CN103585223B - The application of Herba Elephantopi Mollis extract in preparation treatment AIDS-treating medicine - Google Patents
The application of Herba Elephantopi Mollis extract in preparation treatment AIDS-treating medicine Download PDFInfo
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Abstract
The present invention relates to a kind of novelty teabag of Herba Elephantopi Mollis extract, this novelty teabag is the application of Herba Elephantopi Mollis extract in preparation treatment AIDS-treating medicine.Described Herba Elephantopi Mollis extract is prepared as follows: take Herba Elephantopi Mollis as raw material, uses 50%-90% ethanol extraction, is obtained by purification such as high speed adverse current chromatogram, polyamide chromatographs.Herba Elephantopi Mollis extract provided by the present invention is significant to preparation treatment AIDS-treating medicine.
Description
Technical field
The invention belongs to biomedicine field, relate to the application of a kind of Chinese medicinal material extract in preparation treatment AIDS-treating medicine, specifically relate to the application of Herba Elephantopi Mollis extract in preparation treatment AIDS-treating medicine.
Background technology
Acquired immune deficiency syndrome (AIDS) is the new virus sexually transmitted disease found in 1981, and the traditional Chinese medical science past is not on the books to it.The traditional Chinese medical science is dialectical to acquired immune deficiency syndrome (AIDS), thinks that acquired immune deficiency syndrome (AIDS) belongs to epidemic disease category.The cause of disease is the scorchingly hot heresy invasion and attack of epidemic disease, deficiency of vital QI and causing.Pathogenesis is mainly weakened defensive QI, and yang-energy damages, and internal organs are got involved, and causes that the deficiency of the lung is insufficiency of the spleen suffers from a deficiency of the kidney, secondary infection.Thermal burn venation, coagulation of QI-blood, Xing Cheng mass in the abdomen lump tumor.
Syndrome Differentiation of Chinese Medicine " card ": mainly distinguish ebb and flow between pathogen and vital QI and deficiency syndrome excess syndrome.HIV (human immunodeficiency virus) is evil poison, and the change of disease in evil poison enters, the dynamic change of just resisting with the heresy of body healthy energy is advancing of disease process.Infect early stage based on the domination of pathogen, mid-term " causes void because of real ", late period " excess caused by deficiency ".The acquired immune deficiency syndrome (AIDS) state of an illness copies changeable, and acquired immune deficiency syndrome (AIDS) and secondary infection thereof all can be formed " disease ", and has difference " type " and " phase ".The traditional Chinese medical science has dialectical differential diagnosis of diseases to acquired immune deficiency syndrome (AIDS), the typing different diagnosis and treatment project such as by stages.
The Chinese medicine of acquired immune deficiency syndrome (AIDS) still belongs to conceptual phase at present, at home and abroad there is no at present generally acknowledged curative effect certainly go through produce AIDS virus resisting Chinese medicine or Chinese medicine monomer compound.AIDS virus resisting plant amedica and Chinese medicine research mainly divide 3 kinds of approach, and the AIDS virus resisting Chinese medicinal plant medicine that smoking set obtains and composition thereof, some is in clinic trial.
Since HIV (human immunodeficiency virus) finds, use HIV (human immunodeficiency virus) cell culture and reverse transcriptase in the world, the ill vitro test method such as protease and intergrase, while extensive SCREENED COMPOUND, also plant and Chinese herbal medicine extract and composition thereof is screened in a large number, also studied some compound Chinese medicinal preparation, find the active drug of inhibiting HIV in vitro, the viremia of the suppressed simian immunodeficiency virus had (SIV) infected monkey.Some is in clinic trial, not yet clear and definite effect.
China removes Africa treatment AIDS patients from 1987 Nian Pai combination of Chinese and Western medicine medical teams.Domestic before 2002, lack specially good effect Anti-AIDS Drugs, Chinese and western medicine, combines with dialectical to AIDS patients differential diagnosis of diseases according to theory of Chinese medical science clinical, with heat-clearing and toxic substances removing, strengthening the body resistance, or removing heat from blood the kidney invigorating, the rule for the treatment of such as blood circulation promoting and blood stasis dispelling, by the property of Chinese materia medica, select Chinese medicine, prescribed treatment.By clinical practice, think that some Medical Complex Prescription in Treating AIDS can improve symptom, mitigate the disease.In recent years to the AIDS patients that laboratory diagnosis is made a definite diagnosis, adopt treatment by Chinese herbs, if matched group, at treatments period, periodic detection blood viral charge (HIV-RNA) and cd4 cell level, evaluation effect.There is report to improve symptom, improve immunity, mostly still in clinical verification.Enter clinical AntiHIV1 RT activity single medicinal material or plant amedica and Chinese medicine compound as follows:
Glycyrrhizin: glycyrrhizin (glycyrrhizin, GL) be the glycyrrhizin that glycyrrhiza uralensis fisch (Glycyrrhiza glabra L) extracts, there is broad-spectrum disease resistance toxic action, vitro inhibition HIV1-RT, HIV cell fusion is suppressed in cell culture, also resisiting influenza virus and SARS virus, the dead and pneumonopathy change of the pneumonia that experiment in vivo proves to protect influenza infection mice to cause; The CCl of protection mice
4toxic liver injury; Induce gamma interferon; There is parahormone effect.Japan's its intravenous administrations chronic hepatitis B conventional, also once tried out treatment AIDS patients, can suppress serum HIV-1-P24 antigen, raised cd4 cell.Lv Weibai professor 1988-1992 can treat AIDS patients African Tanzania application glycyrrhizin oral formulations gram Chinese mugwort can improve symptom, and cd4 cell slightly rises.Its effect is not yet verified.
Trichosanthin: trichosanthin (Trichosanthin, TCS, GLQ223, Q material) belong to Cucurbitaceae (Cucurbitaceae) for Chinese medicine Fructus Trichosanthis (Trichosanthes kirilowii), the main Chinese medicine ingredients of China's Chinese medicine compound for miscarrying among the people is Fructus Trichosanthis, the effective ingredient of China scientist isolation identification device antiearly pregnancy and induced labor is trichosanthin, molecular weight is 27kD, one, secondary structure and three dimensional structure are understood fully completely, for the toxalbumin of Profilin synthesis, ribosome inactivating protein (the Ribosome Inactivating Protein of strand, RIP).The U.S. is when screening anti-HIV-1 medicines, find that it suppresses HIV-1 cell fusion in lymphocyte is cultivated, thereafter find P24 antigen and the RNA of HIV-1 in mononuclear phagocyte is cultivated, blood plasma HIV-1 can be suppressed in the intravenous injection of 3 simian acquired immunodeficiency syndrome virus (SIV) infected monkeys.The U.S. uses intravenous administrations AIDS patients in forest farm, and HIV-1-P24 antigen in the T lymphocyte that peripheral blood HIV-1 can be suppressed to infect and mononuclear phagocyte, makes CD4/ CD8 ratio slightly raise.Due to anaphylactic reaction can be caused dead, neurovirulent Mental Subnormality and nephrotoxicity reaction, clinical experiment stops.From Radix Trichosanthis, isolate other albumen TAP29 in recent years, molecular weight, at 29 kD, also have the effect suppressing HIV-1, but toxicity is less, not in clinic trial.In addition the bitter melon protein of Fructus Momordicae charantiae (MOmordica charantia) and the Radix Phytolaccae albumen (Pokeweed protein) etc. of Radix Phytolaccae (Phytolacca americana) have the effect suppressing HIV.
Hypericin: plant Herba Hyperici Monogyni (St. John Wort, Hypericum perforatum) its extract for treating depression of folk tradition, the active component proposed from flower is hypericin (hypericin), it is the multi-ring dimerization anthraquinone analog compound of aromatic series, for anti-depression drug, complete synthesis.Hypericin has multiple antiviral activity, in cell culture, suppress HIV, and its mechanism of action is undressing of blocking virus, and assembling and release, the effect of killing the virus, by blood brain barrier.Murine leukemia virus can be suppressed in cell culture to breed.The splenomegaly that murine leukemia virus infecting mouse causes can be suppressed in vivo, duck hepatitis B virus infection Sanguis Anas domestica also can be suppressed to ask viral DNA, and have broad spectrum antibiotic active.Once in treatment on probation of clinical I/II phase HIV sufferers, test had been stopped because there being light sensitization.
1992 year report the native compound that from Malaysian rainforest plant Calophyllum lanigerum be separated dextrorotation lane element A: dextrorotation lane element A(+Calanolide A), Mus coumarin kind compound, suppress HIV1-RT, but invalid to HIV-2.Belong to non-nucleoside HIV1-RT inhibitor, in cell culture, multi-to-multi lives in the responsive and drug resistance HIV-1 of the AZT of clinical separation effectively, and and AZT, NVP have synergism.In HIV hollow fiber pipe Mice Body, prove there is AIDS virus resisting effect in experimental model (HIV hollow fiber mouse model), can drug effect be strengthened with AZT use in conjunction.Complete synthesis, having carried out its clinical trial of I-II proves there is the therapeutic effect suppressing HIV-1 to infect, and toxicity is little, and side effect is light, has headache and inappetence etc., effective with AZT drug combination.Be the single plant component uniquely entering II phase clinical experiment at present, observing in checking curative effect, not yet ratify to produce.
Herba Sidae Rhombifoliae soup: Herba Sidae Rhombifoliae soup (Radix Bupleuri, Radix Ginseng, the Rhizoma Pinelliae, Radix Scutellariae, Radix Glycyrrhizae, Rhizoma Zingiberis Recens, Fructus Jujubae)
It is Zhang Zhongjing Treatise on Febrile Diseases side.Hepatitis B is treated with it by Japan, finds that it suppresses HIV in cell culture.The U.S. is used for the treatment of numerical example AIDS patients clinical, can reduce blood-serum P 24 antigen.We prove that it has the effect suppressing HIV1-RT and suppress HIV-1 in cell culture.Study the effect of its 7 kinds of composition Chinese medicines respectively, find that only Radix Scutellariae water extract and composition baicalin (baicalin) thereof have and suppress HIV-1 effect, HIV1-RT is suppressed in vitro in cell free system, HIV-1 cytopathy is suppressed in human T-cell and peripheral blood lymphocytes cultivate, FLuorescent antigen and P24 antigen, in body, oral splenomegaly and the blood leukocytes of suppressing of murine leukemia virus infecting mouse raises.Japan Ono reports that noroxylin (baicalien) suppresses HIV1-RT.
In grind No. I: in grind No. I commodity such as (principal agent has Herba Violae, Radix Trichosanthis composition) and be called: Chinese mugwort
Gram electuary take heat-clearing and toxic substances removing as the rule for the treatment of, by Chinese medicine research institute of China prescription 1992-1993 in African Tanzania to the clinical practice of early metaphase acquired immune deficiency syndrome (AIDS), think to have and necessarily improve symptom, improve the effect of immunity.Laboratory proves that this medicine vitro inhibition hiv reverse transcriptase is active, simian acquired immunodeficiency syndrome poison infected monkey experimental therapy result is shown in body, plasma viral titre can be reduced, and cd4 cell number and CD4/CD8 ratio and T can be improved, bone-marrow-derived lymphocyte numerical value, inducement interferon and generation immunoloregulation function.Once in Africa and China's clinic trial, not yet clinical verification was ratified.
Zhongyan No. 2: Zhongyan No. 2 (Radix Astragali, Radix Ginseng, Radix Angelicae Sinensis, Fructus Lycii and Radix Glycyrrhizae etc.) is China's traditional Chinese medical science
Academy medical team prescription.1992 at African Tanzania treatment AIDS patients.1998-1999 is at African special outpatient clinic to 29 routine AIDS patients treatments, and 2/16 routine HIV virus load declines, and the routine CD4 of CD4 8/29 raises.2001-2002 helps the routine AIDS patients of peace hospitalize 15 in Beijing, 3/15 routine virus load declines, and the routine CD4 of CD4 1/15 raises.Be improved the effect of symptom.Experiment proves that this medicine shows simian acquired immunodeficiency syndrome poison infected monkey experimental therapy in human body, and medication 4-12 week can reduce plasma viral titre, P27 antigen, and can improve cd4 cell number, has activation to infected monkey lymphocyte.
Herba Elephantopi Mollis: be the dry aerial parts of feverfew Herba Elephantopi Mollis (Picris japonica Thunb.).Summer, autumn two gather when season, the flowers are in blossom, and removing impurity, dries.Record in 1998 " Drug Standard of Ministry of Public Health of the Peoples Republic of China mongolian medicine fascicle ".Cool in nature, rough, bitter in the mouth.Kill " gluing ", pain relieving, heat clearing away, detumescence, removing toxic substances.For pestilence, Adam's apple, mastitis, breast twinge, parotitis, battle array twinge.The common drug of the minority areas such as illiteracy is hidden for China.But about the basic research of Herba Elephantopi Mollis is still very limited, the follow-up promotion and application of this medical material are restricted.Modern study shows, Herba Elephantopi Mollis contains Fols Picridis fuscipilosae glycosides (picriside) A, B, C, lactucin (lactucin), 11 β-13-dihydro lactucins (11 β-13-dihydrolactucin), false Radix Crepidis elongatae glycoside (caepidiaside) A and Herba Ixeritis Denticulatae lactone (ixerin) F etc., also containing polysaccharide and flavone compound, other chemical compositions are not quite clear; Pharmacological research shows, Herba Elephantopi Mollis water extraction, and cross D101 macroporous resin, water elution part has blood sugar lowering, regulating blood lipid action.Refer to document: Xi Xiaohu, Ge Rui, Gao Jianping.Picris japonica Thunb washing position blood sugar lowering, Lipid-regulating effect research, Chinese Medicine Leader, 2011,8(13): 34-36.Other pharmacological actions have no report.
Domestic patent search result, has no Herba Elephantopi Mollis Patents.
Above-mentioned document and patent etc., there is not yet Herba Elephantopi Mollis or the Herba Elephantopi Mollis extract report for the preparation for the treatment of AIDS-treating medicine.
Summary of the invention
The object of the present invention is to provide the application in preparation treatment AIDS-treating medicine of Herba Elephantopi Mollis extract.
The present invention is achieved through the following technical solutions:
The present invention's Herba Elephantopi Mollis used is the dry aerial parts of feverfew Herba Elephantopi Mollis (Picris japonica Thunb.).
Herba Elephantopi Mollis extract of the present invention prepares as follows:
(1) Herba Elephantopi Mollis, with concentration 50%-90% ethanol as solvent, extract at 10 DEG C of-35 DEG C of warm macerating, extraction time is 3-8 time, and each extraction time is 12 ~ 48 hours, and each solvent load is 21-25 times of Herba Elephantopi Mollis weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Herba Elephantopi Mollis extract A;
(2) the Herba Elephantopi Mollis extract A that step (1) obtains is added 10-20 times amount water dissolution, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography, with petroleum ether-ethyl acetate-methanol-water (2:2:1:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, obtain Herba Elephantopi Mollis extract B;
(3) by the medicinal liquid A that step (2) obtains, by polyamide column post, wash with water, then carry out gradient elution with the alcoholic solution of concentration 10%-70%, collect different concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains Herba Elephantopi Mollis extract C;
(4) above-mentioned Herba Elephantopi Mollis extract B, Herba Elephantopi Mollis extract C, wherein one or both mix by a certain percentage, obtain Herba Elephantopi Mollis extract of the present invention.
The preparation method of Herba Elephantopi Mollis extract is:
(1) Herba Elephantopi Mollis, with concentration 55% ethanol as solvent, extract at 25 DEG C of warm macerating, extraction time is 5 times, and each extraction time is 24 hours, and each solvent load is 23 times of medical material weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Herba Elephantopi Mollis extract A;
(2) extract A that step (1) obtains is added 15 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A.By precipitate A, adopt high-speed countercurrent chromatography, with petroleum ether-ethyl acetate-methanol-water (2:3:1:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, namely obtain Herba Elephantopi Mollis extract B;
(3) by the medicinal liquid A that step (2) obtains, by polyamide column post, wash with water, then carry out gradient elution with the alcoholic solution of concentration 60%, collect 60% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains Herba Elephantopi Mollis extract C;
(4) above-mentioned Herba Elephantopi Mollis extract B and the mixing of Herba Elephantopi Mollis extract C, obtain Herba Elephantopi Mollis extract of the present invention.
Herba Elephantopi Mollis extract of the present invention mainly contains: zaluzanin C (Zaluzanin C); 9 Alpha-hydroxy-11 β, the other glycosides of 13-dihydro zaluzanin C 3-O-β-pyrans (9 α-Hydroxy-11 β, 13-dihydrozaluzanin C 3-O-β-allopyranoside); Luteolin-7-O-β-D-Glucose glycosides; Hypericin (hypericin); Totally 4 main components.
Herba Elephantopi Mollis extract of the present invention, by adding the various adjuvants that pharmaceutics allows, can make the various peroral dosage forms on pharmaceutics.
The present invention's exploratory development is first that raw material extracts preparation treatment acquired immune deficiency syndrome (AIDS) extract with the dry aerial parts of feverfew Herba Elephantopi Mollis (Picris japonica Thunb.).This experimentation shows, Herba Elephantopi Mollis extract of the present invention can be used for preparation treatment AIDS-treating medicine, in MAGI cell, have obvious inhibitory action to HIV-1 IIIB strain, when concentration is 50 μ g/ml, and suppression ratio more than 90%; Obvious inhibitory action is had to HIV-1 IIIB strain in MT4 cell, when concentration is 100 μ g/ml, suppression ratio more than 95%.
Herba Elephantopi Mollis extract of the present invention and chemical drugs or Chinese medicine or natural drug form treats AIDS-treating medicine.
Detailed description of the invention
Below by specific experiment example and embodiment, the application of Herba Elephantopi Mollis extract in preparation treatment AIDS-treating medicine is described further, but is not limited to the present invention.
Embodiment 1: Herba Elephantopi Mollis extract and monomeric compound preparation
(1) Herba Elephantopi Mollis 10kg, with concentration 55% ethanol as solvent, extract at 25 DEG C of warm macerating, extraction time is 5 times, and each extraction time is 24 hours, and each solvent load is 23 times of medical material weight; Filter, merge extractive liquid, reclaim ethanol, concentrate drying, obtains Herba Elephantopi Mollis extract A;
(2) the Herba Elephantopi Mollis extract A that step (1) obtains is added 15 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with petroleum ether-ethyl acetate-methanol-water (2:3:1:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, namely obtain Herba Elephantopi Mollis extract B;
(3) by the medicinal liquid A that step (2) obtains, by polyamide column post, wash with water, then carry out gradient elution with the alcoholic solution of concentration 60%, collect 60% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains Herba Elephantopi Mollis extract C;
(4) above-mentioned Herba Elephantopi Mollis extract B and the mixing of Herba Elephantopi Mollis extract C, obtain Herba Elephantopi Mollis extract of the present invention.
Method described above prepares Herba Elephantopi Mollis extract B, again through 200-300 order silica gel column chromatography, with petroleum ether-ethyl acetate gradient elution (10:0.5-5:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, acetone-ethane (1:2) recrystallization, obtains zaluzanin C (Zaluzanin C) respectively; 9 Alpha-hydroxy-11 β, the other glycosides of 13-dihydro zaluzanin C 3-O-β-pyrans (9 α-Hydroxy-11 β, 13-dihydrozaluzanin C 3-O-β-allopyranoside) etc. composition, the chemical constitution of each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation above, and purity detects through high performance liquid chromatography and is all greater than 98.5%.
Method described above prepares Herba Elephantopi Mollis extract C, again through 200-300 order silica gel column chromatography, with chloroform-methanol elution gradient (100:1-2.5:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, recrystallizing methanol, obtain luteolin-7-O-β-D-Glucose glycosides respectively, the compositions such as hypericin (hypericin), the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.
Embodiment 2: Herba Elephantopi Mollis extract and monomeric compound preparation
(1) Herba Elephantopi Mollis 5kg, with concentration 50% ethanol as solvent, extract at 35 DEG C of warm macerating, extraction time is 3 times, and each extraction time is 12 hours, and each solvent load is 21 times of Herba Elephantopi Mollis weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Herba Elephantopi Mollis extract A;
(2) extract A that step (1) obtains is added 10 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with petroleum ether-ethyl acetate-methanol-water (2:2:1:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, namely obtain Herba Elephantopi Mollis extract B;
(3) by the medicinal liquid A that step (2) obtains, by polyamide column post, wash with water, then carry out gradient elution with the alcoholic solution of concentration 10%, collect 10% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains Herba Elephantopi Mollis extract C;
(4) above-mentioned Herba Elephantopi Mollis extract B, Herba Elephantopi Mollis extract C, wherein one or both mix by a certain percentage, obtain Herba Elephantopi Mollis extract of the present invention.
Method described above prepares Herba Elephantopi Mollis extract B, again through 200-300 order silica gel column chromatography, with petroleum ether-ethyl acetate gradient elution (10:0.5-5:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, acetone-ethane (1:2) recrystallization, obtains zaluzanin C (Zaluzanin C) respectively; 9 Alpha-hydroxy-11 β, the other glycosides of 13-dihydro zaluzanin C 3-O-β-pyrans (9 α-Hydroxy-11 β, 13-dihydrozaluzanin C 3-O-β-allopyranoside) etc. composition, the chemical constitution of each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation above, and purity detects through high performance liquid chromatography and is all greater than 98.5%.
Method described above prepares Herba Elephantopi Mollis extract C, again through 200-300 order silica gel column chromatography, with chloroform-methanol elution gradient (100:1-2.5:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, recrystallizing methanol, obtain luteolin-7-O-β-D-Glucose glycosides respectively, the compositions such as hypericin (hypericin), the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.
Embodiment 3: Herba Elephantopi Mollis extract and monomeric compound preparation
(1) Herba Elephantopi Mollis 10kg, with concentration 90% ethanol as solvent, extract at 10 DEG C of warm macerating, extraction time is 8 times, and each extraction time is 48 hours, and each solvent load is 25 times of Herba Elephantopi Mollis weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Herba Elephantopi Mollis extract A;
(2) extract A that step (1) obtains is added 20 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography (HSCCC), with petroleum ether-ethyl acetate-methanol-water (2:2:1:1) for solvent system, carry out separation and purification, recycling design, concentrate drying, namely obtain Herba Elephantopi Mollis extract B;
(3) by the medicinal liquid A that step (2) obtains, by polyamide column post, wash with water, then carry out gradient elution with the alcoholic solution of concentration 70%, collect 70% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains Herba Elephantopi Mollis extract C;
(4) above-mentioned Herba Elephantopi Mollis extract B, the mixing of Herba Elephantopi Mollis extract C, obtain Herba Elephantopi Mollis extract of the present invention.
Method described above prepares Herba Elephantopi Mollis extract B, again through 200-300 order silica gel column chromatography, with petroleum ether-ethyl acetate gradient elution (10:0.5-5:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, acetone-ethane (1:2) recrystallization, obtains zaluzanin C (Zaluzanin C) respectively; 9 Alpha-hydroxy-11 β, the other glycosides of 13-dihydro zaluzanin C 3-O-β-pyrans (9 α-Hydroxy-11 β, 13-dihydrozaluzanin C 3-O-β-allopyranoside) etc. composition, the chemical constitution of each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation above, and purity detects through high performance liquid chromatography and is all greater than 98.5%.
Method described above prepares Herba Elephantopi Mollis extract C, again through 200-300 order silica gel column chromatography, with chloroform-methanol elution gradient (100:1-2.5:1), thin layer tracing detection, merge same stream part, leave standstill crystallize, sucking filtration, recrystallizing methanol, obtain luteolin-7-O-β-D-Glucose glycosides respectively, the compositions such as hypericin (hypericin), the chemical constitution of above each compound is all through the wave spectrum such as mass spectrum and nuclear magnetic resonance, NMR means confirmation, and purity detects through high performance liquid chromatography and is all greater than 98%.
Experimental example 1: Herba Elephantopi Mollis extract Anti-HIV-1 Active is tested.
1. material
Cell: MT-4 cell, MAGI test(Multinuclear Activation of Galactosidase Indicator) also known as Single Life Cycle, cell used is Hela-CD4-LTR-β-gal.Beijing University of Technology's life sciences and Biological Engineering College cultivate and preserve.
Strain: HIV-1
b Beijing University of Technology life sciences and Biological Engineering College cultivate and preserve.
Medicine: the embodiment of the present invention 1, embodiment 2, embodiment 3 gained Herba Elephantopi Mollis extract, be numbered: Herba Elephantopi Mollis extract 1, Herba Elephantopi Mollis extract 2, Herba Elephantopi Mollis extract 3.
2. Herba Elephantopi Mollis extract anti-HIV-1 in MT4 cell
b activity test
Collected by centrifugation 5,000,000 MT-4 cell, adds the RPMI RPMI-1640 of lml containing 10000 TCID50 HIV-1 IIIB, 37 DEG C, 5% CO
22 hours are hatched in incubator.After 1500rpm is centrifugal, the washing of RPMI RPMI-1640 is once, centrifugal, and it is resuspended to add 10ml RPMI RPMI-1640, and the 96 every holes of orifice plate add 100 u1 cell virus suspensions (containing 5 × 10
4cells/well).Maximum drug concentration is selected to be 50 μ g/ml, 5 doubling dilution 4 concentration, the multiple hole of each concentration 4, every hole 100u1 (medicine final concentration reduces by half).Set up virus control group (only add virus, do not add medicine), cell controls group (do not add virus, do not add medicine) simultaneously.In 37 DEG C, 5% CO
2continue cultivation in incubator 5 days, period, within the 3rd day, change medicinal liquid.Day by day observation of cell growth and metamorphosis.Cell controls group cell growth state is good, and virus control group occurs syncytium in 3-4 days after cultivation.Cultivate the 5th day, results culture supernatant, to measure the method for p24 antigen, detect culture supernatant p24 antigenic content, to show the proliferative conditions of virus.Administration group and virus control group are compared, the suppression ratio with following formulae discovery medicine:
Suppression ratio=(virus control group p24 content-administration group p24 content) ÷ virus control group p24 content
Measure medicine to the inhibitory action of HIV-1 IIIB strain with P24 Detection of antigen method, the results are shown in Table 1.With the logarithm of drug level for abscissa, with to the suppression ratio of virus for vertical coordinate curve plotting, utilize rectilinear regression method to calculate and return and appoint a line, calculate each sample and suppress virus concentration (IC to 50% of HIV-1 IIIB strain
50).
Table 1 Herba Elephantopi Mollis extract in MT4 cell to the inhibitory action of HIV-1 IIIB strain.
3. the anti-HIV-1 IIIB activity test of Herba Elephantopi Mollis extract in MAGI cell
MAGI test is also known as single life cycle, reaction virus only carries out copying of one-period in this cell, copying of virus is synchronous, Different periods represents certain stage entering viral life cycle, therefore after virus inoculation, Different periods adds medicine, observe the inhibitory action of the different phase of drug on viral replicative cycle, can judge that drug effect is in the stage of virus function.One of cell used is derived by HeLa cell line.This law can the titre of quantitative assay HIV, to calculate the suppression ratio of drug on viral, and the phase of generation.
Get the every hole inoculation of 96 porocyte culture plate 0.6 ten thousand cell MAGI cells.Maximum drug concentration is selected to be 100 μ g/ml, 5 doubling dilution 5 concentration, the multiple hole of each concentration 4, every hole 100 ul.Set up virus control group (only add virus, do not add medicine), cell controls group (do not add virus, do not add medicine) simultaneously.By the sucking-off of each Kong Zhongpei base, in virus control group and drug test group, every hole virus inoculation liquid (2000 TCID
50) 100 u1.The medicinal liquid (medicine final concentration reduces by half) of the above-mentioned dilution of 100u1 is added while virus inoculation.37 DEG C, 5%CO
2hatch 40-48h, fixing, dyeing.Locus coeruleus number (see table 2) is counted under inverted microscope.
Table 2 Herba Elephantopi Mollis extract in MAGI cell to the inhibitory action of HIV-1 IIIB strain
Result shows: the maximum concentration of Herba Elephantopi Mollis extract can reach more than 95% to the suppression ratio of HIV-1 IIIB.
Claims (2)
1. containing zaluzanin C, 9 Alpha-hydroxy-11 β, the application of Herba Elephantopi Mollis extract in preparation treatment AIDS-treating medicine of the other glycosides of 13-dihydro zaluzanin C 3-O-β-pyrans, luteolin-7-O-β-D-Glucose glycosides, hypericin, described Herba Elephantopi Mollis extract prepares as follows:
(1) Herba Elephantopi Mollis, with concentration 55% ethanol as solvent, extract at 25 DEG C of warm macerating, extraction time is 5 times, and each extraction time is 24 hours, and each solvent load is 23 times of Herba Elephantopi Mollis weight; Filter, merge extractive liquid, reclaim ethanol, concentrated, dry, obtain Herba Elephantopi Mollis extract A;
(2) extract A that step (1) obtains is added 15 times amount water dissolutioies, filter, obtain medicinal liquid A and precipitate A; By precipitate A, adopt high-speed countercurrent chromatography, with the petroleum ether-ethyl acetate-methanol-water of portion rate 2:3:1:1 for solvent system, carry out separation and purification, recycling design, concentrate drying, namely obtain Herba Elephantopi Mollis extract B;
(3) by the medicinal liquid A that step (2) obtains, by polyamide column, wash with water, then carry out gradient elution with the alcoholic solution of concentration 60%, collect 60% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains Herba Elephantopi Mollis extract C;
(4) above-mentioned Herba Elephantopi Mollis extract B and the mixing of Herba Elephantopi Mollis extract C, obtain Herba Elephantopi Mollis extract.
2. application according to claim 1, is characterized in that the treatment AIDS-treating medicine that described Herba Elephantopi Mollis extract and chemical drugs or Chinese medicine or natural drug form.
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