata, and preparation method and application thereof Download PDF

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Publication number
CN104496974A
CN104496974A CN201410716459.1A CN201410716459A CN104496974A CN 104496974 A CN104496974 A CN 104496974A CN 201410716459 A CN201410716459 A CN 201410716459A CN 104496974 A CN104496974 A CN 104496974A
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ethyl acetate
volume ratio
eluate
extract
preparation
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周同永
周国泰
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XINING YIGE INTELLECTUAL PROPERTY ADVISORY SERVICES Co Ltd
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XINING YIGE INTELLECTUAL PROPERTY ADVISORY SERVICES Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to the field of natural medicines, and specifically relates to a novel diterpene lactone compound in andrographis paniculata, a preparation method of the compound and a medical application of the compound in preparation of anti-adenovirus and anti-HIV virus medicines. The compound is a novel diterpene lactone compound and capable of inhibiting adenovirus and HIV virus through an in-vitro test, and has an application prospect of being developed into anti-adenovirus and anti-HIV virus medicines.

Description

A kind of new diterpene ginkgolide, its preparation method and purposes in Herba Andrographis
Technical field
The present invention relates to natural medicine field, be specifically related to a kind of new diterpene ginkgolide in Herba Andrographis, and the preparation method of this compound and medicinal use.
Background technology
Herba Andrographis andrographis paniculata(Burm. f.) Nees. is Acanthaceae (Acanthaceae) punching Nelumbo andrographiswall.ex Nees. plant, has another name called Herba Andrographitis, eel grass, longleaf rabdosia stem, India's grass
Deng, main product is in the ground such as Guangdong, Fujian, and dry aerial parts are as medicinal." Chinese Pharmacopoeia " version one in 2010 records its cold in nature, bitter, has effect of clearing heat and detoxicating, cool blood, detumescence, is widely used in treatment
The diseases such as bacillary dysentery, acute tonsillitis, urinary tract infections, influenza and venomous snake bite.Herba Andrographis is described as " Chinese medicine microbiotic " with its significant curative effect to influenza, epidemic meningitis, acute bacillary dysentery, pneumonia etc.
In Herba Andrographis, the very important chemical composition of a class is lactone composition, is namely the serial diterpene ginkgolide of representative with rographolide.This constituents has pharmacologically active widely, obtains and studies widely and pay attention to.For rographolide, this compound has the effects such as antipyretic, anti-inflammatory, antiviral, hepatic cholagogic, is mainly used in treatment infantile pneumonia and upper respiratory tract infection clinically.Recently study and also find that rographolide has tempting application prospect in immunomodulatory, antitumor, anti-diabetic etc.
At present, from Herba Andrographis, the diterpene ginkgolide representative configurations obtained is separated as follows:
We are in Herba Andrographis chemical constitution study, and to adopt the silica gel column chromatography diterpene ginkgolide that isolation identification one is new from acetic acid ethyl ester extract, this compound is reported first.We have carried out multiple external activity screening to this compound, and the selection result shows, this compound significantly can suppress adenovirus activity and HIV virus activity in vitro.Therefore, this compound can develop the medicine for preparing anti-adenovirus and HIV virus further.
Summary of the invention
The object of this invention is to provide and a kind ofly from Herba Andrographis, be separated a kind of new diterpene ginkgolide I obtained.
Another object of the present invention is to provide a kind of preparation method of separation and purification chemical compounds I from Herba Andrographis.
Another object of the present invention is to provide the application of chemical compounds I preparing anti-adenovirus and HIV virus drugs.
According to an aspect of the present invention, the compound with the following chemical structure formula I is provided:
Be to provide a kind of preparation method of separation and purification chemical compounds I from Herba Andrographis according to another object of the present invention, its preparation process comprises:
A. Herba Andrographis alcohol or alcoholic solution are extracted one or many, filter, collect filtrate, then concentrating under reduced pressure is dry, obtain alcohol extract;
B. alcohol extract is added water and make suspension, use sherwood oil, extraction into ethyl acetate successively, acetic acid ethyl acetate extract concentrating under reduced pressure, obtain ethyl acetate extract medicinal extract;
C. ethyl acetate extract medicinal extract is carried out column chromatography separating purification, obtain pure chemical compounds I.
Step C comprises in detail: 1. by ethyl acetate extract MCI micro-porous resin column chromatography, methanol-water gradient elution, collects methanol-water (volume ratio 9:1) eluate; 2. get methanol-water (volume ratio 9:1) eluate and carry out silica gel column chromatography, with n-hexane-ethyl acetate gradient elution (10:1,8:1,5:1), collect n-hexane-ethyl acetate (volume ratio 5:1) eluate; 3. get n-hexane-ethyl acetate (volume ratio 5:1) eluate and carry out silica gel column chromatography, with n-hexane-ethyl acetate gradient elution (7:1,6:1,5:1), collect n-hexane-ethyl acetate (volume ratio 6:1) eluate and concentrate; 4. get step 3. enriched material preparative high performance liquid chromatography be separated, water-methanol gradient elution, obtains pure chemical compounds I from methanol-water (volume ratio 85:15) wash-out position.
According to another object of the present invention, provide the application of the compounds of this invention I preparing anti-adenovirus and HIV virus drugs.
Embodiment
Embodiment 1: the preparation method of chemical compounds I
Herba Andrographis (10kg, Anhui, the place of production) is extracted 3 times with 85% ethanolic soln, each 30L, filters, collect filtrate, concentrating under reduced pressure obtains ethanol extraction (385g).This alcohol extract is added water and makes suspension, use sherwood oil, extraction into ethyl acetate successively, acetic acid ethyl acetate extract concentrating under reduced pressure, obtain acetic acid ethyl ester extract 150g; Acetic acid ethyl ester extract is carried out column chromatography separating purification: 1. by acetic acid ethyl ester extract MCI micro-porous resin column chromatography, methanol-water gradient elution, collect methanol-water (volume ratio 9:1) eluate; 2. get methanol-water (volume ratio 9:1) eluate and carry out silica gel column chromatography, with n-hexane-ethyl acetate gradient elution (10:1,8:1,5:1), collect n-hexane-ethyl acetate (volume ratio 5:1) eluate; 3. get n-hexane-ethyl acetate (volume ratio 5:1) eluate and carry out silica gel column chromatography, with n-hexane-ethyl acetate gradient elution (7:1,6:1,5:1), collect n-hexane-ethyl acetate (volume ratio 6:1) eluate and concentrate; 4. get step 3. enriched material preparative high performance liquid chromatography be separated, water-methanol gradient elution, obtains pure chemical compounds I (26mg) from methanol-water (volume ratio 85:15) wash-out position.
Chemical compounds I structural identification: off-white powder; HR-ESIMS shows [M+H] +for m/z 333.2512; 1h NMR (CDCl 3, δ ppm, jin Hz, 500 MHz) and 13c NMR (CDCl 3, δ ppm, 100 MHz) and data are in table 1.Its two dimensional structure and relative configuration can be determined in conjunction with DEPT, HSQC, HMBC, NOESY spectrum and this compounds nuclear magnetic data.Absolute configuration is confirmed by ECD.
table 1 1 h NMR and 13 c NMR signals assignment
Embodiment 2: the external Antiadenovirus of chemical compounds I
1, virus and cell: AdV3 virus strain, Hep-2 cell are provided by Jiangsu Prov. Disease Preventing and Controlling Center.
2, reagent: MEM dry powder, foetal calf serum and EDTA-pancreatin are purchased from Gibco company; Penicillin, Streptomycin sulphate are purchased from Huabei Pharmaceutic Co., Ltd; Glutamine, Merk company.
3, medicine: chemical compounds I, self-control (purity is greater than 98%); Ribavirin injection, Kang Yuan medicine company.
4, test method:
1. virus multiplication: virus stock solution used is inoculated on individual layer Hep-2 cell, 35 DEG C, CO 2cell maintenance medium is supplemented after incubator internal adsorption 2h.Every day observes, and when pathology appears in 75 % ~ 100% cells, multigelation 2 times, puts in centrifuge tube, and 4 DEG C of 12500 centrifugal 20 min of r/min, Aspirate supernatant ,-80 DEG C of freezen protective are for subsequent use.
2. virus virulence measures: after increasing poison, AdV3 suspension is by half-log 12 concentration (1 × 10 -0.5~ 1 × 10 -6), be inoculated on 96 orifice plate individual layer Hep-2 cells respectively, each concentration inoculates 4 multiple holes, and viruses adsorption 2h, discards virus liquid, and every hole adds 100 μ L cell maintenance mediums, puts it into 35 DEG C, 5% CO 2in incubator, Continuous Observation result 7d, makes normal cell controls (only adding maintenance medium) simultaneously, reaches more than 50% for positive hole with last 1d cytopathy, without pathology or less than 50% as negative hole, and cultivate median infective dose (TCID with ReedMuench formulae discovery histocyte 50), experiment virus attack amount is 100 TCID 50.
3. drug cytotoxicity measures: Hep-2 cell is seeded to 96 orifice plates, grow up to after individual layer until cell, 2 times are cleaned by washing lotion, each hole liquid is abandoned in suction, is started to dilute 11 concentration by 2 times of gradient cell maintenance mediums by each medicine from mother liquor, and each concentration establishes 4 multiple holes, every hole 100 μ L, separately establish 4 hole normal cell controls, put 37 DEG C, 5% CO 272 h cultivated by incubator, adopt the cytotoxicity of CPE method drugs.
Cytopathy political reform (CPE method): inverted microscope is observed and records cell growth status and metamorphosis lower every day, determines the TC of medicine to Hep-2 cell 0and TC 50.Judge CPE standard: occur cell shrinkage after dosing, intercellular substance becomes large, kytoplasm is coarse, opaque, have the morphologic change such as more particle in part cell detachment, cell, " 0 " indicates without CPE, " 1 " represents that CPE is 0% ~ 25 %, " 2 " represent that CPE is 25% ~ 50%, " 3 " represent that CPE is 50% ~ 75%, " 4 " represent that CPE is 75% ~ 100%, take normal cell as contrast, do not occur that the maximum drug concentration that cellular form changes is medicine maximal non-toxic concentration TC 0, according to Reed-Muench formulae discovery medicine median toxic concentration TC 50.
4. Drug inhibition AdV3 proliferation experiment: Hep-2 cell is inoculated on 96 orifice plates, cell counting 5 × 10 4individual/mL, by virus by 100 TCID 50diluted for use, treats that Hep-2 cell grows up to individual layer, cleans 2 times by washing lotion, and every hole adds 100 μ L virus liquids, adsorbs 2 h, discards virus liquid after taking-up, cleans 2 times, selects each medicine maximal non-toxic dosage TC 0be the first hole initial concentration, and by 2 times of dilutions, 8 concentration, add each concentration medicine 100 to be measured μ L/ hole, the multiple hole of each medicine 4, be provided with virus positive control and each 4 the multiple holes of normal cell negative control simultaneously, continue to put into 35 DEG C, CO 2cultivate 7 d in incubator, observe and record cytopathy situation, when the lesion degree of virus control wells reaches " 3 " ~ " 4 ", stop experiment when cell controls is " 0 ", the cytopathy degree in observation experiment hole.
5, test-results:
1. virus virulence: according to Reed-Muench formulae discovery AdV3 virus median infective dose TCID in Hep-2 cell 50be 1 × 10 -3/ 100 μ L.
2. drug cytotoxicity (CPE method): observe each medicine group and each porocyte of normal cell controls group under inverted microscope.According to morphologic observation and statistical study the data obtained, adopt Reed-Muench method calculating chemical compounds I and the TC50 of ribavirin to Hep-2 cell to be respectively 1. 4617g/L, 10.88 g/L determine TC 0respectively at 1:32 and 1:16.
3. medicine restraining effect that AdV3 is bred: 7d observes the cytopathic restraining effect that medicine causes AdV3 continuously, there is obvious pathology in virus group Hep-2 cell 24 h cells after infection AdV3, show as cellular swelling, become circle, there is gauffer, thickening in cell walls, pyknosis, mutually fusion form thyrsiform, come off and break.Medicine group, the cytopathy that chemical compounds I can significantly suppress AdV3 to cause.
Comprehensive above-mentioned experimental result, the cytopathy that prompting chemical compounds I can significantly suppress AdV3 to cause, has the effect of anti-AdV3 adenovirus.
Embodiment 3: the external AntiHIV1 RT activity virus function of chemical compounds I
HIV1-RT inhibit activities 3h mark detection method: by demarcate the genetically engineered HIV1-RT (p 66/51) of concentration, the liquid of different concns, reaction buffer and 3after H-dTTP adds, mixing, 37 DEG C of reaction 30 min, 0 DEG C of termination reaction.Reaction solution drips on filter paper, and cold trichoroacetic acid(TCA) washes 3 times, dries after ethanol dehydration.Liquid scintillation instrument measures cpm value, establishes enzyme contrast and blank and drug control simultaneously.
HIV-1 integrase inhibiting activities ELISA detection method: by donor substrate bag by test board, adds the gene hiv integrase of reaction buffer, different concns liquid and demarcation concentration after washing plate, 37 DEG C of reaction 1h.Add target substrate, mixing, 37 DEG C of reaction 1h.Wash plate, add BSA (bovine serum albumin), room temperature 30 min.Wash plate, add the avidin of alkali phosphatase enzyme mark, room temperature reaction 1 h.Wash plate, add chromogenic substrate colour developing 30min, add 0.1 mol/L NaOH color development stopping reaction.Microplate reader 405 nm measures absorbance.Establish enzyme to contrast and blank simultaneously.
Test-results shows, chemical compounds I significantly can suppress HIV1-RT activity and HIV-1 intergrase activity, IC 50be respectively 13.24 ± 2.37 μ g/mL and 9.33 ± 1.51 μ g/mL, illustrate that chemical compounds I has the effect of anti HIV-1 virus.

Claims (6)

1. a diterpene ginkgolide I, its chemical structure is as follows:
。
2. the preparation method of the chemical compounds I described in claim 1, its preparation process comprises:
A. Herba Andrographis alcohol or alcoholic solution are extracted one or many, filter, collect filtrate, then concentrating under reduced pressure is dry, obtain alcohol extract;
B. alcohol extract is added water and make suspension, use sherwood oil, extraction into ethyl acetate successively, acetic acid ethyl acetate extract concentrating under reduced pressure, obtain ethyl acetate extract medicinal extract;
C. ethyl acetate extract medicinal extract is carried out column chromatography separating purification, obtain pure chemical compounds I.
3. preparation method according to claim 2, the detailed step of step C comprises:
1. by ethyl acetate extract MCI micro-porous resin column chromatography, methanol-water gradient elution, collects methanol-water (volume ratio 9:1) eluate;
2. get methanol-water (volume ratio 9:1) eluate and carry out silica gel column chromatography, with n-hexane-ethyl acetate gradient elution (10:1,8:1,5:1), collect n-hexane-ethyl acetate (volume ratio 5:1) eluate;
3. get n-hexane-ethyl acetate (volume ratio 5:1) eluate silica gel column chromatography, with n-hexane-ethyl acetate gradient elution (7:1,6:1,5:1), collect n-hexane-ethyl acetate (volume ratio 6:1) eluate and concentrate;
4. get step 3. enriched material preparative high performance liquid chromatography be separated, water-methanol gradient elution, obtains pure chemical compounds I from methanol-water (volume ratio 85:15) wash-out position.
4. the application of the chemical compounds I described in claim 1 in the anti-adenovirus medicine of preparation.
5. application according to claim 4, adenovirus is AdV3 type.
6. the chemical compounds I described in claim 1 is preparing the application in anti HIV-1 virus medicine.
CN201410716459.1A 2014-12-02 2014-12-02 Novel diterpene lactone compound in andrographis paniculata, and preparation method and application thereof Pending CN104496974A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104387400A (en) * 2014-12-06 2015-03-04 西宁意格知识产权咨询服务有限公司 Novel carbazole alkaloid in clausena and preparation method and application of carbazole alkaloid
CN104387352A (en) * 2014-11-20 2015-03-04 西宁意格知识产权咨询服务有限公司 New sesquiterpene compound as well as preparation method and medical application thereof
CN104997840A (en) * 2015-07-15 2015-10-28 中国科学院西北高原生物研究所 Dracocephalum heterophyllum Benth pentacyclic triterpene component sample pretreatment method and use of Dracocephalum heterophyllum Benth pentacyclic triterpene component
CN113321631A (en) * 2020-08-13 2021-08-31 云南西力生物技术股份有限公司 Biandrographolide G, preparation method and application thereof in medicines

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CN101671322A (en) * 2009-09-30 2010-03-17 合肥工业大学 Method for separating and purifying andrograholide

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CN101671322A (en) * 2009-09-30 2010-03-17 合肥工业大学 Method for separating and purifying andrograholide

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104387352A (en) * 2014-11-20 2015-03-04 西宁意格知识产权咨询服务有限公司 New sesquiterpene compound as well as preparation method and medical application thereof
CN104387400A (en) * 2014-12-06 2015-03-04 西宁意格知识产权咨询服务有限公司 Novel carbazole alkaloid in clausena and preparation method and application of carbazole alkaloid
CN104997840A (en) * 2015-07-15 2015-10-28 中国科学院西北高原生物研究所 Dracocephalum heterophyllum Benth pentacyclic triterpene component sample pretreatment method and use of Dracocephalum heterophyllum Benth pentacyclic triterpene component
CN113321631A (en) * 2020-08-13 2021-08-31 云南西力生物技术股份有限公司 Biandrographolide G, preparation method and application thereof in medicines
CN113321631B (en) * 2020-08-13 2022-02-01 云南西力生物技术股份有限公司 Biandrographolide G, preparation method and application thereof in medicines

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