1、天然产物研究与开发Nat Prod Res Dev 2008，20：974-982文章编号：1001-6880(2008)06-0974-09几种常用中草药抗氧化活性研究李华涛，苏东海，尚 涛，潘为高，高 平+匹t)ll大学生命科学学院生物资源和生态环境教育部重点实验室，成都610064摘要：当归、黄芪、银杏叶、益母草、野甘草是中国传统中药材，几千年来一直为中国人民所认可，在中国和世界都具有重要的科研和药用价值。本研究采用HO清除及对肝微粒体和亚油酸脂质过氧化抑制的方法，测定五种草药精油和水煮提取物的抗氧化活性；采用Folin-Cioealtea试剂法测定它们的总酚含量；用肝细胞体外培养法测定
2、它们的细胞毒性并对它们的作用机制进行分析。结果发现五种草药提取物都具有一定的抗氧化活性，尤其是益母草、野甘草、银杏叶的水煮提取物活性较强，其抗氧化活性与总酚含量存在较好的线形关系。此外，本论文还为研究这些草药的一些抗病机制提供参考依据。关键词：抗氧化活性；当归；黄芪；银杏叶；益母草；野甘草中图分类号：R285；Q58；TQ041 文献标识码：AAntioxidant Activity of the Essential Oils and Aqueous ExtractsObtained from Five Chinese Herbal MedicinesLI Huatao，SU Donghai，
3、SHANG Tao，PAN Weigao，GAO Ping+Ministry of Education Key Laboratoryfor Bio-resource and EcoEnvironment，College of Life Science，Sichuan University，Chengdu 610064，ChinaAbstract：The present work examined the antioxidant activity of Motherwort(Leonurus heterophyllus Sweet)，Broomwort(Scoparia dulcis L)，Gi
4、nkgo leaf(Ginkgo biloba L)，AngeUca(Angelica sinensis Diels)，and Milkvetch(Astragalusmembranaceu Bunge)Dried herbs samples were submitted to extraction with steam distillationThe antioxidant activitiesof aqueous extracts and essential oils were assessed by the measurement of activities of HOscavengin
5、ginhibition oflipid peroxidationTotal phenolic constituents of the aqueous extracts and essential oils of five herbs were determined bythe methods involving the Folin-Ciocalteu reagent and gallic acid a8 standardThe cytotoxieity of the aqueous extractsand essential oils were measured by MTr and LDH
6、cytotoxicity assaysAll extracts of five herbal medicines were deter-mined to possess antioxidative activity to some extentThe aqueous extracts of Motherwort，Broomwort and Ginkgo leafshowed hiigh antioxidant activity in the performed testsThe antioxidant activity of essential oils and aqueous extract
7、s obtmned from the five herbs displayed a significant relationship with the total phenols in themKey words：antioxidant activity；Motherwort(Leonurus heterophyllus Sweet)；Broomwort(Scoparia dulcis L)；Ginkgo leaf(Ginkgo bdoba L)；Angelica(Angelica sinensis Diels)；Milkvetch(Astragalusnerz占ranaceu Bunge)I
8、ntroductionAntioxidants have been of interest to pharmacologists，biochemists and other health professionals because theyare supposed to reduce oxidative damage caused by reactive oxygen species，reactive nitrogen species，and reactive chlorine species(Zhu，et al，2004)There isReceived May 28。2007；Accept
9、ed June 12，2007Corresponding author Tel：86-2885251989；E-mail：gaopin999hotmailcomgrowing interest in replacing synthetic antioxidants withnatural ingredients because of山e concern over tle possible carcinogenic effects(Ito et al，1 986；Chen，Shi&Ho，1992)and toxic activity(Martin&Gilbert，1968；Branen AL，1
10、 975；Reische，Lillard&eitenmiller，1 998)of synthetic antioxidants in foodsPlant phenolicand polyphenolic compounds are widespread components of the human diet，which found in vegetables，fruits，soybean，cereals，tea，coffee，red wine and naturalherb extractsAnd Lugasi et al(1994，1996)had reported that extr
11、acts from medicinal plant and culinary万方数据V0120 LI Hua-tao，et al：Antioxidant Activity of the Essential Oils and Aqueous Extracts Obtained from Five Chinese Herbal Medicines 975herb sources possess obvious antioxidant activity invitroMany herbs grown in China have been found to possessantioxidant act
12、ivityMotherwort(Sun，et al，2005；Nam&Kang，2004)，Ginkgo leaf(Lugasi，Horvahovich&Dworschak，1999；Zheng&Wang，2001；Akiba，et al，2004)，Angelica(Wu，Ng&Lin，2004；Hoult&Paya，1996)and Milkvetch(Shirataki et al，1997；Wang&Feng，2000)had always been used in ancient China，and they still demonstrate their important sci
13、entific andmedicinal value all around the world todayHowever，these plants were mostly investigated from some otherpoints regarding their medicinal properties，avonoidcomposition and other material of purificationThe literatures focused on antioxidant activity of aqueous extract and essential oils，cyt
14、otoxicity seemed scarce inthe past few yearsIn this paper，we have investigated the antioxidanteffect and cytotoxicity of extracts from aqueous extractsand essential oils obtained from Motherwort，Broomwort，Ginkgo leaf，Angelica and MilkvetchThe effect ofeach extract was compared with that of butylated
15、hydroxytoluene(BHT)，vitamin E(VE)，vitamin C(Vc)and BzOHThe experiments were repeatedthriceWhen the differences between the replicateswere more significant，the measurements were repeatedMaterials and MethodsMaterialsThe following reagents were used：synthetic antioxidant2，6-ditertbutyl-4一methylphenol(
16、BHT)，linoleic acid，gallic acid(AR，Shanghai Chemical Reagent Factory，China)，EDTA-2NaFe(II)，safranine(AR，Chengdu Kelong Chemical Reagent Factory，China)，cysteine，FeS04(AR，Tianjin Guangfu Fine ChemicalResearch Institute，China)，trichloroacetic acid(AR，Shanghai SSS Reagent Co，LTD，China)，thiobarbituric aci
17、d(TBA)(AR。，Sinopharm Chemical ReagentCo，Ltd，China)，Folin-Ciocaheu reagent(Sanlandchem International Inc)，DMEMF12，eollagenasetype IV，standard FBS，(GIBCO)，HEPES(Lvshengyuan biotechnology)A1l of other chemicals were arialytical gradeAerial paxts of Motherwort(Leonurus heterophyllusSweet)and Broomwort(S
18、coparia dulcis L)，root ofAngelica(Angelica sinensis Diels)and Milkvetch(Astragalus membranaceu Bunge)were obtained fromChengdu Pharmaceuticals market of ChinaGinkgoleaves(Ginkgo biloba L)were harvested in the campus of Sichuan University(Chengdu，China，October2005)The herbs were dried at loom tempera
19、ture andground(max particle size 032 mm)MethodsPreparation of essential oil and aqueous extractsIsolation of the essential oil(EO)Dried and ground herbs were subjeeted to steam distillation in a Clavenger apparatus for 4 h1he essentialoils were dried over anhydrous sodium sulfate and kepti12 sealed
20、botdes in tle dark and stored in a freezer below一18 oC until usePreparation of the aqueous extracts(AE)After completion of steamdistillation，tiquid retentatewas collected and concentrated using a SHZ-D()vacuum pump(Gongyi，China)，a RE52 CS rotavapour(Shanghai，China)with a water B-220 bath(Shanghai，Ch
21、ina)(60)All extracts were finaHy dried in a DZF一200 vacuumdrier(Shanghai，China)at(30-4-2)oC and 008 MPaDry extracts were kept in sealed bottles in the dark andstored in a freezer below一18 oC until useMeasurement of the antioxidative actwitiesHydroxyl radical(He)scavenging assayThe ability of essenti
22、al oil and aqueous extracts to scavenge the hydroxyl radical generated by the Fenton reaction was measured according to the modified method ofChung et al(1997)In brief50汕L of0945 mM EDTA-2Na-Fe(II)and 520 IxgmL safranine were mixedwith 50灿L of 015 mM phosphate buffer(pH 74)and 25 ppm extract in a 96
23、-well cell culture plateAb-sorbance was read at 520 nm using a microplate reader(Model 680，Japan)before 50肛L of 3H202 wasadded to the mixtureThereafter，the plates were incu-bated for 30 min at 37Laterabsorbance was measured at 520 nm in the microplate reader，()=100(A1Ao)万方数据976 Nat Prod Res Dev V012
24、0Where，Ao was the absorbance before 50 IxL of 3H，O，was added to the mixture and A 1 was the absor-bance after 96一well cell culture plate were incubatedfor 30 rain at 37Evaluation of the inhibition of lipid peroxidation in lin-oleic acidLinoleic acid in aqueous ethanolic solution which added 004of ex
25、tract solution was incubated in an ovenat 45in the darkThe inhibition of lipid peroxidation in Linoleic acid was assayed by ferric thiocyanate(FTC)and thiobarbituric acid(TBA)test according tothe method described by Kikuzaki&Nakatani(1 993)Maneuver as described was repeated every 24 h untilone day a
26、fter absorbance of the control reached maximumFTC testIn brief，to take 01 mL the sample，prepared and incubated as described97 mL 75ethanol and 01 mL30ammonium thiocyanate were added one by onePrecisely 3 min after adding 01 mL 210。2 M ferrouschloride in 35hydrochioric acid to the reactionmixture，the
27、 abIsorbance of red color was measured at500 nm with a WFJ一7200 spectrophotometer(Shanghai，China)刀姒testIn brief，to take 01 mL the sample，1 mL 20trichloroacetic acid and 2 mL 03Thiobarbituric acid wasadded one by oneThis mixture was placed in boilingwater bath for 15 minAnd it cooled at room temperat
28、ure before adding 4 mL nbutan01The mixture with nbutanol was centfifugated at 3000 rmin for 20 rainAbsorbance of supernatant was measured at 532 nmwith a WFJ-7200 spectrophotometerInhibition(f)oflipid peroxidation in percent was calculated by follow-ing equation：，()=100(1一AlAo)Where，Ao was the absor
29、bance of the control reaction(=full reaction，containing no test compound)and A1was the absorbance of the sample on the day after absorbance of the control reached maximumEvaluation of the inhibition of lipid peroxidation in liverliposomesLiver microsomes prepared from male Wistar rats werestored as
30、pellets at1 8until requiredFor each experiment they were suspended in 005 M TrisHCIbuffer(pH 74)，to give a stock suspension at a finalconcentration of 1 5 mg of proteinmLProtein contentwas determined by the method of Lowry et al(1951)The inhibition of lipid peroxidation in liver liposomeswas measure
31、d according to the modified method of Andrew JFSearle and Robin LWillson(1983)Inbrief15 mL of a l mr：mL microsomal suspension inTrisHCl was stimulated by the addition of portions offreshly prepared stock solutions of cysteine(5 mM)andFeS04(50 IxM)in doubledistilled waterThese gavefinal concentration
32、s of 500 and 5 IxM respectivelyTheextract solution was added to the microsomal suspension before the addition of cysteine and FeS04Sampleswere incubated at 37and the peroxidation wasstopped after 30 min by the addition of 3 mL of icecold 10(WV)trichloracetic acidAfter centrifugation，2 mL samples of
33、the supernatant fluid were removed and added to 2 mL of 067(wv)thiobarbituric acid in distilled waterAbsorbance was measuredat 535 nm in a 1 am cuvetteInhibition(I)ratio in percent was calculated by follow-ing equation：，()=100(1一A1Ao)Where，An was the absorbance of the control reactionand A 1 was the
34、 absorbance of the sampleAssay for total phenolicsTotal phenolic constituents of the essential oil and a-queous extracts of five herbs were determined by theliterature methods involving the FolinCiocalteu reagentand gallic acid as standard(Chandler&Dodds，1 983；Bektas Tepe et al，2005)01 mL of extract
35、 solutibn，containing l mg extract，was taken in a volumetricflask46 mL distilled water and 1 mL FolinCiocaheureagent were added，and flask was shaken thoroughlyAfter 3 min，3 mL of a solution of 2Na2C03 wereadded and the mixture was allowed to stand for 2 h withintermittent shakingAbsorbance was measur
36、ed at 760nmThe same procedure was repeated for a11 standardgallic acid solutions and a standard curve was obtainedby the equation given below：Absorbance：000 1 5Gallic acid(斗g)+00085万方数据V0120 LI Huatao，et al：Antioxidant Activity of the Essential Oils and Aqueous Extracts Obtained from Five Chinese He
37、rbal Medicines 977Assay for cytotoxicityMTT cytotoxicity assayThe conversion of the tetrazolium salt Mqq“3一(4，5dimethyhhiaz01-2一y1)-2，5一diphenyltetrazolium bromideinto a blue colored formazan by the mitoehondrial enzyme succinate dehydrogenase is very useful for quantifying cell survival and prolife
38、ration by detecting onlyliving ceils(Mosmann，1983)Hepatocyte，which usedto evaluate cytotoxicity of all essential oils and extractsin foods，were obtained from normal female mice(4一week-old)In brief，cells were cultured in DMEMF12with 1 0fetal bovine se31m，seeded at a density of 51 05 cellsmLAfter addi
39、ng 100 txL per well of cellsuspension into a 96一well cell culture plates，the plateswere incubated at 37 oC in a humidified atmospherecontaining 5C02 for 10 hAfter adding 100 txL perwell of different concentrations of test materials(4，3。2，1，05，025，0 mg of dry material)dissolved in 1mL of tridistilled
40、 water or DMSO(in less than 1)，the plates were further incubated for 24 hAfter super-natant fluid were soundly take suction and conserve(forLDH cytotoxicity assay)，175斗L DMEMF 12 with 10fetal bovine serum and 25 IxL per well MTY reagentwere added，and the plates were further incubated under tle same
41、conditions for 4 hAfter removing untransformed MTY reagent，1 00 txL of DMSO was addedto each well to dissolve MTFformazan crystals，and ab-sorbance was read at 490 nm using Model 680 Microplate readerThe estimation of cytotoxicity()wasbased on the difference between the numbers in experimental and co
42、ntrol wells，expressed as a percent of thecontr01LDH cytotoxicity assayLeakage of the liver enzyme lactate dehydrogenase(LDH)is very commonly used for measuring cytotoxicity of test agentsCytotoxicity was measured spectrophotometrically as the activity of LDH leaked into theincubation medium(Jiang an
43、d Acosta，1 993；Sahu andSapienza，2005)and was expressed as a percentage ofthe total LDH activity released from the ceils treated(Barsig et al，1 998；Michael adler and Geoffrey lee，1999)ResultsHydroxyl radical(HO)scavenging assayHydroxyl radical scavenging of the Ginkgo leaf EO andBroomwort AE exhibite
44、d a higher activities(6739and 6913)than other extracts，BHT and BzOHNoextracts had a higher activity than that of Vc in HOscavenging(Table 1)Table 1 Antioxidant activiti两of the essential oils and aqueous extracts from five herbsSample。Activity ofHOscavenging(25 ppm)Lipid peroxidationin linoleic acid(
45、FFC。004)Llipid peroxidationin linoleic acid(TBA，004)Inhibition of lipidperoxidation inliposomes(004)BHTVitamin EVitamin CBzOHbenzoicumGinkgo leaf EOGinkgo leaf AEAngelica EOAngelica AEMilkvetch EOMilkvetch AEMotherwort E0Motherwort AEBroomwort EO6425b058c 9303072 9500士082 8500097一 一 一 82410929610089
46、614005567390605792049555051595107l568304380870589309 4-0698279O5945930324805O3853290478906061928307378050296488O35966707970830545360491321012363102682140509714士0883535024388903224100464630397555O426446033380801212780134541031768505454250463740038Broomwort AE 6913 4061 9207士064 9512 4-086 8262082Note
47、：4EO，essential oil；AE，aqueous extracts；bValues ale the means of three replicates；eStandard deviation万方数据978 NatProdResDw V0120Inhibitory effects of essential oils and extracts onlipid peroxidation in linoleic acidBase on FFC methods，I()of all the EOs and AEsshowed some linoleic acid stabilising effe
48、ct in general(Table 1)The Ginkgo leaf AE were found to be theto BHTThe absorbance of the sample AE of Ginkgoleaf after 6 d of storage was approximately 01 08，whereas in blank samples it increased to 1563，in thesamples with BHT to 0109(Table 2)I()of thesamples with AE of Ginkgo leaf after 6 d of storage wasmost effective natural antioxidants which was superior 9309，whereas in BHT it was 9303Table 2 Absorbency of the essential oils and aqueous extracts from five herbs by FTC methodsNote：
