Characterization of the antifungal activity against botrytis cinerea of sclareol and 13-epi-sclareol, two labdane-type diterpenoids

Article abstract of DOI:10.4067/S0717-97072015000300010

The antifungal activity of Sclareol and 13-epi-Sclareol, two labdane-type diterpenoids, on mycelial growth of the phytopathogenic fungus Botrytis cinerea was evaluated. Diterpenoid fungitoxicity was assessed using the radial growth test method. Both diterpenoids inhibited the mycelial growth of B. cinerea in solid medium; however the inhibitory activity of Sclareol was slightly higher than 13-epi-Sclareol with IC₅₀ value of 237.6 μg/mL and 268.8 μg/mL, respectively. On the other hand, both labdane-type diterpenoids did not alter the plasmatic membrane integrity; however the oxygen consumption of B. cinerea germinating conidia was affected. The evidence suggests that action mechanism of these molecules would be related to the uncoupling of mitochondrial oxidative phosphorylation. Finally, biotransformation of Sclareol by B. cinerea was analysed and the main biotransformed metabolite was identified as 3β-hydroxysclareol.

Full text of DOI:10.4067/S0717-97072015000300010

J. Chil. Chem. Soc., 60, Nº 3 (2015)
CHARACTERIZATION OF THE ANTIFUNGAL ACTIVITY AGAINST BOTRYTIS CINEREA OF SCLAREOL AND
13-EPI-SCLAREOL, TWO LABDANE-TYPE DITERPENOIDS
LEONORA MENDOZA*, CAROLINA SEPÚLVEDA, RICARDO MELO, MILENA COTORAS*
Laboratorio de Micología, Facultad de Química y Biología, Universidad de Santiago de Chile. Alameda 3363, Santiago, Chile
ABSTRACT
The antifungal activity of Sclareol and 13-epi-Sclareol, two labdane-type diterpenoids, on mycelial growth of the phytopathogenic fungus Botrytis cinerea
was evaluated. Diterpenoid fungitoxicity was assessed using the radial growth test method. Both diterpenoids inhibited the mycelial growth of B. cinerea in solid
medium; however the inhibitory activity of Sclareol was slightly higher than 13-epi-Sclareol with IC50 value of 237.6 µg/mL and 268.8 µg/mL, respectively. On
the other hand, both labdane-type diterpenoids did not alter the plasmatic membrane integrity; however the oxygen consumption of B. cinerea germinating conidia
was affected. The evidence suggests that action mechanism of these molecules would be related to the uncoupling of mitochondrial oxidative phosphorylation.
Finally, biotransformation of Sclareol by B. cinerea was analysed and the main biotransformed metabolite was identified as 3β-hydroxysclareol.
Keywords: Sclareol, 13-epi-Sclareol, antifungal activity, Botrytis cinerea
dihydro-15-chloro-14-hydroxy-8,9-dehydro-Sclareol suggesting the epoxide
formation as an reaction intermediate15.
1.
INTRODUCTION
In this study, the fungitoxic effect against B. cinerea of the natural
diterpenoids Sclareol and 13-epi-Sclareol was characterized. Also, the effect
of both diterpenoids on oxygen consumption and on plasmatic membrane
integrity of B. cinerea was studied. Finally, biotransformation of Sclareol by
B. cinerea was analysed.
Botrytis cinerea, the agent of gray mold, is a facultative phytopathogenic
fungus that affects fruits, leaves, stems and flowers of more than 250 plant
species1. Chilean climatic conditions such as high relative humidity, wind
speed and low temperatures promote a high incidence of this disease, causing
serious pre- and postharvest losses in the grape production in Chile2.
Currently, the B. cinerea control is realized with hydroxyanilides,
2. EXPERIMENTAL
anilinopyrimidines,
dicarboximides,
carboxamides,
strobilurins,
phenylpyrroles and some inhibitors of ergosterol synthesis3. These families of
fungicides differ in their mechanism of action on the fungus. The prolonged
use of these synthetic compounds in the field has caused the development of
2.1. General experimental procedures.
The NMR spectra were acquired using a Bruker Avance 400 MHz
spectrometer (400,133 MHz for 1H, 100.624 MHz for 13C). All the measurement
were done in CDCl to 300 K. Chemical shifts (in ppm) for 1H and 13C spectra,
were calibrated to 3solvent signal, CHCl3 7.26 ppm (residual signal solvent)
and 77.16 ppm, respectively, and reported relative to Me4Si. Thin-layer
chromatography was performed on Merck Kiesegel 60 F254, 0.2 mm thick
and semi-preparative thin layer chromatography on Merck Kieselgel 60 F254
20x 20cmx 0.25 mm. Silica gel (Merck) was used for column chromatography.
The melting point uncorrected was determined on a Kofler hot-stage apparatus.
2.2. Chemicals reagents and compounds used in this study.
resistance to these botryticides4-6
. Therefore, it is imperative to find new and
effective molecules with antifungal activity. Secondary metabolites produced
by plants are an alternative to control phytopathogenic fungi; some of these
constitutive or inducible compounds have shown antimicrobial activity.
Among them, diterpenoids are an interesting group of secondary metabolites
due to their biological activities. These compounds are widely distributed in
nature and it has been described that some have antibacterial7, antiviral8 and
antifungal activities9.
Cotoras et al. (2001) reported the effect of the diterpenoid 3β –
hydroxykaurenoic acid on the mycelial growth and conidia germination of
B. cinerea10. Later, the same group reported that the mechanism of action of
3β–hydroxykaurenoic acid was based on the permeabilization of plasmatic
membrane11. On the other hand, Mendoza et al. (2009)12 investigated the
inhibitory effect of the natural diterpenoids salvic acid, acetylsalvic acid,
and three hemisynthetic diterpenoids against B. cinerea. All diterpenoids,
with the exception of isopentenoylsalvic acid, inhibited the mycelial growth
of B. cinerea in solid media. Studies on a possible action mechanism of
natural diterpenoids, salvic acid and acetylsalvic acid, showed that these
diterpenoids exerted their effect by a different mechanism. Salvic acid did
not alter cytoplasmic membrane or cause respiratory chain inhibition. Instead,
acetylsalvic acid affected the integrity of the cytoplasmic membrane12. These
results suggest that compounds, as diterpenoids might be good candidates for
the design of antifungal compounds.
On the other hand, B. cinerea has the ability to biotransform secondary
metabolites, producing chemical changes in these compounds13. It has
been reported that this fungus biotransforms different types of compounds
as: steroids, sesquiterpenoids, flavones, diterpenoids, etc.13. The main
biotransformation reactions involve oxidations and hydroxylations, when B.
cinerea metabolizes phytoalexins or antifungal compounds as a detoxification
mechanism, although reduction reactions have been also described13.
Labdane-type diterpenes are examples of natural products with important
biological activities. Some of these compounds possess antifungal, antibacterial,
antimutagenic, cytotoxic, anti-inflammatory or analgesic activities14. On the
other hand, the fungal biotransformation of Sclareol has also been studied.
The main formed products are 18-hydroxysclareol, 3β-hydroxysclareol and
6α-hydroxysclareol14. It has been reported that B. cinerea biotransforms Sclareol
to epoxysclareol, and this later to a halogenated product called 8-deoxy-14,15-
Technical grade fungicide iprodione [3-(3,5-dichlorophenyl)-N-isopropyl-
2,4-dioxoimidazolidine-1-carboxamide] was provided from INIA (Santiago,
Chile).
Diterpenoid structures used in this study are shown in Figure 1. Sclareol
(Figure 1A) was obtained commercially, Sigma Chemical Co. (St. Louis, MO),
whereas 13-epi-Sclareol (Figure 1B) was isolated and purified from resinous
exudates of Pseudognaphalium cheiranthifolium as has been previously
described with some modifications16. Briefly, the resinous exudates were
obtained by dipping P. cheiranthifolium leaves and stems in CH2Cl2 during
30s at room temperature. Extracts were concentrated by solvent evaporation
in a rotary evaporator to a sticky residue. Residue was redissolved in CH2Cl2
and applied to a silica gel column (30 x 200 mm). The column was eluted
successively with hexane and hexane with increasing amount of ethyl acetate
(9:1 to 1:1 hexane:ethyl acetate). 13-epi-Sclareol contained in the column
fractions was further purified by thin layer chromatography using hexane:ethyl
acetate 3:1. The last step of purification was by recrystallization using methanol
as solvent. Finally, purified 13-epi-Sclareol was analyzed by comparison of
their physical and spectroscopic (1H NMR) data with literature16.
2.3. Fungal isolate and culture conditions.
During this study G29 isolate of B. cinerea was used. This strain was
originally isolated from a naturally infected grape (Vitis vinifera) from Chile
17 and was maintained on malt-yeast extract agar (2% (w/v) malt extract, 0.2%
(w/v) yeast extract and 1.5% (w/v) agar) at 4 °C. The fungus was grown in
the dark on malt-yeast extract agar medium [2% (w/v) malt extract, 0.2%
(w/v) yeast extract and 1.5% (w/v) agar) or soft agar (2% (w/v) malt extract,
0.2% (w/v) yeast extract and 0.6 % (w/v) agar]. In studies related to action
mechanism, liquid minimum medium was used. This medium was composed
by KH2PO4 (1 g/L), K2HPO4 (0.5 g/L), MgSO4 x 7H2O (0.5 g/L), KCl (0.5 g/L),
e-mail: leonora.mendoza@usach.cl
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J. Chil. Chem. Soc., 60, Nº 3 (2015)
FeSO4 x 7H2O (0.01 g/L) pH 6.5, 25 (mol/L) ammonium tartrate as a nitrogen
source, and supplemented with 1% (w/v) glucose as carbon source.
2.4. Effect of the compounds on the mycelial growth of B. cinerea in
solid media.
oxygen consumption by germinating conidia was evaluated using a one way
analysis of variance (Genstat 5, Realease 4.1). Means were separated using the
least significant difference test (P ≤ 0.05).
2.7. Biotransformation of Sclareol by B. cinerea.
Fungitoxicity of both diterpenoids and the commercial fungicide iprodione
was assessed using the radial growth test on malt-yeast extract agar11. Sclareol
and 13-epi-Sclareol were dissolved in methanol at different final concentrations
(150, 200 and 250 μg/mL). The diterpenoids were completely soluble at all
concentration tested. One hundred µL of this solution were added to 20 mL
of malt-yeast extract agar. The final methanol concentration was identical in
controls and treatment assays. The medium with or without diterpenoids or
iprodione was poured into 9 cm diameter Petri dishes. Dishes were left open
in a biosecurity hood for 40 min to remove methanol. After evaporation of
the solvent, the Petri dishes were inoculated with 0.5 cm agar discs with thin
mycelium of B. cinerea. Cultures were incubated in the dark at 22 ºC during
several days. Mycelial growth diameters were measured daily. Results were
also expressed as IC (the concentration that reduced mycelial growth by
50%) determined by r5e0gressing the inhibition of radial growth values (percent
control) against the values of compound concentration. Each experiment was
done at least in triplicate.
The fungus was grown in liquid minimum medium for three days. Three
days old mycelia were transferred to flasks with liquid minimum medium
supplemented with 1% (w/v) glucose in the presence of 150 μg/mL Sclareol.
As control, cultures containing only Sclareol at 150 μg /mL in the absence of
the fungus and cultures of the fungus in the absence of Sclareol were used.
All cultures were incubated during one week at 22 ºC and stirred at 180 rpm.
After this time, the mycelia were filtered and the fermentation broths were
extracted three times with ethyl acetate, chloroform or hexane. The extracts
were treated with sodium chloride to obtain a homogeneous solution and
dried over anhydrous sodium sulphate, and the solvent was then evaporated.
The extracts were analysed by thin layer chromatography (TLC) eluting with
chloroform: ethyl acetate mixture (95:5, v/v). The purification of the main
compound obtained from Sclareol biotransformation was carried out by using
semi-preparative thin layer chromatography with chloroform:ethyl acetate
(95:5) as an eluent system.
2.5 Effect of the compounds on germination of B. cinerea conidia.
Conidial germination assays were carried out on microscope slides coated
with soft agar medium (2 mm thickness). Sclareol and 13-epi-Sclareol were
added dissolved in methanol at a final concentration of 40 μg/mL. Methanol
was allowed to evaporate prior to inoculation. The slides were inoculated with
dry conidia obtained from sporulated mycelia (1 week old), placed in a humid
chamber (90% relative humidity), and incubated in the dark at 22 °C for 7 h.
Conidial germination was determined directly on the slides at intervals of 1
hour. The percentage of germination was estimated by counting the number of
germinated conidia in five microscope fields each containing approximately 40
conidia. Conidia were judged to have germinated when the germ tube length
was equal to or greater than conidial diameter. Each experiment was done at
least in triplicate.
2.6. Mode of action of both labdane-type diterpenoids on B. cinerea.
2.6.1. Determination of the effect of diterpenoids on the membrane integrity
of B. cinerea. To analyze if Sclareol and 13-epi-Sclareol are able to produce
alteration of the integrity of B. cinerea cytoplasmic membrane, SYTOX Green
uptake assay according to described by Cotoras et al. (2011)10 was used. B.
cinerea conidia at a final concentration of 1x105 conidia / mL were inoculated
in 24-well plates (lined with 12-mm glass coverslips) containing 1 mL of liquid
minimum medium. Cultures were incubated at 22 ºC for 15 h to permit the
germination of the conidia. After this time, liquid medium was removed and
same medium with 70% (v/v) ethanol (positive control), 8% (v/v) methanol
(negative control), or 150 μg/mL of compounds was added to each well. The
mixtures were incubated at 22 ºC for one or four hours in the case of diterpenoids
and methanol or for 10 min when ethanol was used. B. cinerea hyphae adhered
to glass coverslips were washed three times with liquid minimum medium and
were stained with 50 nmol/L SYTOX Green. After 10 min of incubation, the
hyphae were washed with minimum medium and glass coverslips containing
hyphae were mounted in slides. For the assembly of the samples in the slides,
15 µL of DABCO (1,4-diazabicyclo[2.2.2]octane) (Sigma Chemical Co., St.
Louis, MO, USA) was used. The fluorescence of B. cinerea hyphae stained
with SYTOX Green was observed under a confocal microscope (Carl Zeiss
LSM 510) at an excitation wavelength of 488 nm and an emission wavelength
of 540 nm. These experiments were done at least in triplicate.
2.6.2. Determinationoftheeffectofditerpenoidsontheoxygenconsumption
of B. cinerea conidia. Oxygen consumption was determined polarographically
at 25 °C with a Hansatech oxygen electrode by using germinating conidia
in a total volume of 1 mL according the protocol previously described10. To
obtain conidia in suspension, Murashige and Skoog´s basal medium at 4.4
(g/L) (Phytotechnology Laboratories, Lenexa, KS, USA) was added to Petri
dishes containing conidia. The conidia were harvested by scraping with a
sterile spatula. To eliminate mycelium, the suspension was filtered through
glass wool. The conidia concentration was adjusted to 1 x 107 conidia/mL with
minimum liquid media, in the presence of 2% (w/v) glucose. Conidia were
incubated for 2 hours at 22 °C. The measurement of basal oxygen consumption
was carried out for 2 min in the same minimum liquid medium. After this time,
10 μg/mL carbonyl cyanide m-chloro-phenylhydrazone (CCCP), 650 μg/mL
KCN or the diterpenoids, dissolved in methanol, at a final concentration of 150
and 250 μg / mL were added. Oxygen consumption was determined for eight
more minutes.
Labda-14-en-3β, 8α, 13β triol (compound 3, Figure 6) was obtained as a
white solid, mp 162º. δ (CDCl3) 0.76 (3H, s, H-19), 0.80 (3H, s, H-20), 0.99
(3H, s, H-18),1.16 (3H, s, H-17), 1.28 (3H, s, H-16), 3.23 (1H, dd, J=4.2 and
11.2 Hz, H-3α), 5.04 (1H, dd, J=1.3 and 10.6 Hz, H-15), 5.23 (1H, dd, J=1.3
and 17.4 Hz, H-15), 5.94 (1H, dd, J=10.8 and 17.4 Hz, H-14). 13C NMR data
(Table 1).
Table 1: 13C NMR data determined of Sclareol (Compound 1) and its
biotransformation product 3β-hydroxysclareol (Compound 3).
Carbon atom
Compound 1
δc (ppm)
39.9
Compound 3
δc (ppm)
39.4
1
2
18.6
27.4
3
42.2
79.2
4
33.4
39.2
5
56.2
55.3
6
20.7
20.5
7
44.3
45.2
8
74.9
74.9
9
61.8
61.6
10
11
12
13
14
15
16
17
18
19
20
39.4
38.2
19.2
19.5
45.1
45.2
73.8
74.0
146.2
111.3
27.4
147.5
111.8
28.5
24.4
24.6
33.5
30.0
21.6
20.6
15.5
15.9
3. RESULTS AND DISCUSSION
3.1 Antifungal activity of diterpenoids on B. cinerea.
The antifungal activity of Sclareol (Compound 1) and 13-epi-Sclareol
(Compound 2) (Figure 1), two labdane-type diterpenoids, against B. cinerea
was analyzed. The difference among these compounds is the configuration in
the C-13. The Sclareol has configuration S and 13-epi-Sclareol configuration
R.
Statistical Analyses. The effect of diterpenoids on mycelium growth and
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J. Chil. Chem. Soc., 60, Nº 3 (2015)
(IC50) the mycelial growth was calculated (Table 2). Sclareol was slightly more
active than 13-epi-Sclareol, since it presented a lower IC50 value. Although
both diterpenoids were able to reduce the hyphal growth, the IC values were
highest than those obtained for the commercial fungicide, Iprodi5o0ne.
Table 2: Effect of Sclareol and 13- epi-Sclareol on in vitro mycelial
growth of B. cinerea.
*
IC50
Compounds
(μg/mL)
Sclareol
13-epi-Sclareol
Iprodione
237.6 ± 12.2a
268.8 ± 9.8b
35.0 ± 16.8c
Figure 1. Structure of labdane-type diterpenoids, Sclareol (1) and 13-epi-
Sclareol (2).
*Estimation of IC was based on colony diameter measurements after 4
days of incubation. Di5f0ferent letters represent significant differences (P ≤ 0.05)
To determine the antifungal activity of the diterpenoids, the effect of
Sclareol and 13-epi-Sclareol on mycelium growth in solid media was evaluated
(Figure 2).
Complementary to this, the effect of Sclareol and 13-epi-Sclareol on
conidia germination was evaluated. The Figure 3 shows the kinetics of
germination of B. cinerea conidia in the presence of both diterpenoids at 40
μg/mL.
Figure 2. Effect of Sclareol and 13-epi-Sclareol on the mycelium growth of
isolate G29 of B. cinerea. Sclareol (A) and 13-epi-Sclareol (B) were dissolved
in methanol and were added at a final concentration of 150 μg/mL (dashed
line). As a control: G29 in the absence (black line) or in presence of methanol
(gray line) at the same concentration that treatments were used. Every point
represents the average of three independent assays ± standard deviation.
Figure 3. Effect of the diterpenoids on conidia germination of B. cinerea.
Sclareol (A) and 13-epi-Sclareol (B) were dissolved in methanol and added
at a final concentration of 40 µg/mL (dashed line). G29 incubated in the
absence (black line) or in the presence of methanol at the same concentration
that treatments were used (grey line) as control. The germination kinetic was
followed during 10 hours of incubation at 22 ºC. Each point represents the
average of three independent assays ± standard deviation.
Both diterpenoids produced inhibition of the B. cinerea mycelium
growth. In control, mycelium growth reached a maximum after seven days
of incubation. Meanwhile, in the presence of Sclareol at 150 μg/mL maximum
mycelium growth was reached after 11 days. On the other hand, in the presence
of 13-epi-Sclareol at the same concentration (150 μg/mL) the maximum
mycelium growth was reached after 12 days. Similar results were obtained
when 200 or 250 μg/mL of Sclareol or 13-epi-Sclareol were used (data not
shown). The diterpenoids decreased the rate of mycelium growth.
The diterpenoids were not able to inhibit the germination. However,
13-epi-Sclareol produced a delay in germination between the 4 and 7 hours
of incubation, but later, a 100 % of germination was obtained (Figure 3B).
In presence of the diterpenoids, the appearance of cytological abnormalities
The inhibition percentages of myceliaum growth were calculated after four
days of incubation and the concentration of the compounds that inhibited a 50%
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J. Chil. Chem. Soc., 60, Nº 3 (2015)
was not observed (data not shown). This result was different to those obtained
with resveratrol, a natural phytoalexin; since it has been reported that this
compound produced curved germ tubes and cytological abnormalities in B.
cinerea conidia18.
It is important to emphasize that this is the first work in which the
antifungal effect against the fungus B. cinerea of two epimers was compared.
3.2 Mode of action of Sclareol and 13-epi-Sclareol on B. cinerea.
To analyze the possible mechanism of action of Sclareol and 13-epi-
Sclareol on B. cinerea, the effect of these diterpenoids on the plasmatic
membrane integrity or conidia oxygen consumption was determined.
To determine if the diterpenoids cause damage in the integrity of the
fungal plasmatic membrane, the assay of SYTOX green uptake was performed.
This assay is based in the uptake of the dye SYTOX green, that fluoresces upon
interaction with nucleic acids and it is able to penetrate cells with compromised
plasmatic membrane; but it does not cross the membranes of non-compromised
living cells19. The results of these experiments are shown in the Figure 4.
Figure 5: Effect of the diterpenoids on oxygen consumption of B. cinerea
germinating conidia. Diterpenoids were aggregated at a final concentration
of 150 or 250 μg/mL to a conidia suspension (1x107 conidia/mL) in minimal
media with 2 % w/v glucose; the oxygen consumption were measured at room
temperature using a Clark electrode. Each bar represents the average of at least
three independent experiments ± standard deviation. The symbol (*) represents
significant differences P ≤ 0.05
In presence of CCCP, an uncoupler of oxidative phosphorylation, also an
increase of the oxygen consumption was observed, reaching to 124%. Instead,
in presence of KCN, a respiratory chain inhibitor, the oxygen consumption
diminished to 15.9%.
These results suggest that the diterpenoids are able to cross the plasmatic
membrane without alter it, then they reach to the mitochondria causing
uncoupling of the oxidative phosphorylation. An overlapping study between
Sclareol and cholesterol based on their corresponding polar and hydrophobic
molecular segments, calculated from their lypophilic profiles, revealed their
space similarities, both molecules are amphipathic and they possess lipophilic
and hydrophilic segments that can be easily superimposed. It was shown that
Sclareol was incorporated in phospholipid membrane model imitated the
thermal effect from the cholesterol20. Based on the structural similarity among
cholesterol and ergosterol, a fungal membrane steroid, it could be suggested
that Sclareol or 13-epi-Sclareol would interact with the fungal mitochondrial
membrane, being able to dissipate the protonic gradient indirectly, acting as
uncoupler of the oxidative phosphorylation.
Different results were obtained in Gram positive bacteria treated with
13-epi-Sclareol21 Tapia et al. (2004) reported that this compound inhibited
oxygen consumption in intact Gram-positive. In addition, the authors described
that the diterpenoid was able to inhibit NADH oxidase and cytochrome C
reductases activities suggesting that the target site of 13-epi-Sclareol was
located between coenzyme Q and cytochrome C of the bacterial respiratory
chain21.
Figure 4: Effects of Sclareol and 13-epi-Sclareol on plasmatic membrane
integrity of B. cinerea Conidia, at a final concentration of 1 × 105 conidia/
mL, were incubated in liquid minimum medium in the presence of 10% (v/v)
methanol (A), 70% (v/v) ethanol (B), 150 µg/mL of Sclareol during 4 h (C) or
150 µg/mL of 13-epi-Sclareol during 4 h (D) The fluorescence of B. cinerea
hyphae stained with SYTOX Green was observed under a confocal microscope.
These photographs are representatives of three independent experiments
3.3 Biotransformation of Sclareol by B. cinerea.
In this work, the biotransformation of Sclareol was analyzed. For this,
B. cinerea mycelium was incubated with Sclareol, at 150 μg/mL, in a culture
medium supplemented with 1% glucose. After seven days of incubation,
compounds were extracted from culture medium by liquid-liquid extraction
using solvents with different polarity.
It is possible to observe that no fluorescence was detected in presence
of methanol (negative control, Figure 4A), by the contrary, in presence of
ethanol, a positive control, fluorescent nuclei were observed (Figure 4B). The
fluorescence detected in presence of ethanol is due this compound generates
dehydration of the cellular membrane producing damage in the membrane
integrity of the fungus. In presence of Sclareol or 13-epi-Sclareol, fluorescence
in the nuclei was not observed (Figure 4C y 4D). Therefore, it is possible to
conclude that these diterpenoids did not alter plasmatic membrane integrity.
On the other hand, to analyse the effect of the diterpenoids on
mitochondrial respiratory chain, the oxygen consumption by germinating
conidia was determined. Both diterpenoids, at a concentration of 150 μg/mL,
increased the oxygen consumption of germinating conidia respecting to the
basal consumption, therefore these compounds would act as uncoupler of the
oxidative phosphorylation. Indeed, the percentages of oxygen consumption
in the presence of Sclareol or 13-epi-Sclareol were 156.7% or 162.2%,
respectively. Similar results were obtained at a concentration of 250 μg/mL of
both diterpenoids where the oxygen consumption in the presence of Sclareol or
13-epi-Sclareol increased to 155.7% or 140.8%, respectively (Figure 5).
Thin layer chromatography analysis of the extracts revealed the presence
of two main metabolites with higher polarities than Sclareol, but only one
biotransformed compound was isolated. This compound was purified and
analyzed through 1H and 13C, and HSQC, NMR spectroscopy, 1H NMR
spectrum of the compound exhibited signals for H-3 (δ 3.23, dd J3-2a = 11.2 Hz,
J3-2b = 4.2 Hz). The 13C NMR showed a resonance at δ 79.2 and a methylene
resonance at δ 27.4. The signal corresponded to C-3 appears now displaced to
lower fields suggesting that the original position at δ 42.2 and after at δ 79.2
was displaced by the presence of an (–OH) group in this carbon atom. The 2D
spectrum, HSQC, displayed an important correlation between H-3 (δ 3.23)/
C-3 (δ 79.2). The 1H assignments of biotransformed compound were compared
with the values of labda-14-en-3β, 8α, 13-β-triol obtained by fermentation
of Sclareol with Cephalosporium aphidicola22 and it was concluded that
Sclareol was transformed to labda-14-en-3β, 8α, 13β triol, commonly called
3β-hydroxysclareol (Figure 6).
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J. Chil. Chem. Soc., 60, Nº 3 (2015)
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Figure 6: Product obtained by Sclareol biotransformation by B. cinerea.
22. J. Hanson, P.B. Hitchcock, H. Nasir, A. Truneh. Phytochem. 36, 903,
(1994)
23. A. Wolf-Rainer. Phytochem. 36, 1421, (1994)
It has been reported that Sclareol is biotransformed to more oxidized
molecules by fungi and bacteria. Supporting this observation, Farooq and
Tahara (2007)15 reported the biotransformation of two cytotoxic terpenes
(α-santonin and Sclareol) by B. cinerea. Sclareol was metabolized to
epoxysclareol
and
to
8-deoxy-14,15-dihydro-15-chloro-14-hydroxy-
8,9-dehydrosclareol, a rare example of halogenation15. In addition, it has
been reported that Sclareol can be biotransformed to 3β-hydroxysclareol,
6α-hydroxysclareol or 18-hydroxysclareol by Aspergillus alliaceus, A. spergillus
niger, A. spergillus ochraceus, Cunninghamella echinulata, Cunninghamella
elegans, Cunninghamella species, C. urvularia lunata, M. ortierella ramanniana,
M. ortierella isabellina, Rhizopus arrhizus, R. hizopus stolonifer, and
Sporotrichum exile14. In addition, several oxidized metabolites were obtained
in the biotransformation of Sclareol by the fungus Cephalosporium aphidicola.
Among the main products, the compounds labda-14-en-8α,13-β,18-triol,
labda-14-en-3β,8α,13β triol were produced22. In the case of hydroxylation of
Sclareol by Bacillus sphaericus the 3β-hydroxysclareol was obtained with
a 40% yield and 18-hydroxysclareol (10% yield). It has been informed that
the hydroxylation in the C-3 position of the Sclareol it is the most frequently
reaction23. However, this is the first study that reports that 3β-hydroxysclareol
was produced as biotransformation product from Sclareol by B. cinerea.
Finally, the results from this work contribute to the evaluation of natural
products Sclareol as potential preventive treatments against B. cinerea.
4. CONCLUSIONS
It was possible to conclude that the diterpenoids, Sclareol and 13-epi-
Sclareol were able to reduce the mycelial growth of B. cinerea. The inhibitory
effect would be related to uncoupling of fungal oxidative phosphorylation. In
addition, Sclareol was biotransformed by Botrytis to 3β-hydroxysclareol.
ACKNOWLEDGMENTS
This research was supported of the Universidad de Santiago de Chile,
FONDECYT (1130389) and FONDEF (CA12i10054) Grants.
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