CN103588834B - Extraction preparation method and the application thereof of onychin - Google Patents
Extraction preparation method and the application thereof of onychin Download PDFInfo
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Abstract
The invention discloses a kind of extraction preparation method of onychin, and treat the application in AIDS-treating medicine in preparation. Described onychin is prepared as follows: taking sivery aleuritopteris herb as raw material, use 10%-80% alcohol extract, obtain by macroporous absorbent resin, silica gel isochromatic spectrum purifying. Onychin provided by the present invention is significant to preparation treatment AIDS-treating medicine.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of extraction preparation method of onychin and the application in preparation treatment AIDS-treating medicine thereof.
Background technology
AIDS, i.e. acquired immunodeficiency syndrome, English name AcquiredImmuneDeficiencySyndrome, AIDS. The mankind because cause immune deficiency after infecting human immunodeficiency virus (HumanImmunodeficiencyVirus, HIV), concurrent a series of opportunistic infections and tumour, severe patient can cause dead syndrome. At present, AIDS has become the public health problem of serious threat people of the world health. Nineteen eighty-three, the mankind find HIV first. At present, AIDS becomes a kind of controlled chronic disease from a kind of fatal disease. HIV belongs to the mankind slow virus group in Retroviridae lentivirus, is divided into 1 type and 2 types. Major Epidemic HIV-1 in world wide at present. HIV-1 is diameter 100 ~ 120nm spheric granules, is made up of core and coating two parts. Core comprises two single-stranded RNA chains, core texture albumen and the necessary enzyme of virus replication, contains reverse transcriptase, integrase and protease. HIV-1 is the virus that a kind of variability is very strong, and nonstandard antiviral therapy is the major reason that causes viral resistance. HIV-2 is mainly present in West Africa, is all found at present on the U.S., Europe, South Africa, India and other places. The ultra microstructure of HIV-2 and cell tropism are similar to HIV-1, and its nucleotides is compared obviously different from HIV-1 with amino acid sequence.
The WHO report whole world in 2010 survival HIV carriers and AIDS patient totally 3,400 ten thousand, newly infect 2,700,000, annual dead 1,800,000 people. Have every day and exceed the new infection of 7000 people, district from all parts of the world all has popular, but more than 97% in, low income country, especially attach most importance to Africa. Expert's estimation, Asia may be shifted to from Africa in Global prevalence severely afflicated area. China CDC estimates, ends to the end of the year 2011 China survival HIV carriers and AIDS patient approximately 780,000 people, annual new the infected 4.8 ten thousand people, dead 2.8 ten thousand people. Epidemic situation has covered national all provinces, autonomous regions and municipalities, and China faces AIDS morbidity and dead peak period at present, and has been started to spread to population by people at highest risk such as drug abuse, unlicensed prostitutes.
The medical scholar of countries in the world is all devoted to research and discovery anti-AIDS new drug, constantly has new medicine try out or come out. Anti-AIDS drug mainly contains following a few class: ucleosides reverse transcription inhibitor, as retrovir (AZT, Zidovudine), Lamivudine etc.; Non-nucleoside reverse transcriptase inhibitor, as delavirdine, Nai Weila etc.; Protease inhibitors, as inverase, Ritonavir, that Wei of indoles etc.; Integrase inhibitor, as Zidovudine mononucleotide etc.; Invade depressant, as T20 etc. Retrovir be 1987 at the clinical inverase using at first, single or bigeminy treatment HIV infects, and because obvious drug resistance appearred in this treatment conventionally in 6 months, and not good to the immunologic reconstitution of body, thereby seldom obtains curative effect. Zidovudine, Lamivudine are dideoxyribonucleoside class, and primary treatment AIDS and related syndromes thereof reduce opportunistic infections, but still cannot effect a radical cure AIDS, and great majority have serious adverse reaction, the long-term or lifelong medication of need. That Wei of inverase, Ritonavir and indoles and ucleosides coupling can effectively suppress HIV and copy, and reduce bad reaction.
In addition, immunoregulation medicament, as interferon, interleukin 2 and gamma globulin etc., all has antiviral, bacterial-infection resisting and the immunoregulatory effect of enhancing. Wherein interleukin 2 also can make patient's lymphocyte number increase, and improves immune function of human body.
At present, treating AIDS has the cytostatics T20 that enters of United States advanced to treat, and T20 is a kind of peptide class, can not be oral, can only inject, and expensive. The annual medical expense of every patient is all more than 20000 dollars, and the costliness of medicine valency all cannot be effectively treated most AIDS patients.
Traditional Chinese medicine is a great treasure-house, the to succeed and develop motherland traditional medicine of several thousand, excavates medical treasure-house, utilizes modern compartment analysis means, filter out Chinese medical extract or the active ingredient of effective treatment AIDS, develop the active drug for the treatment of AIDS. The active component extracting from Chinese medicine and the composition reported at present have pinecone polysaccharide, lentinan, ginsenoside, trichosanthin, camptothecine, papaverine etc.
Sivery aleuritopteris herb is Sinopteridaceae plant aleuritopteris argentea Fee Aleuritopterisargentea(Gmel.) the dry herb of Fee. Gather autumn, removes impurity and dry. Record in 1998 " Drug Standard of Ministry of Public Health of the Peoples Republic of China anaesthetic fascicle ". Its property is flat, and mildly bitter flavor, has the effects such as improving eyesight, callus, hemostasis, promoting blood circulation for regulating menstruation, cough-relieving, dampness removing, removing toxicity for detumescence, can be used for hot eyes, hypopsia, thoracic injury, bone fracture, pulmonary tuberculosis cough, haematemesis. Hide the common drug of the minority areas such as illiteracy for China. But still very limited about the basic research of sivery aleuritopteris herb, the follow-up promotion and application of this medicinal material are restricted. Modern study shows, sivery aleuritopteris herb contains alepterolic acid (alepterolicacid), sucrose (sucrose) and flavone compound, and other chemical compositions are not quite clear; Pharmacological research shows, sivery aleuritopteris herb flavones is that a kind of natural effective free radical scavenger sivery aleuritopteris herb flavones has very strong scavenging action to hydroxyl radical free radical, ultra-oxygen anion free radical, hydrogen peroxide, its EC50Be respectively 0.0099,0.0060 and 0.0190mg/mL, and be concentration dependence, other pharmacological actions have no report.
Number of patent application is that the patent of CN96102241.8 discloses a kind of specific medicament prescription for the treatment of liver and bile stone and anti-recurrence, wherein contains sivery aleuritopteris herb. Number of patent application be CN200310110176.4 Patent Application Publication a kind of medicament composing prescription for the treatment of nasosinusitis is disclosed, wherein contain sivery aleuritopteris herb. Number of patent application is that the patent of CN200310110531.8 discloses a kind of herbal mediciment that is used for the treatment of alpastic anemia, and it is formulated that it adds white wine by nearly 20 kinds of Chinese herbal medicines such as HUISHENGCAO, Radix Rhodiolae, sivery aleuritopteris herbs through water infusion. Number of patent application is that the patent of CN97107327.9 discloses a kind of Chinese herbal medicine for the treatment of dysmenorrhes, and the composition of formula comprises: sivery aleuritopteris herb, rhizoma atractylodis, the membrane of a chicken's gizzard etc. Number of patent application is that the patent of CN201210440734.2 discloses a kind of medicine for the treatment of the optimum myositis of children acute, and its composition of raw materials and weight proportion are: 6～10 parts of sivery aleuritopteris herbs, 8～15 parts of luffas, 8～15 parts of laciniate blumea herbs etc. Number of patent application is that the patent of CN201210462718.3 discloses a kind of medicine for the treatment of breast disease, it is characterized in that component and weight proportion are: sivery aleuritopteris herb 1: Chinese lantern 2: the bighead atractylodes rhizome 1.5 etc.
Above-mentioned document and patent etc., there is not yet to extract from sivery aleuritopteris herb and prepare onychin and the report for the preparation for the treatment of AIDS-treating medicine.
Summary of the invention
The object of the invention is to the extraction preparation method of open a kind of onychin and the application in preparation treatment AIDS-treating medicine thereof.
The present invention is achieved through the following technical solutions:
The raw materials used sivery aleuritopteris herb of the present invention is Sinopteridaceae plant aleuritopteris argentea Fee (Aleuritopterisargentea(Gmel.) Fee) dry herb.
Sivery aleuritopteris herb extract of the present invention is to prepare as follows:
(1) sivery aleuritopteris herb, with concentration 10%-80% ethanol, as solvent, 30 DEG C of-90 DEG C of extractions, extraction time is 1-3 time, and each extraction time is 1-4 hour, and the 6-15 that each solvent load is sivery aleuritopteris herb weight is doubly; Filter, merge extract, reclaim ethanol, concentrated, dry, obtain sivery aleuritopteris herb extract A;
(2) sivery aleuritopteris herb extract A step (1) being obtained adds 10-20 times of water gaging and dissolves, filter, obtain liquid A, liquid A by large pore resin absorption column, washes with water, then carries out wash-out with the ethanolic solution of concentration 50%-70%, collect different ethanol eluates, reclaim ethanol, concentrate drying, obtains sivery aleuritopteris herb extract;
(3) sivery aleuritopteris herb extract step (2) being obtained is again through 200-300 order silica gel column chromatography, with chloroform-methyl alcohol (100:1-3:1) gradient elution, thin layer is followed the tracks of and is detected, merge same stream part, collect chloroform-methyl alcohol (10:1) gradient eluent, leave standstill crystallization, suction filtration, recrystallizing methanol, obtains onychin.
The chemical constitution of onychin is through the wave spectrum such as mass spectrum and nuclear magnetic resonance means confirmation, and purity detects and is all greater than 98% through high performance liquid chromatography.
Onychin: C27H32O13, M564, white, needle-shaped crystals (methyl alcohol), mp.279-280 DEG C; UV λ in ethanolic solutionmax: 213,286,330nm; IR:3420,3360,1655,1647,1632,1574,1500,994,978. EIMS:564.447,418.400(M-rhamnose), 368,354,256(M-rhamnose-glucose) and, 238,179,153,152,129,104,85,77,71.55;1HNMR(DMSO-d6,δ):12.459（s），7.548（d，7.6），7.548（d，7.6），7.412（t，7.6），7.412（t，7.6），7.364（t，7.6），6.731（d，1.6），6.672（d，1.6），6.377（s），5.696（d，7.6），5.40（dd，12.8），2.8，3.20（dd，17.2,12.8），2.888（dd，17.2,12.8）；13CNMR(DMSO-d6, δ): 196.52(s), 166.22(s), 164.43(s), 163.32(s), 79.40(d), 43.45(t), 97.85(d), 96.23(d), 104.35(s), 139.24(s), 126.75(d), 129.07(d), 128.98(d), 129.07(d), 126.75(d), 99.40(d), 77.80(d), 79.07(d), 74.01(d), 78.75(d), 62.03(t), 102.36(d), 72.31(d), 72.68(d), 71.10(d), 69.81(d), 18.78(q). The onychin (onychin) of above data and bibliographical information is consistent; TLC inspection, Rf value is identical with onychin standard items.
Preferred sivery aleuritopteris herb method for preparing extractive of the present invention:
(1) sivery aleuritopteris herb, with concentration 60% ethanol, as solvent, 70 DEG C of extractions, extraction time is 2 times, and each extraction time is 2 hours, and each solvent load is 12 times of sivery aleuritopteris herb weight; Filter, merge extract, reclaim ethanol, concentrated, dry, obtain sivery aleuritopteris herb extract A;
(2) sivery aleuritopteris herb extract A step (1) being obtained adds 15 times of water gagings and dissolves, filter, obtain liquid A, liquid A by HPD600 macroporous absorbent resin, washes with water, then carries out wash-out with the ethanolic solution of concentration 70%, collect 70% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains sivery aleuritopteris herb extract;
(3) sivery aleuritopteris herb extract step (2) being obtained is again through 200-300 order silica gel column chromatography, with chloroform-methyl alcohol (100:1-3:1) gradient elution, thin layer is followed the tracks of and is detected, merge same stream part, collect chloroform-methyl alcohol (10:1) gradient eluent, leave standstill crystallization, suction filtration, recrystallizing methanol, obtains onychin.
The chemical constitution of onychin is through the wave spectrum such as mass spectrum and nuclear magnetic resonance means confirmation, and purity detects and is all greater than 98% through high performance liquid chromatography.
Onychin: C27H32O13, M564, white, needle-shaped crystals (methyl alcohol), mp.279-280 DEG C; UV λ in ethanolic solutionmax: 213,286,330nm; IR:3420,3360,1655,1647,1632,1574,1500,994,978. EIMS:564.447,418.400(M-rhamnose), 368,354,256(M-rhamnose-glucose) and, 238,179,153,152,129,104,85,77,71.55;1HNMR(DMSO-d6,δ):12.459（s），7.548（d，7.6），7.548（d，7.6），7.412（t，7.6），7.412（t，7.6），7.364（t，7.6），6.731（d，1.6），6.672（d，1.6），6.377（s），5.696（d，7.6），5.40（dd，12.8），2.8，3.20（dd，17.2,12.8），2.888（dd，17.2,12.8）；13CNMR(DMSO-d6, δ): 196.52(s), 166.22(s), 164.43(s), 163.32(s), 79.40(d), 43.45(t), 97.85(d), 96.23(d), 104.35(s), 139.24(s), 126.75(d), 129.07(d), 128.98(d), 129.07(d), 126.75(d), 99.40(d), 77.80(d), 79.07(d), 74.01(d), 78.75(d), 62.03(t), 102.36(d), 72.31(d), 72.68(d), 71.10(d), 69.81(d), 18.78(q). The onychin (onychin) of above data and bibliographical information is consistent; TLC inspection, Rf value is identical with onychin standard items.
Sivery aleuritopteris herb extract of the present invention, its preparation method is characterised in that: the macroporous absorbent resin adopting is DM130 macroporous absorbent resin, D101 macroporous absorbent resin, HPD600 macroporous absorbent resin.
Sivery aleuritopteris herb extract of the present invention, can, by the various auxiliary materials that add pharmacy to allow, make the various peroral dosage forms in pharmacy.
The present invention's exploratory development is first taking Sinopteridaceae plant aleuritopteris argentea Fee (Aleuritopterisargentea(Gmel.) Fee) dry herb prepare onychin as raw material extracts, and be used for the treatment of AIDS. This experimental study shows, onychin prepared by the inventive method has obvious inhibitory action to HIV-1IIIB strain in MAGI cell, and when concentration is 50 μ g/ml, inhibiting rate is more than 90%; In MT4 cell, HIV-1IIIB strain is had to obvious inhibitory action, when concentration is 100 μ g/ml, inhibiting rate is more than 95%.
Onychin of the present invention and chemical drugs or Chinese medicine or natural drug composition treatment AIDS-treating medicine.
Detailed description of the invention
Below by specific experiment example and embodiment, the extraction preparation method to onychin and the application in preparation treatment AIDS-treating medicine thereof are described further, but are not limited to the present invention.
Embodiment 1: the preparation of sivery aleuritopteris herb extract and onychin
Taking Sinopteridaceae plant aleuritopteris argentea Fee (Aleuritopterisargentea(Gmel.) Fee) dry herb as raw material, adopt the preparation of following steps methods:
(1) sivery aleuritopteris herb 10kg, with concentration 60% ethanol, as solvent, 70 DEG C of extractions, extraction time is 2 times, and each extraction time is 2 hours, and each solvent load is 12 times of sivery aleuritopteris herb weight; Filter, merge extract, reclaim ethanol, concentrated, dry, obtain sivery aleuritopteris herb extract A;
(2) sivery aleuritopteris herb extract A step (1) being obtained adds 15 times of water gagings and dissolves, filter, obtain liquid A, liquid A by HPD600 large pore resin absorption column, washes with water, then carries out wash-out with the ethanolic solution of concentration 70%, collect 70% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains sivery aleuritopteris herb extract;
(3) sivery aleuritopteris herb extract step (2) being obtained is again through 200-300 order silica gel column chromatography, with chloroform-methyl alcohol (100:1-3:1) gradient elution, thin layer is followed the tracks of and is detected, merge same stream part, collect chloroform-methyl alcohol (10:1) gradient eluent, leave standstill crystallization, suction filtration, recrystallizing methanol, obtains onychin.
Chemical constitution is confirmed through the wave spectrum such as mass spectrum and nuclear magnetic resonance means:
Onychin: C27H32O13, M564, white, needle-shaped crystals (methyl alcohol), mp.279-280 DEG C; UV λ in ethanolic solutionmax: 213,286,330nm; IR:3420,3360,1655,1647,1632,1574,1500,994,978. EIMS:564.447,418.400(M-rhamnose), 368,354,256(M-rhamnose-glucose) and, 238,179,153,152,129,104,85,77,71.55;1HNMR(DMSO-d6,δ):12.459（s），7.548（d，7.6），7.548（d，7.6），7.412（t，7.6），7.412（t，7.6），7.364（t，7.6），6.731（d，1.6），6.672（d，1.6），6.377（s），5.696（d，7.6），5.40（dd，12.8），2.8，3.20（dd，17.2,12.8），2.888（dd，17.2,12.8）；13CNMR(DMSO-d6, δ): 196.52(s), 166.22(s), 164.43(s), 163.32(s), 79.40(d), 43.45(t), 97.85(d), 96.23(d), 104.35(s), 139.24(s), 126.75(d), 129.07(d), 128.98(d), 129.07(d), 126.75(d), 99.40(d), 77.80(d), 79.07(d), 74.01(d), 78.75(d), 62.03(t), 102.36(d), 72.31(d), 72.68(d), 71.10(d), 69.81(d), 18.78(q). The onychin (onychin) of above data and bibliographical information is consistent; TLC inspection, Rf value is identical with onychin standard items.
It is 99.7% that onychin purity detects through high performance liquid chromatography.
Embodiment 2: the preparation of sivery aleuritopteris herb extract and onychin
Taking Sinopteridaceae plant aleuritopteris argentea Fee (Aleuritopterisargentea(Gmel.) Fee) dry herb as raw material, adopt the preparation of following steps methods:
(1) sivery aleuritopteris herb 10kg, with concentration 10% ethanol, as solvent, 90 DEG C of extractions, extraction time is 1 time, and each extraction time is 4 hours, and each solvent load is 15 times of sivery aleuritopteris herb weight; Filter, merge extract, reclaim ethanol, concentrated, dry, obtain sivery aleuritopteris herb extract A;
(2) sivery aleuritopteris herb extract A step (1) being obtained adds 10 times of water gagings dissolves, and filters, and obtains liquid A, liquid A by DM130 large pore resin absorption column, washes with water, then carries out wash-out with the ethanolic solution of concentration 50%, collect ethanol eluate, concentrate drying, obtains sivery aleuritopteris herb extract;
(3) sivery aleuritopteris herb extract step (2) being obtained is again through 200-300 order silica gel column chromatography, with chloroform-methyl alcohol gradient elution (100:1-3:1), thin layer is followed the tracks of and is detected, merge same stream part, collect chloroform-methyl alcohol (10:1) gradient eluent, leave standstill crystallization, suction filtration, recrystallizing methanol, obtains onychin.
Chemical constitution is confirmed through the wave spectrum such as mass spectrum and nuclear magnetic resonance means:
Onychin: C27H32O13, M564, white, needle-shaped crystals (methyl alcohol), mp.279-280 DEG C; UV λ in ethanolic solutionmax: 213,286,330nm; IR:3420,3360,1655,1647,1632,1574,1500,994,978. EIMS:564.447,418.400(M-rhamnose), 368,354,256(M-rhamnose-glucose) and, 238,179,153,152,129,104,85,77,71.55;1HNMR(DMSO-d6,δ):12.459（s），7.548（d，7.6），7.548（d，7.6），7.412（t，7.6），7.412（t，7.6），7.364（t，7.6），6.731（d，1.6），6.672（d，1.6），6.377（s），5.696（d，7.6），5.40（dd，12.8），2.8，3.20（dd，17.2,12.8），2.888（dd，17.2,12.8）；13CNMR(DMSO-d6, δ): 196.52(s), 166.22(s), 164.43(s), 163.32(s), 79.40(d), 43.45(t), 97.85(d), 96.23(d), 104.35(s), 139.24(s), 126.75(d), 129.07(d), 128.98(d), 129.07(d), 126.75(d), 99.40(d), 77.80(d), 79.07(d), 74.01(d), 78.75(d), 62.03(t), 102.36(d), 72.31(d), 72.68(d), 71.10(d), 69.81(d), 18.78(q). The onychin (onychin) of above data and bibliographical information is consistent; TLC inspection, Rf value is identical with onychin standard items.
Onychin purity detects and is all greater than 98.8% through high performance liquid chromatography.
Embodiment 3: the preparation of sivery aleuritopteris herb extract and onychin
Taking Sinopteridaceae plant aleuritopteris argentea Fee (Aleuritopterisargentea(Gmel.) Fee) dry herb as raw material, adopt the preparation of following steps methods:
(1) sivery aleuritopteris herb, with concentration 80% ethanol, as solvent, 30 DEG C of extractions, extraction time is 3 times, and each extraction time is 1 hour, and each solvent load is 6 times of sivery aleuritopteris herb weight; Filter, merge extract, reclaim ethanol, concentrated, dry, obtain sivery aleuritopteris herb extract A;
(2) sivery aleuritopteris herb extract A step (1) being obtained adds 20 times of water gagings and dissolves, filter, obtain liquid A, liquid A by D101 large pore resin absorption column, washes with water, then carries out wash-out with the ethanolic solution of concentration 70%, collect 70% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains sivery aleuritopteris herb extract;
(3) sivery aleuritopteris herb extract step (2) being obtained is again through 200-300 order silica gel column chromatography, with chloroform-methyl alcohol (100:1-3:1) gradient elution, thin layer is followed the tracks of and is detected, merge same stream part, collect chloroform-methyl alcohol (10:1) gradient eluent, leave standstill crystallization, suction filtration, recrystallizing methanol, obtains onychin.
Chemical constitution is confirmed through the wave spectrum such as mass spectrum and nuclear magnetic resonance means:
Onychin: C27H32O13, M564, white, needle-shaped crystals (methyl alcohol), mp.279-280 DEG C; UV λ in ethanolic solutionmax: 213,286,330nm; IR:3420,3360,1655,1647,1632,1574,1500,994,978. EIMS:564.447,418.400(M-rhamnose), 368,354,256(M-rhamnose-glucose) and, 238,179,153,152,129,104,85,77,71.55;1HNMR(DMSO-d6,δ):12.459（s），7.548（d，7.6），7.548（d，7.6），7.412（t，7.6），7.412（t，7.6），7.364（t，7.6），6.731（d，1.6），6.672（d，1.6），6.377（s），5.696（d，7.6），5.40（dd，12.8），2.8，3.20（dd，17.2,12.8），2.888（dd，17.2,12.8）；13CNMR(DMSO-d6, δ): 196.52(s), 166.22(s), 164.43(s), 163.32(s), 79.40(d), 43.45(t), 97.85(d), 96.23(d), 104.35(s), 139.24(s), 126.75(d), 129.07(d), 128.98(d), 129.07(d), 126.75(d), 99.40(d), 77.80(d), 79.07(d), 74.01(d), 78.75(d), 62.03(t), 102.36(d), 72.31(d), 72.68(d), 71.10(d), 69.81(d), 18.78(q). The onychin (onychin) of above data and bibliographical information is consistent; TLC inspection, Rf value is identical with onychin standard items.
Onychin purity detects and is all greater than 99.5% through high performance liquid chromatography.
Experimental example 1 onychin Anti-HIV-1 Active test
1, material
Cell: MT-4 cell, MAGItest(MultinuclearActivationofGalactosidaseIndicator) claim that again SingleLifeCycle, cell used are Hela-CD4-LTR-β-gal. Beijing University of Technology's life science and Biological Engineering College cultivate and preserve;
Strain: HIV-1B Beijing University of Technology life science and Biological Engineering College cultivate and preserve;
Medicine: the embodiment of the present invention 1 gained onychin.
2, onychin anti-HIV-1 B activity test in MT4 cell
Centrifugal collection 5,000,000 MT-4 cells, add lml to contain the RPMI1640 nutrient solution of 10000TCID50HIV-1IIIB, in 37 DEG C, 5%CO2 incubator, hatch 2 hours. After 1500rpm is centrifugal, RPMI1640 nutrient solution washs once, centrifugal, adds 10mlRPMI1640 nutrient solution resuspended, and the 96 every holes of orifice plate add 100u1 cell virus suspension (containing 5X104Cells/well). Selecting medicine maximum concentration is 50 μ g/ml, 54 of doubling dilutions concentration, 4 multiple holes of each concentration, every hole 100u1 (medicine final concentration reduces by half). Set up virus control group (only add virus, do not add medicine), cell control group (do not add virus, do not add medicine) simultaneously. In 37 DEG C, 5%CO2 incubator, continue to cultivate 5 days, during this time, within the 3rd day, change liquid. Day by day observation of cell growth and metamorphosis. Cell cellular control unit growth conditions is good, and plasomidum appears for 3-4 days in virus control group after cultivating. Cultivate the 5th day, results culture supernatant, to measure the method for p24 antigen, detects culture supernatant p24 antigenic content, to show viral propagation situation. Administration group and virus control group are compared, calculate the inhibiting rate of medicine with following formula:
Inhibiting rate=(virus control group p24 content-administration group p24 content) ÷ virus control group p24 content
Measure the inhibitory action of medicine to HIV-1IIIB strain with P24 antigen detection method, the results are shown in Table 1. Taking the logarithm of drug concentration as abscissa, taking the inhibiting rate to viral as ordinate curve plotting, utilize linear regression method to calculate and return and appoint a line, calculate each sample and suppress virus concentration (IC to 50% of HIV-1IIIB strain50)。
Table 1 onychin is the inhibitory action to HIV-1IIIB strain in MT4 cell
3, the anti-HIV-1 IIIB activity test of onychin in MAGI cell
MAGItest claims again singlelifecycle, reaction virus is only carried out copying of one-period in this cell, copying of virus is synchronous, the different periods represent to enter certain stage of viral life cycle, therefore after virus inoculation, the different periods add medicine, observe the inhibitory action of the different phase of medicine to the virus replication cycle, can judge that drug effect is in the stage of virus function. One of cell used is to be derived by HeLa clone. This law can quantitative assay HIV titre, to calculate medicine to viral inhibiting rate. The time phase occurring.
Get the every hole inoculation of 96 porocyte culture plate 0.6 ten thousand cell MAGI cells. Selecting medicine maximum concentration is 100 μ g/ml, 55 of doubling dilutions concentration, 4 multiple holes of each concentration, every hole 100ul. Set up virus control group (only add virus, do not add medicine), cell control 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 (2000TCID50) 100u1. In virus inoculation, add the liquid (medicine final concentration reduces by half) of the above-mentioned dilution of 100u1. 37 DEG C, 5%CO2Hatch 40-48h, fixing, dyeing. Under inverted microscope, count locus coeruleus number (in table 2).
Table 2 onychin is the inhibitory action to HIV-1IIIB strain in MAGI cell
Shown by locus coeruleus count results: onychin has obvious inhibitory action to HIV-1IIIB.
Claims (4)
1. the application of onychin in preparation treatment AIDS-treating medicine.
2. application according to claim 1, is characterized in that described onychin is to prepare as follows:
(1) sivery aleuritopteris herb, with concentration 10%-80% ethanol, as solvent, 30 DEG C of-90 DEG C of extractions, extraction time is 1-3 time, and each extraction time is 1-4 hour, and the 6-15 that each solvent load is sivery aleuritopteris herb weight is doubly; Filter, merge extract, reclaim ethanol, concentrated, dry, obtain sivery aleuritopteris herb extract A;
(2) sivery aleuritopteris herb extract A step (1) being obtained adds 10-20 times of water gaging and dissolves, filter, obtain liquid A, liquid A by macroporous absorbent resin, washes with water, then carries out wash-out with the ethanolic solution of concentration 50%-70%, collect different concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains sivery aleuritopteris herb extract;
(3) sivery aleuritopteris herb extract step (2) being obtained is again through 200-300 order silica gel column chromatography, with chloroform-methyl alcohol gradient elution of portion rate 100:1-3:1, thin layer is followed the tracks of and is detected, merge same stream part, collect chloroform-methyl alcohol gradient eluent of portion rate 10:1, leave standstill crystallization, suction filtration, recrystallizing methanol, obtains onychin.
3. application according to claim 1, is characterized in that described onychin is to prepare as follows:
(1) sivery aleuritopteris herb, with concentration 60% ethanol, as solvent, 70 DEG C of extractions, extraction time is 2 times, and each extraction time is 2 hours, and each solvent load is 12 times of sivery aleuritopteris herb weight; Filter, merge extract, reclaim ethanol, concentrated, dry, obtain sivery aleuritopteris herb extract A;
(2) sivery aleuritopteris herb extract A step (1) being obtained adds 15 times of water gagings and dissolves, filter, obtain liquid A, liquid A by HPD600 macroporous absorbent resin, washes with water, then carries out wash-out with the ethanolic solution of concentration 70%, collect 70% concentration ethanol eluent, reclaim ethanol, concentrate drying, obtains sivery aleuritopteris herb extract;
(3) sivery aleuritopteris herb extract step (2) being obtained is again through 200-300 order silica gel column chromatography, with chloroform-methyl alcohol gradient elution of portion rate 100:1-3:1, thin layer is followed the tracks of and is detected, merge same stream part, collect chloroform-methyl alcohol gradient eluent of portion rate 10:1, leave standstill crystallization, suction filtration, recrystallizing methanol, obtains onychin.
4. application according to claim 2, the macroporous absorbent resin that the preparation method of the sivery aleuritopteris herb extract described in it is characterized in that adopts is DM130 macroporous absorbent resin, D101 macroporous absorbent resin or HPD600 macroporous absorbent resin.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4873250A (en) * | 1988-07-14 | 1989-10-10 | The University Of Mississippi | Antimicrobial compound and compositions particulary effective against candida albicans |
| CN1210715A (en) * | 1997-09-09 | 1999-03-17 | 衡阳医学院 | Medicine for curing malignant tumour |
| CN1430979A (en) * | 2002-01-10 | 2003-07-23 | 张国林 | Prescription combined with Chinese herbal medicine for treating AIDS |
| CN102093459A (en) * | 2011-01-10 | 2011-06-15 | 中国人民解放军第二军医大学 | Penthorum chinense pursh extract and preparation method and application thereof |
-
2013
- 2013-10-31 CN CN201310525386.3A patent/CN103588834B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4873250A (en) * | 1988-07-14 | 1989-10-10 | The University Of Mississippi | Antimicrobial compound and compositions particulary effective against candida albicans |
| CN1210715A (en) * | 1997-09-09 | 1999-03-17 | 衡阳医学院 | Medicine for curing malignant tumour |
| CN1430979A (en) * | 2002-01-10 | 2003-07-23 | 张国林 | Prescription combined with Chinese herbal medicine for treating AIDS |
| CN102093459A (en) * | 2011-01-10 | 2011-06-15 | 中国人民解放军第二军医大学 | Penthorum chinense pursh extract and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 通经草叶中粉背蕨酸的分离及含量测定;赵迎春 等;《沈阳药科大学学报》;20080131;第25卷(第1期);第52-54页 * |
| 金粉蕨素体外抗人卵巢癌作用研究;唐华贵 等;《医药论坛杂志》;20080815;第29卷(第15期);第4-6页 * |
| 金粉藏素抑制氧化应激诱导的血管内皮细胞凋亡;庹勤慧 等;《中国动脉硬化杂志》;20040530;第12卷(第3期);第283-286页 * |
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