全 文 ：　　　　 天然产物研究与开发　 　　　　　
　　　　　　　　　　 NAT URAL PRODUCT RESEARCH AND DEVELOPMENT　 2004　Vol.16　No.3
　
　
　
　
　　Received 24 S eptember 2003;Accepted 18 Feb ruary 2004
Foundation Item:National Natu ral Science Foundat ion of China
(30270020)
　 ＊Corresponding author Tel:86-10-62893012;Fax:86-10-62891013;
E-mai l:lgzhou@cau.edu.cn
ENDOPHYTIC FUNGI OF PARIS POLYPHYLLA
VAR.YUNNANENSIS AND STEROID
ANALYSIS IN THE FUNGI
ZHOU Li-gang＊ ,CAO Xiao-dong ,YANG Cheng-zong ,WU Xue-hong ,ZHANG Li-qun
(College of Agronomy and Biotechnology , China Agricultural University , Beijing 100094 , China)
Abstract　Fifty endophytic fungal isolates were separated and purified from the rhizomes and seeds of Paris poly-
phylla var.yunnanensis collected in Yunnan P rovince of China.More endophy tic fungi were separ ated from the
phloem than from the seed or x ylem.Detection and analysis methods for steroids w ere established.The steroids were
detected in the fermentation cultures of thirty-two fungal isolates.It was found that five fungal isolates could produce
steroids with a yield exceeded 50 mg/ L.
Key words　endophy tic fungi;Paris polyphylla var.yunnanensis;steroids
滇重楼的内生真菌及真菌中甾体化合物的分析
周立刚＊　曹晓冬　杨成宗　吴学宏　张力群
(中国农业大学农学与生物技术学院　北京　100094)
摘　要　从采自云南的滇重楼(Paris polyphylla var.yunnanensis)根状茎韧皮部 、木质部和种子中分离出 50
株内生真菌 , 其中从根状茎的韧皮部中分离出内生真菌最多。建立了甾体类化合物的检测和分析方法。检测
到 32株内生真菌的发酵培养物中含有甾体化合物 ,其中 5 株内生真菌的发酵培养物中甾体化合物的产率超
过了 50 mg/ L。
关键词　内生真菌;滇重楼;甾体化合物
Introduction
The most widely accepted term `endophyte was defined
as all organisms which inhabit plant organs , and can colo-
nize internal plant tissues without causing apparent harm
to their host at some time in their life cycles[ 1].Recent re-
search concerning endophytes has been focused on the ef-
fects of nitrogen fixing bacteria , plant grow th promoting
rhyzobacteria (PGPR), resistance inducing endophytes ,
and also on secondary metabolites of endophytes[ 2].Stierle
et al (1993) found a fungal endophyte Taxomyces an-
dreanae produced taxol and related compounds , which
were synthesized by its host Taxus brevi folia (Pacific
yew)[ 3].
Par is polyphylla var.yunnanensis (T rilliaceae), a valu-
able herb exclusively distributed in the Southwest of China
and the North of Burma , is used as Chinese folk medicine
for a few hundred years.Its rhizome contains steroids
which are of g reat medical importance and is adapted to a
variety of complicated human diseases such as heart and
blood vessel diseases
[ 4].Recently ,we found that heptasac-
charide and octasaccharide isolated from P.polyphylla
var.yunnanensis possessed plant g row th-regulatory activi-
ty[ 5].The major objective of this work is to isolate endo-
phytic bacteria and fungi capable of synthesizing steroids
similar to those of the host plant P.polyphylla
var.yunnanensis.Our previous study showed that the en-
dophytic fungi Hormomyces par idiphilus secreted extra-
cellular polysaccharide that was correlated to the col-
loidization of rhizomes and increase of polysaccharide of
198
P.polyphylla var.yunnanensis[ 6] .In this paper , we re-
port the separation of the endophytic fungi from this plant
and their steroid analysis.
Materials and methods
Plant materials
The plant sample Paris polyphyl la var.yunnanensis
(Franch.) Hand.-Mazz.was collected in Kunming ,
China.Nine-year-old rhizomes (or called underg round
stem)and seeds were harvested in October 2002 , and
stored in sealed plastic bags at 4 ℃.
Media
Four media were adapted to the isolation and purification
of endophytic fungi f rom the rhizomes and seeds of
P.polyphylla var.yunnanensis.Potato-dextrose agar
(PDA)[ 8] , cornmeal-malt-ext ract agar(CMM)and M102
media[ 7] were employed.The rhizomes of P.polyphylla
var.yunnanensis were lyophilized and powdered.About
20 g of rhizome powder was boiled for 20 min with 200
m L distilled water , filtrated with 3 layers of gauze to ob-
tain liquid tissue ext ract (LTE).100 m L of LTE was sup-
plemented to 1 liter of PDA media to obtain a modified
PDA media (M-PDA).150 mg/L of streptomycin was
added into the media when separation of endophy tic fungi.
Separation and purification of endophytic fungi
Endophytic fungi were separated from the samples
through the following two methods.
Method 1 Both healthy seeds and rhizomes were surface
disinfected by immersing in 70% ethanol solution for 10
s , followed by soaking them in 0.2%of HgCl2 solution for
20 min.The sterilized material w as washed with sterilized
distilled water for five times.The seeds were cut equal-
ly.Rhizomes were separated into xylem and phloem tissue
and cut into cubes (0.5 cm ×0.5 cm ×0.5 cm).Then
the material was inoculated in Petri dishes with CMM ,
M102 ,PDA and M-PDA media.
Method 2 One seed , 1 g of fresh xylem or phloem tissue
of rhizomes were surfaced-disinfected through the same
procedure mentioned in method 1 , then separately triturat-
ed with 10 mL of sterile distilled water.The obtained liq-
uid was diluted 10 times.Then 50μL of diluted liquid was
separately incubated on CMM , M102 , PDA and M-PDA
media.Once fungal isolates were recovered ,mycelium tips
were transferred to PDA medium to get pure culture.
Extraction of components
Endophytic fungi were incubated in f lasks containing 30
m L of liquid PDA for 7 d at 28 ℃ under shaking at 120
rpm in a rotating shaker (DH2-D , Jiangsu , China).For
the initial extraction , the fermentation culture broth of the
fungal isolates was filtered with one layer of pa-
per.Mycelia were grinded with quartz sands first in 15 mL
of distilled water then in 15 mL of petroleum ether which
was exhaustively filt rated and added to the filtered
broth.About 30 mL of pet roleum ether was employed to
extract metabolites from the mixed solution.Petroleum
ether layer was concent rated at 50 ℃ in vacuum to yield a
residue.Water layer was then passed through a small
macroporous adsorption resin (Diaion HP-20)column e-
luted first with water then with methanol to
flush.Methanol effluent was then concent rated under vac-
uum at 50 ℃ to yield a residue.Both the petroleum ether
extracts and methanol extracts were analyzed.
Analysis of steroids
In this study , a Liebermann-Burchard visualization was
used to detect steroidal compounds from the endophy-
tes[ 9] .10 mg of pure diosgenin was dissolved in 50 mL
chloroform , t he solution w as then diluted into different
concent ration.3 mL chloroform solution containing dios-
genin was mixed with 2 mL sulfuric acid-acetic anhydride
(19∶1 , v/ v).After incubation for 40 min at 50 ℃, the
mixed solution was measured for its light absorption at the
wavelength 460 nm with a spectrophotometer (LNI-
CAM ,Helios Alpha ,England).The linear regressive equa-
tion was y=1.9879x-0.001 with correlated coefficient R
=0.9983 , here y is light absorption at the wavelength
460 nm , and x the concentration(mg/mL)of diosgenin.3
mL chloroform with 2 mL sulfuric acid-acetic anhydride
was used as a cont rol.
Both the petroleum ether ext racts and methanol extracts
of the fungal cultures were dissolved in 3 mL chloroform ,
and mixed with 2 mL sulfuric acid-acetic anhydride solu-
tion (19∶1 , v/ v).The spectrophotometric analysis was
carried out as that of diosgenin.Based on the light absorp-
tion , the relative contents of the steroids in fungal cultures
were estimated.
Fresh liquid PDA was used as a negative control with the
same extraction procedure described above.1mg of crude
steroidal saponin from P.polyphylla var.yunnanensis
was dissolved in chloroform as a positive control.
Results
Separation and purification of the endophytic fungi
Fifty fungal isolates were recovered and purified(Table
1).Compared with the xylem and phloem of the rhizome ,
Table 1　Fungal isolates separated from dif ferent materials
Isolation
methods
Seeds Xylem of rhizome Phloem of rhi zome
PDA M-PDA CMM M102 PDA M-PDA CMM M102 PDA M-PDA CM M M102
Method 1 0 0 0 0 0 2 2 2 0 7 6 0
Method 2 0 2 0 1 1 2 1 0 11 5 5 3
1992004　Vol.16　No.3 周立刚等:滇重楼的内生真菌及真菌中甾体化合物的分析 　　　　　　　　　
few er fungal endophytes were separated from the
seeds which accounted for only 6% of the total num-
ber.20% of fungal endophy tes w ere separated f rom
the xy lem , and 74% of fungal endophytes from the
phloem.Compared w ith the method 1 , more fungal
endophy tes were separated with method 2 from seed
and phloem materials.
Analysis of steroids in endophytic fungal cultures
Steroids were detected in 32 of 50 isolates.The yields
were shown in Table 2.Comparing with petroleum ether
extracts , more steroidal compounds existed in methanol
extracts.It w as found that fungal isolates No.6 , 26 , 30 , 46
and 48 could produce more steroidal compounds with
yields exceeded 50 mg/L.
Table 2　Steroid yields(mg/ L)of the endophytic fungi in fer-
mentation culture
Code of
fungal isolates
Steroid yield
(petroleum
ether ext ract s)
S teroid yield
(methanol
ex tracts)
Total
yield
1 4.472 18.894 23.366
2 3.326 29.208 32.534
4 2.096 10.313 12.409
6 8.413 88.881 97.294
7 4.444 17.692 22.136
8 2.320 12.969 15.289
10 1.593 41.507 43.100
13 3.689 34.658 38.347
17 2.404 24.456 26.860
21 2.068 21.186 23.254
22 3.801 41.226 45.027
24 3.550 14.199 17.749
26 4.165 64.816 68.981
27 1.733 19.230 20.963
28 3.522 21.969 25.491
29 1.761 33.540 35.301
30 4.081 45.922 50.003
32 12.885 27.978 40.863
35 3.550 11.180 14.730
36 3.186 10.565 13.751
37 2.767 11.208 13.975
38 5.199 9.699 14.898
40 7.966 20.264 28.230
42 4.221 42.484 46.705
43 1.649 8.329 9.978
44 3.242 15.208 18.450
45 12.047 23.674 35.721
46 2.991 68.869 71.860
47 2.236 8.441 10.677
48 2.543 62.357 64.900
49 2.907 16.267 19.174
50 2.767 25.630 28.397
Discussion
The results show ed that many endophytic fungal iso-
lates separated from P.polyphy lla var.yunnanensis
could produce steroidal compounds.Of them , f ive iso-
lates could produce more steroidal compounds than
other isolates.There w ere mo re fungal endophy tes
separated from the phloem than from the xy lem or
seeds.It could be concluded that endophy tic fungi
mainly exist in the phloem of the rhizome , which is
very similar to the results reported
[ 3 ,10] .Most of the
steroidal compounds present in methanol ext racts , in-
dicating that most of steroidal compounds are polar
ones.If the steroids in endophy tes resemble to those
in their host , it w ould be an ef fective way to obtain
steroidal compounds from the endophytes of P.poly-
phylla var.yunnanensis[ 11] .
Acknowledgements This work was supported by Nation-
al Natural Science Foundation of China(No.30270020)
The authors are grateful to Prof.Shilin Wang and
Prof.Heng Li in Kunming Institute of Botany for collect-
ing plant materials , and identifying plant specimen , respec-
tively.Diosgenin w as kindly provided by Dr.Jiaru Li in
Wuhan University.
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