全 文 ：Activity of Zanthoxylum Species and their Compounds A-
gainst Oral Pathogens
CJNM
SHI Yao＊ , LI Ding-Xiang ,MIN Zhi-Da
Department of Natural Medicinal Chemistry , China Pharmaceutical University , Nanjing 210009 , China
【ABSTRACT】　 Ethanolic extracts obtained from six different Zanthoxylum species and four compounds isolated from Z.
dimorphyllum were screened for their antibacterial activity against oral pathogens(Streptococcus mutans ATCC 25175 , Actino-
myces naeslundii ATCC 12104 , Actinobacillus actinomycetemicomtans ATCC 43717).Antibacterial activity was tested using a-
gar diffusion method and minimum inhibitory concentration(MIC).The results revealed that Z.lactum showed the strongest
inhibitory effect , whereas Z.nitidum and Z.dimorphyllum exhibited moderate antibacterial activity.Further analysis via
bioassay-guided fractionation and purification of the plant Z.dimorphyllum resulted in the isolation of four known compounds
identified as Scoparone(1);6 , 7 , 8-Trimethoxycoumarin(2);Canthin-6-one (3);Syringaresinol (4).Canthin-6-one(3)
exhibited strong activity against the tested bacteria with MIC values between 15.6 and 62.5μg/mL.
【KEY WORDS】　Zanthoxylum;Z.dimorphyllum;Z.lactum;Oral pathogens;Antibacterial activity;Canthin-6-one
【CLCNumber】　R965.1　　【Document code】　A　　【Ariticle ID】　1672-3651(2005)04-0249-05
【Received date】　2005-01-04
【＊Corresponding author】　Tel:025-85322441, E-mail:sshi2003@
hotmail.com
1　Introduction
Chewing sticks for oral hygiene is widely practiced
in Africa.Some of the chewing sticks are made from the
plants of genus Zanthoxylum.It is generally believed
that the beneficial effect is attributed to the mechanical
cleaning effect and antimicrobial substances present in
their chewing sticks.For example , fagaronine , a com-
pound isolated from a chewing stick , Z.fagara , has
been shown to aid improvement of oral hygiene of some
rural natives[ 1] .Similarly , in China the extract of Z.
nitidum has been added to toothpaste due to the strong
antibacterial activity of its benzo[ c] phenanthridine al-
kaloids
[ 2].
The purpose of this study was to compare the an-
tibacterial activities of six Zanthoxylum plant extracts a-
gainst oral pathogens S.mutans , A.naeslundii , A.
actinomycetemicomtans.S.mutans is a bacterium re-
sponsible for the development of dental caries. A.
naeslundii is an early colonizer to form dental plaque , a
biofilm on the tooth surface which is considered impact
oral hygiene.A.actinomycetemicomtans is a specific
pathogen causing certain serious type of periodontal dis-
ease in adolescents.Study of those bacteria is of impor-
tance for potential application to improve oral health.
The antibacterial effect of the extracts was evaluated via
conventional microbiological methods.Through further
bioassay-guided fractionation and purification , four
known compounds , Scoparone(1);6 ,7 , 8-Trimethoxy-
coumarin(2);Canthin-6-one (3);Syringaresinol (4)
were isolated from Z.dimorphyllum.Their antimicro-
bial activities were also evaluated.
2　Materlal &methods
Plant material.　Six species of Zanthoxylum
Plants , Z.esquirolii , Z.dimorphyllum.Z.nitidum ,
Z.armatum , Z.scandens , Z.lactum , were collect-
ed from Guangxi province , China in 2001 and identified
by Prof.Lai Mao-xiang Voucher specimens have been
deposited at the Dept.of Natural Medicinal Chemistry ,
China Pharmaceutical University , Jiangsu.
Preparation of extracts　Extracts were prepared
by mincing it into pieces , and grinding it in a mill into
coarse powder.150 g of each powderwas extracted with
1 000 mL of EtOH for 3 h at 60 ℃ and repeated for 2
times.The resultant suspension was passed through a
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中国天然药物　2005年 7月　第 3卷　第 4期
gauze filter to remove large debris.Solvent was evapo-
rated under reduced pressure to obtain an extraction so-
lution , and then centrifuged at 3 000 r/min for 10 min.
The supernatant fraction was carefully decanted and
passed through a 0.22 μm(pore size)membrane filter
under vacuum to remove insoluble materials.The corre-
sponding concentrations(garm/milliliter:the weight of
dry plant per extraction solution)for all the extracts
were 1.0 g/mL.The extracts were kept at -4 ℃until
tested.
Compounds(1 ～ 4)isolated from Z.dimor-
phyllum　The powdered , air-dried roots (10 kg)were
extracted with 15 L of hot EtOH for 5 h.The extraction
was repeated 3 times.Solvent was evaporated under re-
duced pressure to obtain 759 g of a dark viscous residue
(crude extract).To this extract , H2O was added and
acidified with 2mol/L HCl to pH 3.The water insoluble
phases were combined and not analyzed.The aqueous
acidic solution was basified with NaOH to pH 8.0 and
extracted with CHCl3 until Dragendorff s test was nega-
tive.The organic phases were combined and the solvent
evaporated to 24 g(yield:0.24%)of a dark residuewas
obtained.The concentrated CHCl3 fraction was subjected
to repeatedly flash-chromatography on silica (40 ～ 63
μm)eluted with increasing amounts of acetone in n-hex-
ane to afford compound 1(43 mg), 2(19 mg), 3(202
mg), 4(24 mg).The structures of compounds(1 ～ 4)
were identified by their 1D and 2D NMR spectrum and
comparisons with published data[ 3-6] .
　　Microorganisms　The cariogenic bacteria Strep-
tococcus mutans(ATCC 25175), and Actinomyces naes-
lundii (ATCC 12104), and periodontopathic bacteria
A.actinomycetemicomtans(ATCC 43717)were used for
the agar diffusion assay and the minimum inhibitory
concentration (MIC).These strains were maintained
anaerobically on blood agar plates containing trypticase
soy agar(TSA , Becton Dickinson Microbiology System ,
Cockeysville , MD)supplemented with 10%defibrinat-
ed horse blood.
Agar diffusion assays 　The disc-diffusion as-
say
[ 7]
was used to determine the growth inhibition
caused by plant extracts against the above oral bacterial
strains.Base plates were prepared by pouring 15 mL
trypticase soy agar into sterile petri dishes (9 cm)and
allowed to set.MoltenMH agar held at 48 ℃was inoc-
ulated with a broth culture (106 ～ 108 bacteria per
milliliter)of the test organism and poured over the base
plates forming a homogenous top layer.Filter paper
discs(WhatmanNo.3 , 6mm diameter)were sterilized
by autoclaving.Twenty milliliters of plant extract (1
mg/mL)was applied per filter paper disc so that each
disc contained 2 mg of material.The discs were air-
dried and placed onto the seeded top layer of the MH a-
gar plates.Each extract was tested in quadruplicate
(four discs per plate), with a 2 , 4 , 4′-trichloro-2′-hy-
droxydiphenyl ether (commercial name known as Tri-
closan
TM)(1 mg/mL)disc as reference or positive con-
trol.Air-dried solvent (ethanol)saturated discs were
used as negative controls.The plates were evaluated af-
ter incubation at 37.0 ℃±1.0 ℃ for 18 h after which
the zones of inhibition around each disc were measured.
The ratio of the inhibition zone(mm)produced by
the plant extract and the inhibition zone around the Tri-
closan reference(mm)was used to express antibacterial
activity[ 8].The activity of Triclosan was included in this
equation to adjust for plate-to-plate variations in the
sensitivity of a particular bacterial strain
[ 9].
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Chin J Nat Med　July.2005　Vol.3　No.4　249　
Table 1　Zone(mm)of growth inhibition of bacteria by EtOH extracts from Zanthoxylum species and MIC(mg/mL)
Plant species
A.naeslundii
Zone(mm) MIC(mg/mL)
S.mutans
Zone(mm) MIC (mg/mL)
A.actinomycetemicomtans
Zone(mm) MIC (mg/mL)
Z.esquirolii (Stem) 4.5 3.1 0 >25.0 3.0 >25.0
Z.dimorphyllum (Root) 5.5 3.1 0 >25.0 2.0 >25.0
Z.nitidum (Root) 5.0 3.1 0 >25.0 3.0 >25.0
Z.nitidum (Stem) 4.0 6.2 0 >25.0 2.0 >25.0
Z.armatum (Root) 1.0 >25.0 0 >25.0 1.0 >25.0
Z.armatum (Stem) 1.5 >25.0 0 >25.0 1.0 >25.0
Z.armatum (Leaves) 1.0 12.5 0 >25.0 0 >25.0
Z.scandens(Root) 3.0 6.2 0 >25.0 3.0 >25.0
Z.scandens(Stem) 3.0 6.2 0 >25.0 3.0 >25.0
Z.scandens(Stem/ Leaves) 4.0 12.5 0 >25.0 3.0 >25.0
Z.lactum (Root) 5.0 1.6 0 6.2 5.0 6.2
mg/mL:dry weight of powdered plant per residue solution in mi lliliter
　　MIC assays.　The MIC of the respective plant
extracts and isolated pure compounds against oral bacte-
ria were determined with liquid cultures in 96-well cul-
ture plates according to a modification of the method de-
scribed by Shapiro et al.[ 10] Trypticase soy broth(TSA ,
Becton Dickinson Microbiology System , Cockeysville ,
MD)was used for S.mutans , A.naeslundii and A.
actinomycetemicomtans , Todd Hewitt broth supplement-
ed with 1% yeast extract (Difco Laboratories , Detroit ,
MI)was used.Serial dilutions (1.0%～ 0.002%)of
each extracts and compounds were prepared in each cul-
ture medium.Aliquots(200μL)of each dilution were
dispensed in 96-well cell culture plates.Subsequently ,
10
5 ～ 106 test bacteria that had been cultured overnight
in each culture medium were inoculated into each well ,
and cultured at 37.0 ℃±1.0 ℃ for 1 ～ 2 days under
anaerobic conditions.Then the absorbance was mea-
sured at 630 nm(Bio-tek , ELX808).The highest dilu-
tion at which no growth (OD630≤0.05)was observed ,
was defined as minimum inhibitory concentration(MIC)
.(See Table 2)
Table 2　MIC of compounds 1 ～ 4 against oral bacteria
Compounds
MIC(μg/mL)
A.naeslundii S.mutans A.actinomyce-
temicomtans
Scoparone (1) >250 >250 >250
6 ,7 , 8-Trimethoxy-coumarin(2) >250 >250 >250
Canthin-6-one(3) 31.2 62.5 15.6
Syringaresinol(4) >250 >250 >250
3　Results &discussion
In the agar diffusion assay , Z.lactum showed the
strongest anti-bacterial activity against the two tested o-
ral pathogens , the rest of the extracts revealed moderate
activity against A. naeslundii and A.actinomycete-
micomtans.In the MIC assay , Z.lactum appeared to
be the most active extract against all 3 oral bacteria.Z.
dimorphyllum and Z.nitidum exhibited activity against
A.naeslundii.Based on the results of antibacterial ac-
tivity , we further studied the chemical constituents of
Z.dimorphyllum and Z.nitidum.This paper reports
4 isolated compounds (1 ～ 4)from Z.dimorphyllum
CHCl3 fraction.In the six compounds , Canthin-6-one(
3)showed MIC values of 62.5 , 31.2 and 15.6μg/mL
for S.mutans , A.naeslundii , and A.actinomycete-
micomtans , respectively , whereas the MIC values of the
other 5 compounds were above 250 μg/mL (Table 3).
Canthin-6-one(3)appears to be the most effective an-
tibacterial extract obtained from Z.dimorphyllum.The
results suggest that Cathin-6-one contributes to the an-
tibacterial activity of Z.dimorphyllum.Canthin-6-one
is a well known antimicrobial compound with a broad
antimicrobial spectrum.However , the activity of Can-
thin-6-one has not been studied on oral bacteria.Fur-
thermore , it is necessary to understand its toxicity in
addition to its antimicrobial property.Study is in
progress to explore its other potential activity , i.e.anti-
inflammatory activity.
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花椒属植物对口腔致病菌的抗菌活性
施　瑶＊ ,李定祥 ,闵知大
中国药科大学天然药物化学教研室 ,江苏 南京 , 210009
【摘　要】　目的:比较 6 种花椒属(Zanthoxylum)植物乙醇提取物对口腔致病菌的抗菌活性 , 从中寻找抗菌的活性物
质。方法:用于抗菌活性实验的口腔致病菌是变形链球菌(Streptococcus mutans ATCC 25175), 放线菌(Actinomyces naeslundii
ATCC 12104)和嗜血放线伴生杆菌(Actinobacillus actinomycetemicomtans ATCC 43717)。抗菌实验为琼脂扩散法(agar diffusion
assays)和最低抑菌浓度法(MIC)。结果:拟蚬壳花椒(Z.lactum)的根对这些口腔致病菌表现出最强的抗菌活性 ,其次是两
面针(Z.nitidum)和异叶花椒(Z.dimorphyllum)的根。进而对异叶花椒(Z.dimorphyllum)的根进行化学成分研究 ,分离到
4 个已知化合物 ,其中化合物Canthin-6-one对这三种口腔致病菌均有很强的抗菌能力 , 最低抑菌浓度(MIC)在 15.6 和 62.5
μg/mL之间。结论:Canthin-6-one可能是异叶花椒抗菌活性的物质基础。
【关键词】　花椒属;异叶花椒;拟蚬壳花椒;口腔致病菌;抑菌;Canthin-6-one
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