全 文 ：天然产物研究与开发 NatProdResDev2008, 20:888-891
文章编号:1001-6880(2008)05-0888-04
ReceivedJune14, 2007;AcceptedJuly7, 2007
FoundationItem:ThisworkwassupportedbyGansuProvincialAdmin-
istrationBureauofChineseMedicalSciencesFund(No:2003-GZK-
17)andGansuProvincialNaturalSciencesFund(No:YS-011-A25-
003).
＊CorrespondingauthorE-mail:cuiyan369@sina.com
藏药砂生槐种子提取物抑菌和细胞毒活性的初步研究
马兴铭 1, 2 ,于红娟 2 ,雒艳萍 2 ,崔 燕 1＊
1甘肃农业大学动物医学院 , 兰州 730070;2兰州大学基础医学院 , 兰州 730000
摘 要:砂生槐是我国西藏高原一种特有植物 , 是一种极为宝贵的药用植物资源。我们首次从砂生槐种子中获
得氯仿 、 95%乙醇 、75%乙醇和水提取物 , 用琼脂扩散实验测定其抑菌活性和用 MTT法测定其对肿瘤细胞的细
胞毒效应 , 表明氯仿提取物和 95%乙醇提取物具有广谱的抑菌活性 , 抑菌活性较强的是氯仿提取物。 各种提取
物显示出浓度依赖性的细胞毒效应 , 氯仿 、95%乙醇 、75%乙醇和水提取物对胃癌 SGC-7901细胞系的 LC50分别
是 4.5、1.4、 1.9和 41.7 mg/mL, 抑制胃癌 SGC-7901细胞增殖作用较强的成分是砂生槐种子 95%乙醇提取物。
这一发现为开发砂生槐这一宝贵植物资源的药用价值提供了重要的依据。
关键词:砂生槐;抑菌;细胞毒;MTT法;人类植物学
中图分类号:R931.71;Q946.91;R285.5 文献标识码:A
AntimicrobialandCytotoxicActivityofExtractsfrom
SophoramoorcroftianaSeedsinVitro
MAXing-ming1, 2 , YUHong-juan2 , LUOYan-ping2 , CUIYan1＊
1FacultyofVeterinaryMedicine, GansuAgriculturalUniversity, Lanzhou730070 , China;
2SchoolofBasicMedicine, LanzhouUniversity, Lanzhou730000 , China
Abstract:Sophoramoorcroftiana(Walich)isanendemicshrubspeciesinTibet, China, andisaplantwithagreatval-
ueinfolkmedicine.Chloroform, ethanolanddistilled-waterextractsoftheplantweretestedforantimicrobialactivityu-
singtheagar-weldiffusiontechniqueandforcytotoxicactivityusinganMTTassayinvitro.Thechloroformand95% al-
coholextractswereshowntopossessabroadspectrumofastrongantimicrobialactivityagainstaltestedbacteriaandthe
chloroformextractofS.moorcroftianaseedsismuchmoreeffectiveinantimicrobialactiveness.After24 h, allcrudeex-
tractssignificantlyshowedthecytotoxicactivityonhumanstomachcancerSGC-7901 celllineinaconcentration-depend-
entmanner.Themedianlethalconcentrations(LC
50
)ofchloroformextracts, 95% ethanolextracts, 75%ethanolextracts
anddistilled-waterextractsinthehumanstomachcancerSGC-7901 cellinewerefoundtobe4.5, 1.4, 1.9 and41.7
mg/mL, respectively.The95% ethanolextractofS.moorcroftianaseedsismuchmoreeffectivethanotherextractsin
termsofprovidingantiproliferativeefectsinthehumanstomachcancerSGC-7901 celline.
Keywords:Sophoramoorcroftiana;antimicrobialactivity;cytotoxicity;MTT;ethnobotany
Introduction
Sophoramoorcroftiana(Walich.)Benth.exBakeris
anendemicshrubspeciesinTibet, China, andismain-
lydistributedinthewidevaleysinthemiddlereaches
ofseveralmaintributariesoftheYaluTsangboRiver
(NianchuandLhasaRivers).S.moorcroftianaseeds
havebeenusedforalongtimeinChinesefolkmedi-
cine.ThedecoctionoftheseedhasbeenusedinChi-
nesefolkmedicinefordephlogistication(translated, in
general, asanti-inflammationinmodernmedicine), de-
toxication, emeticprocesses, andtreatinginfectiousdis-
easesandverminosis[ 1] .ThealkaloidsobtainedfromS.
moorcroftianahaveaprotoscolicidal, anti-inflammatory
efect[ 2] andinduceapoptosisofthehumanstomach
cancerSGC-7901 cellineinvitro[ 3] .Thesophorastil-
beneAand(+)-α-viniferinfromS.moorcroftianain-
hibitedcopper-inducedproteinoxidativemodification
invitro[ 4] .TheprenylflavanonesfromSophoratomento-
saL.andS.moorcroftianashowtumor-specificcytotox-
icactivityandantimicrobialactivity[ 5] .However, the
efectofchloroform, alcoholandwaterextractsfromS.
moorcroftianaseedsonmicroorganismsandoncancer
celshasnotyetbeenexamined.Inordertodevelopthe
traditionalmedicinefurther, thisinvitrostudywasun-
dertakentodemonstratetheefectofdiferentextracts
ofS.moorcroftianaseedsonmicroorganismsandona
humanstomachcancercellineandalsodetermine
whichtypeofextracthadthemostefectivecomposition
intermsofantimicrobialandcytotoxicactivityinvitro.
MaterialsandMethods
Plantmaterialandcrudeextracts
TheseedsofS.moorcroftianawerecolectedinOctober
2001fromthebanksoftheYaluTsangboRiverinthe
Tibetanaltiplanoandair-driedatroomtemperaturefor
3 weeks.Oneherbariumvoucherspecimen(M2001-
1005)oftheplanthasbeenpreservedinourlaboratory
forfuturereference.Theseedswerepowdered(422.8
g)usingablenderandthenChloroform(11.2 g, yield
2.6%), 95% ethanolextracts(10.6 g, adarkbrown
solidmass, yield2.6%), 75% ethanolextracts(15.1
g, adarkbrownsolidmas, yield3.8%)anddistiled-
waterextracts(20.6g, adarkbrownsolidmass, yield
5.2%)ofseedswerepreparatedbyYANGYunshang
DoctorofGansuTechnologicalUniversity.Thedry
crudeextractsofS.moorcroftianaweredissolvedinto
therequiredconcentrationsindimethylsulphoxide
(DMSO), thefinalconcentrationofwhichwas0.1%.
Agar-weldifusiontechnique
ThebacteriaStaphylococuaureus(ATCC 25923,
A209, CMCC26003), Bacilussubtilis(ATCC6633),
Escherichiacoli(ATCC25922, CMCC44102), Shigel-
laflexneri(51571), Pseudomonasaeruginosa(ATCC
27853)andSalmonelatyphi(H901)wereobtained
fromtheSchoolofBasicMedicine, LanzhouUniversity,
China.Thediskdifusionmethodwasusedforthede-
terminationoftheantimicrobialactivityofthecrudeex-
tracts[ 7] .Briefly, asuspensionofthetestedmicroorgan-
isms(0.1 mLof108 CFUs/mL)wasspreadonthe
solidmediaplates.TheMHnutritivemedium(Hang-
zhouMicrobialReagentLimitedCompany, China)was
used.Nutritivemediawerepreparedaccordingtothe
manufacturer sinstructions.Alagarplateswerepre-
paredin90-mmPetridisheswith22 mLofagar, giving
afinaldepthof4mm.Sterilefilter-paperdisks(6mm
indiameter)wereimpregnatedwith10 μLperdiskof
thecrudeextractsandplacedoninoculatedplates.
Theseplates, afterstandingat4 ℃ for2 h, wereincu-
batedat37 ℃ for24 htodevelopbacteria.Standard
disksofcidomycin(10 μgperdisk)wereusedasa
positivecontroland0.1% DMSOdisks(10 μLper
disk)wereusedasanegativecontrol.Thediametersof
theinhibitionzonesweremeasuredinmilimetresusing
averniercaliper.Eachtestwasperformedintriplicate
andrepeatedthreetimes.
Invitrocytotoxicityassay
Thecytotoxic efectsofcrude extractsfrom S.
moorcroftianaseedstoprotectagainsthumanstomach
cancerweredeterminedusinga3-(4, 5-dimethylthia-
zol-2-yl)-2, 5-diphenyltetrazoliumbromide(MTT)
colorimetricassay[ 7] .ThehumanstomachcancerSGC-
7901 cellineinthelogarithmicgrowthphasewas
washedoncewithphosphate-buferedsaline(PBS),
andresuspendedtoafinalconcentrationof1.5 ×105
cels/mLinfreshRPMI1640medium(Sino-American
BiotechnologyCompany, China), andsupplemented
with10% inactivatedfetalbovineserum(FBS, Sino-
AmericanBiotechnologyCompany, China), penicilin
(100 U/mL)andstreptomycin(100 μg/mL).The
celsuspension(1.5 ×104)wasplacedina96-wel
tissue-cultureplateandhumidifiedat37 ℃ and5%
CO2 atmosphere.Afterthelogarithmicgrowthphaseof
celswasreached, thesupernatantwasdiscarded.Fol-
lowingthis, thechloroformextracts, 95% ethanolex-
tracts, 75%ethanolextracts, andthedistiled-waterex-
tractswithfinalconcentrationsof0.31, 0.63, 1.25,
2.50 and5.00mg/mLwereaddedtothewels, respec-
tively, suchthateach3 welswereincubatedwithone
oftheseconcentrations.CelsthatweretreatedwithEt-
oposide(VP-16, QindaoHaierPharmaLimitedCompa-
ny, China), anacytotoxicdrug, at0.08 μmol/L, were
usedasapositivecontrol, and0.1% DMSOwasused
asanegativecontrol.After24h, viablecelgrowthwas
889Vol.20 MAXing-ming, etal:AntimicrobialandCytotoxicActivityofExtractsfromSophoramoorcroftianaSeedsinVitro
determinedusingtheMTTassay.Foreach200 μl
growthmedium, 20 μLMTT(Sino-AmericanBiotech-
nologyCompany, China)solutionwasaddedandincu-
batedfor4 h.Afteralgrowthmediahadbeenre-
moved, thecrystalparticlesofMTTweredissolvedby
adding200μLDMSOandshakingplatesfor2-3 min.
Theabsorbanceofformazandyewasreadat570 nmu-
singanELISAplatereader.Thewholeprocedurewas
repeatedthreetimes.Theacytotoxicitywascalculated
asfolows:%acytotoxicity=(1-ODextracttreated)
/ODnegativecontrol×100.
Statistics
DatawereexpressedasMean±SD.Statisticalsignifi-
cancewasdeterminedbyone-wayANOVAwithSPSS
(StatisticalPackagefortheSocialSciences, 10.0)for
Windows(SPSSIncorporationinChicago, IL, USA).
ResultsandDiscussion
AlthoughmanytraditionalChinesemedicineshowthe
antitumorefectsoncancercels[ 8] andthealkaloids
andprenylflavanonesfromS.moorcroftianaalsoshow
anantitumoreficacyinvitro[ 2, 4] , nopreviousliterature
hasshowntheefectivenessofcrudeextractsofS.
moorcroftianaseedsonbacteriaandSGC-7901 cel
lines.Ourdatademonstratedthatthechloroformextract
andthe95% ethanolextractfrom S.moorcroftiana
seedsweresignificantlyactiveagainstthetestedbacte-
riaandtheactivitywasdose-dependent.The75% eth-
anolextracthadslightantibacterialactivity.However,
thedistiled-waterextractdidnotshowactivityagainst
thetestedbacteria.Althecrudeextractsdidnotshow
activityagainstCandidaalbicans(notreportedinTa-
ble1).ThechloroformextractsoftheS.moorcroftiana
seedsshowedmuchmoreefectiveingredientstopro-
moteantimicrobialactivityinvitro(Table1).
Table1 AntimicrobialactivityofcrudeextractsfromSophoramoorcroftianaseeds
Microor
ganism
Zonesofinhibition(mm, n=9, x±s)a
Chloroform(mg/disk) 95%Ethanol(mg/disk) 75% Ethanol(mg/disk) Water(mg/disk) Ref.b
2 4 8 2 4 8 2 4 8 2 4 8 (10μg)
S.aATCC
25923
9±0.9 11±1.8＊ 16±1.7■ 9±0.9 9±0.6 10±1.1■ 7±0.5 8±0.6 8±0.4＊ 0 0 0 18
S.aA209 9±0.4 12±1.3＊ 16±2.2■ 8±0.4 9±0.4＊ 10±1.4■ 7±0.4 8±0.4 8±0.3＊ 0 0 0 18
S.aCMCC
26003
9±0.6 11±1.1＊ 15±1.9■ 8±0.4 9±0.4 10±0.9■ 7±0.2 8±0.6 9±0.9＊ 0 0 0 18
B.sATCC
6633
8±1.0 12±1.3＊ 17±2.3■ 7±0.5 8±0.2 10±0.8■ 7±0.4 8±0.4 9±0.8＊ 0 0 0 21
E.cATCC
25922
8±0.4 9±0.4＊ 11±1.4■ 9±0.8 10±1.3＊ 11±1.2■ 0 0 7±0.3 0 0 0 20
E.cCMCC
44102
8±0.3 9±1.6 11±1.3＊ 7±0.4 8±0.7 8±0.6 0 0 7±0.4 0 0 0 20
S.f51571 8±0.7 10±1.6＊ 12±1.0■ 7±0.6 8±0.4＊ 10±0.8■ 0 7±0.6 9±1.1 0 0 7±0.3 21
P.aATCC
27853
8±0.8 9±0.6＊ 11±1.1■ 7±0.3 8±0.8 9±1.3＊ 8±0.6 8±1.0 9±1.0＊ 0 0 0 17
S.tH 901 8±0.7 9±0.9＊ 11±0.9■ 7±0.4 8±1.0 9±1.1＊ 0 7±1.0 8±0.7 0 0 0 16
Note:S.a=Staphylococcusaureus, B.s=Bacilussubtilis, E.c=Escherichiacoli, S.f=Shigelaflexneri, P.a=Pseudomonasaeruginosa, S.t=Sal-
monelatyphi.aValues, includingdiameterofthewel(6.0mm), arethemeanofthreereplicates.bCidomycinStatisticaldataarereportedasmean±
SD:＊P< 0.05, ■P< 0.01vslowconcentration(2mg/disk).
Fig.1showsthatalcrudeextractssignificantlyinhibi-
tedtheproliferationofthehumanstomachcancerSGC-
7901cellineatalconcentrations.Thepositivecyto-
toxiccontrolagent, VP-16, ataconcentrationof0.08
μmol/Lcancauseacytotoxicitylevelof16%.Incom-
parisonwiththepositivecytotoxiccontrolagent, VP-
16, ahighercytotoxicitylevelintheSGC-7901celline
wasobservedaftertreatmentwith0.63 mg/mLofthe
crude extracts.The median lethalconcentrations
890 NatProdResDev Vol.20
(LC50)ofthechloroformextracts, the95% ethanolex-
tract, the75% ethanolextractandthedistiled-water
extractsonthecytotoxicityoftheSGC-7901 celline
were4.5, 1.4, 1.9 and41.7 mg/mL, respectively.In
thepresenceof2.50 (60%)and5.00 (87%)mg/
mL95% ethanolextract, astrongercytotoxicactivityin
theSGC-7901cellinewasfound.Theseresultssuggest
thatthe95% ethanolextractfractionofS.moorcrofti-
anaseedsismuchmoreefectivethanotherextracts
Fig.1 Growthinhibitioncurvesofcrudeextractsfrom
SophoramoorcroftianaseedsontheSGC-7901
celllinefor24hinvitro(n= 9)
Thereforeitcouldbeanewsourceofnaturalantimicro-
bialandcytotoxicactivityinhumanstomachcancer
SGC-7901celline.However, intermsofhowthisfrac-
tioncandiferentiatebetweenthecrudeextractsofS.
moorcroftianaseedsforantimicrobialandcytotoxicac-
tivity, furtherstudiesareneededtoexplorethisand
otherrelatedimportantissues.
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891Vol.20 MAXing-ming, etal:AntimicrobialandCytotoxicActivityofExtractsfromSophoramoorcroftianaSeedsinVitro