CN101011435A - Shanxiangyuan leaf extract, preparation method and uses thereof - Google Patents
Shanxiangyuan leaf extract, preparation method and uses thereof Download PDFInfo
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Abstract
The invention relates to a method for preparing the suaveolic round leaf extractive which contains celery element or the chromocor compound with celery element as corn, or relative salt. The invention also provides relative quality control method, while said extractive can be used to prepare the drug for treating virus B hepatitis, thrombus, cancer, or the like.
Description
Technical field
The present invention relates to a kind of Chinese medicine extract; be specifically related to a kind ofly contain apigenin or be the flavonoid glycoside chemical constituent of aglycon and leaf of Turpinia pomifera (Roxb) D O. extract and this preparation method of extract of medically acceptable salt thereof with the apigenin; the method of quality control of extract; and this extract is antibiotic in preparation; antiviral; antioxidation; mutation; antitumor; antithrombotic; arteriosclerosis; liver protecting; anti-hepatitis virus; antidepressant; application in the medicine such as neuro-protective and immunomodulating is such as treating pharyngolaryngitis in preparation; oral ulcer; gynecological inflammation; hepatitis; viral influenza; application in tumor and the cardio-cerebrovascular diseases medicine.
Background technology
Leaf of Turpinia pomifera (Roxb) D O. is the fresh or dried leaves of Staphyleaceae plant Turpinia pomifera(Roxb) D O. Turpinia arguta Seem. or Turpinia indochinensis, has clearing heat, purging intense-heat, the effect of relieving sore throat and diminishing swelling, up to the present, its chemical constituent of the definite report of research document mainly contains 2,2 α, 19 alpha-dihydroxy-ursolic acids, 19a-Hydroxyursolic Acid, 2 α-hydroperoxyl ursolic acid, ursolic acid, α-Amyrin, myristic acid, (square Zhapu such as daucosterol, Deng, the leaf of Turpinia pomifera (Roxb) D O. The Chemical Constituents. Chinese herbal medicine, 1987.18 (7): 6), mostly be little polarity part; Patent of invention " a kind of treatment chronic pharyngitis; the preparation method of the effective ingredient in Chinese Turpinia pomifera(Roxb) D O. total flavones of chronic tonsillitis (application number: 200510038205.X) " disclose a kind of preparation method of Turpinia pomifera(Roxb) D O. total flavones and treated chronic pharyngitis, the application of chronic tonsillitis, but the extract of its preparation gained is indeterminate, it is impossible to be with acute turpinia leaf A that index components is measured the Turpinia pomifera(Roxb) D O. total flavones, bibliographical information acute turpinia leaf A separates huge pillar alkyl compound (the Qian Yu that obtains in SANYE Turpinia pomifera(Roxb) D O. (Turpinia ternate) leaf, HideakiOTSUKA, Eiji HIRATA, Takakazu SHUNZATO, Yoshio TAKEDA.TurpinionsidesA-E:Megastigmane Glucisides from Leaves of Turpinia ternateNAKIA.Chem.Pharm.Bull.50 (5) 640-644 (2002)), not flavone compound.The leaf of Turpinia pomifera (Roxb) D O. preparation that has gone on the market at present has Turpinia pomifera(Roxb) D O. granule, Caulis et Folium Hyptidis suaveolentis disk, Turpinia pomifera(Roxb) D O. buccal tablet etc., be the sore throat quasi drugs, the clinical exuberant lung-stomach heat that is used for, pharyngitis, acute tonsillitis, the treatment of laryngopharynx swelling and pain, good curative effect is arranged, do not see the report that leaf of Turpinia pomifera (Roxb) D O. and preparation untoward reaction thereof are arranged at present.
The invention provides a kind of leaf of Turpinia pomifera (Roxb) D O. extract and this preparation method of extract; method of quality control; especially extract fingerprint pattern quality control method; and the application of this extract in medicines such as antibiotic, the antiviral of preparation, antioxidation, mutation, antitumor, antithrombotic, arteriosclerosis, liver protecting, anti-hepatitis virus, antidepressant, neuro-protective and immunomodulating, such as the application in preparation treatment pharyngolaryngitis, oral ulcer, gynecological inflammation, hepatitis, viral influenza, tumor and cardio-cerebrovascular diseases medicine.
Summary of the invention
The object of the present invention is to provide a kind of extract of effective leaf of Turpinia pomifera (Roxb) D O., reach preparation method of extract, method of quality control, medical usage.
Leaf of Turpinia pomifera (Roxb) D O. extract of the present invention, it is characterized in that the main flavonoid chemical constituent that contains in the extract, the flavonoid chemical constituent contain apigenin and (or) be the flavonoid glycoside composition of aglycon with the apigenin, and the medically acceptable salt of this flavonoid chemical constituent or compositions, as sodium salt, potassium salt, ammonium salt, magnesium salt etc., the preparation of medically acceptable salt as being reacted with an amount of corresponding dilute alkaline soln and be obtained by extract, and alkali can be sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, ammonia, magnesium oxide etc.
The mixture of this extract mainly is made up of apigenin and the glycosides that contains apigenin, particularly, extract contains chemical constituents such as being mainly A, B, C, D, E, F, G, H, wherein A is an apigenin, B is apigenin-7-O-beta-D-glucoside, C is apigenin-7-O-neohesperidoside, D is apigenin-7-O-2 '-rhamanopyranosyl rutinoside, E, F, G, H are buff powder, E, F, G, H composition are further carried out the acid hydrolysis (alcoholic solution of E, F, G, H, add equal-volume 2mol/L hydrochloric acid solution, filling N
2The heating in water bath hydrolysis is 3 hours in the environment), fling to ethanol, and use the ethyl acetate extraction hydrolyzed solution, obtain acetic acid ethyl ester extract respectively, use dissolve with methanol, with the apigenin is contrast, thin layer chromatography (the photograph thin layer chromatography (" 2005 editions one appendix VI B of Chinese pharmacopoeia) test, lamellae: silica gel G plate, developing solvent: toluene-chloroform-acetone (4: 2.5: 3.5), developer: 1% aluminum chloride alcoholic solution, inspect under the ultraviolet 365nm), the result shows that on the corresponding position of apigenin reference substance chromatograph acetic acid ethyl ester extract all shows the fluorescence speckle of a same color; High performance liquid chromatography is differentiated and (is measured according to high performance liquid chromatography (2005 editions appendix VID of Chinese Pharmacopoeia), Tianjin, island LC-10Atvp high performance liquid chromatograph, chromatographic column: Hypersil ODS post (4.6 * 250mm, 5 μ m), mobile phase: acetonitrile-0.1% aqueous acetic acid (30: 70), detect wavelength: 335nm, sample size: 10 μ l) show, in the acetic acid ethyl ester extract chromatograph, all show a chromatographic peak, and consistent with apigenin reference substance chromatograph, determine that compd E, F, G, H are for being the flavonoid glycoside composition of aglycon with the apigenin.
Different according to extraction process and the medical material place of production and collecting season, main component and content thereof can be different in the extract, in embodiment 1, it is apigenin-7-O-neohesperidoside that 65% ethanol elution owner wants composition, apigenin-7-O-2 '-rhamanopyranosyl rutinoside, apigenin-7-O-beta-D-glucoside, apigenin-7-O-neohesperidoside wherein, apigenin-7-O-2 ' rhamanopyranosyl rutinoside content can reach respectively more than 1.0%, even more than 3%, further silica gel column chromatography (extract: silica gel (200-300 order)=1: 20, with chloroform methanol 9: 1,8: 2,7: 3 gradient elutions, every gradient elution 2-3BV collects stream part with every 0.5BV) can get apigenin-7-O-neohesperidoside respectively, apigenin-7-O-2 '-rhamanopyranosyl rutinoside, apigenin-7-O-beta-D-glucoside and compd E, F, G, monomer components such as H.
Extract calculates with total flavones, can contain flavones ingredient more than 20%, preferred more than 50%, total flavones can contain compositions such as apigenin, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside, apigenin-7-O-beta-D-glucoside and other flavonoids, as with the apigenin being the flavonoid glycoside composition of aglycon, as above-mentioned E, F, G, H composition.
Behind the oral flavonoid glycoside compound of bibliographical information animal, in vivo through intestinal microflora (Walle T, etal.Quercetin glucosides are completely hydrolyzed in ileostomy patients beforeabsorption.J Nutr, 2000,130:2658) or (the Doostdar H of enzyme system such as corresponding liver biotransformation enzyme, etal.Bioflavonoids:selective substrates and inhibitors for cytochrome P450 CYP1Aand CYP1B1.Toxicology, 2000,144:31; Lambert N, Kroon PA, Faulds CB, PlumbGW, McLauchlan WR, Day AJ, Williamson G.Purification of cytosolic beta-glucosidasefrom pig liver and its reactivity towards flavonoid glycosides.Biochim BiophysActa.1999 Nov 16; 1435 (1-2): 110-6) be separately converted to corresponding aglycon.As being converted into aglycon (Day AJ after apigenin-7-O-beta-D-glucose deglycosylation, Du Pont MS, Ridley S, Rhodes M, Rhodes MJ, MorganMR, Williamson G.Deglycosylation of flavonoid and isoflavonoid glycosides byhuman small intestine and liver beta-glucosidase activity.FEBS Lett.1998 Sep25; 436 (1): 71-5), then described extract also can be the hydrolysate of flavone, as always getting the hydrolysate that thing or medical material crude extract method for hydrolysis obtain, hydrolysate is after making with extra care, can obtain containing the apigenin amount and be the extract more than 35%, method for hydrolysis can be acid hydrolysis or enzyme hydrolysis, as the alcoholic solution of extract, add equal-volume 2mol/L hydrochloric acid solution, heating hydrolysis can get the apigenin aglycon in the N2 environment; Or the pH5.0 acetate buffer of extract, add beta-glucosidase or emulsin, 37 ℃ of placements, hydrolysis obtains the apigenin aglycon; Process for purification can be got ethyl acetate layer for the acid hydrolysis liquid ethyl acetate extraction, reclaims solvent, promptly; Or hydrolyzed solution adjusting pH value 1～3, filter, get precipitation, that is, or ethanol is refining repeatedly, gets apigenin.
Can contain the phenolic acid compound except that flavonoid in the extract, main component is a gallic acid, also can contain p-Coumaric Acid, vanillic acid etc., and a small amount of or micro-pyrogallol, extract generally contains total phenolic acid and can reach more than 20% in total phenolic content, and constituent of different process phenolic acid compound and content are different, mainly be meant flavone compound as embodiment 1 total phenolic acid, contain gallic acid hardly; As total phenolic content in embodiment 3 extracts, can reach more than 35%, wherein gallic acid content is more than 0.8%, even can reach 1.5%, wherein apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside content is all greater than 1%, and same process is because crude drug source difference and content difference to some extent.
The preparation method of leaf of Turpinia pomifera (Roxb) D O. extract of the present invention is to get fresh or exsiccant leaf of Turpinia pomifera (Roxb) D O., adopt in the technologies such as solvent extraction method, solvent extraction, resin adsorption method, column chromatography one or more and make.Extract solvent and can be one or more mixing or combination of water, sour water, aqueous alkali, alcohol-water solution, methanol, ethanol, hexane (as cyclohexane extraction, normal hexane), petroleum ether, chloroform, dichloromethane, acetone, ether, Ethyl formate, ethyl acetate etc.; Extractant can be one or more mixing or combination of hexane, petroleum ether, chloroform, dichloromethane, ether, ethyl acetate, butanols (as n-butyl alcohol), amylalcohol, hexanol etc.; Adsorbent resin can be in phenylethylene, esters of acrylic acid, the nitrogen oxide type adsorbent resin one or more; The column chromatography filler can be silica gel, aluminium oxide, active carbon, polyamide, adsorbent resin, polydextran gel etc.
The preparation method of leaf of Turpinia pomifera (Roxb) D O. extract of the present invention contains the extraction refining step briefly, and solvent extraction method is an abstraction process, and solvent extraction, resin adsorption method, column chromatography are refining step.Solvent extraction is as getting fresh or exsiccant leaf of Turpinia pomifera (Roxb) D O., adopt water, methanol, ethanol, the method of acetone isopolarity solvent and extraction with aqueous solution thereof, extract of the present invention be flavone or (with) liposoluble ingredient, extract solvent and can be diluted acid water or dilute alkaline aqueous solution, example hydrochloric acid, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, ammonia spirit etc., extracting method can be microwave, ultrasonic, reflux, leave standstill immersion (containing warm macerating), percolation etc., be further process for refining, extraction process can contain the primary purification impurity removal process, as water extract-alcohol precipitation, alcohol extracting-water precipitating, solvent degreasing, technologies such as clarifier and gelatin precipitation (are edited referring to Fan Biting, pharmacy of Chinese materia medica, Shanghai science tech publishing house: 1997; The Xiao Chonghou chief editor, Chemistry for Chinese Traditional Medicine, Shanghai science tech publishing house: 1997), the aqueous extract precipitate with ethanol is as getting leaf of Turpinia pomifera (Roxb) D O., add water and decoct extraction 2～3 times, each 1.5 hours, concentrating under reduced pressure (about crude drug: water=1: 0.8～2) for 16 times, concentrated solution is put cold, add ethanol and make the alcohol amount of containing 40%～85%, stir, leave standstill, filter, get filtrate; The alcohol extraction water precipitating adds 75% ethanol and decocts extraction 2 times for 16 times as getting leaf of Turpinia pomifera (Roxb) D O., each 1.5 hours, reclaims ethanol, concentrating under reduced pressure (about crude drug: water=1: 0.8～2), add water an amount of (about 0.5～1 times), stir, filter, get filtrate; Clarifier can be gelatin, also can be chitosan, as ZTC clarifier series (Tianjin became the clarification technique company limited in positive day), as get leaf of Turpinia pomifera (Roxb) D O., decoct with water, and the extracting solution concentrating under reduced pressure (about crude drug: water=1: 5), in 80 ℃ of ZTC-II type clarifier B components (being mixed with 1% concentration) that add medicine liquid volume 2%, stir, be incubated 30 minutes,, be incubated 30 minutes in 60 ℃ of A components (being mixed with 0.5% concentration) that add medicine liquid volume 2%, standing over night is got supernatant; Because leaf of Turpinia pomifera (Roxb) D O. contains oil-soluble impuritieses such as chlorophyll, and the target extract be apigenin and (or) apigenin is the flavonoid glycoside composition of aglycon, and other liposoluble ingredients, as gallic acid, polarity is relatively big, solvent extraction can contain the process that non-polar organic solvent extracts or impurity is removed in extraction, as adopt non-polar solvens such as petroleum ether (as hexane, chloroform, dichloromethane, ether) extracts (dipping, reflux) extract medical material or extract extracting solution and remove impurity such as chlorophyll, safer, economically viable method as: 1) get leaf of Turpinia pomifera (Roxb) D O., measure reflux 2 times for 15 times with petroleum ether, get petroleum ether layer, reclaim petroleum ether, remove impurity such as chlorophyll, leaf is flung to petroleum ether, continues with 75% ethanol (or 60% acetone) reflux, extract, 2 times merge extractive liquid,, reclaim solvent, promptly get crude extract; 2) get leaf of Turpinia pomifera (Roxb) D O., decoct with water, merge aqueous extract, concentrating under reduced pressure (about crude drug: water=1: 0.5～2), use petroleum ether extraction 2～4 times, merge, reclaim petroleum ether, remove impurity such as chlorophyll, the water intaking layer.Refining step also can be other suitable methods, as the extraction solution (or through certain purified crude extract) that solvent extraction method obtains, adds 0.1% sodium hydroxide solution regulator solution pH to alkalescence, as regulator solution pH to 7.5～8.5, ultrafiltration, filtrate concentrates, spray drying; Or filtrate 1mol/L hydrochloric acid solution adjusting pH as regulating pH to 4.0～7.0, concentrates spray drying to acid or neutral.
Solvent extraction is that refining step is a kind of, the method that can obtain extract with ethyl acetate, n-butanol extraction for the extracting solution or the crude extract solution of above-mentioned solvent extraction operation, the aqueous solution of extraction generally can reach better effect under acidity or slant acidity condition, as using hydrochloric acid conditioning solution pH value 2～6.5, as regulate pH to 4.2, ethyl acetate, butanol extraction liquid continue to reclaim solvent, concentrate, be drying to obtain that (or extract is further used dissolve with ethanol, filter, reclaim ethanol, concentrate, be drying to obtain); Or extract concentrated solution, mix thoroughly with kieselguhr, add ethanol or acetone reflux, extract,, merge extractive liquid, reclaims solvent, concentrates, and is drying to obtain; Or extract concentrated solution, with the active carbon powder mixing, add ethanol, stir, filter, concentrate, reclaim ethanol, be drying to obtain.
Resin adsorption method is a kind of safety and economic process for purification, and resin can be ion exchange resin and non-ionic resin, the crude extract solution that obtains as above-mentioned solvent extraction method or the extract solution of solvent extraction gained, by macroporous adsorbent resin (or medicinal liquid concentrated solution and low amounts of resin absorption, mixing joins in the resin column), the water elution remove impurity, low-concentration ethanol solution (0～20%) eluting remove impurity, the higher concentration ethanol elution is collected eluent respectively, concentrates, drying, promptly; Higher concentration eluting concentration of alcohol can be for more than 20%, more suitable eluting alcoholic solution concentration is as 40%～95%, and adsorbent resin can be one or more combinations in phenylethylene, polyacrylate, acrylamide copolymer, vinyl cyanide, the nitrogen oxide type copolymer etc.; As model AB-8 (Chemical Plant of Nankai Univ.), D101 (Tianjin pesticide limited company), HPD100 (Cangzhou, Hebei precious grace chemical industry company limited), HPD400, HPD500, HPD600, ADS-21 (Tianjin Nankai Hecheng S﹠T Co., Ltd.), ADS-7, ADS-F8 etc., adsorbent resin also can be changed to polyamide granules.
Resin column is made with extra care processing steps such as generally being divided into the adsorption-edulcoration eluting, the absorption of last sample can with thick extract concentrated solution directly with a small amount of (as account for resin total amount 5%～15%) treated adsorbent resin mixing, low temperature is flung to solvent, adds the resin column top that has installed and gets final product; Or solution is at a slow speed by sample absorption on the resin column, general low flow velocity is advisable, as 1-4BV/hr, preferred 1-2BV/hr, can leave standstill some times behind the end of the sample, as 3 hours, for reaching adsorption effect preferably, the all right according to circumstances saline solns of medicinal liquid, as contain the medicinal liquid of 3% sodium chloride, directly go up sample relatively with medicinal liquid, acid medicinal liquid can reach better absorption and concentration effect, the general control 2～6.5 of last sample medicinal liquid pH value, preferred pH value 3～5, last sample liquor strength can be medical material: water (1: 1～12), amount of resin can medical material: resin (1: 0.5～5), above sample effluent has just detected flavones ingredient for fully loaded, generally be advisable with fully loaded 70%-80%, (detection method was once attempted easier hydrochloric acid-magnesium powder reaction to the detection method of flavone component detection method such as embodiment 1, but chromogenic reaction is not obvious) or thin layer chromatography (according to " the about 25ml water bath method of afterflow fluid is got in 2005 editions one appendix VI B of Chinese pharmacopoeia test, and residue adds methanol 1～2ml makes dissolving, get 5 Cheng, point is on the silica gel g thin-layer plate with 0.5% sodium hydroxide solution preparation, with ethyl acetate-butanone-formic acid-water (6: 3: 1: 1) be developing solvent, expansion, take out, dry, spray is put under the ultra-violet lamp (365nm) and is inspected with the aluminum chloride test solution, positive with obvious apparent fluorescence speckle), post footpath height is generally 1: 3～10.Impurity removal process can be used the pure water eluting, also can use the alcoholic solution eluting remove impurity of low concentration, the alcohol of low concentration such as the ethanol of 0-20%, different to different resins because adsorption property is different, as among the embodiment 1 with 15% ethanol elution remove impurity, the remove impurity elution amount is generally 2-4BV.Eluting is refining can be the ethanol of 15%-95%, also can be the aqueous solution of methanol, acetone, as 60% acetone soln, and alkaline solution (pH value 8～13), alkali such as sodium hydroxide, potassium hydroxide, ammonia etc. are as the aqueous solution or the Diluted Alcohol solution of 0.05% sodium hydroxide, elution speed such as 0.6-2BV/hr, elution amount 3-6BV, the eluting endpoint can be carried out flavones ingredient such as the detection of above-mentioned method to continuous eluent, is not advisable there to be flavones ingredient.The adsorbent resin of above-mentioned technology can be changed to polyamide granules, or polyamide and diatomaceous hybrid particles.
Column chromatography can obtain more high-load total flavones or (with) total phenolic acid extract, the column chromatography filler can be silica gel, active carbon, aluminium oxide, polydextran gel etc.As embodiment 3 n-butyl alcohol extracts, by silica gel column chromatography (extract: silica gel=1: 15), respectively with chloroform-methanol with 95: 5,9: 1,8: 2,7: 3,1: 1 eluting, the Fractional Collections eluent, reclaim each stream part solvent, respectively silica gel G plate thin layer chromatography (chloroform-methanol launched with 95: 5,9: 1,8: 2, the colour developing of 10% ethanol solution of sulfuric acid), merge same stream part, the merging stream of 9: 1 and 8: 2 eluting part can contain gallic acid and reach 50% above extract; Merging stream part of 7: 3 eluting can contain apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside reaches more than 15% respectively, contain total flavones more than 80%, even more than 95%, continued silica gel column chromatography and sephadexLH20 post respectively, chloroform-methanol and methanol-eluted fractions can obtain monomer components such as gallic acid, apigenin, apigenin-7-O-beta-D-glucoside, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside and compd E, F, G, H respectively.
Process for refining can be the multinomial combination of said method, as solvent extraction-solvent extraction-resin column program, solvent extraction resin column-silica gel column chromatography program, solvent extraction-resin column-solvent extraction program, solvent extraction-resin column-solvent extraction-silica gel column chromatography program etc., also can be according to extract character in conjunction with solvent, the purified technology of adsorbent, as crude extract with ethanol or acetone solution, activated carbon decolorizing and recrystallization method; Regulate the pH method,, filter with 0.05% sodium hydroxide solution (or its dilute alcohol solution) dissolving as crude extract, the 1mol/L dilute hydrochloric acid is regulated pH to 5, filters, and concentrates, and drying must be made with extra care extract.Concrete extraction process for purification is according to the action mode of concrete extract medicinal usage and difference, as for oral solid formulation, extractive total flavone content is generally greater than 50%, for the injection preparation, general flavone content requires more than 90%, as obtaining the extract of general flavone content more than 93% in conjunction with above-mentioned solvent extraction, solvent extraction, resin absorption, column chromatography, single stream part can obtain gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside, apigenin monomer component.
According to medicinal plants relationship scientific principle opinion (Xiao Peigen. the dependency between medicinal plants form, chemical constituent and the curative effect, Acta Pharmaceutica Sinica .1978,13: 1-5; Chen Sibao, Peng Yong, Chen Shilin, Xiao Peigen. natural resources of Chinese medicinal materials sustainable use-medicinal plants relationship, World Science technology-Chinese medicine modernization, 2005 (6): 97-103), leaf of Turpinia pomifera (Roxb) D O. extract of the present invention is except that deriving from sharp sharp Turpinia pomifera(Roxb) D O. Turpinia arguta (Lindl.) Seem., also can derive from other kinds that Turpinia pomifera(Roxb) D O. belongs to, comprise sharp sharp Turpinia pomifera(Roxb) D O. (former mutation) Turpinia arguta (Lindl.) Seem.Var.arguta, Turpinia simplicifolia Merr.var.longipes C.Y.Wu Turpiniasimplicifolia Merr.Var.longipes C.Y.Wu, Turpinia robusta Craib Turpinia robusta Craib, Turpinia pomifera (Roxb) DO. Turpinia pomifera (Roxb.) DC., Turpinia pomifera (Roxb) DO. (former mutation) Turpinia pomifera (Roxb.) DC.var.pomifera, big seed Turpinia pomifera(Roxb) D O. Turpinia macrosperma C.C.Huang, light Turpinia pomifera(Roxb) D O. Turpinia montana (Rl.) Kurz var.glaberrima (Merr.) T.Z.Hsu, bright leaf Turpinia pomifera(Roxb) D O. Turpiniasimplicifolia Merr., bright leaf Turpinia pomifera(Roxb) D O. (former mutation) Turpinia simplicifolia Merr.var.simplicifolia, ovum leaf Turpinia pomifera(Roxb) D O. Turpinia ovalifolia Elmer, the sharp sharp Turpinia pomifera(Roxb) D O. Turpiniaarguta of fine hair (Lindl.) Seem.var.pubescens T.Z.Hsu, SANYE Turpinia pomifera(Roxb) D O. Turpinia ternata Nakai, mountain Jatropha curcas Turpinia pomifera (Roxb.) DC.var.minor C.C.Huang, Turpinia pomifera(Roxb) D O. Turpiniamontana (Bl.) Kurz, Turpinia pomifera(Roxb) D O. (former mutation) Turpinia montana (Bl.) Kurz var.montana, dredge arteries and veins Turpinia pomifera(Roxb) D O. Turpinia indochinensis Merr., Taiwan Turpinia pomifera(Roxb) D O. Turpinia formosana Nakai, narrow leaf Turpinia pomifera(Roxb) D O. Turpinia montana (Bl.) Kurz var.stenophylla (Merr.et Perry) T.Z.Hsu, lobus cardiacus Turpinia pomifera(Roxb) D O. Turpinia subsessilifolia C.Y.Wu, bristle Turpinia pomifera(Roxb) D O. Turpinia affinis Merr.etPerry, Vietnam Turpinia pomifera(Roxb) D O. Turpinia cochinchinensis (Lour.) Merr. etc.
Get the extract of the embodiment of the invention 1 and embodiment 3, carry out extracorporeal bacteria inhibitor test respectively, the inductive mice ear inhibition test of dimethylbenzene, the inductive rat paw edema inhibition test of mice acetic acid twisting inhibition test and carrageenin, the result shows that extract of the present invention has significant antiinflammatory, analgesic activity.
Extracorporeal bacteria inhibitor test: get embodiment 1 and embodiment 3 extracts, adopt test tube method and flat band method to measure, show that it all has obvious inhibitory action to staphylococcus aureus, streptococcus pneumoniae, Salmonella typhimurium, escherichia coli, pseudomonas aeruginosa, streptococcus salivarius, micrococcus scarlatinae, staphylococcus haemolyticus etc., it is 0.062g～0.250g crude drug/ml that the MIC scope is converted to crude drug, and the bacteriostasis of embodiment 3 extracts is better than the extract of embodiment 1.
The inductive mice ear inhibition test of xylol: get the test of embodiment 1 and embodiment 3 extracts, 40 of Kunming mouses are divided into 4 groups at random, 10 every group.Gastric infusion, for three days on end, every day 1 time, extract divides 4g/kg, 8g/kg, three of 12g/kg (all being converted to the crude drug meter) are promptly low, in, high dose group, irritating the stomach normal saline is the blank group, be coated with dimethylbenzene 0.05ml in last administration 1 hour at the mouse right ear exterior feature, after 2 hours mice being taken off neck puts to death, two ears about cutting along the ear baseline, draw materials in the punching of left and right sides auricle same area with diameter 9mm card punch, weigh, with left and right sides ear weight difference as the swelling degree, the result show embodiment 1 extract in, the low dosage of low dosage and embodiment 3 extracts all can significantly suppress mice caused by dimethylbenzene xylene ear swelling (p＜0.05), in the high dose of embodiment 1 extract and embodiment 3 extracts, high dose all can suppress mice caused by dimethylbenzene xylene ear swelling (p＜0.01) by highly significant.
Mice acetic acid twisting inhibition test: get the test of embodiment 1 and embodiment 3 extracts, 40 of Kunming mouses are divided into 4 groups at random, 10 every group.Gastric infusion, for three days on end, every day 1 time, extract divides 4g/kg, 8g/kg, three of 12g/kg (all being converted to the crude drug meter) are promptly low, in, high dose group, irritating the stomach normal saline is the blank group, in the last administration after 30 minutes, each lumbar injection 0.5% acetum 0.2ml, and observed and recorded is injected 30 minutes mouse writhing reaction times behind 0.5% acetum, calculate, the result show embodiment 1 extract in, the low dosage of low dosage and embodiment 3 extracts all can significantly reduce the acetic acid induced mice and turn round body number of times (p＜0.05), in the high dose of embodiment 1 extract and embodiment 3 extracts, high dose all can highly significant minimizing acetic acid induced mice be turned round body number of times (p＜0.01).
The inductive rat paw edema inhibition test of on Carrageenan: get the test of embodiment 1 and embodiment 3 extracts, 40 of SD rats are divided into 4 groups at random, 10 every group.It is basic, normal, high dosage group that gastric infusion, extract divide 4g/kg, 8g/kg, three of 12g/kg (all being converted to the crude drug meter), and irritating the stomach normal saline is the blank group.The standardized line in top, the right back ankle of every Mus joint as a token of, measure this foot corpus unguis long-pending (draining milliliter number) with the capillary tube amplifying method, every Mus right hind foot sole of the foot subcutaneous injection 1% carrageenin 0.1ml of portion causes inflammation, cause scorching back 0.5 hour with 4 hours respectively by above-mentioned dosage gastric infusion (or normal saline), measure after the administration 1 with method, 2,3,4,6,8h should amass by the foot corpus unguis, with the change of its volume (the administration front and rear row water yield poor) expression inflammation swelling degree, the result show embodiment 1 extract in, the low dosage of low dosage and embodiment 3 extracts all can significantly suppress the inductive rat paw edema of carrageenin (p＜0.05), in the high dose of embodiment 1 extract and embodiment 3 extracts, high dose all can suppress the inductive rat paw edema of carrageenin (p＜0.01) by highly significant.
Extract main component of the present invention be apigenin and (or) with the apigenin be aglycon the flavonoid glycoside composition and (or) liposoluble ingredient, document shows, flavonoid glycoside compound is separately converted to corresponding aglycon through enzymes such as intestinal microflora or corresponding liver biotransformation enzyme system in animal body, can be converted into apigenin as apigenin-7-O-beta-D-glucoside; Document shows that apigenin has many-sided effect such as antiinflammatory, antioxidation, antiviral, inhibition tumor, antithrombotic arteriosclerosis, antidepressant, neuro-protective, immunomodulating (Sun Bin, Deng. the Advance on Pharmacological Activities of apigenin. Chinese crude drug, 2004,27 (7): 531; Liu Chan, etc. apigenin is to the neurergic progress of maincenter. foreign medical science neurological neurosurgery fascicle .2005,32 (3): 264; Go through by force, etc. the progress of apigenin function of tumor inhibition. Shaanxi medical journal, 2003,32 (5): 439; Patel D, Shukla S, Gupta S.Apigenin and cancer chemoprevention:progress, potential and promise (review) .Int J Oncol.2007 Jan; 30 (1): 233-45.); As AIDS virus resisting effect (Critchfield JW, Butera ST, Folks TM.Inhibition of HIVactivation in latently infected cells by flavonoid compounds.AIDS Res HumRetroviruses.1996 Jan 1; 12 (1): 39-46.); Apigenin-7-O-beta-D-glucoside has hepatoprotective effect (Zheng QS, Sun XL, Xu B, Li G, Song M.Mechanisms of apigenin-7-glucoside as ahepatoprotective agent.Biomed Environ Sci.2005 Feb; 18 (1): 65-70.); Gallic acid has antiinflammatory; antioxidation; mutation; antitumor; antiviral; pharmacologically active (the Li Xiaoling of liver protecting and anti-hepatitis virus etc.; Deng. the progress of gallic acid biological action. Chinese pharmacist; 2004; 7 (10): 767); extract of the present invention and medically acceptable salt thereof can be applicable to prepare antibiotic; antiviral; antioxidation; mutation; antitumor; liver protecting; anti-hepatitis virus; antithrombotic; medicines such as arteriosclerosis are such as treating pharyngolaryngitis in preparation; oral ulcer; gynecological inflammation; hepatitis; tumor; application in viral influenza and the cardio-cerebrovascular diseases medicine.
Leaf of Turpinia pomifera (Roxb) D O. extract of the present invention and medically acceptable salt thereof are used for preparation antibiotic, antiviral, antioxidation, mutation, antitumor, liver protecting, anti-hepatitis virus, antithrombotic, the method of medicines such as arteriosclerosis, for making the various pharmaceutical preparatioies that are suitable for clinical practice with pharmaceutically acceptable excipient, described various pharmaceutical preparatioies made from acceptable carrier or excipient, be as the criterion with the explanation of the dosage form under 2005 editions one appendix I rules of preparations of the Chinese Pharmacopoeia item, as mixture, tablet, capsule, granule, drop pill, injection, suppository, gel, spray or the like, as according to apigenin, good Transdermal absorption effect (the Merfort I of apigenin-7-O-beta-D-glucoside etc., Heilmann J, Hagedorn-LewekeU.Lippold BC.In vivo skin penetration studies of camomile flavones.Pharmazie1994 Jul; 49 (7): 509-11; Li B, Birt DF.In vivo and in vitro percutaneousabsorption of cancer preventive flavonoid apigenin in different vehicles inmouse skin.Pharm Res.1996 Nov; 13 (11): 1710-5.) and the transdermal absorption formulation etc. of preparation.Be bioavailability of the oral formulations that improves leaf of Turpinia pomifera (Roxb) D O. extract of the present invention and medically acceptable salt thereof etc., can increase the absorption except that extract being hydrolyzed into aglycon, can adopt advanced preparation technique and reach good medical science effect, as inclusion technique, solid dispersion technology, micronization, cosolvent, emulsifying technology etc., as with polyvinylpyrrolidone (PVP), carriers such as Polyethylene Glycol adopt the solid dispersion of solvent method or fusion method preparation, as the solid dispersion that makes than melting mixing with 1: 0.6～6 quality with Macrogol 4000 (PEG4000); Or the phosphatide complexes that forms with phospholipid; Clathrate that forms with mass ratio 1: 1～16 preparations with beta-schardinger dextrin-, HP-or part clathrate etc.
The method of quality control of extract of the present invention and medically acceptable salt thereof, be determination of total flavonoids method (referring to embodiment 1), total phenolic content assay method (referring to embodiment 3), with gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside, apigenin-7-O-beta-D-glucoside and apigenin and compd E, F, G, H etc. are the content assaying method of index components, as existing document portable injection chemiluminescence method (Liu Zemin, Deng. the portable injection chemiluminescence method is measured apigenin, Southwestern Normal University's journal: natural science edition, 2006 (3): 89), capillary zone electrophoresis method (Wang Yueling, Deng. the capillary zone electrophoresis method is measured apigenin luteolin and Isoquercitrin, physical and chemical inspection: chemical fascicle, 2005 (8): 544), high performance liquid chromatography (Tan Xingqi, Deng. the content of apigenin in the rp-hplc determination Caulis Trachelospermi-7-O-β-neohesperidoside, CHINA JOURNAL OF CHINESE MATERIA MEDICA: 2005,30 (24): 1958; Liu Guangtian. 51) apigenin in Solid-Phase Extraction-rp-hplc determination marchantia, chemistry and biological engineering: 2004 (6): method such as.
The fingerprint pattern quality control method of extract of the present invention, containing with gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside, apigenin-7-O-beta-D-glucoside and apigenin etc. is the method for quality control of index components, and concrete high-performance liquid chromatogram determination as contrasts such as gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside, apigenin-7-O-beta-D-glucoside and apigenins: with the octadecylsilane chemically bonded silica is filler; With the acetonitrile is mobile phase A, is Mobile phase B with 0.1% phosphate aqueous solution; At 0～70 minute, mobile phase A was from 10%～25%, and Mobile phase B is from 90%～75%; Detect wavelength: 269 ± 1nm or 335 ± 1nm.Or be filler with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A, is Mobile phase B with 0.1% phosphate aqueous solution; At 0～30 minute, mobile phase A was from 5%～15%, and Mobile phase B is from 95%～85%, and at 30～70 minutes, mobile phase A was from 15%～25%, and Mobile phase B is from 85%～75%; Or at 0～30 minute, mobile phase A was from 5%～15%, and Mobile phase B is from 95%～85%, and at 30～90 minutes, mobile phase A was from 15%～25%, and Mobile phase B is from 85%～75%; Detect wavelength: 269 ± 1nm or 350 ± 1nm.Concrete reference substance preparation, test sample preparation and assay method example are referring to embodiment 1, embodiment 3.
The specific embodiment
Following examples just are described more specifically the present invention, and the present invention is not limited in the content of following examples.
Embodiment 1: get leaf of Turpinia pomifera (Roxb) D O., decoct with water 2 times, measured 2 hours for 16 times for the first time, measured 1.5 hours for 14 times for the second time, merge decoction liquor, being evaporated to relative density is the clear paste of 1.08～1.18 (80 ℃ of heat are surveyed), and adding ethanol is 75% to containing the alcohol amount, stirs, left standstill 24 hours, filter, filtrate recycling ethanol is evaporated to 1:0.8～1.6 (W/V), thin up is to 3 times (V/W) of medical material amount, dilute hydrochloric acid is regulated pH value to 4, the AB-8 macroporous adsorptive resins of having handled well by (about 1BV/hr) 2 times of medical material amounts (W/W) slowly, water elution 3BV (about 2BV/hr), 15% ethanol elution 3BV (about 2BV/hr), 65% ethanol elution is collected 65% ethanol elution, reclaims ethanol, concentrating under reduced pressure, spray drying gets leaf of Turpinia pomifera (Roxb) D O. extract, and yield is about 2.3%.
1, leaf of Turpinia pomifera (Roxb) D O. extract determination of total flavonoids:
Measure according to UV-VIS spectrophotometry (2005 editions one appendix V A of Chinese Pharmacopoeia).
The preparation precision of reference substance solution takes by weighing 105 ℃ of control substance of Rutin that are dried to constant weight, adds 70% ethanol and makes the solution that every 1ml contains 0.2mg, and is standby.
The preparation precision of need testing solution takes by weighing leaf of Turpinia pomifera (Roxb) D O. extract, adds 70% ethanol and makes the solution that every 1ml contains 0.3mg, and is standby.
Standard curve prepares that precision is measured reference substance solution 0,1,2,3,4,5,6ml puts respectively in the 25ml measuring bottle, respectively add water 10ml, add 5% sodium nitrite solution 1ml, shake up, placed 6 minutes, add 10% aluminum nitrate solution 1ml, shake up, placed 6 minutes, and added 4% sodium hydroxide solution 10ml, add water to scale, shake up, placing 15 minutes, is blank with corresponding reagent solution, measures trap at 500nm wavelength place, with trap A is vertical coordinate, with reference substance concentration is abscissa, asks linear equation, and the result shows in 0～0.062mg/ml scope internal linear relation good.
The sample determination precision is measured need testing solution 2ml, and to the 25ml measuring bottle, method under the sighting target directrix curve preparation with the method operation, is measured trap, calculates general flavone content by equation of linear regression, and this product general flavone content is 63%.
The determination of total flavonoids method also can use apigenin, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside etc. to measure for reference substance, assay method can be with the UV-VIS spectrophotometry of above-mentioned principle, also can be without colour developing, directly measure trap as reference substance solution and need testing solution at 279nm or 335nm, calculate and promptly get general flavone content, general flavone content is and to some extent difference different with assay method owing to the difference of reference substance.
2, HPLC measures:
Measure according to high performance liquid chromatography (2005 editions appendix VID of Chinese Pharmacopoeia).
Chromatographic condition and system suitability are filler with the octadecylsilane chemically bonded silica; With the acetonitrile is mobile phase A, and 0.1% phosphate aqueous solution is a Mobile phase B; Carry out gradient elution by table 1, the detection wavelength is 270nm.Number of theoretical plate calculates by apigenin-7-O-neohesperidoside peak should be not less than 2000, flow velocity 1ml/min, column temperature: room temperature.
Adopt Tianjin, island LC-10Atvp high performance liquid chromatograph, UV-detector, Hypersil ODS post (4.0 * 250mm, 5 μ m).
Table 1 eluent gradient eluting allocation table
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～90 | 10→25 | 90→75 |
Apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-reference substances such as rhamanopyranosyl rutinoside are got in the preparation of reference substance solution, add methanol respectively to make the solution that every 1ml contains 25～100 μ g, in contrast product solution.
It is an amount of that leaf of Turpinia pomifera (Roxb) D O. extract is got in the preparation of need testing solution, prepares with method with the reference substance solution preparation method, gets need testing solution.
Accurate above-mentioned need testing solution, each the 10 μ l of reference substance solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
The HPLC collection of illustrative plates is seen Fig. 1,72.532min be apigenin-7-O-neohesperidoside chromatographic peak, 64.665min be apigenin-7-O-2 '-rhamanopyranosyl rutinoside chromatographic peak, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside content are respectively 5.42%, 4.18%.This assay method also can be used for the finger printing quality control of extract.
Embodiment 2: with embodiment 1 decocting in water alcohol precipitation process, get extracting solution, the polyamide column of having handled well by (about 1BV/hr) 2 times of medical material amounts (W/W) slowly, water elution 4BV (about 2BV/hr), 95% ethanol elution, collect eluent, reclaim ethanol, concentrating under reduced pressure, spray drying, get leaf of Turpinia pomifera (Roxb) D O. extract, yield is about 3.4%.
Embodiment 3: with embodiment 1 decocting in water alcohol precipitation process, get extracting solution, dilute hydrochloric acid is regulated pH value to 4, water saturated n-butanol extraction 4 times, and combining extraction liquid, the reclaim under reduced pressure n-butyl alcohol concentrates, spray drying, promptly; Or n-butanol extraction concentrated solution and an amount of kieselguhr mixing, dry, add ethanol extraction 2 times, merge ethanol extract, reclaim ethanol, concentrating under reduced pressure, spray drying, that is, yield is 3.6%.
1, total phenolic content is measured:
Measure according to ultraviolet visible spectrophotometry (2005 editions one appendix V A of Chinese Pharmacopoeia).
The preparation precision of reference substance solution takes by weighing the gallic acid reference substance, adds dehydrated alcohol and makes the solution that every 1ml contains 20 μ g, and is standby.
Leaf of Turpinia pomifera (Roxb) D O. extract fine powder 60mg is got in the preparation of need testing solution, and accurate the title decides, and puts in the 50ml measuring bottle, adds the about 40ml of dehydrated alcohol, supersound process 15 minutes adds dehydrated alcohol to scale, mixing, the accurate 1ml that draws, put in the 25ml measuring bottle, add dehydrated alcohol to scale, standby.
Standard curve prepares precision and measures reference substance solution 0.5,1.0,1.5,2.0,2.5ml, put respectively in the 25ml measuring bottle, each dehydrated alcohol is to 5ml, add 0.3% sodium dodecyl sulfate solution 2ml and 0.6% ferric chloride-0.9% potassium ferricyanide (1: 9) mixed solution 1ml, mixing, in the dark placed 5 minutes, add the 0.1mol/L hydrochloric acid solution to scale, in the dark placing 20 minutes, is blank with corresponding reagent solution, measures trap at 720nm wavelength place, with trap A is vertical coordinate, with reference substance concentration is abscissa, asks linear equation, and the result shows in 10.16～50.80 μ g/ml scope internal linear relation good.
The sample determination precision is measured need testing solution 1ml, and to the 25ml measuring bottle, method under the sighting target directrix curve preparation with the method operation, is measured trap, calculates total phenolic content by equation of linear regression, and the total phenolic content of this product is 53.08%.
2, total flavones, gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside are measured: according to embodiment 1 total flavones assay method, extractive total flavone content is 43%; Press embodiment 1HPLC content assaying method, with gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside is reference substance, contains gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside and is respectively 2.18%, 3.22%, 2.37%.
The HPLC collection of illustrative plates is seen Fig. 2, and 5.632min is the gallic acid chromatographic peak, and 65.865min is apigenin-7-O-2 '-rhamanopyranosyl rutinoside chromatographic peak, and 73.598min is apigenin-7-O-neohesperidoside chromatographic peak.This assay method also can be used for the finger printing quality control of extract.
Embodiment 4: get leaf of Turpinia pomifera (Roxb) D O., decoct with water 2 times, measured 1.5 hours for 16 times for the first time, measured 1.5 hours for 14 times for the second time, merge decoction liquor, be evaporated to crude drug: water=1: 3 (W/V), in 80 ℃ of ZTC-II type clarifier (Tianjin became the clarification technique company limited in positive day) B components (being mixed with 1% concentration) that add medicine liquid volume 2%, stir, be incubated 30 minutes, in 60 ℃ of A components (being mixed with 0.5% concentration) that add medicine liquid volume 2%, be incubated 30 minutes, standing over night is got supernatant, dilute hydrochloric acid is regulated pH value to 4, the HPD-600 macroporous adsorbent resin of having handled well by (about 2BV/hr) 2 times of medical material amounts (W/W) slowly, water elution 3BV (about 2BV/hr), 15% ethanol elution 3BV (about 2BV/hr), 75% ethanol elution, collect 75% ethanol elution, reclaim ethanol, concentrating under reduced pressure, spray drying gets leaf of Turpinia pomifera (Roxb) D O. extract; Or the sedimentary supernatant of clarifier, concentrating under reduced pressure, dilute hydrochloric acid is regulated pH value to 3～4, ethyl acetate extraction 4 times, combining extraction liquid, the reclaim under reduced pressure ethyl acetate concentrates, spray drying, promptly.
Embodiment 5: get leaf of Turpinia pomifera (Roxb) D O., add 75% alcohol reflux 2 times, measured 1.5 hours for 16 times for the first time, measured 1.5 hours for 14 times for the second time, merge extractive liquid, reclaims ethanol, concentrating under reduced pressure filters, and dilute hydrochloric acid is regulated pH value to 3～4, n-butanol extraction 4 times, combining extraction liquid, the reclaim under reduced pressure n-butyl alcohol concentrates, spray drying, promptly; Or ethanol extract, be evaporated to 1: 1 (W/V), filter, continue to be concentrated into thick paste, add the small amount of ethanol dissolving, filter, add the ADS-21 macroporous adsorbent resin mixing of 0.6 times of amount of medical material, fling to ethanol, be added on the ADS-21 macroporous adsorptive resins top of having handled well, water elution 3BV (about 2BV/hr), 10% ethanol elution 3BV (about 2BV/hr), 75% ethanol elution, collect 75% ethanol elution, reclaim ethanol, concentrating under reduced pressure, spray drying gets leaf of Turpinia pomifera (Roxb) D O. extract.
Embodiment 6: get leaf of Turpinia pomifera (Roxb) D O., add the petroleum ether reflux, extract, 2 times, each 1.5 hours, merge extractive liquid, reclaimed petroleum ether, fling to medical material PetroChina Company Limited. ether, add acetone (or ethanol) reflux, extract, 2 times, each 1.5 hours, merge extractive liquid,, decompression and solvent recovery concentrates, drying, promptly; Or get the aqueous solution of acetone (or ethanol) extract, with embodiment 1 or embodiment 5 methods, get leaf of Turpinia pomifera (Roxb) D O. extract with legal system.
Embodiment 7: get leaf of Turpinia pomifera (Roxb) D O., pulverize, add 30% ethanol or 75% soak with ethanol 24hr, percolation, collect percolate (about 10～18 times of medical material amounts), reclaim ethanol, be evaporated to 1: 1～3, filter, filtrate gets leaf of Turpinia pomifera (Roxb) D O. extract with embodiment 1 or embodiment 5 methods with legal system.
Embodiment 8: get leaf of Turpinia pomifera (Roxb) D O., pulverize, add 0.04% sodium hydroxide solution and soak 24hr, percolation is collected percolate, and dilute hydrochloric acid is regulated pH value to 4～6, concentrating under reduced pressure, adding ethanol is 75% to containing the alcohol amount, leaves standstill 24hr, gets supernatant, reclaim ethanol, concentrate, concentrated solution gets leaf of Turpinia pomifera (Roxb) D O. extract with embodiment 1 or embodiment 5 methods with legal system.
Embodiment 9: get the leaf of Turpinia pomifera (Roxb) D O. extract of embodiment 1～8, add ethanol and make dissolving, add equal-volume 2mol/L hydrochloric acid solution, reflux is 5 hours in the N2 environment, puts coldly, and decompression recycling ethanol filters, and gets precipitation, with low amounts of water washing, drying, promptly; Or hydrolyzed solution recovery ethanol, adding ethyl acetate (or n-butyl alcohol) extraction 4 times, combining extraction liquid reclaims solvent, promptly.
Embodiment 10: gets the leaf of Turpinia pomifera (Roxb) D O. extract of embodiment 1～9, adds low amounts of water dissolving or suspendible, add 1% sodium hydroxide (or potassium hydroxide, ammonia etc.) solution, regulate pH value 5.0～7.5, concentrate, and drying, promptly.
Embodiment 11: get leaf of Turpinia pomifera (Roxb) D O., add 75% alcohol reflux 2 times, measured 1.5 hours for 16 times for the first time, measured 1.5 hours for 14 times for the second time, merge extractive liquid, reclaims ethanol, filters, 1% sodium hydroxide solution is regulated pH value to 8, ultrafiltration, filtrate is regulated pH value to neutral with dilute hydrochloric acid, concentrating under reduced pressure, spray drying, promptly.
Embodiment 12: get embodiment 3 leaf of Turpinia pomifera (Roxb) D O. extract 50g, add methanol and make dissolving in right amount, mix with 35g silica gel (200-300 order), fling to methanol, porphyrize, be added on the 800g 200-300 order silicagel column, chloroform-methanol gradient (9: 1,8: 2,7: 3,1: 1) eluting, every gradient elution 2BV, with 0.5BV Fractional Collections eluent, reclaim solvent, thin layer chromatography is inspected (silica gel g thin-layer plate respectively, with chloroform-methanol 9: 1,8: 2, be developing solvent at 7: 3,10% sulphuric acid ethanol is developer), merge same composition, get respectively in right amount in 200g gel (sephadex LH20) post column chromatography repeatedly, collect stream part (10ml/ flows part), get gallic acid, apigenin, apigenin-7-O-beta-D-glucoside, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside and compd E, F, G, monomer components such as H.
Wherein 9: 1 and 8: 2 eluting merge the main gallic acid (content can reach more than 35%) that contains of section part; Eluting merged main apigenin-7-O-neohesperidoside, the apigenin-7-O-2 '-rhamanopyranosyl rutinoside composition (content can reach more than 15%) of containing of section part in 7: 3.
Embodiment 13: gets leaf of Turpinia pomifera (Roxb) D O. extract 150g, pulverizes, add starch 50g, and lactose 50g, micropowder silica gel 1g, mixing is made 1000 capsules, promptly altogether.
Embodiment 14: gets leaf of Turpinia pomifera (Roxb) D O. extract 150g, pulverizes, and standby; Other gets beta-schardinger dextrin-450g, adds suitable quantity of water, grinds, and adds above-mentioned leaf of Turpinia pomifera (Roxb) D O. extract, grinds, and adds mannitol 100g, starch 80g mixing, and drying is pulverized, and the system granule adds aspartame 6g, magnesium stearate 2g, and mixing is pressed into 1000, promptly altogether.
Embodiment 15: get leaf of Turpinia pomifera (Roxb) D O. extract 100g, add the about 900ml of injection water and make dissolving, 1% sodium hydroxide solution is regulated 1pH value to 5～7.5, adds needle-use activated carbon 2g, heating, filter,, merge washing liquid with a small amount of water for injection detergent active charcoal, add the injection water to 1000ml, fill becomes 200, sterilization, promptly.
Embodiment 16: get leaf of Turpinia pomifera (Roxb) D O. extract 100g, mannitol 50g adds the about 900ml of injection water and makes dissolving, 1% sodium hydroxide solution is regulated pH value to 5～7.5, add needle-use activated carbon 2g, heating filters, with a small amount of water for injection detergent active charcoal, merge washing liquid, add the injection water to 1000ml, fill becomes 200, lyophilization, promptly.
Description of drawings:
Fig. 1 is the HPLC collection of illustrative plates of embodiment 1 extract;
Fig. 2 is the HPLC collection of illustrative plates of embodiment 3 extracts.
Claims (10)
1. leaf of Turpinia pomifera (Roxb) D O. extract is characterized in that: contain flavonoid chemical constituent and medically acceptable salt thereof, the flavonoid chemical constituent contains apigenin and is the flavonoid glycoside composition of aglycon with the apigenin.
2. leaf of Turpinia pomifera (Roxb) D O. extract according to claim 1 is characterized in that: the flavonoid glycoside composition that with the apigenin is aglycon is one or more in apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside, the apigenin-7-O-beta-D-glucoside.
3. leaf of Turpinia pomifera (Roxb) D O. extract according to claim 1 and 2, it is characterized in that: general flavone content is more than 50%, and it is equal more than 1% or apigenin content more than 1% to contain apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside amount.
4. leaf of Turpinia pomifera (Roxb) D O. extract according to claim 1 is characterized in that: contain phenolic acid compound except that flavones ingredient and medically acceptable salt thereof, described phenolic acid compound contains gallic acid.
5. leaf of Turpinia pomifera (Roxb) D O. extract according to claim 4 is characterized in that: contain total phenolic acid more than 20%.
6. leaf of Turpinia pomifera (Roxb) D O. extract according to claim 4 is characterized in that: contain gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside content and be not less than 0.8%, 1.0%, 1.0% respectively.
7. leaf of Turpinia pomifera (Roxb) D O. extract according to claim 4 is characterized in that: contain gallic acid, apigenin-7-O-neohesperidoside, apigenin-7-O-2 '-rhamanopyranosyl rutinoside content and be not less than 2.5%, 3.0%, 3.0% respectively.
8. according to the preparation method of the described leaf of Turpinia pomifera (Roxb) D O. extract of claim 1～7, it is characterized in that getting fresh or dry leaf of Turpinia pomifera (Roxb) D O., adopt in the technologies such as solvent extraction method, solvent extraction, resin adsorption method, column chromatography one or more and make.Extract solvent and can be one or more mixing or combination of water, sour water, aqueous alkali, alcohol-water solution, methanol, ethanol, hexane, petroleum ether, chloroform, dichloromethane, acetone, ether, Ethyl formate, ethyl acetate etc.; Extractant can be one or more mixing or combination of hexane, petroleum ether, chloroform, dichloromethane, ether, ethyl acetate, n-butyl alcohol, amylalcohol etc.; Adsorbent resin can be in phenylethylene, polyacrylate, vinyl cyanide, the nitrogen oxide type adsorbent resin one or more; The column chromatography filler can be in silica gel, aluminium oxide, active carbon, polyamide, adsorbent resin, the polydextran gel etc. one or more.
9. the preparation method of leaf of Turpinia pomifera (Roxb) D O. extract according to claim 8 is characterized in that:
Get fresh or dry leaf of Turpinia pomifera (Roxb) D O., decoct with water 2 times, each 1.5 hours, merge extractive liquid,, concentrating under reduced pressure, concentrated solution add ethanol to containing alcohol amount 40%～85%, leave standstill, filter filtrate recycling ethanol, concentrating under reduced pressure, dilute hydrochloric acid is regulated pH value 3～5, slowly by macroporous adsorbent resin, with 0～20% ethanol elution remove impurity, 40%～95% ethanol elution merges eluent, reclaims ethanol, concentrating under reduced pressure, spray drying, promptly.
10. according to the described leaf of Turpinia pomifera (Roxb) D O. extract of claim 1～7, the application in the medicines such as, antiviral antibiotic, antioxidation, mutation, antitumor, antithrombotic, arteriosclerosis, liver protecting, anti-hepatitis virus, antidepressant, neuro-protective and immunomodulating in preparation.
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| CNA2008103003913A CN101249118A (en) | 2007-02-25 | 2008-02-25 | Chinese medicine extract and medicine use thereof |
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