Microbial transformation of zaluzanin-D

Article abstract of DOI:10.1016/S0031-9422(02)00667-2

Microbial transformation of zaluzanin-D using different fungi gave 11,13-dihydrozaluzanin-C, zaluzanin-C, 4,16,11,13 -tetrahydro zaluzanin-C, estafiatone, dihydroestafiatol and dihydroestafiatone.

Full text of DOI:10.1016/S0031-9422(02)00667-2

Phytochemistry 62 (2003) 1101–1104
www.elsevier.com/locate/phytochem
Microbial transformation of zaluzanin-D
G.N. Krishna Kumari*, S. Masilamani, M.R. Ganesh, S. Aravind
Centre for Natural Products, SPIC Science Foundation, 111 Mount Road, Chennai 600 032, India
Received 8 April 2002; received in revised form 18 September 2002
Abstract
Microbial transformation of zaluzanin-D using different fungi gave 11,13-dihydrozaluzanin-C, zaluzanin-C, 4,16,11,13 - tetra-
hydro zaluzanin-C, estafiatone, dihydroestafiatol and dihydroestafiatone.
#
2003 Elsevier Science Ltd. All rights reserved.
Keywords:Vernonia arborea ; Zaluzanin; Estafiatone; Rhizoctonia solani; Botrytis cinerea; Curvularia lunata; Colletotrichum lindemuthianum;
Botryodiplodia theobromae; Fusarium equiseti; Fusarium oxysporum; Alternaria alternata; Sclerotinia sclerotiorum; Phyllosticta capsici
1
. Introduction
12 h dark). Culture filtrates were withdrawn at different
intervals, extracted with ethyl acetate and the organic
extract was analyzed by TLC. Fungal cultures in the
medium alone were maintained under similar conditions
and extracted for comparison. Chemical structures of
transformed compounds were deduced from compar-
ison of their spectroscopic data with that of zaluzanin-
D. The yield of products formed with different micro
organisms were presented in Table 1.
Hydrogenation of the double bonds was the common
transformation observed with all the fungi tested
(Scheme 1). Rhizoctonia solani was the only fungus,
which afforded oxidation products. Out of the 3 exo-
cyclic double bonds in zaluzanin-D (1), the 10–14 dou-
ble bond present in the 7-membered ring, being non-
conjugated to a carbonyl groupwas found to be resis-
tant to reduction.
Sesquiterpene lactones are known to possess insect
antifeedant, insect growth regulatory, antifungal, cyto-
toxic, and anti-tumor activities (Mabry and Gill, 1979;
Wedge et al., 2000).
Biotransformation of naturally occurring sesqui-
terpene lactones using fungi were attempted in order to
obtain regio and stereo selective modifications to
enhance the activity (Becher et al., 1981) and to develop
structure activity models. A number of biotransforma-
tions of sesquiterpene lactones using fungi has been
attempted earlier (Aleu et al., 1999; Barrero et al., 1999).
In pursuit of sesquiterpene lactones that possess insect
antifeedant activity, phytochemical investigation of
plants belonging to Indian verbenaceae and compositae
were attempted in this laboratory. Vernonia arborea
(
Compositae), a species endemic to southern India,
Botrytis cinerea was able to reduce the exocyclic dou-
ble bond conjugated to g-lactone stereospecifically to
give the major product 11,13-dihydrozaluzanin-D (2),
yielded considerable quantitites of zaluzanin-D (1), a
bioactive sesquiterpene lactone of guaianolide skeleton.
Biotransformation of zaluzanin-D was attempted in the
present investigation utilizing several fungal plant
pathogens and the results are presented.
with
11,13-dihydrodeacetylatedzaluzanin-D
(dihy-
drozaluzanin-C (3)) as minor product. Six fungi effected
total conversion of zaluzanin-D to 3 (Scheme 1). With
Sclerotinia sclerotiorum only deacetylation was observed
giving 4 as an exclusive product without effecting any
reduction. With Rhizoctonia solani, in addition to the
reduction of double bonds, further oxidation products
were obtained, the products obtained being 11,13-dihy-
drozaluzanin-C (3), dihydroestafiatone (5), dihy-
droestafiatol (6) and estafiatone (7).
2
. Results and discussion
Zaluzanin-D was incubated with the liquid cultures
under normal laboratory light conditions (i.e. 12 h light/
1
Comparison of H NMR spectrum (Table 2) of 2 with
that of zaluzanin-D (1) indicated the hydrogenation of
*
Corresponding author.
E-mail address: gnkrishna@hotmail.com (G.N. Krishna Kumari).
0031-9422/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00667-2

1102
G.N. Krishna Kumari et al. / Phytochemistry 62 (2003) 1101–1104
Table 1
Yields of products obtained
spectrum of 2. The orientation of the 13-methyl group
was assigned as a by the NOE observed between 6-H
and 11-H and also from the coupling constant of 11 Hz
between 7-H and 11-H. Thus compound 2 was identified
as 11,13-dihydrozaluzanin-D.
1H NMR spectrum of compound 3 showed the
absence of acetyl methyl signal. Also the upfield shift of
signal at ꢀ 5.56 corresponding to –CHOAc in 1 to ꢀ 4.54
in 3 indicated deacetylation. This was further supported
by the absence of acetyl carbonyl signal at ꢀ 170.6 in the
C-13 spectrum. In addition, the secondary methyl signal
at ꢀ 1.23 (d, J=6.9 Hz) showed the hydrogenation of
Name of the fungus
Age of the
culture (days) time (days)
Fermentation Product
(% yield)
A
Botrytis cinerea
Fusarium equiseti
Curvularia lunata
3
5
3
3
3
3
5
3
5
3
2
2 (85)+3 (15)
2 (33)+3 (66)
3 (96)
4
3
Collet. lindemuthianum
Botryodiplodia theobromae
Fusarium oxysporum
Alternaria alternata
Phyllosticta capsici
Sclerotinia sclerotiorum
Rhizoctonia solani
3
3 (94)
3 (94)
3
3
3 (93)
3 (86)
5
5
3 (88)
4 (98)
B
C
5
24
3 (<1)+5 (42)
+6 (47)+7 (6)
11–13 double bond. The 13-methyl orientation was
confirmed as a as in 2. Thus compound 3 was identified
as 11,13-dihydrozaluzanin-C. Both the products were
found to be formed simultaneously (by TLC) ruling out
the possibility of initial formation of 2 and its sub-
sequent conversion to 3.
1
1,13- double bond as shown by the presence of one
secondary methyl at ꢀ 1.24 (d, J=6.9 Hz) and the
absence of olefinic protons at ꢀ 6.22 and ꢀ 5.51 (corre-
sponding to 13-H). This was further confirmed by
replacement of the quaternary carbon, C-11, in 1 at ꢀ
Compound 4 was identified as zaluzanin-C from
the H and 13C NMR data (Tables 2 and 3), which is
deacetyl derivative of zaluzanin-D.
1
1
39.5 by the methine carbon at ꢀ 42.0 in the 13C NMR
Scheme 1. Transformation of zaluzanin-D (1) by different fungi (A, B and C mentioned in Table 1).

G.N. Krishna Kumari et al. / Phytochemistry 62 (2003) 1101–1104
1103
Table 2
1H NMR spectral data for compounds 1–7 (200 MHz, values in CDCl
3
)
H
1
2
3
4
5
6
7
1
3
6
3.05 m
3.05 m
5.56 m
4.07 t (9.3)
–
5.55 dd (3.5, 5.9)
3.99 t (9.4)
2.12 dq (11, 6.9)
4.54 t (7.4)
4.02 t (9.5)
2.14 qd (11, 6.9)
4.57 tt (7.5, 3)
4.10 t (9.2)
–
3.71 m
3.93 t (10)
2.12 m
3.96 t (9.2)
2.12 m
3.97 t (8.6)
–
1
1
1
1
1
1
1
1
a
a
a
a
3 a
3 b
4 a
4 b
5 a
5 b
6.22 d (3.5)
5.51d (3.5)
4.95 s
1.24 d (6.9)
1.23 d (6.9)
6.21 d (3.5)
5.49 d (3.5)
5.00 s
1.23 d (6.7)
1.21 d (6.8)
6.29 d (3.2)
5.57 d (3.2)
5.01 s
4.93 s
4.91 s
4.96 s
4.93 s
4.98 s
4.66 s
1.26 d (6.1)
4.95 s
4.94 s
4.68 s
1.24 d (6.3)
a
a
a
5.49t (2)
5.29 t (1.9)
2.11 s
5.41 t (2.1)
5.27 t (2.1)
2.10 s
5.38 t (1.9)
5.29 t (1.9).
–
5.46 t (1.8)
5.33 t (1.8)
–
1.24 d (6.3)
CH
3
-CO-O
–
–
–
a
Methyl doublets integrating for 3 H.
Table 3
13C NMR spectral data for compounds 1–6 (50 MHz, values in
CDCl
which was earlier reported from Centaurea webbiana
(
Antonia et al., 1972).
Relative to 5, compound 6 contained two additional
3
)
Carbon no.
1
2
3
4
5
6
protons (C H O ) as determined by EIMS. The lack
15 22 3
of 5-membered ring ketone carbon signal at ꢀ 219.2 in
1
2
3
4
5
6
7
8
9
44.5
36.4
43.9
36.2
74.6
148.6
49.8
83.6
50.4
32.2
36.1
148.2
42.0
178.3
13.1
113.2
113.4
170.7
21.1
43.5
38.7
73.5
153.2
49.5
83.7
50.8
32.3
35.9
148.8
42.0
179.8
13.1
113.5
111.0
–
44.1
39.0
73.5
153
39.7
44.0
219.2
47.2
50.9
88.5
48.6
32.9
39.0
149.1
41.7
178.2
13.3
112.5
14.0
–
42.1
38.3
78.3
47.0
50.6
85.9
52.9
32.7
37.0
149.2
42.0
178.6
13.0
112.5
14.1
–
1
3
the C NMR and the presence of additional methine
carbon bearing –OH at ꢀ 78.3, suggested the possibility
of a secondary hydroxyl groupat po sition 3. This was
further confirmed by the characteristic hydroxyl
74.6
147.7
45.2
83.7
45.5
83.8
49.9
30.5
34.1
147.9
139.6
169.9
120.1
114.3
111.2
–
À1
absorption at 3200 cm in the IR spectrum. Hence
50.2
30.5
compound 6 was identified as dihydroestafiatol.
1H NMR of compound 7 was similar to that of com-
pound 5 which showed the absence of peak at ꢀ 5.41 and
34.4
1
1
1
1
1
1
0
1
2
3
4
5
148.0
139.5
169.8
120.2
113.4
114.3
170.6
21.1
ꢀ
5.27 corresponding to H-15. The presence of second-
ary methyl signal at ꢀ 1.24 (d, J=6.8 Hz) confirmed the
reduction of a 4–15 double bond. But the methylene
protons (C-13) seen at ꢀ 6.29 and ꢀ 5.57 suggested that
the 11–13 double bond was intact. Thus 7 was identified
as estafiatone.
CH
CH
3
–CO–O–
–CO–O–
3
–
–
–
–
3. Experimental
With Rhizoctonia solani two major products 5 and 6
were obtained in addition to the minor products 3 and 7.
Compound 5 had the molecular formula C H O .
Leaves of Vernonia arborea (50 kg) were collected
from Kolli hills, Tamilnadu, during November 1999.
Their identity was confirmed by Dr. K.V.Krishna-
murthy, Department of Botany, Bharathidasan Uni-
versity, and a voucher specimen (No. BDU 556) was
deposited in their herbarium. The dried and crushed
material was extracted exhaustively with n-hexane and
methanol. One hundred grams of the residue from the
hexane extract was chromatographed over silica gel
column using n-hexane–EtOAc mixtures (1–100%) as
eluents.
1
5
20
3
1H NMR of 5 showed absence of acetyl methyl signal
and presence of two secondary methyl signals at ꢀ
1
.23 (d, J=6.7 Hz), ꢀ 1.26 (d, J=6.1 Hz) indicating
deacetylation as well as reduction of two exocyclic
double bonds. 13C NMR of compound 5 also con-
firmed the absence of acetyl methyl and acetyl car-
bonyl. Instead, a signal at ꢀ 219.2 indicated the
presence of a 5-membered ring ketone. IR band at
À1
1
738 cm
supported this. Upfield shift of the 5-H
signal to ꢀ 2.2 (which appeared at ꢀ 2.78 in 1) sug-
gested the reduction of a 4–15 double bond. The NOE
observed between 6-H, 4-H and 11-H indicated the a
orientation of the methyls at C-4 and C-11. Thus
compound 5 was identified as dihydroestafiatone,
3.1. Isolation of compound 1
Fractions 133–150 on crystallization from hexane–
ꢀ
ethyl acetate gave compound 1 (4 g) mp103–104
;

1104
G.N. Krishna Kumari et al. / Phytochemistry 62 (2003) 1101–1104
2
5
ꢀ
CHCl3
max
[
2
(
a] +21.43 (CHCl ; c 0.28); UV l
nm 246 ("
13
3.6. Compound 4
D
3
5302); IR ꢁmax cm ; 1756, 1732; 1H, C NMR
Tables 2 and 3); [M+1] 289.
KBr
À1
+
ꢀ
25
D
KBr
CHCl3
nm
max
Mp95–96 ; [a] +50o (CHCl ; c 0.1); UV l
3
À1
+
1
13
242 (" 59290); IR ꢁmax cm ; 3200, 1748; H, C NMR
(Tables 2 and 3); [M+1] 247.
3
.2. Microorganisms and culture media
Microorganisms Rhizoctonia solani (SPICFCC-18),
Botrytis cinerea (SPICFCC-12), Curvularia lunata
3.7. Compound 5
ꢀ
25
ꢀ
CHCl3
Mp82–83 ; [a] +66.67 (CHCl ; c 0.12); UV l
D max
(
SPICFCC-29),
SPICFCC-38), Botryodiplodia theobromae (SPICFCC-
1), Fusarium equiseti (SPICFCC-31), Fusarium oxy-
sporum (SPICFCC-9), Alternaria alternata (SPICFCC-
5), Sclerotinia sclerotiorum (SPICFCC-24), Phyllosticta
Colletotrichum
lindemuthianum
3
KBr
À1
1
13
(
4
nm 243 (" 12052); IR ꢁmax cm ; 1771, 1738; H, C NMR
(Tables 2 and 3); NOE-diff experiments: Proton irradiated
(NOEs observed) H-6 (H-4, H-11); [M+1]+ 249.
1
capsici (SPICFCC-46) used in the experiments were
obtained from Mycology Department, Centre for Nat-
ural Products, SPIC Science Foundation.
3.8. Compound 6
2
5
ꢀ
CHCl3
max
[a] +25 (CHCl ; c 0.08); UV l
nm 242 ("
D
3
75927); IR ꢁmax cm ; 3200, 1738; 1H, C NMR
KBr
À1
13
Czapek-Dox liquid medium containing NaNO (2 g),
3
+
KH PO (1 g), KCl (0.5 g), MgSO .7H O (0.5 g),
2
(Tables 2 and 3); [M+1] 251.
4
4
2
Sucrose (30 g) with traces of FeSO .7H O in 1000 ml of
4
2
water was used for transformation experiments.
3.9. Compound 7
CHCl3
max
KBr
À1
cm ; 1763,
max
3
.3. Biotransformation of zaluzanin-D
UV l
nm 243 (" 60507); IR ꢁ
+
1
1
737; H NMR (Table 2); [M+1] 247.
The organism was inoculated in sterilized media and
kept in a shaker (100 rpm) for 3–5 days (Table 1) at
ꢀ
2
6
C. The substrate (40 mg) dissolved in 2 ml of
Acknowledgements
acetone was added to the broth (100 ml) and the
incubation continued till complete conversion of
starting material. The reaction was monitored using
TLC. On completion of the reaction, the broth was
filtered from the biomass, which was repeatedly
washed with water and EtOAc. The filtrate was then
extracted with EtOAc, dried over Na SO and con-
We wish to thank Professor K.V. Krishnamurthy,
Department of Botany, Bharathidasan University, Tri-
chy, for collection and identification of the plant mate-
rial. One of the authors (M.R. Ganesh) wishes to thank
CSIR, India for the research fellowship.
2
4
centrated under vacuum. The crude product obtained
in each case was purified by Silica-gel column chro-
matography using n-hexane and increasing quantities
of EtOAc (1–100%).
References
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3
.4. Compound 2
ꢀ
25
ꢀ
KBr
Mp126–128 ; [a] +54.02 (CHCl ; c 0.06); UV
D
nm 246 (" 129533); IR ꢁmax cm ; 1771, 1733; H,
3
À1
CHCl3
max
1
l
1
3
¨
Becher, E., Schuep, W., Matzinger, P. K., Teisseire, P. J., Ehert, Ch.,
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6
), H-6 (H-15a, H-11) [M+1]+ 291.
3
.5. Compound 3
2
5
ꢀ
KBr
CHCl3
nm 257 ("
max
[
a] +83.33 (CHCl ; c 0.24); UV l
D
3
Wedge, D.E., Galindo, J.C.G., Macias, F.A., 2000. Fungicidal activity
of natural and synthetic sesquiterpene lactone analogs. Phytochem-
istry 53, 747–757.
1225); IR ꢁmax cm ; 3197, 1747; 1H, C NMR
À1
13
3
+
(Tables 2 and 3); [M+1] 249.