Ecological Roles of Tryptanthrin, Indirubin and N-Formylanthranilic Acid in Isatis indigotica: Phytoalexins or Phytoanticipins?

Article abstract of DOI:10.1002/cbdv.201800579

Leaves of the plant species Isatis indigotica Fortune ex Lindl. (Chinese woad) produce the metabolites tryptanthrin, indirubin and N-formylanthranilic acid upon spraying with an aqueous solution of copper chloride but not after spraying with water. The antifungal activities of these metabolites against the phytopathogens Alternaria brassicicola, Leptosphaeria maculans and Sclerotinia sclerotiorum established that tryptanthrin is a much stronger growth inhibitor of L. maculans than the phytoalexin camalexin. The biosynthetic precursors of tryptanthrin and N-formylanthranilic acid are proposed based on the deuterium incorporations of isotopically labeled compounds. The overall results suggest that tryptanthrin is a phytoalexin and indirubin and N-formylanthranilic acid are phytoanticipins in the plant species I. indigotica and that chemical diversity and biodiversity are intimately connected.

Full text of DOI:10.1002/cbdv.201800579

DOI: 10.1002/cbdv.201800579
FULL PAPER
Ecological Roles of Tryptanthrin, Indirubin and N-Formylanthranilic
Acid in Isatis indigotica: Phytoalexins or Phytoanticipins?
a
a
a
a
M. Soledade C. Pedras,* Abbas Abdoli, Q. Huy To, and Chintamani Thapa
a
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon SK S7N 5C9, Canada,
e-mail: s.pedras@usask.ca
Leaves of the plant species Isatis indigotica FORTUNE EX LINDL. (Chinese woad) produce the metabolites
tryptanthrin, indirubin and N-formylanthranilic acid upon spraying with an aqueous solution of copper chloride
but not after spraying with water. The antifungal activities of these metabolites against the phytopathogens
Alternaria brassicicola, Leptosphaeria maculans and Sclerotinia sclerotiorum established that tryptanthrin is a much
stronger growth inhibitor of L. maculans than the phytoalexin camalexin. The biosynthetic precursors of
tryptanthrin and N-formylanthranilic acid are proposed based on the deuterium incorporations of isotopically
labeled compounds. The overall results suggest that tryptanthrin is a phytoalexin and indirubin and N-
formylanthranilic acid are phytoanticipins in the plant species I. indigotica and that chemical diversity and
biodiversity are intimately connected.
Keywords: Brassicaceae, Isatis indigotica, alkaloid, antifungal activity, bioactive compounds, crucifer, isotopic
labeling, phytoalexin, phytoanticipin.
Introduction
The genus Isatis L. (Brassicaceae family) comprises
[1]
more than seventy species, several of which have
been investigated for their pharmacological properties.
Indeed, numerous reports dealing with the isolation
and structure elucidation of metabolites of Isatis Figure 1. Chemical structures of tryptanthrin (1) and indirubin
(
2) isolated from dried roots and leaves of Isatis indigotica and I.
species have been published, however, the ecological
roles of such metabolites in their natural or cultivated
environments remain unknown. For example, Isatis
indigotica FORT. (Chinese woad), a biennial crucifer
tinctoria.
widely cultivated and used in traditional Chinese their pharmacological activities including antimicro-
medicine,[ and I. tinctoria L., an indigo dye plant, bial, anti-inflammatory and anticancer.
2]
[3,5–8]
Nonethe-
produce a number of metabolites with various bio- less, their roles in the producing organisms remain
logical activities.[ Interestingly, air drying leaves of I. unknown to date.
2,3]
tinctoria and I. indigotica was shown to change their
Phytoalexins are elicited antimicrobial metabolites
metabolite profiles causing the loss of glucosinolates produced de novo in plants under stress, but not
[
9–11]
and indigo precursors and the formation of tryptan- produced under normal conditions.
By contrast,
thrin (1) and indirubin (2; Figure 1).[ Both tryptanthrin phytoanticipins are antimicrobial metabolites constitu-
1) and indirubin (2) are compounds well-known for tively produced in amounts that usually increase under
4]
(
[
12,13]
stress.
ecological roles that include involvement in molecular
Supporting information for this article is available on the plant defense mechanisms against microbial patho-
These two groups of metabolites have
[
10,11]
WWW under https://doi.org/10.1002/cbdv.201800579
gens.
Cruciferous phytoalexins encompass a
Chem. Biodiversity 2019, 16, e1800579
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Chem. Biodiversity 2019, 16, e1800579
Figure 2. Chemical structures of the cruciferous phytoalexins brassinins and camalexins, derived from L-tryptophan, and
nasturlexins, derived from L-phenylalanine.
group of alkaloids derived from L-tryptophan such as 2.1 min (HPLC-DAD method B; Figure 4) that were not
[
11]
the brassinins and camalexins,
or from L-phenyl- observed in control leaves. The UV and ESI-MS spectra
alanine such as the nasturlexins (Figure 2).[ To date, of these components were not available in our virtual
no phytoalexins or other elicited metabolites have libraries, suggesting that these compounds had not
been reported from I. indigotica or any other Isatis been detected in any of the crucifer species previously
14]
[
15–19]
species.
investigated.
After isolation and identification of
In continuation of previous studies on the structure the three elicited metabolites (t =2.1, 15.4, 20.7 min),
R
and biosynthesis of cruciferous phytoalexins,[
14–18]
we calibration curves were built for quantification by
investigated and report herein the metabolites iso- HPLC-DAD (amounts calculated based on peak areas),
lated from leaves of elicited Chinese woad (I. indigo- as summarized in Table 1.
tica) and their antifungal activities against three fungal
species pathogenic to crucifers. Furthermore, the
biosynthetic precursors of tryptanthrin (1) and N-
formylanthranilic acid (3) were investigated using
isotopically labeled compounds.
Results and Discussion
Time-Course Analyses of Metabolite Production in
Elicited Leaves of Isatis indigotica
Whole plants of I. indigotica were grown for three
weeks in a growth chamber and elicited by spraying
with an aqueous solution of CuCl . Leaves were
2
excised after 1, 3 and 5 days of incubation, frozen in
liquid nitrogen and ground, and the resulting leaf
materials were extracted to yield non-polar (CH Cl )
2
2
and polar (MeOH) extracts, as described in the
Experimental Section. Control plants were sprayed with
water and treated similarly. The HPLC-DAD chromato-
Figure 3. HPLC-DAD chromatograms (method A, detection at
20 nm) of extracts of leaves of Isatis indigotica after elicitation
2
grams of the CH Cl2 extracts of elicited leaves with CuCl : A) 1-day post-elicitation; B) 3-day post-elicitation; C)
2
2
displayed two peaks at t =15.4 and 20.7 min (HPLC- 5-day post-elicitation; D) control; tR =15.4 min, tryptanthrin (1);
R
tR =20.7 min, indirubin (2).
DAD method A; Figure 3) and another peak at t =
R
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Chem. Biodiversity 2019, 16, e1800579
by analyses of spectroscopic data and confirmed by
comparison with authentic samples, as described
below.
The HR-EI-MS spectrum of the elicited metabolite
at t =15.4 min suggested a molecular formula of
R
C H N O that implied 13 degrees of unsaturation (m/
1
5 8 2 2
+
1
z M 248.0581). The H-NMR spectrum (500 MHz,
CDCl ) displayed eight aromatic protons (δ(H) 8.60–
3
1
3
7
.41), whereas the C-NMR data showed two low field
signals (δ(C) 182.7, 158.3) and twelve additional sp
2
Figure 4. HPLC-DAD chromatograms (method B, detection at
20 nm) of extracts of leaves of Isatis indigotica after elicitation
with CuCl : A) 1-day post-elicitation; B) control; t =2.1 min, N-
2
carbons. A literature search using these data sug-
gested the structure of tryptanthrin (1). This assign-
ment was unambiguously confirmed by direct compar-
ison of all spectroscopic data with those of an
authentic standard of tryptanthrin (1; purchased from
2
R
[20]
formylanthranilic acid (3); t =24.6 min, isatan B (4).
R
Chemical Structure Determination of Unknown Accela ChemBio Inc., San Diego, CA).
Metabolites with t =2.1, 15.4 and 20.7 min
The HR-ESI-MS spectrum of the elicited metabolite
at t =20.7 min suggested a molecular formula of
R
R
To isolate the metabolites responsible for the HPLC C H N O that implied 13 degrees of unsaturation
1
6 10 2 2
+
1
peaks observed at t =15.4 and 20.7 min (Method A) (m/z [M+H]
263.0828). The H-NMR spectrum
R
and at t =2.1 min (method B), a larger scale experi- (500 MHz, (D )DMSO) showed two broad singlets (δ(H)
R
6
ment was carried out as described in the Experimental 11.0 and 10.9) and eight aromatic protons, suggesting
Section. In brief, leaves (ca. 100 g of elicited leaves, two that the compound contained two indolyl moieties. A
days post elicitation) were extracted (MeOH) and literature search indicated that the spectroscopic data
filtered; the filtrate was concentrated and partitioned obtained for this compound were consistent with the
[
21]
with AcOEt/H O. The AcOEt extract was concentrated structure of indirubin (2).
The structure was con-
2
and fractionated using multiple chromatographic col- firmed by direct comparison of all spectroscopic data
umns to yield two elicited non-polar metabolites with with those of an authentic sample of indirubin (2)
tR =15.4 min (1) and 20.7 min (2), and the polar prepared as detailed in the Supporting Information.
metabolite at t =2.1 min (3). The chemical structures
The polar metabolite at t =2.1 min (method B)
R
R
of these compounds (Figure 1 and 5) were established displayed an HR-EI-MS spectrum consistent with the
molecular formula C H NO (five degrees of unsatura-
8
7
3
+
tion, m/z M 165.0412) and its structure was assigned
as N-formylanthranilic acid (3) by direct comparison of
its spectroscopic data with those of a synthetic sample
[
22]
prepared as previously reported.
Previously, both
tryptanthrin (1) and indirubin (2) were reported to be
formed spontaneously in post-harvested leaves and
Figure 5. Chemical structures of N-formylanthranilic acid (3),
isatan B (4) and isatin (5) isolated from elicited leaves of Isatis
indigotica.
roots of I. indigotica, whereas N-formylanthranilic acid
[4]
(3) was reported to be constitutive only in I. tinctoria.
During the fractionation process, an additional
compound with t =24.6 min (method B) was obtained
R
in a relatively larger amount (ca. 190 mg) than any of
the elicited metabolites. This compound displayed an
Table 1. HPLC-DAD analyses of elicited metabolites in the leaves of Isatis indigotica.
[
a]
Metabolites, t (HPLC method)
Amounts (μmole/100 g of fresh tissue � SD )
R
1
-day post-elicitation
3-day post-elicitation
5-day post-elicitation
Tryptanthrin (1), t =15.4 min (method A)
12�11
12�10
32�13
32�14
65�17
33�15
149�43
165�71
R
Indirubin (2), t =20.7 min (method A)
R
N-Formylanthranilic acid (3), t =2.1 (method B)
201�74
R
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Chem. Biodiversity 2019, 16, e1800579
Scheme 1. Preparation of indirubin (2).
HR-FD-MS spectrum that suggested a molecular for- Antifungal Activity of Elicited Metabolites: Tryptanthrin
mula of C H N O , conveying eight degrees of (1), Indirubin (2) and N-Formylanthranilic Acid (3)
1
4 15 1 6
+
1
unsaturation (m/z M 293.0904). The H-NMR spec-
trum ((D )DMSO) displayed an exchangeable singlet at The antifungal activities of the elicited metabolites
6
δ(H) 10.65, three doublets at δ(H) 7.57 (1H, d, J=8.0), tryptanthrin (1), indirubin (2) and N-formylanthranilic
7
.30 (1H, d, J=8.5) and 7.14 (1H, d, J=2.5), and two acid (3) were tested against three fungal species:
triplets at δ(H) 7.07 (1H, t, J=7.0) and 6.96 (1H, t, J= Alternaria brassicicola (SCHWEIN) WILTSHIRE and Leptos-
.5), suggesting an indole nucleus substituted at C-2 phaeria maculans (DESM.) CES. et de NOT. (asexual stage
or C-3. In addition, nine protons observed at δ(H) 5.74 Phoma lingam (TODE ex FR.) DESM.), both specific
3.33, three of which were exchangeable, suggested pathogens of crucifers and the generalist pathogen
7
–
1
3
the presence of a glycosyl moiety. The C-NMR Sclerotinia sclerotiorum (LIB.) de BARY. These species
spectrum displayed a signal at δ(C) 206.0, suggesting a cause economically important losses in various crucifer
ketone group, plus nine signals at δ(C) 136.7–104.7 species. The cruciferous phytoalexin camalexin was
and four signals at δ(C) 76.7–60.8, which were used for comparison as the positive control. As
consistent with the presence of a glycosyl moiety. summarized in Table 2, tryptanthrin (1) was strongly
Altogether these data suggested an indole attached to antifungal against L. maculans, causing complete
a ketohexose. A literature search indicated that the growth inhibition at 0.10 mM and 66% inhibition at
spectroscopic data of isatan B (4) were identical to the lowest tested concentration (0.010 mM). However,
[23]
those of the metabolite with t =24.6 min.
There- tryptanthrin (1) showed lower inhibitory activity
R
fore, the structure of metabolite at t =24.6 min was against A. brassicicola (31% inhibition at 0.50 mM) and
R
assigned as isatan B (4), previously reported to be a S. sclerotiorum (12% inhibition at 0.50 mM). Tryptan-
precursor of indigoids in I. tinctoria.[ Isatin (5) was thrin appears to have a more selective inhibitory effect
also obtained during the chromatographic separations on fungal growth than camalexin, which completely
and the structure was confirmed by comparison of its inhibited the mycelial growth of the three species at
spectroscopic data with those of a commercially 0.50 mM. Furthermore. indirubin (2) exhibited higher
23]
available sample.
inhibitory activity than tryptanthrin (1) against S.
sclerotiorum (20% inhibition at 0.50 mM), but substan-
tially lower against L. maculans, whereas N-formylan-
thranilic acid (3) displayed inhibitory activity only
Preparation of Indirubin (2)
A number of approaches have been developed to against A. brassicicola.
synthesize indirubin (2). In this work, a procedure
similar to that used by Ichimaru et al. to prepare
Biosynthesis of Tryptanthrin (1) and N-Formylanthranilic
Acid (3)
indirubin derivatives was used to prepare indirubin
[
24]
(
2),
as detailed in the Supporting Information. In
brief, isatin (5) was allowed to react with phosphorus Tryptanthrin (1) was first isolated from a natural source
[
25]
pentachloride (PCl ) in benzene at reflux, and the in 1971.
Cultures of the yeast Candida lipolytica
5
resulting precipitate was filtered and dissolved in grown in a L-tryptophan (Trp) rich medium produced
[
25]
toluene; oxindole (6) was added to the solution and tryptanthrin (1),
substituted tryptanthrins if incu-
the reaction mixture was heated at reflux for 1 h, bated with Trp and substituted anthranilic acids, or
[
26]
followed by work-up and chromatography to yield substituted Trps and anthranilic acid.
It was then
indirubin (2) in reasonable yield (84%; Scheme 1).
proposed that tryptanthrin (1) was biosynthesized
from anthranilic acid (7) and indolyl-3-pyruvic acid in
[
25]
C. lipolytica.
Since then, tryptanthrin (1) has been
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Chem. Biodiversity 2019, 16, e1800579
Table 2. Antifungal activity of tryptanthrin (1), indirubin (2), N-
that anthranilic acid (7) and N-formylanthranilic acid
formylanthranilic acid (3) and the phytoalexin camalexin against (3) are the primary biosynthetic precursors of tryptan-
Alternaria brassicicola, Leptosphaeria maculans and Sclerotinia
thrin (1; Figure 6). To the best of our knowledge, no
sclerotiorum
[a]
Concentration [mM] % Inhibition � S.D.
A. L.
brassicicola[ maculans sclerotiorum
S.
b]
[c]
[d]
Tryptanthrin (1)
f
f
f
e
e
e
g
0
0
0
0
0
0
.50
.20
.10
.050
.020
.010
31�3
22�3
20�0
c.i.
c.i.
c.i.
12�5
g
g
f
n.i.
n.i.
n.i.
n.i.
n.i.
f
f
f
e
n.i.
n.i.
n.i.
88�0
e
e
e
e
78�2
66�2
Indirubin (2)
g
f
f
0
0
0
.50 M
.20 M
.10 M
13�3
33�2
20�2
8�0
20�3
h
g
g
f
f
7�3
14�2
12�2
g
g
n.i.
Figure 6. Retrobiosynthetic analysis: potential precursors of
tryptanthrin (1) in the leaves of Isatis indigotica.
N-Formylanthranilic
acid (3)
g
g
n.i.h
n.i.
n.i.
0
0
0
.50
.20
.10
17�4
6�3
g
h
h
g
11�3
n.i.
n.i.
g
n.i.
formal isotope labeling studies to uncover such
precursors have been reported to date.
Camalexin – positive
control
For this reason, to clarify the biosynthetic origin of
tryptanthrin (1) in I. indigotica, the isotopically labeled
e
e
e
f
c.i.e
0
0
0
0
.50
.20
.10
.050
c.i.
c.i.
c.i.
1
3
15
f
e
compounds [3,5-D ]anthranilic acid (7a), [ C , N ]Trp
41�2
c.i.
2
11
2
e
f
e
76�0
24�3
n.i.
c.i.
(8a) and [4,5,6,7-D ]indole (9a; Figure 7) were adminis-
4
e
e
25�5
81�6
tered separately to elicited leaves of I. indigotica.
[
a]
Tetradeuterated indole (9a; ca. 96% D ) was synthe-
Percentage of inhibition=100 – [(growth on amended/
4
[
31]
growth in control) ×100]; control plates contained PDA
medium and 1% DMSO; c.i.=complete inhibition; n.i.=no
inhibition. Values are averages of two independent experiments
conducted in triplicate; for statistical analysis, one-way ANOVA
tests were performed followed by Tukey’s test with adjusted α
set at 0.05; n=6; different letters in the same row (e–h) indicate
sized as previously reported,
and dideuterated
anthranilic acid (7a; ca. 96% D ) was prepared by
2
proton/deuteron exchange, as described in the Sup-
13
15
13
15
porting Information. [ C , N ]Trp (8a; C , N ꢀ
11
2
11
2
9
8%) was commercially available. The whole plants
[b]
were elicited, incubated, the leaves were excised, and
the petioles were immediately immersed in aqueous
solutions of the labeled compounds. After further
incubation, leaves were frozen and then extracted, as
described in the Experimental Section. Control samples
significant differences (P<0.05). Incubated for 60–70 h
[c]
under continuous light. Incubated for 4–5 days under
continuous light. [ Incubated for ca. 20 h in the dark.
d]
isolated from different organisms including various (elicited leaves fed with non-labeled compounds or
[7]
plant species; however, in plants no information
regarding the biosynthesis of tryptanthrin (1) has been
reported. Indirubin (2), a natural component of indigo
dyes, was reported to derive from isatans A and B (4)
and indican in Isatis species,[
23,27]
whereas N-formylan-
thranilic acid (3) was reported to derive from the
degradation of gramine via indole-3-carboxylic acid in
seedlings of Hordeum vulgaris L. (Poaceae).[
28]
Current knowledge on the shikimate and L-Trp Figure 7. Structures of isotopically labeled compounds adminis-
biosynthetic pathways in plants,[
29,30]
together with a tered to the leaves of Isatis indigotica: [3,5-D2]anthranilic acid
1
3
15
(
7a), L-[ C , N ]Trp (8a) and [4,5,6,7-D ]indole (9a).
retrobiosynthetic analysis of tryptanthrin (1), suggests
11 2 4
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Chem. Biodiversity 2019, 16, e1800579
Table 3. Metabolism of [3,5-D ]anthranilic acid (7a) in elicited
leaves of Isatis indigotica
or N-formylanthranilic acid (3). In addition, the
amounts of labeled Trp present in the leaf extracts
were substantially lower (55�7%) than those present
in the fed solutions (ca. 98%), whereas [4,5,6,7-
2
Deuterated metabolites detected in leaf
extracts
% of
Deuterium
D ]indole (9a) was not detected in any of the leaf
4
[
3,5-D ]Anthranilic acid (7a)
ca. 95%
2
extracts. Indirubin (2) was detected only in trace
amounts, therefore, no incorporations were detected
(Table 3).
The overall results of the incorporation of isotopi-
cally labeled compounds into tryptanthrin (1) suggest
that anthranilic acid (7) and N-formylanthranilic acid
(3) are its precursors, as proposed in Scheme 2.
N-[3,5-D ]Formylanthranilic acid (3a)
30�18%
2
[a]
[
[
D ]Tryptanthrin (1a)
D ]Tryptanthrin (1b)
86�3%
2
[a]
2.3�1.4%
4
[
a]
Isotope incorporations calculated from HPLC-ESI-MS (peak
intensities in positive and negative modes); % of incorpora-
+
+
+
tion={[M�H+n] /([M�H] +[M�H+2] +[M�H+4]
+
}
×100, n represents D atoms, n=2 for 1a, n=4 for 1b; values
represent the mean and standard deviation of triplicate
samples.
[
D ]Tryptanthrin (1a) can be formed from two different
2
precursor pools: either by i) coupling of [3,5-
D ]anthranilic acid (7a) and natural abundance 3
2
(highly abundant, higher probability event) or ii)
solvent) were treated similarly. All leaf extracts were coupling of [D ]-3a and natural abundance 7. How-
2
analyzed by HPLC-DAD-ESI-MS and the percentage of ever, [D ]tryptanthrin (1b) can only be formed from
4
isotope incorporation into each metabolite was deter- one precursor pool by coupling of [D ]-7a and [D ]-3a,
2
2
mined using the peak intensities of ESI-MS spectra both very low abundance, hence the probability of
(
positive and negative ion modes, according to the forming [D ]-1b is much lower than that of [D ]-1a.
4
2
equation reported in Table 3).
Furthermore, the higher incorporation of deuterium
After administration of [3,5-D ]anthranilic acid (7a) into [D ]tryptanthrin (1a) reflects the low dilution of
2
2
to elicited leaves of I. indigotica and incubation, both [D ]tryptanthrin (1a; 86%) and [3,5-D ]anthranilic
2
2
tryptanthrin (1) was detected in leaf extracts and the acid (7a; 95%; Table 3) with their natural abundance
deuterium incorporations were quantified. The ESI-MS counterparts 1 and 7, respectively (because 1 and 7
spectrum of tryptanthrin showed a major ion peak at are produced in very small amounts, Table 1). Similarly,
+
m/z 251.1 [M+H+2] (100%), corresponding to these deuterium incorporation results are consistent
[
[
(
D ]tryptanthrin (1a), and minor ion peaks at m/z 253.0 with the relatively lower incorporation of deuterium
2
+
M+H+4] (4.7%), corresponding to [D ]tryptanthrin into [D ]tryptanthrin (1b; 2.3%; Table 3). By contrast,
4
4
+
1b) and at m/z 249.1 [M+H] (7.6%), corresponding N-[3,5-D ]formylanthranilic acid (3a; 30%; Table 3) is
2
to natural abundance tryptanthrin (1), which were produced in larger amounts than 1a and has a high
further confirmed by HR-ESI-MS. Similarly, the ESI-MS dilution with the natural pool of 3, which is produced
spectrum of N-formylanthranilic acid (3) showed two in large amounts (Table 1). Because no isotope incor-
+
major ion peaks at m/z 163.9 [MÀ H] (100%) and m/z porations from either indole (9a) or Trp (8a) into
+
1
65.9 [MÀ H+2] (35%), corresponding to natural tryptanthrin (1) were detected in planta, these com-
abundance N-formylanthranilic acid (3) and to N-[3,5- pounds might have undergone enzyme catalyzed
D ]formylanthranilic acid (3a), respectively. By contrast, transformations unrelated to the biosynthesis of 1.
2
1
3
15
after feeding experiments using [ C , N ]Trp (8a) Furthermore, since substantial incorporation of deute-
11
2
and [4,5,6,7-D ]indole (9a), no incorporation of either rium from [3,5-D ]anthranilic acid (7a) into N-
4
2
13
15
C, N or D atoms were detected in tryptanthrin (1) [D ]formylanthranilic acid (3a) was detected, anthra-
2
Scheme 2. Biosynthetic precursors of tryptanthrin (1) produced in Isatis indigotica.
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Chem. Biodiversity 2019, 16, e1800579
nilic acid (7) appears to be the closest precursor of 3. Conclusions
Alternatively, 3 could result from oxidation of indole
(
9) or catabolism of Trp (8), however, since no isotope In conclusion, our work on the elicited metabolites
incorporations from these compounds were detected, produced in leaves of I. indigotica has uncovered three
these hypotheses appear less likely. elicited antifungal metabolites: tryptanthrin (1), indir-
We have shown that elicitation of whole plants of I. ubin (2) and N-formylanthranilic acid (3). These
indigotica and isolation of elicited metabolites pro- metabolites are likely to act as plant defenses in the
duced in leaves yielded tryptanthrin (1), indirubin (2) interaction of I. indigotica with microbial pathogens
and N-formylanthranilic acid (3), which were not and perhaps other stresses. Importantly, although
detected in non-elicited leaf extracts. Consequently, these metabolites have been known for decades to
considering that metabolites 1–3 display antifungal display important pharmacological activities, this is the
activity against phytopathogenic fungi and are pro- first time that an ecological defensive role can be
duced only in elicited leaves, the question arises assigned to these compounds. These findings substan-
whether their ecological role fits in the phytoalexin or tiate what is currently indisputable, i.e., secondary
phytoanticipin class. Because tryptanthrin (1) displays metabolites are produced for the benefit of the
strong antifungal activity and is biosynthesized via producing organism, not for the benefit of human
intermediates derived de novo from anthranilic acid kind. As our investigation on cruciferous phytoalexins
(
7), we suggest that it is a phytoalexin in I. indigotica. expands into different plant genera, the variety of
However, indirubin (2) and N-formylanthranilic acid (3) chemical structures continues to increase, consistent
might fit better in the phytoanticipin class due to their with the current understanding that chemical diversity
biosynthetic origin from very close precursors. This is and biodiversity are intimately connected.
because phytoalexins, by definition, are biosynthesized
‘de novo’, i.e., from remote precursors that may require
several enzymatic transformations, whereas phytoanti- Experimental Section
cipins either are constitutive (pre-formed) or result
from single transformations such as hydrolysis, dimeri-
General
zation, or oxidation. Indeed, in spite of significant All solvents were of HPLC grade and used as such,
endeavors to clarify definitions,[
9,10,12]
many secondary except for those used in chemical syntheses, as noted.
or specialized plant metabolites that would be more Unless otherwise noted, materials were obtained from
appropriately considered phytoanticipins are reported commercial suppliers and used without further purifi-
as phytoalexins.[ Not surprisingly, like for many other cation. Flash column chromatography (FCC): silica gel,
classifications, there are gray areas and cases where grade 60, 230–400 μm. NMR spectra were recorded on
13]
1
the distinction between phytoanticipins and phytoa- Bruker Avance 500 MHz spectrometers. For H-NMR
1
3
lexins is not so obvious. In such cases, examination of (500 MHz) and
C-NMR (125.8 MHz) spectra, the
biosynthetic intermediates might be required to more chemical shifts (δ) are reported in parts per million
accurately classify those plant metabolites as phytoan- (ppm) relative to TMS. MS data (high resolution (HR))
ticipins or phytoalexins. Notwithstanding the caveat, were obtained on a Jeol AccuToF GCv 4G mass
in our view tryptanthrin (1) represents a new group of spectrometer (field desorption (FD)) or on a Qstar XL
cruciferous phytoalexins in which anthranilic acid (7) is MS/MS System (ESI) by direct insertion.
its primary biosynthetic precursor, whereas previously
known phytoalexins derive from protein amino acids
HPLC Analysis
via the corresponding glucosinolates.[ Importantly,
11]
because Brassica species do not appear to produce HPLC-DAD analyses were carried out with Agilent 1100
tryptanthrin (1), it would be worthwhile to transfer this and 1200 series systems all equipped with quaternary
pathway to Brassica species susceptible to L. maculans, pump, autosampler, diode array detector (DAD, wave-
to investigate its effect on plant disease resistance length range 190–600 nm) and degasser; used a
levels to this important pathogen.
Zorbax Eclipse XDBÀ C18 column (5 μm particle size
silica gel, 150×4.6 mm i.d.), equipped with an in-line
filter, with the mobile phase H O/MeCN from 75:25 to
2
2
1
5:75, linear gradient for 35 min and a flow rate of
.0 mL/min (method A for phytoalexins and non-polar
metabolites) or H O/MeCN from 100:0 to 10:90 in
2
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Chem. Biodiversity 2019, 16, e1800579
2
0 min, with a flow rate of 1.0 mL/min (method B for plants were sprayed with H O. The leaves (ca. 0.8 g
2
polar metabolites).
fresh weight per sample) were excised 1, 3 and 5 days
after elicitation, frozen in liquid N and ground, and
2
HPLC-DAD-ESI-MS analysis was carried out with an the resulting leaf materials were individually extracted
Agilent 1100 series HPLC system equipped with an with MeOH (5 mL). The extracts were filtered, the
autosampler, binary pump, degasser and a diode array filtrates were concentrated and rinsed with CH Cl .
2
2
detector connected directly to a mass detector The CH Cl extracts were concentrated, dissolved in
2
2
(Agilent G2440 A MSD-Trap-XCT ion trap mass spec- MeCN/MeOH (1:1, 1 mL) and analyzed by HPLC-DAD
trometer) with an electrospray ionization (ESI) source. using methods A and B, and by HPLC-DAD-ESI-MS
Chromatographic separations were carried out at using method C. For isolation of the unknown
room temperature using an Eclipse XDB-C-18 column metabolites detected in elicited extracts, 3-week-old
(
5 μm particle size silica gel, 150 mm×4.6 mm i.d.). plants were sprayed with an aqueous CuCl solution
2
The mobile phase (method C) consisted of a linear (10 mM) and plants were kept in growth chamber.
gradient of H O (with 0.2% HCO H)/MeCN (with 0.2% After two days, elicited leaves (ca. 100 g) were frozen
2
2
HCO H) from 75:25 to 25:75 in 25 min and a flow rate in liquid N , ground and rinsed with hexane (2×
2
2
of 1.0 mL/min. Data acquisition was carried out in 300 mL). The leaf materials were shaken in MeOH for
positive and negative polarity modes in a single LC 1 h and filtered. The filtrate was concentrated and
run and data processing was carried out with Agilent partitioned in AcOEt/H O. The organic extract was
2
Chemstation Software.
concentrated to dryness to yield the crude AcOEt
extract (ca. 1.9 g), which was fractionated as described
HPLC-ESI-HR-MS was performed on an Agilent below.
HPLC 1100 series directly connected to QSTAR XL
Systems mass spectrometer (Hybrid Quadrupole-TOF
Isolation of Metabolites
LC/MS/MS) with turbo spray ESI source. Chromato-
graphic separations were carried out using a Hypersil The AcOEt extract (ca. 1.9 g) was fractionated using
ODS C-18 column (5 μL particle size silica gel, 200 mm flash column chromatography (FCC, gradient MeOH/
×
2.1 mm i.d.) or a Hypersil ODS C-18 column (5 μL CH Cl , 0:100–20:80) to give seven fractions, which
2 2
particle size silica gel, 100 mm×2.1 mm i.d.). The were further fractionated by FCC and the eluting
mobile phase consisted of a linear gradient of H O solvents were summarized in Figure 8.
2
(
0.1% HCO H)/MeCN (0.1% HCO H) from 75:25 to
2 2
2
0
5:75 in 35 min, to 0:100 in 5 min and a flow rate of
.25 mL/min. Data acquisition was carried out in either
Antifungal Bioassays
positive or negative polarity mode in a single LC run The antifungal activity of compounds against three
and data processing was carried out with Analyst QS fungal species was investigated using a mycelial radial
Software.
growth bioassay (PDA media and DMSO solutions of
each compound, 0.50, 0.25 and 0.10 mM; control
solutions contained 1% DMSO in PDA media). A.
brassicicola isolate UAMH 7474 and L. maculans isolate
Plant Material and Extractions
Seeds of Isatis indigotica FORT. (Chinese woad) were UAMH 9410 were obtained from the University of
purchased from Sand Mountain Herbs (321 County Alberta Microfungus Collection and Herbarium and S.
Road 18 Fyffe, AL 35971, www.sandmountainherbs.- sclerotiorum clone #33 was obtained from the AAFC
com). The seeds were placed on a filter paper in a Petri Saskatoon Research Center. Spores of A. brassicicola
dish and wet with distilled water. After germination and L. maculans were spotted onto potato dextrose
occurred (6–7 days), seedlings were transplanted to agar plates (PDA) and allowed to grow for seven days
perlite and nutrient free LG-3 soil (Sun Gro Horticulture under constant light at 23�1°C. Similarly, sclerotia of
Canada) in small pots (15 cm diameter) in a growth S. sclerotiorum were placed on PDA plates and allowed
chamber at 16 h of light/8 h of dark, 20°C day/18°C to germinate and grow mycelia for four days incu-
À 2 À 1
night, 350–360 μEm
s
and with ambient humidity. bated in the dark. Plugs (3 mm) were cut from the
edges of growing mycelia and placed inverted onto
For time-course HPLC analyses, 3-week-old plants 12-well plates containing compounds in DMSO mixed
were sprayed with an aqueous CuCl solution (10 mM) into PDA. These 12-well culture plates containing
2
and plants were kept in the growth chamber. Control compounds to be tested were allowed to grow under
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Chem. Biodiversity 2019, 16, e1800579
Figure 8. Isolation of metabolites from elicited leaves of Isatis indigotica.
constant light or dark (Table 2) at 23�1°C up to 96 h. MeOH. The MeOH extracts were filtered, concentrated
The diameter of the mycelial mat was measured and and rinsed with CH Cl . The CH Cl extracts were
2
2
2
2
compared to control mycelia grown on plates contain- concentrated and analyzed by HPLC-DAD-ESI-MS using
ing DMSO, using the formula in Table 2 (the diameter method C. Control samples included elicited leaves fed
of the mycelial plug was subtracted from the total with solvent and, otherwise, treated similarly.
mycelial growth).
Feeding Experiments Using [3,5-D ]Anthranilic Acid (7a), Supplementary Material
2
13
15
[
C , N ]Trp (8a) and [4,5,6,7-D ]Indole (9a)
11 2 4
HPLC-DAD chromatograms (method B, detection at
For feeding experiments, three-week old plants were 220 nm) of extracts of leaves of Isatis indigotica after
sprayed with an aqueous CuCl solution (10 mM) and elicitation with CuCl ; preparation of new compound
2
2
1
13
incubated under fluorescent light for 48 h. Leaves [3,5-D ]anthranilic acid (7a) and its H- and C-NMR
2
were cut and immediately immersed in an aqueous spectra; preparation of indirubin (2).
1
3
15
solution of [3,5-D ]anthranilic acid (7a), [ C , N ]Trp
(
2
11
2
À 3
8a) and [4,5,6,7-D ]indole (9a; 10 M dissolved in H O
4 2
for 7a and 8a, or in H O/MeOH (1:9, v/v) for 9a).
Similar experiments were carried out with the corre-
2
Acknowledgements
sponding natural abundance compounds (anthranilic Support for the authors’ work was obtained from the
acid (7), Trp (8) and indole (9)). When most of the Natural Sciences and Engineering Research Council of
solutions were uptaken, the tubes were refilled with Canada (Discovery Research Grant to M. S. C. P.), the
distilled H O and leaves were further incubated under Canada Research Chairs Program (awarded to M. S. C.
2
continuous fluorescent light. After 24 h, the leaves P.), and the University of Saskatchewan (CPGS gradu-
were frozen in liquid N , ground and extracted with ate scholarship to Q. H. T.). We acknowledge the
2
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Chem. Biodiversity 2019, 16, e1800579
technical assistance of K. Thoms, Department of [12] H. D. VanEtten, J. W. Mansfield, J. A. Bailey, E. E. Farmer,
‘
“
Two classes of plant antibiotics: phytoalexins versus
phytoanticipins”’, Plant Cell 1994, 6, 1191.
Chemistry and Saskatchewan Sciences Structural
Centre, in HR-ESI-MS determinations.
[
[
13] M. S. C. Pedras, E. E. Yaya, ‘Plant chemical defenses: are all
constitutive antimicrobial metabolites phytoanticipins?’,
Nat. Prod. Commun. 2015, 10, 209–218.
14] M. S. C. Pedras, Q. H. To, ‘Interrogation of biosynthetic
pathways of the cruciferous phytoalexins nasturlexins with
isotopically labelled compounds’, Org. Biomol. Chem. 2018,
Author Contribution Statement
M. S. C. Pedras designed and supervised this study
and wrote and revised the manuscript. A. Abdoli
synthesized indirubin, carried out the antifungal assays
with indirubin and tryptanthrin and performed all
statistical analyses. Q. H. To carried out all elicitation
and isotope-feeding experiments, isolated, identified
and characterized all plant metabolites (1–5), pre-
pared dideuterated anthranilic acid and carried out
the antifungal assays with N-formylanthranilic acid. C.
Thapa analyzed, purified and spectroscopically charac-
terized dideuterated anthranilic acid. All authors read
and reviewed the manuscript.
1
6, 3625–3638.
[
[
15] M. S. C. Pedras, E. E. Yaya, ‘Tenualexin, Other Phytoalexins
and Indole Glucosinolates from Wild Cruciferous Species’,
Chem. Biodiversity 2014, 11, 910–918.
16] M. S. C. Pedras, Q. H. To, ‘The first non-indolyl cruciferous
phytoalexins: Nasturlexins and tridentatols, a striking
convergent evolution of defenses in terrestrial plants and
marine animals?’, Phytochemistry 2015, 113, 57–63.
17] M. S. C. Pedras, M. Alavi, Q. H. To, ‘Expanding the nasturlex-
in family: nasturlexins C and D and their sulfoxides are
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[18] M. S. C. Pedras, Q. H. To, ‘Unveiling the first indole-fused
thiazepine: structure, synthesis and biosynthesis of cyclo-
nasturlexin, a remarkable cruciferous phytoalexin’, Chem.
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Zheng, M. Jha, M. G. Sarwar, ‘Detection, characterization
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Received November 2, 2018
Accepted December 17, 2018
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