CN1958057A - Application of extractive of Daliangjiang, and application in medication - Google Patents
Application of extractive of Daliangjiang, and application in medication Download PDFInfo
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Abstract
An application of the extract of alpinia galanga rhizome in preparing the Rev protein depressant for preventing and treating HIV infection and the medicine for preventing and treating the infection of tuberculous bacillus is disclosed. A process for preparing said extract is also disclosed.
Description
Technical field
The present invention relates to alcohol extract, the supercritical CO of Zingiberaceae Zingiberaceae Alpinia plants Fructus Galangae Alpinia galanga Willd. rhizome Rhizoma Alpiniae Galangae
2Extraction extract and water vapour distillation thing and extraction preparation method, the preparation that the main component of Rhizoma Alpiniae Galangae extract and method of quality control and Rhizoma Alpiniae Galangae extract are made and the application in preparation novel targets anti-HIV infection medicine of Rev protein inhibitor and anti-tubercle bacillus infection medicine.
Background technology
Rhizoma Alpiniae Galangae has another name called Rhizoma Alpiniae Galangae, Rhizoma Alpiniae Officinarum, Alpinia japonica (Thunb.) Miq., Alpinia japonica (Thunb.) Miq. head, Rhizoma alpiniae chinensis, is Zingiberaceae Zingiberaceae Alpinia plants Fructus Galangae Alpinia galanga Willd. rhizome.Rhizoma Alpiniae Galangae is grown in the torrid zone, subtropical climate, mainly is distributed in the provinces and regions such as Guangdong, Guangxi, Hainan, Taiwan, Yunnan of China.
Rhizoma Alpiniae Galangae (Fructus Galangae) is recorded in " property of medicine opinion " the earliest, calls it and cures mainly: " cold air stomachache, the miasma that disappears mist poison removes dyspepsia, and warm abdomen intestinal is vomited and diarrhoea dysentery.Bitter, hot, it is thick that polyphagia makes us tongue, anorexia." put down in writing according to book on Chinese herbal medicine afterwards; have the situation that Rhizoma Alpiniae Galangae (Fructus Galangae) and this platymiscium (particularly with Rhizoma Alpiniae Officinarum) are used with ancient times; book on Chinese herbal medicine thinks that Fructus Galangae and Rhizoma Alpiniae Officinarum derive from same plant more; Fructus Galangae is a fruit; Rhizoma Alpiniae Officinarum is a root; as: " Kaibao Bencao " said: " the living South Sea Zhu Gu of Fructus Galangae, Rhizoma Alpiniae Officinarum is also ... ", " Zhenglei Bencao " said." Fructus Galangae ... cloud is a FRUCTUS GALANGAE ... " etc., thus Compendium of Material Medica is merged into one to Fructus Galangae and Rhizoma Alpiniae Officinarum.
According to textual criticism, use Rhizoma Alpiniae Galangae (Fructus Galangae) and this platymiscium (particularly with Rhizoma Alpiniae Officinarum) ancient times with, modern relevant survey result also shows, at present Rhizoma Alpiniae Galangae also with beak Zingiber zerumbet Rhynchanthus beesinanus, Fructus Alpiniae Oxyphyllae Alpinia oxyphylla Miq., often together mixed up Rhizoma Alpiniae Officinarum apart from Zingiber zerumbet A Calcarata Rosc. rhizome and used.But modern relevant result of study shows that Rhizoma Alpiniae Galangae and above easy adulterant no matter are aspect tissue morphology, appearance character, microscopical identification, physicochemical identification, main component and content or all there were significant differences at aspects such as pharmacologically actives.
In China, do " Fructus Galangae " except being used for picking fruit on a small quantity, Rhizoma Alpiniae Galangae is not seen Rhizoma Alpiniae Galangae standardized planting in blocks as yet mainly by the making that is used for diet flavoring agent among the people, and main dependence is born in shady wet sylvan life of hill cheuch or the bushes and the Rhizoma Alpiniae Galangae wild resource in the thick grass." Guangxi Chinese medicinal herbal " record Rhizoma Alpiniae Galangae has " warming stomach for dispelling cold analgesic effect.Be used to control heart peratodynia, the cold and dyspeptic vomiting and diarrhea of cold syndrome of the stomach." China is among the people with Rhizoma Alpiniae Galangae treatment digestive tract disease such as gastric ulcer, dyspepsia, large intestine disease etc.; but mostly be as a kind of special flavor seasoning product and the composition of drug matching, lack systematic studys such as its extract and preparation thereof, analysis of effective component, pharmacological effect, safeties always.
Contain volatile oil, flavones ingredient and starch, protein, fat, inorganic salt, minor metallic element etc. in the Rhizoma Alpiniae Galangae.Flavone component Zhu in the Rhizoma Alpiniae Galangae is Yaoed Wei Quercetin, Quercetin-3-methyl ether, nimbecetin, kaempferol, isorhamnetin, galangin, galangin-3-methyl ether etc.Volatile oil component in the Rhizoma Alpiniae Galangae is mainly 1 '-acetoxychavicol acetate, 1 '-acetoxyl group Eugenol acetate, 1 '-hydroxyl chavicol-acetate, methyl cinnamate, cineole, Camphora, pinene, eugenol, sesquiterpene, sesquiterpenoid and sesquialter terpene aldehyde etc., wherein main component is 1 '-acetoxychavicol acetate, account for more than 90% of volatile oil relative amount, also be key component (the yang XG that Rhizoma Alpiniae Galangae has unique pungent fragrance, [J] .J Agric FoodChem, 1999,47:1657～1662), has antitumor (Moffatt J, A, etal.[J] .Carcinogenesis, 2000,21:2151-2157; Zheng Q et al.[J] .J Cancer ResClin Oncol, 2002,128:539～546), antibacterium (Janssen A M, [J] .plantaMed, 1985 (6): 507～511), antifungal (Ficker C E, et al.[J] .JEthnophamacal, 2003,85:589～593), antiulcer (Mitsuis, et al.[J] .Chem PhamBull, 1976,24:2377～2382), antioxidation (Kubota K, et al.[J] .Spec Publ R SocChem, 2001,274:601～607), antiinflammatory (Nakamura Y, [J] .Cancer Res, 1998,58:4832-4839) wait biological activity, do not see Rhizoma Alpiniae Galangae as yet and extract the report of anti-HIV infection and tuberculosis effect.
At present, HIV (human immunodeficiency virus) (HIV) infects the Therapeutic Method research that causes acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS)) and mainly concentrates on the limited drug target, i.e. the reverse transcriptase of HIV, hiv protease, hiv integrase.Many known anti-HIV infect chemical compounds and not only have serious toxic and side effects, and the toleration of medicine and compliance are also worse and worse.
According in November, 2006 global epidemiology investigation report demonstration, the HIV sufferers in the whole world has 30% to merge tuberculosis infection approximately, the some areas in Africa more reach 60%, these two kinds of infection can be mutually promoted, M ﹠ M is quite high, and all be prone to situations such as drug resistance, adverse effect be more, make the tuberculosis prevention and treatment difficulty aggravation of HIV sufferers.Report points out that the wide-scale distribution of acquired immune deficiency syndrome (AIDS) and popular rapidly causes the number of tuberculosis and acquired immune deficiency syndrome (AIDS) double infection to increase in recent years, and the control of acquired immune deficiency syndrome (AIDS) and tuberculosis complication should attract great attention.And present antituberculotics toxic and side effects is big, and toleration is poor, influences effective control lungy.Therefore, develop the anti-HIV infection medicine of safe and effective novel targets and prevent and treat the task of top priority that the tuberculosis medicine becomes containment acquired immune deficiency syndrome (AIDS) and tuberculosis complication.
The breeding of HIV needs the effective expression of its gene, and the effective expression of HIV gene depends on its a kind of adjusting albumen-Rev albumen.Rev albumen increases mRNAs the gathering in cytoplasm of HIV, the latter is through from nucleus to cytoplasmic transmission and the structural protein of the HIV that encodes, because Rev albumen is the key factor of HIV breeding, suppress the Rev protein function and can block the HIV breeding, therefore, the Rev protein inhibitor becomes a kind of new treatment strategy with bright prospects, sees Fig. 1.
Because Rev albumen is a kind of albumen that does not have the corresponding body of cell of HIV, so several distinct methods that suppress the Rev protein function are used to suppress the research of HIV breeding.The effect of antisense oligonucleotide inhibition Rev protein function has been used to suppress HIV (Matsukura, M.et al.1989) in the chronic infection cell.The proteic dominance of the Rev of HIV shifts variation (Bevec, D.et al.1992), the different (Bevec of the cloudy transformation of the dominance of the collaborative sub-eIF-5A of the Rev albumen of cell, D.et al.1996), the cell inner expression (Duan that suppresses Rev albumen single-chain antibody structure, L.et al.1994) and RRE mutagenic agent (Lee, L.et al.1992) all show the effect that HIV infects that suppresses in cell.Neomycin is found the combination that can block Rev-RRE and has the Rev protein function effect (Zapp, M.L.et al.1993) of inhibition; XIAONIE mycin B is proved to have that the outer output signal of blocking-up Rev proteic nuclear combines with receptor protein CRM1 and the breeding of HIV is demonstrated powerful inhibitory action (Wolff, B.et al.1997).Although neomycin and XIAONIE mycin have a significant biological activity external, all owing to toxicity is not suitable for developing into medicine especially greatly.Therefore, screening safety.Effectively the anti-HIV infection medicine of Rev protein inhibitor novel targets is extremely important for the containment acquired immune deficiency syndrome (AIDS).
Goal of the invention
Anti-HIV infection medicine of Rev protein inhibitor novel targets and antituberculotics that screening is safe, effective from natural product, anti-drug resistance is strong reach the purpose that overcomes anti-HIV infection and safety of tuberculosis chemicals and toleration difference.
Summary of the invention
For achieving the above object, we have set up a kind of quick, accurate, the micro-anti-HIV infection medicine of fusion yeast Schizosa-ccharomyces pombe (S.pombe) Rev protein inhibitor novel targets screening model.This primary yeast can be expressed the albumen that the outer output signal (NES) of glutathione S-transferase (GST), the antigenic nuclear localization signal of SV40T (NLS), green fluorescent albumen (GFP) and the proteic nuclear of Rev merges.
We adopt this screening model that the extract of more than 700 kinds of marine organisms and natural plants is carried out the anti-HIV infection medicine of Rev protein inhibitor novel targets screening test, found that the extract of Zingiberaceae Zingiberaceae Alpinia plants Fructus Galangae Alpinia galanga Willd. rhizome Rhizoma Alpiniae Galangae has extremely significant anti-HIV infection activity.Further result of the test shows, the Rhizoma Alpiniae Galangae extract not only has remarkable inhibitory action to clinical up-to-date isolating HIV-1 strain and clinical up-to-date isolating HIV-1 variation strain, but also have good synergism, and do not show any cytotoxicity with known anti-HIV infection medicine.Simultaneously, find that also the Rhizoma Alpiniae Galangae extract has the tuberculosis activity.
Rhizoma Alpiniae Galangae extract of the present invention is the ethanol of Zingiberaceae Zingiberaceae Alpinia plants Fructus Galangae Alpinia galanga Willd. rhizome Rhizoma Alpiniae Galangae and methanolic extract, supercritical CO
2Extraction extract and water vapour distillation thing and extraction preparation method thereof, the application of the preparation that the main component of Rhizoma Alpiniae Galangae extract and method of quality control and Rhizoma Alpiniae Galangae extract are made in preparation novel targets anti-HIV infection medicine of Rev protein inhibitor and antituberculotics.
The specific embodiment
1. Rhizoma Alpiniae Galangae ethanol extraction and preparation method
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, extract 2 times with 2～10 times of amount alcohol heating reflux of 50%～90%, and each 1～3 hour, merge secondary raffinate, decompression recycling ethanol gets extracting solution; Extracting solution extracts 1～3 time with the normal hexane-ethyl acetate extraction agent of 1～5 times of amount 2～10: 1, and the reclaim under reduced pressure extractant is collected extract; Extract is an immobile phase with the silica gel column chromatography, and with 2～10: normal hexane-ethyl acetate of 1 is the separation and purification of eluant eluting, collects eluent, and the reduced vacuum drying promptly gets the Rhizoma Alpiniae Galangae ethanol extraction.
2. Rhizoma Alpiniae Galangae methanolic extract and preparation method
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, with 5～10 times of methanol heating and refluxing extraction 2 times, and each 2 hours, merge secondary raffinate, reclaim under reduced pressure methanol must extracting solution; Extracting solution extracts 1～3 time with the normal hexane-ethyl acetate extraction agent of 1～5 times of amount 2～10: 1, and the reclaim under reduced pressure extractant is collected extract; Extract is an immobile phase with the silica gel column chromatography, and with 2～10: normal hexane-ethyl acetate of 1 is the separation and purification of eluant eluting, collects eluent, and the reduced vacuum drying promptly gets the Rhizoma Alpiniae Galangae methanolic extract.
3. Rhizoma Alpiniae Galangae supercritical CO
2Extraction extract and preparation method
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder and exists, and puts supercritical CO
2In the extractor, extraction temperature is 30 ℃～50 ℃, and extracting pressure is 10MPa～20MPa, and separating pressure is 10MPa～20MPa, and separation temperature is 30 ℃～50 ℃, and the extract and separate time is 2～6 hours, collects extract; Extract is an immobile phase with the silica gel column chromatography, and with 2～10: normal hexane-ethyl acetate of 1 is the separation and purification of eluant eluting, collects eluent, and the reduced vacuum drying promptly gets the Rhizoma Alpiniae Galangae supercritical CO
2The extraction extract.
4. Rhizoma Alpiniae Galangae water vapour distillation thing and preparation method
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, adds 2～10 times of water gagings, and heating distillation extraction 1～6 hour is collected distillate; Distillate is an immobile phase with the silica gel column chromatography, and with 2～10: normal hexane-ethyl acetate of 1 is the separation and purification of eluant eluting, collects eluent, and the reduced vacuum drying promptly gets Rhizoma Alpiniae Galangae water vapour distillation thing.
5. the conclusive evidence of main component in the Rhizoma Alpiniae Galangae extract
(1) the refining purification of main component in the Rhizoma Alpiniae Galangae extract
With preparation liquid-phase refining purification main component.Preparation condition is mobile phase for being filler with octadecylsilane chemically bonded silica with methanol-water (50: 50), uses preparative column, gets Rhizoma Alpiniae Galangae extract dissolve with methanol, injects preparative liquid chromatograph, collects separation and purification purification product at 254nm wavelength place, promptly.
(2) conclusive evidence of main component in the Rhizoma Alpiniae Galangae extract
Adopt elementary analysis, ultra-violet absorption spectrum, infrared absorption spectroscopy, high performance liquid chromatography, mass spectrum, nuclear magnetic resonance spectroscopy analysis to resolve, the refining purification product of proving conclusively above-mentioned Rhizoma Alpiniae Galangae extract are 1 '-acetoxychavicol acetate, and structural formula is
Molecular formula is C
13H
14O
4, molecular weight is 234.25, sees Fig. 2～Figure 12.
6. Rhizoma Alpiniae Galangae extraction of substance amount control method
(1) character: the Rhizoma Alpiniae Galangae extract is a grease.In methanol, dissolve.
(2) differentiate: get the need testing solution under the assay item, measure according to an appendix VA of Chinese Pharmacopoeia version in 2005 ultraviolet visible spectrophotometry, need testing solution has absorption maximum at the wavelength place of 204nm and 254nm.
(3) assay: according to an appendix VD of Chinese Pharmacopoeia version in 2005 high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler, is mobile phase with methanol-water (50: 50), and the detection wavelength is 254nm.
Algoscopy: get Rhizoma Alpiniae Galangae extract 2ml, with dissolve with methanol and be transferred in the 10ml measuring bottle, add methanol and be diluted to scale, shake up, as need testing solution, precision is measured need testing solution 20 μ l, inject chromatograph of liquid, the record chromatogram with the peak area sum of two adjacent main peaks, calculates 1 '-acetoxychavicol acetate (C in the Rhizoma Alpiniae Galangae extract by normalization method
13H
14O
4) content, promptly.
7. the preparation made of Rhizoma Alpiniae Galangae extract
Adopt the conventional formulation technology, the Rhizoma Alpiniae Galangae extract is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
8. the application of Rhizoma Alpiniae Galangae extract in the anti-HIV infection medicine of preparation novel targets Rev protein inhibitor
(1) is used to detect the preparation of the inhibiting fusion yeast bacterium of Rev protein function SchizosaccharomycesPombe.
Schizosaccharomyces Pombe (s.pombe) fusion yeast bacterium is used to this research.The pREP1 plasmid that contains wild type nmt1 promoter (this promoter can effectively guide and transcribe) in not having the culture fluid of vitamin B1 is used to the saccharomycetic conversion of s.pombe.In order to see the transmission between the cell caryoplasm in the s.pombe yeast, the protein-contg gene that melts of the outer output signal (NES) of coding for glutathion s-transferring enzyme (GST), the antigenic nuclear localization signal of SV40T (NLS), green fluorescent albumen (GFP) and the proteic nuclear of Rev is directed in the s.pombe yeast.Usually, can both see green fluorescent at nucleus and Cytoplasm, when conveying was suppressed outside the albuminous cell nuclear, green fluorescent concentrated on nucleus, and Cytoplasm almost be cannot see fluorescent, thereby can be used for Rev protein inhibitor in-vitro screening model, sees Figure 13.
(2) Rhizoma Alpiniae Galangae extract blocking-up REV albumen transmission effect
Get introduce fusion rotein the s.pombe yeast in the culture fluid that lacks VitB1 30 ℃ cultivated 3 hours, the dimethyl sulfoxide solution that adds 1% Rhizoma Alpiniae Galangae extract, mixing, under 30 ℃ of conditions, continue to cultivate 3 hours, under luminescence microscope, detect the fluorescent distribution situation of GST-NLS-GFP-REV NES fusion rotein.
(3) the anti-HIV infection effect of Rhizoma Alpiniae Galangae extract
1. the preparation of cell culture and HIV-1 NL4-3, JR-CSF virus liquid: get human T lymphocyte's strain MT
4Cell, H9 cell are put in the RPMI RPMI-1640 of 10% hyclone (FBS), add 50 μ g/mlNormocin, 50 μ g/ml G-418 and cultivate; Other gets human peripheral blood mononuclear cell (PBMC) and cultivates in the RPMI RPMI-1640 that contains 20% hyclone (FBS) and 4 μ g phytohaemagglutinin (PHA).After three days, the PBMC that PHA was excited washes 3 times with PBS (phosphate buffer), cultivates in the culture fluid that contains 20%FBS and 10U/ml interleukin-2 then.Plasmid PNL4-3, π SVJR-CSF express the full-length gene of HIV-1 in people's epithelium 293T cell.After plasmid pNL4-3, π SVJR-CSF change the 293T cell over to three days, the collecting cell culture fluid was measured the generation of virus with P24 ELISA (elisa).
2. P24 ELISA: the HIV-1 P24 ELISA reagent of producing with Perkin Elmer company is measured the amount of the HIV-1 P24 in the cell culture fluid.
3. antiviral activity is measured: MT
4Cell, H9 cell, PBMC cell be infected by HIV-1 IIIB, NL4-3 and JR-CSF virus respectively.Wash cell 2 times with PBS, add the viral liquid of fresh cultured and the Rhizoma Alpiniae Galangae extract solution of variable concentrations then, cultivate the generation that detects P24 in the cell culture fluid after 7 days, and observe the synergism of Rhizoma Alpiniae Galangae extract and its anti-HIV infection medicine of base.
4. Rhizoma Alpiniae Galangae extract cytotoxic assay: the Cell Titer 96aqueous cell proliferstion assay that produces with Promega company detects the cytotoxicity of Rhizoma Alpiniae Galangae extract.Every hole adds 1 * 10 in 96 hole plate culture plates
5Individual cell, the Rhizoma Alpiniae Galangae extract solution that adds variable concentrations then, cultivate after 48～72 hours, every hole adds the mixed liquor of 20 μ l MTS (2ml) t phenazine methosulfates (PMS), 100 μ l, continuation was cultivated 3 hours under 37 ℃ of conditions, with the trap of spectrophotometer, calculate cytotoxicity at the wavelength place of 490nm mensuration culture fluid.
9. the application of Rhizoma Alpiniae Galangae extract in preparation anti-mycobacterium tuberculosis infection medicine
(1) minimal inhibitory concentration (MIC) is measured: get tubercule bacillus, bacillus tuberculosis bovis, strong malicious Bacillus tuberculosis (H37RV) culture fluid and be inoculated in the simple synthetic medium test tube of tubercule bacillus, the Rhizoma Alpiniae Galangae extract methanol solution that adds variable concentrations, shake up, cultivated 14 days for 37 ℃, observe each duct ligation nuclear bacillus growing state, calculate the minimal inhibitory concentration (MIC) of Rhizoma Alpiniae Galangae extract each tubercule bacillus.
(2) the Rhizoma Alpiniae Galangae extract is to infecting the protective effect of tubercule bacillus mice: getting body weight is that 16～18g is through the negative mice of tuberculosin result of the test; male and female half and half; random packet behind every intravenous injection inoculation H37RV tubercule bacillus culture fluid 0.2ml; mice gives the Rhizoma Alpiniae Galangae extract at the 3rd day beginning oral administration gavage that vein infects; every day 3 times; for three days on end, observe the death time T of half animal
50, and with normal saline blank group and isoniazid positive controls relatively.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, extract 2 times with 8 times of amount alcohol heating reflux of 75%, and each 2 hours, merge secondary raffinate, decompression recycling ethanol gets extracting solution; Extracting solution extracts 3 times with 10: 1 normal hexane of equivalent-ethyl acetate extraction agent, and the reclaim under reduced pressure extractant is collected extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 10: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 1.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 1 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, extract 2 times with 5 times of amount alcohol heating reflux of 80%, and each 1.5 hours, merge secondary raffinate, decompression recycling ethanol gets extracting solution; Extracting solution extracts 2 times with 5: 1 normal hexane of 2 times of amounts-ethyl acetate extraction agent, and the reclaim under reduced pressure extractant is collected extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 5: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 2.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 2 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, extract 2 times with 10 times of amount alcohol heating reflux of 50%, and each 3 hours, merge secondary raffinate, decompression recycling ethanol gets extracting solution; Extracting solution extracts 3 times with 2: 1 normal hexane of 5 times of amounts-ethyl acetate extraction agent, and the reclaim under reduced pressure extractant is collected extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 2: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 3.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 3 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, extract 2 times with 2 times of amount alcohol heating reflux of 90%, and each 1 hour, merge secondary raffinate, decompression recycling ethanol gets extracting solution; Extracting solution extracts 1 time with 8: 1 normal hexane of 3 times of amounts-ethyl acetate extraction agent, and the reclaim under reduced pressure extractant is collected extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 8: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 4.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 4 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, with 8 times of amount methanol heating and refluxing extraction 2 times, and each 2 hours, merge secondary raffinate, decompression recycling ethanol must extracting solution; Extracting solution normal hexane-ethyl acetate extraction of 10: 1 of equivalent 3 times, the reclaim under reduced pressure extractant is collected extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 10: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 5.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 5 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, with 5 times of amount methanol heating and refluxing extraction 2 times, and each 2 hours, merge secondary raffinate, decompression recycling ethanol must extracting solution; Extracting solution is with normal hexane-ethyl acetate extraction 2 times of 5: 1 of 3 times of amounts, and the reclaim under reduced pressure extractant is collected extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluting eluant with normal hexane-ethyl acetate of 5: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 6.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 6 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, with 10 times of amount methanol heating and refluxing extraction 2 times, and each 2 hours, merge secondary raffinate, decompression recycling ethanol must extracting solution; Extracting solution is with normal hexane-ethyl acetate extraction 3 times of 2: 1 of 5 times of amounts, and the reclaim under reduced pressure extractant is collected extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 2: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 7.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 7 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, with 3 times of amount methanol heating and refluxing extraction 2 times, and each 2 hours, merge secondary raffinate, decompression recycling ethanol must extracting solution; Extracting solution is with normal hexane-ethyl acetate extraction 1 time of 8: 1 of 2 times of amounts, and the reclaim under reduced pressure extractant is collected extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 8: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 8.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 8 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Embodiment 9
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, puts supercritical CO
2In the extractor, extraction temperature is 33 ℃, extracting pressure 15Mpa, and separating pressure 10Mpa, 40 ℃ of separation temperatures, the extract and separate time is 4 hours, collects extract; Extract is immobile phase with the silica gel column chromatography, is the eluant separation and purification with normal hexane-ethyl acetate of 10: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 9.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 9 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, puts supercritical CO
2In the extractor, extraction temperature is 40 ℃, extracting pressure 10Mpa, and separating pressure 15Mpa, 35 ℃ of separation temperatures, the extract and separate time is 2 hours, collects extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 5: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 10.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 10 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Embodiment 11
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, puts supercritical CO
2In the extractor, extraction temperature is 45 ℃, extracting pressure 20Mpa, and separating pressure 20Mpa, 50 ℃ of separation temperatures, the extract and separate time is 5 hours, collects extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 2: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 11.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 11 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Embodiment 12
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, puts supercritical CO
2In the extractor, extraction temperature is 50 ℃, extracting pressure 15Mpa, and separating pressure 20Mpa, 40 ℃ of separation temperatures, the extract and separate time is 6 hours, collects extract; Extract is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 8: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 12.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 12 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Embodiment 13
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, adds 10 times of water gagings, and heating distillation extraction 3 hours is collected distillate; Distillate is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 10: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 13.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 13 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, adds 8 times of water gagings, and heating distillation extraction 1 hour is collected distillate; Distillate is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 5: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 14.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 14 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Embodiment 15
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, adds 5 times of water gagings, and heating distillation extraction 5 hours is collected distillate; Distillate is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 8: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 15.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 15 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Get Rhizoma Alpiniae Galangae, chopping is ground into coarse powder, adds 2 times of water gagings, and heating distillation extraction 6 hours is collected distillate; Distillate is immobile phase with the silica gel column chromatography, is the separation and purification of eluant eluting with normal hexane-ethyl acetate of 2: 1, collects eluent, and the reduced vacuum drying gets good greatly extract 16.Adopt the conventional formulation method that Rhizoma Alpiniae Galangae extract 16 is made soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, injection.
Industrial applicibility
1. the 1 '-acetoxy-chavicol ethyl ester (C in Rhizoma Alpiniae Galangae extract extract yield, extract identification check and the extract
13H
14O
4) assay the results are shown in Table 1.
Table 1. Rhizoma Alpiniae Galangae extract extract yield, discriminating, assay result
2. the anti-HIV infection effect of Rhizoma Alpiniae Galangae extract
(1) inhibitory action of pair cell infected by HIV-1: with HIV standard virus IIIB strain 100TCID
50Virus quantity infects MT
4, three kinds of cells of H9, PBMC.The Rhizoma Alpiniae Galangae extract is as follows with the antiviral result of contrast medicine zidovudine (AZT) variable concentrations:
1. MT
4Medium effective concentration (the IC of cell Rhizoma Alpiniae Galangae extract
50) be 2.2 micromoles, selection index (SI) is 25, sees Figure 14.
2. the IC of H9 cell Rhizoma Alpiniae Galangae extract
50Be 17 micromoles, therapeutic index is 15.9, sees Figure 15.
3. the IC of PBMC cell Rhizoma Alpiniae Galangae extract
50Be 6.2 micromoles, SI is 6.4, sees Figure 16.
(2) influence of pair cell infected by HIV-1 time: with HIV-1 standard virus III-B strain 20TCID
50Virus quantity infects MT
4, H9, PBMC cell.The infective virus different time is measured the antigenic amount of P24 in three kinds of cell culture fluids, sees Figure 17, Figure 18, Figure 19.The result shows that the antigenic amount of P24 has reached stable after infective virus 5-7 days.So the cell culture antivirus test is selected infective virus 5-7 days, measure the antigenic amount of P24 in the cell culture fluid.
With HIV-1 NL4-3, HIV-1 JR-CSF while infection of PBMCs cell, the result shows that the Rhizoma Alpiniae Galangae extract is to X
4HIV-1 and R
5HIV-1 all has extremely strong inhibitory action, and HIV-1 infected in 24 hours, and the Rhizoma Alpiniae Galangae extract shows extremely strong antivirus action, sees Figure 20.
(3) Rhizoma Alpiniae Galangae extract and the anti-HIV infection medicine interaction of removing the hydroxyl glycosides: select the anti-HIV-1 reverse transcriptase inhibitors to remove hydroxyl glycosides (ddI) and Rhizoma Alpiniae Galangae extract, result of the test shows, the Rhizoma Alpiniae Galangae extract obviously strengthens the antiviral effect of ddI, have good synergism, see Figure 21.
(4) cell toxicity test of Rhizoma Alpiniae Galangae extract: the Rhizoma Alpiniae Galangae extract is to MT
4, H9 and PBMC cell toxicity test the results are shown in Figure 22, Figure 23, Figure 24, the result shows that the Rhizoma Alpiniae Galangae extract does not have obvious cytotoxicity to host cell in the micromolar effective antiviral concentration range in 0.5 micromole～4.
3. the effect of Rhizoma Alpiniae Galangae extract anti-mycobacterium tuberculosis
(1) Rhizoma Alpiniae Galangae extract minimal inhibitory concentration (MIC) measurement result shows, the Rhizoma Alpiniae Galangae extract all has remarkable inhibitory action to three kinds of tubercule bacillus, sees Table 2.
Table 2. Rhizoma Alpiniae Galangae extract MIC measurement result
| Strain | Tubercule bacillus | Bacillus tuberculosis (H 37Rv) | Bacillus tuberculosis bovis |
| MIC(μg/ml) | 26.3 | 90.3 | 160.7 |
(2) the Rhizoma Alpiniae Galangae extract is to infecting the protective effect of tubercule bacillus mice: the result shows that Rhizoma Alpiniae Galangae extract significant prolongation infects the death time of tubercule bacillus mice, sees Table 3.
Table 3. Rhizoma Alpiniae Galangae extract is to infecting the protective effect result of the test of tubercule bacillus mice
| Group | Normal saline blank group | The isoniazid positive controls | Rhizoma Alpiniae Galangae extract group |
| T 50(my god) | 11～15 | 38～43 | 40～45 |
The Figure of description explanation
Accompanying drawing 1 is the anti-HIV infection effect of a Rev protein inhibitor mechanism sketch map
Accompanying drawing 2 is 1 '-acetoxychavicol acetate ultraviolet spectrogram
Accompanying drawing 3 is 1 '-acetoxychavicol acetate infrared spectrogram
Accompanying drawing 4 is 1 '-acetoxychavicol acetate high-efficient liquid phase chromatogram
Accompanying drawing 5 is 1 '-acetoxychavicol acetate mass spectrum
Accompanying drawing 6～accompanying drawing 12 is 1 '-acetoxychavicol acetate nmr spectrum
Accompanying drawing 13 is a Rev protein inhibitor S.pombe yeast in-vitro screening model sketch map
Accompanying drawing 14 is that Rhizoma Alpiniae Galangae extract, zidovudine (AZT) are to MT
4The inhibitory action result of the test of cell infection HIV-1
Accompanying drawing 15 is Rhizoma Alpiniae Galangae extract, zidovudine (AZT) the inhibitory action result of the test to H9 cell infection HIV-1
Accompanying drawing 16 is Rhizoma Alpiniae Galangae extract, zidovudine (AZT) the inhibitory action result of the test to PBMC cell infection HIV-1
Accompanying drawing 17 is MT
4The time result of the test of cell infection HIV-1
Accompanying drawing 18 is the time result of the test of H9 cell infection HIV-1
Accompanying drawing 19 is the time result of the test of PBMC cell infection HIV-1
Accompanying drawing 20 is the inhibitory action result of the test of Rhizoma Alpiniae Galangae extract to PBMC cell infection HIV-1 NL4-3, HIV-1JR-CSF
Accompanying drawing 21 is the synergism result of the test that Rhizoma Alpiniae Galangae extract and anti-HIV infection medicine remove hydroxyl glycosides (ddI)
Accompanying drawing 22 is that the Rhizoma Alpiniae Galangae extract is to MT
4The toxicity test result of cell
Accompanying drawing 23 is the toxicity test result of Rhizoma Alpiniae Galangae extract to the H9 cell
Accompanying drawing 24 is the toxicity test result of Rhizoma Alpiniae Galangae extract to the PBMC cell
Claims (9)
1. Rhizoma Alpiniae Galangae extract is characterized in that it is Zingiberaceae Zingiberaceae Alpinia plants Fructus Galangae Alpinia galanga Willd. rhizome Rhizoma Alpiniae Galangae ethanol and methanolic extract, supercritical CO
2Extraction extract, water vapour distillation thing.
2. the described Rhizoma Alpiniae Galangae extract of claim 1 is characterized in that it is to get Rhizoma Alpiniae Galangae, chopping, be ground into coarse powder, extract each 1～3 hour 2 times with 2～10 times of amount alcohol heating reflux of 50%～90%, merge secondary raffinate, decompression recycling ethanol gets extracting solution; Extracting solution extracts 1～3 time with the normal hexane-ethyl acetate extraction agent of 1～5 times of amount 2～10: 1, and the reclaim under reduced pressure extractant is collected extract; Extract is an immobile phase with the silica gel column chromatography, and with 2～10: normal hexane-ethyl acetate of 1 is the separation and purification of eluant eluting, collects eluent, the Rhizoma Alpiniae Galangae ethanol extraction of making after the reduced vacuum drying.
3. the described Rhizoma Alpiniae Galangae extract of claim 1 is characterized in that it is to get Rhizoma Alpiniae Galangae, and chopping is ground into coarse powder, with 5～10 times of methanol heating and refluxing extraction 2 times, and each 2 hours, merge secondary raffinate, reclaim under reduced pressure methanol must extracting solution; Extracting solution extracts 1～3 time with the normal hexane-ethyl acetate extraction agent of 1～5 times of amount 2～10: 1, and the reclaim under reduced pressure extractant is collected extract; Extract is an immobile phase with the silica gel column chromatography, and with 2～10: normal hexane-ethyl acetate of 1 is the separation and purification of eluant eluting, collects eluent, the Rhizoma Alpiniae Galangae methanolic extract of making after the reduced vacuum drying.
4. the described Rhizoma Alpiniae Galangae extract of claim 1 is characterized in that it is to get Rhizoma Alpiniae Galangae, and chopping is ground into coarse powder and exists, and puts supercritical CO
2In the extractor, extraction temperature is 30 ℃～50 ℃.Extracting pressure is 10MPa～20MPa, and separating pressure is 10MPa～20MPa, and separation temperature is 30 ℃～50 ℃, and the extract and separate time is 2～6 hours, collects extract; Extract is an immobile phase with the silica gel column chromatography, and with 2～10: normal hexane-ethyl acetate of 1 is the separation and purification of eluant eluting, collects eluent, the Rhizoma Alpiniae Galangae supercritical CO of making after the reduced vacuum drying
2The extraction extract.
5. the described Rhizoma Alpiniae Galangae extract of claim 1 is characterized in that it is to get Rhizoma Alpiniae Galangae, and chopping is ground into coarse powder, adds 2～10 times of water gagings, and heating distillation extraction 1～6 hour is collected distillate; Distillate is an immobile phase with the silica gel column chromatography, and with 2～10: normal hexane-ethyl acetate of 1 is the separation and purification of eluant eluting, collects eluent, the Rhizoma Alpiniae Galangae water vapour distillation thing that the reduced vacuum drying is made.
6. the described Rhizoma Alpiniae Galangae extract of claim 1, the main component that it is characterized in that it is 1 '-acetoxychavicol acetate, its structural formula is
Molecular formula is C
13H
14O
4, molecular weight is 234.25.
7. the content assaying method of main component 1 '-acetoxychavicol acetate is a high performance liquid chromatography in the described Rhizoma Alpiniae Galangae extract of claim 6, it is characterized in that it chromatographic condition and system suitability test for being filler with octadecylsilane chemically bonded silica, with the methanol-water is mobile phase, and the detection wavelength is 254nm; Algoscopy is for getting Rhizoma Alpiniae Galangae extract 2ml, with dissolve with methanol and be transferred in the 10ml measuring bottle, add methanol and be diluted to scale, shake up, as need testing solution, precision is measured need testing solution 20 μ l, inject chromatograph of liquid, the record chromatogram with the peak area sum of two adjacent main peaks, calculates the method for 1 '-acetoxychavicol acetate content in the Rhizoma Alpiniae Galangae extract by normalization method.
8. the application of Rhizoma Alpiniae Galangae extract in medicine is characterized in that soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, the application of injection in the anti-HIV infection medicine of preparation Rev protein inhibitor made from it.
9. the application of Rhizoma Alpiniae Galangae extract in medicine is characterized in that soft capsule, drop pill, tablet, capsule, suppository, aerosol, spray, powder spray, microcapsule, Liposomal formulation, the application of injection in preparation anti-mycobacterium tuberculosis infection medicine made from it.
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|---|---|---|---|
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102657841A (en) * | 2012-06-08 | 2012-09-12 | 桂林三金药业股份有限公司 | Ginger phenols extract preparation and preparation method thereof |
| CN103040802A (en) * | 2011-10-11 | 2013-04-17 | 叶英 | Method for inhibiting reproduction of HIV-1 (human immunodeficiency virus-1) drug-resistant virus |
| CN104058963A (en) * | 2014-06-30 | 2014-09-24 | 苏州派腾生物医药科技有限公司 | Method for extracting 1'-acetoxychavicol acetate from alpinia chinensis |
| CN104147557A (en) * | 2012-06-08 | 2014-11-19 | 桂林三金药业股份有限公司 | Shogaol extract soft capsule and preparation method thereof |
| CN105287365A (en) * | 2015-11-26 | 2016-02-03 | 徐诚 | Compound podophyllotoxin gel for treating HPV virus and preparation method thereof |
| CN111840230A (en) * | 2020-08-13 | 2020-10-30 | 蚌埠学院 | Ginger volatile oil liposome and preparation method and application thereof |
| WO2022244691A1 (en) * | 2021-05-21 | 2022-11-24 | サントリーホールディングス株式会社 | Method for producing galangal extract containing 1'-acetoxychavicol acetate |
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2006
- 2006-11-16 CN CNB2006101491326A patent/CN100525823C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103040802A (en) * | 2011-10-11 | 2013-04-17 | 叶英 | Method for inhibiting reproduction of HIV-1 (human immunodeficiency virus-1) drug-resistant virus |
| CN102657841A (en) * | 2012-06-08 | 2012-09-12 | 桂林三金药业股份有限公司 | Ginger phenols extract preparation and preparation method thereof |
| CN104147557A (en) * | 2012-06-08 | 2014-11-19 | 桂林三金药业股份有限公司 | Shogaol extract soft capsule and preparation method thereof |
| CN104058963A (en) * | 2014-06-30 | 2014-09-24 | 苏州派腾生物医药科技有限公司 | Method for extracting 1'-acetoxychavicol acetate from alpinia chinensis |
| CN104058963B (en) * | 2014-06-30 | 2016-05-11 | 苏州派腾生物医药科技有限公司 | A kind of method of extracting ACA from the ginger of the Huashan |
| CN105287365A (en) * | 2015-11-26 | 2016-02-03 | 徐诚 | Compound podophyllotoxin gel for treating HPV virus and preparation method thereof |
| CN111840230A (en) * | 2020-08-13 | 2020-10-30 | 蚌埠学院 | Ginger volatile oil liposome and preparation method and application thereof |
| WO2022244691A1 (en) * | 2021-05-21 | 2022-11-24 | サントリーホールディングス株式会社 | Method for producing galangal extract containing 1'-acetoxychavicol acetate |
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|---|---|
| CN100525823C (en) | 2009-08-12 |
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