全 文 ：Received date:2008-05-23
Foundation item:This w ork was supported by National Nature Science Foundation of China(No.30400038) and the
“ XiBuZhiGuang” Project of Chinese Academy o f Sciences
Biography:DU Zhi-zhi(1974-), female ,Han na tionality , associate professor , PhD., research field:natural products chemistry ,
medical and aromatic plants , Tel:(0871)5223224 , Fax:(0871)5150227 , E-mail:duzhizhi@mail.kib.ac.cn.
Article ID:1005-0108(2008)06-0452-05
Secondary metabolites produced by Fusarium sp.2TnP1-2 ,
an endophytic fungus from Trewia nudif lora
DU Zhi-zhi1 , SONG Cheng-zhi1 , 2 ,YU Bu-zhu1 , LUO Xiao-dong1
(1.Kunming Inst itute of Botany , Chinese Academy of Sciences , Kunming 650204 , China;
2.Department of Chem istry ,Zhaotong Teacher′s College , Zhaotong 657000 , China)
Abstract:Aim To study antifungal and antibacterial secondary metabolites of Fusarium sp.
2TnP1-2 , an endophy tic fungus isolated f rom Trewia nudi f lora.Methods PDA fermentation ex-
tract was isolated by bio-assay guided fractionation and different column chromatography methods
including silica gel column ,Sephadex LH-20 column and preparative thin layer chromatog raphy .
Structures of these compounds w ere identified on the basis of spect roscopic analy sis of 1D , 2D-
NMR ,MS and comparison of chemical and physical data wi th authent ic samples reported in li tera-
tures.The antibacterial and antifungal activi ties of the isolated compounds were measured using
the disc diffusion method.Results Three compounds w ere isolated and their st ructures were deter-
mined to be trichosetin(N-demethyl equisetin , 1), lateritin(4-methy l-6-(1-methy lethyl)-3-
phenylmethy l-1 ,4-perhydrooxazine-2 ,5-dione , 2), 5α, 6α-epoxy-24(R)-methy lcholesta-7 , 22-di-
en-3β-ol(3).Conclusion All the compounds were obtained from Fusarium for the first time.Tri-
chosetin and lateritin possess ant ibacterial activi ty against Staphy lococcus aureus.
Key words:Trewia nudi f lora;Fusarium ;antibacteria;tetramic acid
CLC number:R914　　　Document code:A
滑桃树内生真菌 Fusarium sp.2TnP1-2次生代谢产物研究
杜芝芝1 ,宋成芝1 , 2 ,郁步竹1 ,罗晓东1
(1.中国科学院 昆明植物研究所 ,云南 昆明 650204;2.昭通师范高等专科学校 化学系 ,云南 昭通 657000)
摘　要:目的 对滑桃树内生真菌 Fusarium sp.2TnP1-2的抗菌活性次生代谢产物进行研究。方法 采用硅胶
柱色谱 、Sephadex LH-20 、制备性薄层色谱等对次生代谢产物进行分离纯化。根据理化性质和波谱分析进行
结构鉴定。利用纸片扩散法对这些化合物的抗真菌及抗细菌活性进行测试。结果 从该菌株 PDA 培养基发
酵产物中分离得到 3 个化合物 , 分别鉴定为:trichose tin(N-demethyl equisetin , 1), lateritin(4-methyl-6-(1-
methylethy l)-3-phenylmethy l-1 , 4-perhydrooxazine-2 , 5-dione , 2), 5α, 6α-epoxy-24(R)-methy lcholesta-7 , 22-dien-
3β-ol(3)。结论 3 个化合物均为首次从该真菌的代谢物中分离得到 , trichosetin 和 lateritin 具有抗金黄色葡萄
球菌活性。
关键词:滑桃树;镰刀菌属;抗菌;tetr amic acid
第　18 卷　第 6 期
2008 年12月　总86 期
中 国 药 物 化 学 杂 志
Chinese Journal o f Medicinal Chemistry
Vol.18　No.6　p.452
Dec.2008 Sum 86
DOI :10.14142/j.cnki.cn21-1313/r.2008.06.005
　　Endophytes ,microorganisms that reside in the tis-
sues of living plants , are relatively unstudied as potential
sources of novel natural products for exploitation in
medicine , agriculture and indust ry[ 1-2] .Based on
the methods and rationale used to provide the best
opportunities to isolate novel endophy tic microor-
ganisms at the genus o r species[ 3-4] , the t ropical
plant Trewia nudi f lora L.(Euphorbiaceae)was
selected to isolate endophytes and search for bioac-
tive compounds f rom their metabolites.Fungi are
the most commonly isolated endophy tic microor-
ganisms from the plant.During the course of anti-
fungal activity survey of the endophytes from T .
nudi f lora , a fungus Fusarium sp.2TnP1-2 was
selected fo r further investig at ion.Bioassay guided
fractionation led to isolation of three compounds.
Theses compounds were elucidated to be t richosetin
(N-demethyl equisetin , 1), lateritin(4-methy l-6-
(1-methylethy l)-3-pheny lmethyl-1 , 4-perhydroox-
azine-2 ,5-dione ,2), 5α,6α-epoxy-24(R)-methy lc-
holesta-7 , 22-dien-3β-ol(3).Their antibacterial and
antifungal activities w ere tested by disk diffusion
method.
1　Material and methods
1.1　General
Optical rotations w ere determined on a JASCO
DIP370 digital Polarimeter.MS were measured on
a VG AutoSpec 3000 mass spectrometer.All the
NMR data were obtained at room temperature on
AM-400 and DRX -500 spectrometer(TM S as
internal reference , chemical shift in δ).Thin layer
chromatography w as conducted on silica gel F 254
plates(Qingdao Meigao Chemical Co.Ltd.).TLC
developing agent is 5%(V/ V)sulphuric acid in
ethanol.Column chromatography w as carried out
on silica gel(50-71μm;Qingdao M arine Chemical
Factory),Sephadex LH-20(GE Healthcare Bio-Sci-
ences AB),RP-18 gel(40-63 μm;Merck , Darm-
stadt ,Germany).Preparative TLC was carried out
on silica gel F 254(Qingdao Meigao Chemical Co.
Ltd.).
1.2　Biological material
The fungal st rain w as isolated f rom the peti-
oles of Trewia nudi f lora L.(Euphorbiaceae),
which w ere collected f rom greenhouse of Kunming
Insti tute of Botany , People′s Republic of China.
The petioles w ere washed in running tap w ater and
cut into 5 mm pieces.These small pieces w ere sur-
face sterilized successively w ith 0.1%(V/ V)
Tween-20 for 30 s , 1%(V/ V)sodium hypochlo-
rite for 5 min , sterilized w ater for 5 min then 75%
(V/ V)ethanol for 5 min.The surface sterilized
pieces were incubated at 26 ℃ on PDA supple-
mented with 100 mg·L-1 nalidixic acid to suppress
the bacterial g row th and incubated at 26 ℃ until
colony o r mycelium appeared surrounding the
pieces.Hyphal t ips originat ing f rom segments w ere
t ransferred to Petri dishes containing f resh PDA
medium free of antibio tics.Each isolates w as then
grow n and examined to ascertain that it originated
from a sing le organism.During the antifungal
bioassay test , the st rain 2TnP1-2 ,whose ext ract af-
forded ant ifungal act ivity , was selected fo r further
investigation.The strain was then identified as a
Fusarium sp.by Prof.LUO Yun-long , Yunnan A-
g riculture Universi ty , Kunming , China , and de-
posited in Kunming Institute of Bo tany ,Chinese A-
cademy of Science ,Kunming ,China.
453第 6期 　 　　　
1.3　Fermentation , extraction and fractionation
Agar fermentation w as performed w ith PDA
medium(2 L)for 14 d.The fermentation product
w as chopped and ex tracted four times with ethyl
acetate-methanol-formic acid(80∶15∶5 , V/ V)ex-
haustively.The combined ex tract solution was e-
vaporated in vacuo at 45 ℃ to give a residue.The
residue w as ex tracted by methanol to give 1.6 g
crude ext ract.
The crude ex tract(FM)was chromatographed
on vacuum liquid chromatography(VLC)(37 g sili-
ca gel), eluted wi th a PE-EtOAc(20∶1 -0∶100 ,
V/ V)and EtOAc-MeOH gradient system , to af-
ford 12 sub fractions(FM 1 -FM 12).Antifungal
test showed that FM 5 and FM6 were active.Com-
bined FM5 and FM 6(340 mg)was subjected on
V LC RP-18(10 g), eluted w ith a MeOH-H2O gra-
dient system , to afford three main fractions(FM 5c1
-FM 5c3).FM5c1 w as isolated by Sephadex LH-
20 in acetone and further purified by prepared TLC
to obtain compound 1(30 mg).FM 7 was isolated
by V LC RP-18(4 g), eluted w ith a MeOH-H2O(50
∶50-95∶5)to afford compound 2(29 mg).FM8
was purified by Sephadex LH-20 in methanol and
silica gel to give compound 3(4 mg).
1.4　Bioassays
Antifungal activities against Canidia albi-
cans , Saccharomyces cerev isiae , and antibacterial
activi ties against S taphylococcus aureus , Mycobac-
terium tuberculosis were carried out by disk dif fu-
sion assay on agar plates as described
[ 5] .Results
w ere expressed by minimal inhibition amount(μg/
disc)and diameter of inhibi tion zone.Rifampicin
w as used as positive control.
2　Results
Compound 1:white pow der , [ α] 17D =-261°
(c=4.66 ,MeOH).IR(KBr)σ:3443 , 2921 ,1595 ,
1448 cm
-1.ESI-MS m/ z :360[ M +H] +(100),
358[ M -H] +(100).The molecular formula is
C21H29NO4 based on i ts HR ES I-MS data(m / z:
382.1983[ M +Na] + , cal:382.1994).The IR spec-
trum showed the presence of OH(3443 , 2921 cm-1)
and C=C(1595 cm-1)goups.The 13C-NMR spec-
trum showed 21 carbon signals:three CH3 g roups ,
four CH2 , nine CH groups including four double
bonds and five quaternary C-atoms(table 1).Com-
pound similarity search in DNP database indicated
that this newly isolated compound has similar
skeleton to equisetin.Detailed comparison revealed
equiset in has one additional methyl g roup.It sug-
gested the methyl on N-1′may be substi tuted by a
proton in the moiety of 1.Determinat ion of the
structure of 1 was confirmed by HMQC , HMBC
and 1H-1H COSY experiments and comparison of
spectra w ith reference[ 6-7] .NOESY was useful to
establish the relative configuration of stereocenters
for 1.NOESY experiment showed NOE co rrela-
tions betw een H-3(δ3.94)and H-8(δ1.32),H-
12(δ1.32)and H-13(δ5.20), also betw een H-6
(δ1.70)and H-8 and H-12.The negative optical
ro tation of 1 ([ α] 17D =-261°)is very similar to
that of equisetin([ α] 17D =-273°),which suggested
1 has same stereo configuration as N-demethy l eq-
uisetin(trichosetin).
Compound 2:The molecular formula is C15H 19
NO3 , colo rless crystal.[ α] 17D =+22.3°(c =1.1 ,
CHCl3).EI-MS m / z:261[ M +] ,(100), 244(7),
231(3), 217(3), 170(25), 142(36), 134(23), 91
(100).The 13C-NMR spect rum showed 15 carbon
signals:three CH3 , one CH2 , eight CH g roups in-
cluding one oxygenated carbon and three quater-
nary C-atoms including tw o carboxyl carbons(table
2).Compound similarity search in DNP database
indicated that NMR data of 2 was a good match for
lateri tin w hich that isolated f rom Gibberella lateri-
tium IFO 7188
[ 8] .Kagamizono repo rted the st ruc-
ture of lateritin might be w rong
[ 9] .In o rder to con-
f irm the correct st ructure , 2D-NMR experiments
were conducted and the st ructure w as elucidated as
4-methyl-6-(1-methylethy l)-3-phenylmethy l-1 , 4-
perhydrooxazine-2 ,5-dione(laterit in).
454 中 国 药 物 化 学 杂 志 第 18 卷
Table 1　1H and 13C-NMR spectroscopic data for compound 1(in CD3OD)
Position δ(C) δ(H) HMBC(C※H) 1H-1H COSY
1 202.8(s) / H-12 /
2 51.5(s) / H-12 /
3 44.1(d) 3.94(m) H-5 , H-13 H-13
4 130.7(d) 5.28(m) H-3 , H-6 H-3
5 128.9(d) 5.28(m) H-3 , H-6 ,H-11 /
6 40.3(d) 1.70(m) H-4 , H-5 H-11
7 44.1(t) 1.72(m)
0.74(m)
H-5 , H-9 ,H-16 H-7
8 34.9(d) 1.32(m) H-9 , H-16
9 36.8(t) 1.47(m)
0.87(m)
10 30.7(t) 1.26(m)
11 42.1(d) 1.47(m) H-12
12 13.9(q) 1.32(s) H-1 , H-2 ,H-3
13 126.2(d) 5.20(m) H-15 H-3
14 133.8(d) 5.14(m) H-3 , H-15
15 18.4(q) 1.47(d),
3.96
H-13 ,H-14
16 23.2(q) 0.83(d),
4.4
H-7 , H-9
2′ 181.2(s) / / /
3′ 103.5(s) / / /
4′ 192.4(s) / / /
5′ 62.7(d) 3.57(m)
6′ 64.5(t) 3.82(m) H-6′
3.55(m) H-6′
Table 2　1H and 13C-NMR spectroscopic data of compound 2(in CD3OD)
Position δ(C) δ(H) HMBC 1H-1H COSY ROESY
C-2 169.9(s) / H-3 ,H-6 ,H-10 / /
C-3 57.0(d) 5.58(1H , dd , J=4.6 , 11.7 Hz) H-10 ,H-17 H-10 /
C-5 169.7(s) / H-7 ,H-10 ,H-17 / /
C-6 75.6(d) 4.88(1H , d , J=8.5 Hz) H-7 ,H-8 ,H-9 H-7 H-7 ,H-17
C-7 29.7(d) 2.02(1Hm) H-6 H-8 ,H-9 H-6 H-8 ,H-9 H-6 ,H-8 ,H-9
C-8 17.3(q) 0.40(3H , d , J=6.8 Hz) H-7 ,H-9 H-7 H-6 ,H-7 ,H-9
C-9 18.3(q) 0.80(3H , d , J=6.6 Hz) H-6 ,H-8 H-7 H-6 ,H-7 ,H-8
C-10 34.7(d) 3.41(1H , dd , J=4.9 , 14.6 Hz)
2.99(1H , dd , J=12.1 , 14.5 Hz)
H-10 H-3
H-10
C-11 136.5(s) / H-10 ,H-14 ,H-16 / /
C-12 , 16 128.8(d) 7.27(2H ,m) H-10 ,H-14 / H-10
C-13 , 15 128.5(d) 7.27(2H ,m) H-10 ,H-16 / H-10
C-14 126.8(d) 7.19(1H ,m) H-16 / /
N-CH3 32.1(q) 3.01(3H , s) H-3 / H-6 ,H-7
　　Compound 3:Colorless crystal.FAB+-MS
m/ z :413.EI-MS m/ z(%):394 [ M -H2O ] +
(12),376[ M-2H2O] +(53), 361(10), 251[ M -
2H2O-C9H17] +(100).1H-NMR(500 MHz , CD-
Cl3)δ:3.95(1H , m , H-3), 3.52(1H , m , H-6),
5.26(1H ,m , H-7), 0.54(3H , s , H-18), 1.00(3H ,
s , H-19), 0.97(3H , d , J =6.6 Hz , H-21), 5.13
(1H , dd , J =15.3 , 8.0 Hz , H-22), 5.18(1H , dd ,
J =15.3 ,7.3 Hz ,H-23),0.77(3H , d , J =7.2 Hz ,
H-26),0.78(3H , d , J =7.4 Hz ,H-27),0.86(3H ,
455第 6期 　 　　　
d , J =6.8 Hz ,H-28).13C-NMR(CDCl3 ,125 MHz)
δ:32.7(C-1),30.4(C-2),67.2(C-3),38.9(C-4),
75.8(C-5), 73.1(C-6),117.4(C-7), 143.4(C-8),
43.2(C-9), 36.9(C-10), 21.2(C-11), 39.2(C-
12),43.6(C-13), 54.6(C-14), 22.8(C-15), 27.8
(C-16), 55.9(C-17), 12.1(C-18), 18.2(C-19),
40.2(C-20),20.9(C-21), 135.3(C-22), 132.0(C-
23),42.7(C-24), 33.0(C-25), 19.7(C-26), 19.4
(C-27), 17.4(C-28).The st ructure w as deter-
mined as 5α, 6β-epoxy-24(R)-methylcholesta-7 ,
22-dien-3 β-ol by comparison of spectra data w ith
reference
[ 10] .
Compounds 1 and 2 showed antibacterial ac-
tivi ty against Staphylococcus aureus at 2 μg/disc
(inhibition zone =7 mm), but no activity ag ainst
o ther test microorg anisms at 40 μg/disc.Compound
3 was inactive against all test microorg anisms at
40μg/disc.
3　Conclusion
Trichoset in(N-demethyl equisetin)was fi rst
isolated f rom dual culture of Trichoderma
harzianum and Catharanthus roseus callus as a
novel tetramic acid(2 ,4-py rrolidinedione)[ 6] .It has
a remarkable antimicrobial activi ty against the
Gram-posit ive bacterial Staphy lococcus aureus and
Baci llus subti lis.Phy toto xicity assays revealed tri-
choset in inhibited root and shoot g row th of all test-
ed plant species[ 11] .Lateritin w as fi rst isolated f rom
the mycelial cake of Gibberel la lateritium IFO
7188 as a new inhibitior of acy l-CoA:cholesterol a-
cy ltransferase(ACAT)[ 8] .Our bioassay experiment
showed it could also inhibit the g row th of Gram-
posit ive bacterial Staphylococcus aureus.5α, 6β-e-
poxy-24(R)-methy lcholesta-7 , 22-dien-3 β-ol was
first reported as an antitumor sterol from mycelia of
Cordyceps sinensis(dong-chong-xia-cao)[ 10] .All of
three compounds have different biological act ivit ies
and it supported the opinion that endophytes are
potential sources of novel natural products fo r ex-
ploitation in medicine.
Acknowledgements:We are g rateful to the
Analy tical Group , Laboratory of Phy tochemist ry ,
Kunming Institute of Botany , Chinese Academy of
Sciences for the spectral measurements.
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456 中 国 药 物 化 学 杂 志 第 18 卷