Two new antifungal saponins from the Tibetan herbal medicine Clematis tangutica

Article abstract of DOI:10.1055/s-2003-40652

Bioassay-guided fractionation of the ethanol extract of the aerial parts of Clematis tangutica led to the isolation of two new antifungal triterpene saponins. Their structures were determined to be 3-O-α-L-arabinopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl ester (1) and 3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl ester (2) on the basis of spectral data and chemical evidence. Inhibitory activities of the two saponins against seven fungal strains were evaluated. Compounds 1 and 2 showed evident antifungal activity (MIA ≈ 2.5 μg/disc) against Saccharomyces cerevisiae, similar to the positive control amphotericin B and ordinary activities (MIA ≈ 10 μg/disc) against Penicillium avellaneum UC-4376, Candida glabrata, Trichosporon beigelii and Pyricularia oryzae. Compound 2 is a better antifungal agent than compound 1 against most of the fungal strains that were tested.

Full text of DOI:10.1055/s-2003-40652

Zhizhi Du1
Na Zhu1
Na Ze-Ren-Wang-Mu2
Yuemao Shen1
Two New Antifungal Saponins from the Tibetan
Herbal Medicine Clematis tangutica
Abstract
ity (MIA » 2.5 mg/disc) against Saccharomyces cerevisiae, similar
to the positive control amphotericin B and ordinary activities
Bioassay-guided fractionation of the ethanol extract of the aerial (MIA » 10 mg/disc) against Penicillium avellaneum UC-4376,
parts of Clematis tangutica led to the isolation of two new anti- Candida glabrata, Trichosporon beigelii and Pyricularia oryzae.
fungal triterpene saponins. Their structures were determined to Compound 2 is a better antifungal agent than compound 1
be 3-O-a-L-arabinopyranosyl hederagenin 28-O-a-L-rhamnopyr- against most of the fungal strains that were tested.
anosyl ester (1) and 3-O-b-D-glucopyranosyl-(1®4)-a-L-arabi-
nopyranosyl hederagenin 28-O-a-L-rhamnopyranosyl ester (2) Key words
on the basis of spectral data and chemical evidence. Inhibitory Clematis tangutica ´ Ranunculaceae ´ triterpene saponin ´ hedera-
activities of the two saponins against seven fungal strains were genin ´ antifungal activity
evaluated. Compounds 1 and 2 showed evident antifungal activ-
Introduction
genus, few references to antifungal activity were found in the lit-
erature. It is only reported that the crude extracts of C. hirsuta
547
Fungal infections are being recognized with increasing frequency and C. cirrhosa had antifungal activities [6], [7]. But the antifun-
as an important cause of both morbidity and mortality. Current gal constituents of those two species have not been reported yet.
antifungal therapy is less than optimal, and there is evidence The Tibetan herb Clematis tangutica (Maxim.) Korsh. (Ranuncu-
that resistance to available drugs is developing [1], [2], [3]. Nat- laceae) is used to treat indigestion and skin diseases and contains
ural products produced by higher plants have evolved from se- dozens of triterpene saponins [8], [9]. However, no work has
lection for acquisition of improved defense against microbial at- been reported on the antifungal bioactivity of this species [8],
tacks [4], therefore, the pathogens are less likely to incur resist- [9]. In a screening for antifungal agents, the ethanol extract of
ance. Saponins are glycosylated plant secondary metabolites that the aerial parts of C. tangutica showed antifungal activities
have potent antifungal properties and are present in some heal- against Penicillium avellaneum UC-4376, and two new antifungal
thy plants at high concentrations, and those molecules may act triterpene saponins, 3-O-a-L-arabinopyranosyl hederagenin 28-
as preformed chemical barriers to plant pathogenic fungal attack O-a-L-rhamnopyranosyl ester (1) and 3-O-b-D-glucopyranosyl-
[5]. Many species of the genus Clematis which are used in folk or (1®4)-a-L-arabinopyranosyl hederagenin 28-O-a-L-rhamnopyr-
traditional Chinese medicines contain saponins in abundance. anosyl ester (2) were obtained by antifungal assay-guided frac-
Although there are many reports on the phytochemistry of this tionation [10].
Affiliation
1 The State Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, P. R. China
2 Tibetan Institute of Plateau Biology, Lasha, Tibet, P. R. China
Correspondence
Yuemao Shen ´ The State Key Laboratory of Phytochemistry and Plant Resources in West China ´
Kunming Institute of Botany ´ Chinese Academy of Sciences ´ Heilongtan ´ Kunming ´ Yunnan 650204 ´
People's Republic of China. ´ Phone: +86-871-5223111 ´ Fax: +86-871-5150227 ´
E-mail: yshen@public.km.yn.cn
Received November 19, 2002 ´ Accepted March 8, 2003
Bibliography
Planta Med 2003; 69: 547±551 ´ ꢀ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943

Materials and Methods
General procedures
and B1±4 (100 mL). The fraction B1±3was chromatographed
over silica gel (60 g) eluted with EtOAC-MeOH (10:1, v/v, 300
mL, 9:1, v/v, 400 mL) to yield compound 2 (30 mg) which was
Uncorrected melting points were determined using an XRC-1 further purified through recrystallization from MeOH.
micromelting apparatus. Optical rotations were measured with
a JASCO DIP-370 digital polarimeter in MeOH solution. IR spectra Partial hydrolysis and acid hydrolysis of compound 1
were measured on a Bio-Rad FTS-135 infrared spectrometer with Compound 1 (100 mg) was dissolved in 200 mL 2% H2SO4 aqu-
KBr pellets. UV spectra were obtained on a Shimadzu double- eous methanol (80%) solution at ambient temperature for eight
beam 210A spectrophotometer. Mass spectra were measured on days. After being neutralized with saturated aqueous Ba(OH)2
a VG Auto Spec-3000 spectrometer. NMR spectra were recorded and extracted with ethyl acetate, the ethyl acetate extract of the
on INOVA-400, Bruker AM-400 or DRX-500 NMR spectrometers reaction mixture was subjected to column chromatography over
with TMS as internal standard. Silica gel (200±300 mesh) for silica gel (35 g) and eluted with CHCl3-MeOH (30:1, 500 mL 4:1,
column chromatography and precoated TLC plates (Si gel G) 1000 mL to produce 1 (42 mg), 1a (6 mg) and 1d (11 mg), respec-
were purchased from the Qindao Marine Chemical Factory, Qing- tively.
dao, P. R. China. Reversed-phase C18 silica gel for column chroma-
tography and C18-RP-TLC plates were obtained from Merck. Se- Compound 1 (100 mg) was dissolved in 5% HCl-MeOH solution
phadex LH-20 for column chromatography was purchased from (13mL) and refluxed for 2 h. After cooling, the reaction solution
Amersham Biosciences. Amphotericin B was purchased from Sig- was neutralized with 5% aqueous KOH then evaporated under
ma (A-4888).
vacuum to provide a white powdery residue. The residue was ex-
tracted with MeOH and the methanol extract was purified by
column chromatography over Sephadex LH-20 eluted with me-
Plant material
The aerial parts of Clematis tangutica (Maxim) Korsh. were col- thanol to yield the aglycone of 1 (1a) and a mixture of sugars
lected from Dui-Long-De-Qing County of Tibet, P. R. China in Oc- (27 mg). The mixture was further purified by column chromatog-
tober, 2000. A voucher specimen (Kun No. 0758101) is deposited raphy over silica gel (3.5 g) and eluted with CHCl3-MeOH (10:1,
in the Herbarium of Kunming Institute of Botany, Chinese Acad- v/v, 100 mL) to yield two methyl mono-glycosides 1b and 1c,
emy of Sciences.
respectively.
Extraction and isolation
Synthesis of methyl L-arabinoside, D-xyloside and
The air-dried aerial parts of C. tangutica (5 kg) were ground and L-rhamnoside and comparison of the optical rotations with
extracted three times with boiling 95% EtOH. After the removal those of 1b and 1c
of solvents, the residual syrup (about 150 g) was chromato- Rhamnose, arabinose and xylose (50 mg each) was dissolved in
graphed over silica gel (500 g) and eluted with EtOAC, EtOAC- 10 ml of 5% HCl-MeOH and refluxed for 2h, respectively. After
MeOH (4:1, v/v) and MeOH to yield fractions A (44g), B (40 g) being neutralized with 5% aqueous KOH solution and the remov-
and C (60 g). Fraction B (showing activities) was subjected to al of solvents under vacuum, the methylated sugar was exhaus-
MPLC over reversed-phase C18 silica gel (300 g, 5 m) eluted with tively extracted with ethyl acetate and purified by column chro-
acetone-water (3:2, v/v) to afford fractions B1, B1¢, B2, B3, B4 matography over silica gel eluted with CHCl3-MeOH (10:1, v/v).
and B5 (350 mL eluate each). The fraction B3 (1.05 g) was sep- The comparison of the optical rotation values of the above me-
arated by column chromatography over silica gel (100 g) eluted thyl monosaccharides (1b and 1c) obtained from the hydrolysis
with chloroform-methanol (11:2, v/v) to yield B3±1 (160 mL), of compound 1 with the respective values of methyl L-arabino-
548
B3±2 (80 mL), and B3±3 (200 mL). The fraction B3±2 was
purified by recrystallization from MeOH to yield compound 1 1b and 1c was methyl L-arabinoside ([a]D: + 93.3, c 0.3, MeOH;
(0.23g), and further 0.61 gram was obtained from the fraction sys. [a]D: + 92.0, c 1.0, MeOH) and methyl L-rhamnoside ([a]D:
side, methyl D-xyloside and methyl l-rhamnoside indicated that
B4 (2.25 g) by the same method. The fraction B1 was separated + 53.3, c 5.6, MeOH, sys. [a]D: + 53.5, c 5.6, MeOH ), respectively.
by column chromatography on Sephadex LH-20 eluted with Additionally, TLC results indicated that all of those five methyl
MeOH to afford B1±1 (200 mL), B1±2 (180 mL), B1±3(40 mL),
mono-glycosides were epimeric pairs at the anomeric carbon.
Antifungal assay
Inhibitory activities of compounds 1 and 2 against Penicillium
avellaneum UC-4376, Candida albicans, Candida glabrata,
Saccharomyces cerevisiae, Cryptococcus neoformans, Trichosporon
beigelii and Pyricularia oryzae were determined by disk diffusion
assay on agar plates as described [11]. Results were presented in
minimal inhibition amounts (MIA, mg/disc) and diameters of in-
hibition zones (Table 2). Amphotericin B was used as positive
control.
Compound 1: colorless needles (MeOH), m. p. 272±274 8C; [a]2D5:
+ 46.7 (c 0.1, MeOH); UV (MeOH): lmax = 202 nm; IR (KBr):
nmax = 3434, 2941, 1700, 1652, 1559, 1541, 1507, 1457, 1387, 1137,
1052 cm±1; 1H- and 13C-NMR data, see Table 1; HR-FABMS: m/z =
Fig. 1 Structures of compounds 1, 2, and the hydrolysates of 1.
Du Z et al. Two New Antifungal¼ Planta Med 2003; 69: 547±551

Table 1 NMR data of compounds 1 and 2 in C5D5Na
1
2
Position
13C
1Hb
13C
1H
1
2
39.0
1.51 (m), 1.08 (m)
2.12 (2H, m)
39.0
1.48 (br d, 12.8), 0.97 (m)
2.13(2H, m)
26.5
4.24 (m)
43.6
48.5
18.2
33.3
39.8
48.2
37.0
23.7
122.7
/
26.4
4.18 (m)
43.6
47.8
18.2
33.3
39.8
48.2
37.0
23.7
122.7
/
381.1
4
81.1
/
/
5
1.78 (m)
1.74 (m)
6
1.72 (m), 1.34 (m)
2.02 (m), 1.81 (m)
/
1.72 (m), 1.34 (m)
7
1.98 (m), 1.78 (m)
8
/
9
1.75 (m)
1.68 (m)
10
11
12
13144.9
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Ara-1
2
/
/
1.92 (2H, m)
5.45 (br s)
1.89 (2H, br d, 9.2)
5.43(s)
144.9
42.2
28.4
23.9
46.7
42.4
46.5
31.0
32.8
34.3
64.0
14.1
16.1
17.7
26.2
180.3/
33.3
23.9
104.4
75.9
4.10 (m)
/
42.2
28.4
24.0
46.7
42.0
46.5
31.1
32.9
34.3
63.9
14.2
16.2
17.5
26.3
/
2.05 (m), 1.08 (m)
2.08 (2H, m)
/
2.06 (m), 1.10 (H, m)
2.06 (2H, br s)
/
3.29 (dd, 4.0, 13.6)
1.72 (m), 1.23(m)
/
3.26 (dd, 4.0, 13.6)
1.78 (m), 1.23(m)
/
1.54 (m), 1.27 (m)
1.37 (m), 1.23 (m)
4.12 (m), 3.75 (d, 10.4)
1.04 (3H, s)
0.92 (3H, s)
0.99 (3H, s)
1.22 (3H, s)
1.56 (m), 1.23 (m)
1.38 (m), 1.23 (m)
4.11 (m), 3.70 (d, 10.4)
1.05 (3H, s)
0.88 (3H, s)
0.97 (3H, s)
1.19 (3H, s)
180.5
/
549
0.92 (3H, s)
1.00 (3H, s)
5.11 (d, 6.0)
4.58 (t, 6.8)
75.6
33.4
23.9
0.88 (3H, s)
0.95 (3H, s)
4.96 (d, 6.8)
4.52 (t, 7.6)
104.6
76.2
374.8
4
4.02 (d, 8.4)
69.34.16 (m)
80.7
4.14 (m)
5
65.9
4.24 (m), 3.72 (br d, 11.6)
65.7
4.35 (m), 3.63 (br d, 11.6)
5.09 (d, 7.6)
Glc-1
2
106.9
75.34.02 (d, 8.4)
3
78.6
71.2
78.9
62.5
101.8
72.4
72.5
74.2
69.7
18.8
4.20 (m)
4
4.22 (m)
5
3.87 (m)
6
4.48 (br d, 12.8), 4.32 (m)
6.26 (br s)
Rha-1
2
101.7
72.6
72.4
74.2
69.8
18.6
6.22 (br s)
4.71 (m)
4.68 (m)
3
4.64 (dd, 3.2, 9.2)
4.27 (m)
4.62 (dd, 3.2, 9.2)
4.28 (m)
4
5
4.68(m)
4.70 (m)
6
1.63 (3H, d, 6.0)
1.62 (3H, d, 6.0)
a The NMR data for compounds 1 and 2 were recorded on Bruker AM-400 and INOVA-400, respectively.
b Coupling constants are presented in Hz. Unless otherwise indicated, all proton signals integrate to 1H.
749.4497 [M±H], calcd.: 749.4476; FABMS: m/z = 749 [M±H], nmax = 3419, 2940,1699,1634,1558,1540,1506,1457,1386,1260,
631 [M±ara], 603 [M±rha], 585, 525, 499, 471 [M±rha±ara], 423. 1074, 1032 cm±1; 1H- and 13C-NMR data, see Table 1; HR-FABMS:
m/z = 911.5002 [M±H], calcd.: 911.5004; FABMS: m/z = 912
Compound 2: colorless plate crystals (MeOH), m.p. 237±2428C; [M±H], 866, 766 [M±rha], 750 [M±glc], 603[M±rha±glc], 525,
[a]D25: + 33.3 (c 0.1, MeOH); UV (MeOH): lmax = 202 nm; IR (KBr): 499, 471 [M±rha±glc±ara], 423.
Du Z et al. Two New Antifungal¼ Planta Med 2003; 69: 547±551

Table 2 Minimal inhibition amounts of compounds 1 and 2 in agar diffusion assay
Fungal strains
Minimal inhibition amounts (mg/disc)
2a 2b
1a
1b
amphotericin Bb
P. avellaneum
C. albicans
C. glabrata
S. cerevisiae
C. neoformans
T. beigelii
5
10
nt
8
4
8
0.04
0.4
> 80
4
25
10
50
20
5
0.8
1
2
2.5
3.2
> 100
5
nt
10
20
25
5
50
10
10
0.8
0.8
P. oryzae
10
5
0.08
a Diameter of inhibition zone < 6 mm.
b Diameter of inhibition zone = 8 mm.
nt = not tested.
Results and Discussion
those of compound 1 except for the glucosyl moiety. The FABMS
fragments at m/z = 766 [M±rha], 750 [M±glc], 603[M±rha±
Compound 1 gave a positive coloration in the Liebermann- glc] or [M±glc±rha] indicated the presence of two terminal su-
1
Burchard and Molisch tests, suggesting that it was a triterpene gar moieties. The H-NMR singlet at d = 6.26 showed the pres-
glycoside. The IR spectrum revealed the presence of hydroxy ence of C-28 rhamnosyl ester, the same as that of 1, which was
(3434 cm±1), ester carbonyl (1700 cm±1), and double bond (1634 confirmed by the molecular ion peak of the peracetylated prod-
cm±1) functionalities. The HR-FABMS determined the molecular uct of 2 at m/z = 1331. A cross-peak of 1H-13C long-range cou-
formula to be
C41H66O12 (m/z = 749.4497 [M±H], calcd.: pling was observed between the proton signal at d = 4.96 (ara-
749.4476). Inspection of the NMR data revealed that 1 was a tri- H-1) and the carbon signal at d = 81.1 (C-3), indicating that the
terpene saponin having oleanolic acid as the aglycone moiety arabinose was linked at C-3. The proton at d = 5.09 (glc H-1)
(six tertiary methyl groups at d = 0.92, 0.92, 0.99, 1.00, 1.04 and showed an HMBC correlation with the carbon at d = 80.7 (ara-
1.22, two olefinic carbons at d = 122.7 and 144.9 and one carbo- C-4), indicating the glc-C-1/ara-C-4 linkage between the gluco-
nyl at d = 180.3). The presence of two sugar moieties was indicat- pyranosyl and arabinopyranosyl units. Therefore, 2 was elucidat-
ed by the 1H-NMR signals assigned to the two anomeric protons ed to be 3-O-b-D-glucopyranosyl±(1®4)-a-L-arabinopyranosyl
at d = 5.11 and 6.22 together with the observation of two hederagenin 28-O-a-L-rhamnopyranosyl ester.
550
anomeric carbons at d = 101.7 and 104.4, which was further sup-
ported and identified by the production of three compounds 1a, Compounds 1 and 2 showed antifungal activities against seven
1b and 1c in the hydrolysis of 1 with 5% HCl-MeOH. The 13C-NMR fungal strains including pathogenic fungi such as Candida
data (not shown) of 1a were identical with those of 3b,23-dihy- albicans, Candida glabrata, Saccharomyces cerevisiae, Cryptococcus
droxy-12-oleanen-28-oic acid (hederagenin) [12]. The two me- neoformans and Trichosporon beigelii, and the plant pathogenic
thyl monosaccharides 1b and 1c were separated by column chro- fungus Pyricularia oryzae (Table 2). Meanwhile, the antifungal ac-
matography over silica gel and Sephadex LH-20, and determined tivity of the prosaponin of 1, 1d, against P. avellaneum UC-4376
to be methyl L-arabinopyranoside and methyl L-rhamnopyrano- was tested, and the susceptibility amount was over 200 mg (data
side by comparison of their optical rotation and Rf values on TLC not shown). These data indicated that bidesmosides were better
with those of corresponding synthetic methyl monosaccharides, antifungal agents than monodesmosides, which was consistent
respectively. The sugar linkages were determined on the basis of with the report of Bader et al. [14]. The activities observed for com-
1
HMBC experiments. A cross-peak of H-13C long-range coupling pounds 1 and 2 against the plant pathogenic fungus P. oryzae
was observed between the proton signal at d = 5.11 (ara-H-1) showed increasing antifungal activity correlating with the exten-
and the carbon signal at d = 81.1 (C-3), indicating that the arabi- sion of the sugar chain, matching the phenomenon observed in
nose was linked at C-3. The 1H-NMR singlet at d = 6.22 showed the progress of plant defense against pathogens [15].
the presence of a C-28 rhamnosyl ester. The mild acid hydrolysis
of 1 in 2% H2SO4 aqueous methanol (80%) solution at ambient
temperature for eight days afforded 1a and 1d (structure was de- Acknowledgements
termined to be hederagenin 3-O-a-L-arabinopyranoside on the
basis of 13C-NMR data comparison with literature [13]), which This work was supported by the Ministry of Science and Technol-
further confirmed the presence of a C-28 rhamnosyl ester. There- ogy grant 2001-51, the National Natural Science Foundation of
fore, 1 was elucidated to be 3-O-a-L-arabinopyranosyl hedera- China (30 070007) and Natural Science Foundation of Yunnan Pro-
genin 28-O-a-L-rhamnopyranosyl ester.
vince (99B0017G). The authors are grateful to Prof. D. Z. Wang, Mr.
Y. N. He and Ms. H. L. Liang in the State Key Laboratory of Phyto-
The HR-FABMS determined the molecular formula of compound chemistry and Plant Resources in West China, Kunming Institute
2 to be C47H76O17 (m/z = 911.5002 [M±H], calcd.: 911.5004). The of Botany, Chinese Academy of Sciences, for measuring 2D NMR,
1H- and 13C-NMR spectra of 2 showed great similarities with 1D NMR and MS data, respectively, and to Mr. Jianxin Zhang in
Du Z et al. Two New Antifungal¼ Planta Med 2003; 69: 547±551

7 Ali-Shtayeh MS, Abu Ghdeib SI. Antifungal activity of plant extracts
against dermatophytes. Mycoses 1999; 42: 665±72
the Key Laboratory of Natural Products Chemistry of Guizhou Pro-
vince and the Chinese Academy of Sciences, Guiyang 650204, for
obtaining 1D and 2D NMR spectra on an INOVA-400 instrument.
8 Northwest Plateau Institute of Biology, Academia Sinica. Zang Yao Zhi.
Xining: People's Press, 1991: 249
9 Zhong H-M, Chen C-X, Tian X, Chui Y-X, Chen Y-Z. Triterpenoid sapo-
nins from Clematis tangutica. Planta Medica 2001; 67: 484±8
10
Du Z-Z, Zhu N, Shen Y-M. Two novel antifungal saponins from the Ti-
betan herbal medicine Clematis tangutica. Chin Chem Lett 2003; Vol.
14 No. 7: (in press)
Espinel-Ingroff A, White T, Pfaller MA. Antifungal Agents and Suscept-
ibility Tests. Manual of Clinical Microbiology.; 7th Edn (Murray PR,
editor). Washington DC: American Society for Microbiology, 1999:
pp. 1640±52
Kizu H, Tomimori T. Studies on the constituents of Clematis species. V.
On the saponins of the root of Clematis chinensis Osbeck. Chem Pharm
Bull 1982; 30: 3340±6
Kizu H, Hirabayashi S, Suzuki M, Tomimori T. Studies on the constitu-
ents of Hedera rhombea Bean. IV. On the hederagenin glycosides. (2).
Chem Pharm Bull 1985; 33: 3473±8
References
1
Ghannoum MA, Rice LB. Antifungal agents: mode of action, mecha-
11
nisms of resistance, and correlation of these mechanisms with bacte-
rial resistance. Clin Microbiol Rev 1999; 12: 501±17
2 Gilmore MS, Hoch JA. Antibiotic resistance: A vancomycin surprise.
Nature 1999; 399: 524±7
12
13
3 Novak R, Henriques B, Charpentier E, Normark S, Tuomanen E. Emer-
gence of vancomycin tolerance in Streptococcus pneumoniae. Nature
1999; 399: 590±3
4 Dixon RA. Natural products and plant disease resistance. Nature 2001;
411: 843±7
5 Papadopoulou K, Melton RE, Leggett M, Daniels MJ, Osbourn AE. Com-
promised disease resistance in saponin-deficient plants. Proc Natl
Acad Sci USA 1999; 96: 2923±8
14
15
Bader G, Kulhanek Y, Ziegler-Böhme H. Zur Antimyzetischen Wirk-
samkeit von Polygalasäureglycosiden. Pharmazie 1990; 45: 618±20
Grayer RJ, Harborne JB. A Survey of antifungal compounds from higher
plants, 1982±1993. Phytochemistry 1994; 37: 9±42
6 Cos P, Hermans N, Bruyne TD, Apers S, Sindambiwe JB, Berghe DV et al.
Further evaluation of Rwandan medicinal pant extracts for their antimi-
crobial and antiviral activities. J Ethnopharmacol 2002; 79: 155±63
551
Du Z et al. Two New Antifungal¼ Planta Med 2003; 69: 547±551