South African Journal of Botany 137 (2021) 242 248

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Specialized plant metabolites from indolic and polyphenolic biosynthetic

pathways in Wrightia religiosa (Teijsm. & Binn.) Benth. and Wrightia
pubescens R. Br. (Apocynaceae)
Florian Traxlera, Nitkamon Iamprasertkunb,c, Anna Maria Tschigga, Srunya Vajrodayab,*,
Karin Valant-Vetscherac, Lothar Breckera, Johann Schinnerlc,*
a
Department of Organic Chemistry, University of Vienna, Wa €hringer Strasse 38, A-1090 Vienna, Austria
b
Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
c
Department of Botany and Biodiversity Research, University of Vienna, Rennweg 14, A-1030 Vienna, Austria

A R T I C L E I N F O A B S T R A C T

Article History: Phytochemical investigation of different organs and of the latex of Wrightia religiosa (Teijsm. & Binn.) Benth.
Received 2 December 2019 as well as of the stem bark of Wrightia pubescens R. Br. (Apocynaceae), both collected in Thailand, yielded a
Revised 14 September 2020 total of seven known compounds. All are specialized metabolites from indolic and polyphenolic biosynthetic
Accepted 19 October 2020
pathways. From leaves of W. religiosa, 3-indole D-apio-b-D-furanosyl-(1!6)-b-D-glucopyranoside (1) and
Available online 11 January 2021
benzouracil (2) were isolated. Compound 1 and blepharin (3) were purified from the root bark, and the lignan
Edited by I Vermaak 4-pinoresinol D-apio-b-D-furanosyl-(1!2)-b-D-glucopyranoside (5) was isolated from the stem bark of this
Keywords:
species. Chromatographic separation of the hitherto unstudied latex yielded blepharigenin (4). The metha-
Apocynaceae nolic stem bark extract of W. pubescens yielded tryptanthrin (6) and kelampayoside A (7). Radical scavenging
Wrightia religiosa activities of the isolated compounds and of the crude methanolic extracts indicate that the isolated special-
Wrightia pubescens ized metabolites do not contribute appreciable to the antioxidative properties of the plant extracts. The crude
Chromatography methanolic extracts do not show noteworthy anti-feedant response of the insect pest Spodoptera littoralis
DPPH assay Boisduval. Ecological aspects are shortly addressed, and results are discussed in the phylogenetic framework
of the studied species.
© 2020 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
The palaeotropically distributed genus Wrightia R. Br. (Apocyna-
ceae: Apocynoideae) comprises of 33 species (Middleton, 2005), with
ten species occurring in Thailand (Middleton, 1999). Some species
are used by local people for ethnomedicinal purposes throughout
their distribution area. For example, W. arborea (Dennst.) Mabb. (syn
W. tomentosa (Roxb.) Roem. & Schult). is used as an antidote for scor-
pion and snake bites (Chakravarti et al., 2012). Leaf extracts tested on
hamsters showed also antidyslipidemic effects (Maurya et al., 2012).
In India this species is used for healing wounds (Sharma et al., 2014).
W. tinctoria R. Br., a medicinal plant species distributed in Asia, Africa
and Australia, showed anticancer and antioxidant activities
(Fatima et al., 2016). Furthermore, the bark of this species is used in
the treatment of rheumatoid arthritis and to relief pain caused by
various health problems and exhibited antinociceptive activity
* Corresponding author.
E-mail addresses: fscisyv@ku.ac.th (S. Vajrodaya), johann.schinnerl@univie.ac.at
(J. Schinnerl).
(Reddy et al., 2002). The extract of this species also showed hypogly-
cemic effects (Kumar et al., 2011).
So far, only a few Wrightia species of ethnomedical interest were
studied phytochemically. From W. pubescens subsp. laniti (Blanco)
Ngan (syn. W. javanica A. DC.), cytotoxic pregnane alkaloids were
reported (Kawamoto et al., 2003). The xanthone glucoside mangi-
ferin, together with b-amyrin derivatives, were identified as root
constituents of W. arborea (Maurya et al., 2012; Nagarajan et al.,
2012). Its bark was claimed to contain the isoflavone wrightiadione
(Lin et al., 1992), and the same compound was later isolated from W.
religiosa (Teijsm. & Binn.) Benth. (Fig. 1) (Arai et al., 2015). However,
the proposed structure of this isoflavone was recently shown to be a
misidentification, and this compound corresponds structurally to the
isobaric and isostere tryptanthrin (Garcellano et al., 2019), which is
structurally related to indigo. According to Muruganandam and Bhat-
tacharya (2000), indigo and flavonoid derivatives occur in W. tincto-
ria, W. arborea and W. coccinea (Roxb. Ex Hornem.) Sims. The seeds of
W. tinctoria were further demonstrated to be a source of sterols e.g.
cholesterol, 24-ethylcholesterol, 24-methylcholesterol (Akihisa et al.,
1988). The seed oil of W. tinctoria and W. arborea yielded several tri-
terpenes, with W. arborea accumulating squalene, g -tocopherol,

https://doi.org/10.1016/j.sajb.2020.10.019
0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
F. Traxler, N. Iamprasertkun, A.M. Tschigg et al.
Fig. 1. Habitus (A), inflorescence (B) and flower (C) of W. religiosa (photos: N. Iamprasertkun) and W. pubescens (D) (photo: S. Vajrodaya).
campestrol, lupeol and phytol (Nagalakshmi and Murthy, 2015).
Nagalakshmi and Murthy, 2015. Recently, Sahakitpichan et al. (2018)
identified benzoxazinoid and indoxyl glycosides from a combined
leaves and twigs extract of W. religiosa (Teijsm. & Binn.) Benth., and
similar compounds were reported from W. antidysenterica (L.) R.Br.
(Srinroch et al., 2019). Most of the other publications about natural
products from Wrightia species deal with bioactivities based upon
the presence of compound classes rather than upon specific isolated
and identified metabolites (Klomsakul and Chalopagorn, 2019;
Kumar et al., 2011; Maurya et al., 2012; Reddy et al., 2002). For W.
pubescens R.Br. and W. religiosa, the fairly common triterpenoids such
as ursolic acid, oleanolic acid, b-sitosterol and a-amyrin acetate
together with the above-mentioned wrightiadione were described
(Ragasa et al., 2014; De Los Reyes at al., 2018).
The present study deals with a more in-depth analysis, in particu-
lar by taking organ-specific accumulation into account, an often
neglected aspect. Thus, we studied leaves, stem bark and root bark,
as well as latex of W. religiosa separately, complemented by stem
bark analysis of W. pubescens. Additionally, radical scavenger activity
of crude extracts as well as isolated compounds were assessed, by
using the DPPH assay. In addition, we performed an anti-feedant
assay against the insect pest Spodoptera littoralis Boisduval, to deter-
mine the presence of potential antifeedant compounds. The obtained
results are discussed in view of phylogenetic and ecological aspects.
2. Material and methods
2.1. General experimental procedures
HPLC analyses were performed on Agilent 1100 series with UV-
diode array detection using a Hypersil BDS-C18 column,
250 x 4.6 mm, 5 mm particle size, at a flow rate of 1.0 mL/min and an
injection volume of 10 mL. The concentration of the injected crude
extracts were set after evaporation of the extraction solvent at 5 mg/
mL in pure methanol (MeOH). An aqueous solution containing
10 mM ammonium acetate (A) and MeOH (B) were used as eluents.
The following gradient was applied for W. pubescens: From 40 90% B
in A within 12 min, from 90 100% B in A within 0.1 min and 100% B
was kept for 5.9 min. For W. religiosa, the gradient started at 10% B
and reached 100% B at 15 min, and kept for 7.0 min. The wavelength
of detection was set at 230 nm (reference WL 360 nm). MPLC
243
South African Journal of Botany 137 (2021) 242 248
separations were done either over silica gel 60 columns (40 63 mm
particle size, Merck Lobar with dimensions of 240 10 or
310 25 mm) employing various eluents or over reversed phase C-8
column with the dimensions of 240 10 mm, eluted with mixtures of
MeOH and water. TLC analyses were done on silica gel 60 F254 plates,
layer thickness 0.2 mm (Merck) developed with CHCl3/MeOH 90:10
and 80:20. The stationary phases for CC and MPLC were either Sepha-
dex LH-20 (GE Healthcare) or silica gel 60 (Merck) with 0.2 0.5 mm
or 40 63 mm particle size. For preparative TLC, silica gel F254 plates,
layer thickness 0.5 mm (Merck) were used.
For NMR spectroscopic measurements each compound was dis-
solved in deuterated solvent (CD3OD, CDCl3 or acetone-d6) (the iso-
lated amounts (1 5 mg) in 0.6 mL) and transferred into 5 mm high
precision NMR sample tubes. NMR spectra were recorded on a Bruker
AVIII 600 spectrometer at 600.25 MHz (1H) and 150.93 MHz (13C),
respectively. Spectra were processed with Topspin 3.5 software.
Chemical shifts (d) are reported in ppm; for 1H relative to residual
non-deuterated solvent signals in methanol (dH = 3.31 ppm), chloro-
form (dH = 7.26 ppm) and acetone (dH = 2.05 ppm) and for 13C relative
to solvent signals (methanol-d4, dC = 49.0 ppm; CDCl3, dC = 77.0 ppm;
acetone-d6, dC = 29.8 and 206.3). CH3, CH2, CH and Cq are indicated by
the multiplicities (q, t, d, s), respectively, which indicate the signal
form, as if the 13C NMR measurements had been taken without pro-
ton broadband decoupling. HRESIMS spectra were obtained on a
maXis UHR ESI-Qq-TOF mass spectrometer (Bruker Daltonics, Bre-
men, Germany). Samples were dissolved and further diluted in ACN/
MeOH/H2O in the ratio of 99:99:2 (v/v/v) and directly infused into
the ESI source with a syringe pump. The ESI ion source was operated
as follows: capillary voltage: 4.0 4.5 kV, nebulizer: 0.4 bar (N2), dry
gas flow: 4 L/min (N2), and dry temperature: 180 °C. Mass spectra
were recorded in the range of m/z 50 1900 in the positive- and nega-
tive ion mode. The sum formulae of the detected ions were deter-
mined using Bruker Compass DataAnalysis 4.1 based on the mass
accuracy (Dm/z  5 ppm) and isotopic pattern matching (SmartFor-
mula algorithm).
2.2. Plant material
The plant material was collected in Kasetsart University (13° 500
32.96" N, 100° 340 2.98" E), Bangkok, Thailand in 2018. The flowering
W. religiosa was growing in a hedge of around 1.5 m in height. The
F. Traxler, N. Iamprasertkun, A.M. Tschigg et al.
second studied species W. pubescens was a tree of approx. 15 m
height. At the time of collection this tree was without leaves. This
species was determined after flowering and the development of
leaves. The corresponding voucher specimen for W. religiosa (WU
0099055) was deposited at the Herbarium of the University in Vienna
(WU) and Bangkok Forest Herbarium (BKF) for W. pubescens
(WM2018012), respectively. Both plant species were identified by
Prof. S. Vajrodaya using the key provided in the Flora of Thailand (for
reference see Middleton, 1999).
2.3. Extraction and isolation
Wrightia religiosa—Ground air-dried leaves (31 g), stem bark
(29 g), root bark (42 g) and latex (3 mL) were exhaustively extracted
separately with MeOH at room temperature by soaking the plant
material for two days in an excess of solvent. This step was repeated
three times. This extraction process was supported frequently by
sonication. The filtered crude extracts were pooled, and the solvent
was removed in vacuo. The residue was further partitioned between
chloroform (CHCl3) and water. The aqueous phase was subsequently
washed with ethyl acetate (EtOAc) and n-butanol. The alkaloid con-
taining phases were further processed by various chromatographic
techniques. The obtained fractions were analyzed by TLC and HPLC.
Leaves—A portion of ca 200 mg of the aqueous phase was chroma-
tographed by column chromatography (CC) with Sephadex LH-20
eluted with methanol. Twenty-five fractions of 5 mL were collected.
TLC and HPLC analyses revealed the presence of compound 1 in frac-
tions 10 and 11. Pooling these both fractions afforded 8.3 mg of 1.
Chromatographic separation of the ethyl acetate phase (100 mg) con-
taining 1 and 2 over Sephadex LH20 eluted with MeOH yielded
twenty-two fractions, 5 mL each). This step yielded 6.8 mg of pure 1
in fractions 14 and 15. Fractions 17 and 18 showing similar HPLC pro-
files were pooled (4.3 mg) and purified by MPLC (Si 60, 40 60 mm)
eluted with CHCl3/MeOH (95:5) (15 fractions, 5 mL each). The latter
step yielded 1.0 mg of 2 in fractions 3 to 6.
Stem bark—A portion of ca 800 mg from the aqueous phase was
separated by CC over Sephadex LH20 eluted with MeOH (twenty-five
fractions). Fractions 16 to 18 (56.5 mg) contained impure 5 were
pooled and the half of this fraction was subsequently purified by
MPLC (RP-C8 column, eluted with mixtures of H2O/MeOH starting
from 95:5 to 50:50 (twenty fractions, 10 mL each).This step yielded
3.2 mg of 5.
Root bark—A portion of ca 170 mg from the aqueous phase (1.3 g)
was separated by CC over Sephadex LH20 eluted with MeOH. This
step yielded thirty fractions (5 mL each). Fractions 23 and 24 afforded
2.7 mg of 1 and 3.3 mg of 3.
Latex—A volume of 3 mL of polymerized latex was extracted with
MeOH, the solvent evaporated and the residue partitioned between
CHCl3 and water. The whole of the aqueous phase (33 mg) was chro-
matographed by CC over Sephadex LH20 eluted with MeOH. Fraction
5 (10.9 mg) obtained from this separation step, which showed a sin-
gle band on TLC, was further purified by preparative TLC developed
in CHCl3/MeOH (90:10). This step afforded 1.9 mg of 4.
Wrightia pubescens—A portion of 120 g air-dried stem bark
yielded 3.1 g crude methanolic extract. After removing the methanol
under reduced pressure, the brownish residue was suspended in
water and the lipophilic compounds extracted using CHCl3. This gave
800 mg lipophilic extract. This phase was subsequently partitioned
between water and a mixture of 20% EtOAc in petrol ether (PE),
affording 590 mg of organic phase. Separation of this phase over silica
gel 60, 40 60 mm, eluted with mixtures of CHCl3 and MeOH yielded
twenty-eight fractions of approx. 10 mL each. Fraction twenty
(1.2 mg) containing impure compound 7 was finally purified by prep.
TLC developed in CHCl3/MeOH (98:2). This step gave 0.8 mg of 7.
Fraction 13 (7.2 mg) was subjected to CC over Sephadex LH20 eluted
isocratic with methanol, yielding 1.1 mg of 6.
244
South African Journal of Botany 137 (2021) 242 248
2.4. Antioxidant assay
This experiment was performed according to Sudzukovic
et al. (2016). From crude extracts obtained from leaves, stem bark
and root bark, a dilution series starting from ca. 10 mg/mL was pre-
pared (the accurate concentrations are given below). The leaf extract
was tested in the concentration range of 10.165 mg/mL and
4.963 mg/mL, for the stem bark 9.225 mg/mL to 4.504 mg/mL and the
root bark extract 8.8 mg/mL to 4.297 mg/mL. The stem bark extract of
W. pubescens was tested in a conc. range of 20.0 mg/mL to 9.766 mg/
mL. The isolated compounds 1 to 4, 6 and 7 were tested in the range
of 1 mg/mL to 0.5 mg/mL. These compounds were only tested one
time due to the insignificant results obtained from the first run. After
measuring the blank, 50 mL of a freshly prepared methanolic 2,2-
diphenyl-1-picrylhydrazyl (DPPH) solution (0.4 mM) was added to
each well. The microwell plates were measured at 550 nm 30 min
after adding the stable radical solution using a Tecan SunriseÒ well
plate reader. Ascorbic acid was used as positive control.
2.5. Insect feeding assay
The insect assay was performed in triplicate with slight modifica-
tions according to Kornpointner et al. (2018). Briefly, 367 mg of
freeze-dried food powder containing ground white beans, yeast,
ascorbic acid and ethyl 4-hydroxybenzoate as preservative was
spiked with a 1.8 mg/g and 4.5 mg/g food pellet of the crude extracts.
After evaporation of the solvent (MeOH, 16 h), an aqueous solution
containing vitamins and the antibiotic chloramphenicol was added.
This artificial diet was stabilized by adding 1.1 g of a warm agar solu-
tion at a concentration of 5 g agar in 140 mL H2O. Ten freshly hatched
larvae of the cotton leafworm S. littoralis were placed on each food
pellet on separate Petri dishes and these were kept in an incubator at
26 °C and 90% humidity in darkness. The mass of surviving larvae
was evaluated after 96 h, and the percentage of the gained weight
was calculated. Nicotine served as positive control.
2.6. Spectroscopic data of the isolated compounds
Indoxyl 3-O-b-D-apiofuranosyl-(1!6)-b-D-glucopyranoside (1):
Colorless powder; UV max MeOH/H2O 222, 280 nm; HR ESI-MS m/z
450.1376 [M+Na]+ (calcd for C19H25NO10Na, 450.1376), m/z 466.1123
[M+K]+ (calcd for C19H25NO10K, 466.1115); m/z 877.2853 [2M+Na]+
(calcd for C38H50N2O20Na, 877.2854); m/z 426.1408 [M-H]- (calcd for
C19H24NO10, 426.1400); m/z 462.1177 [M+Cl]- (calcd for
C19H25NO10Cl, 462.1167); 1H NMR (CD3OD): dH 7.69 (d, 1H, J = 8.1 Hz,
H-4), 7.27 (d, 1H, J = 8.2 Hz, H-7), 7.09 (s, 1H, H-2), 7.07 (m, 1H, H-6),
6.98 (dd, 1H, J = 8.1, 7.2 Hz, H-5), 5.02 (d, 1H, J = 2.4 Hz, H-1''), 4.67 (d,
1H, J = 7.7 Hz, H-1'), 4.04 (dd, 1H, J = 11.7, 2.0 Hz, H-6'a), 3.96 (d, 1H, J
= 9.6 Hz, H-4''a), 3.95 (d, 1H, J = 2.4 Hz, H-2''), 3.76 (d, 1H, J = 9.6 Hz,
H-4''b), 3.64 (dd, 1H, J = 11.7, 6.5 Hz, H-6'b), 3.59 (s, 2H, H-5''), 3.50
(m, 1H, H-5'), 3.49 (m, 1H, H-2'), 3.43 (dd, 1H, J = 9.1, 8.8 Hz, H-3'),
3.36 (dd, 1H, J = 9.8, 8.8 Hz, H-4'); 13C NMR (CD3OD): dC 138.9 (s, C-3),
135.2 (s, C-9), 122.8 (d, C-6), 121.4 (s, C-8), 119.5 (d, C-5), 118.7 (d, C-
4), 112.4 (d, C-2), 112.3 (d, C-7), 111.0 (d, C-1''), 105.8 (d, C-1'), 80.6
(s, C-3''), 78.1 (d, C-3'), 78.1 (d, C-2''), 77.0 (d, C-5'), 75.1 (d, C-2'), 75.0
(d, C-4''), 71.8 (d, C-4'), 68.9 (t, C-6'), 65.5 (t, C-5'').
Benzouracil (2): Colorless powder; UV max MeOH/H2O 218, 240 (sh),
310 nm; HR ESI-MS m/z 185.0321 [M+Na]+ (calcd for C8H6N2O2Na,
185.0321), m/z 207.0139 [M+2Na-H]+ (calcd for C8H5N2O2Na2,
207.0146); m/z 161.0355 [M-H]- (calcd for C8H5N2O2, 161.0351); 1H
NMR (CD3OD): dH 8.01 (d, 1H, J = 8.0 Hz, H-5), 7.65 (dd, 1H, J = 8.2, 7.5
Hz, H-7), 7.23 (dd, 1H, J = 7.5, 8.0 Hz, H-6), 7.18 (d, 1H, J = 8.2 Hz, H-
8); 13C NMR (CD3OD): dC 165.2 (s, C-4), 142.3 (s, C-10), 136.5 (d, C-5),
128.4 (d, C-7), 124.1 (d, C-6), 116.5 (d, C-8), 115.9 (s, C-9). The signal
relative to C-2 was not observable using CD3OD as solvent.
F. Traxler, N. Iamprasertkun, A.M. Tschigg et al.
1
H NMR (acetone-d6): dH 8.00 (d, 1H, J = 7.4 Hz, H-5), 7.66 (dd, 1H,
J = 7.8, 7.4 Hz, H-7), 7.29 (d, 1H, J = 7.4 Hz, H-8), 7.22 (dd, 1H, J = 7.8,
7.4 Hz, H-6); 13C NMR (acetone-d6): dC 163.4 (s, C-4), 150.8 (s, C-2),
142.0 (s, C-10), 135.9 (d, C-7), 128.3 (d, C-5), 123.5 (d, C-6), 116.2 (d,
C-8), 115.8 (s, C-9).
Blepharin (3): Colorless powder; UV max MeOH/H2O 208, 205, 276
(sh) nm; HR ESI-MS m/z 350.0847 [M+Na]+ (calcd for C14H17NO8Na
350.0852), m/z 677.1802 [2M+Na]+ (calcd for C28H34N2O16Na,
677.1805); m/z 326.0881 [M-H]- (calcd for C14H16NO8, 326.0876); 1H
NMR (CD3OD): dH 7.09 (m, 1H, H-8), 7.00 (m, 2H, H-6 and H-7), 6.93
(m, 1H, H-5), 5.75 (s, 1H, H-2), 4.68 (d, 1H, J = 7.9 Hz, H-1'), 3.85 (dd,
1H, J = 11.8, 2.0 Hz, H-6'a), 3.68 (dd, 1H, J = 11, 8, 4.8 Hz, H-6'b), 3.35
(m, 1H, H-5'), 3.32 (m, 1H, H-3'), 3.30 (m, 1H, H-4'), 3.19 (dd, 1H, J =
8.9, 7.9 Hz, H-2'); 13C NMR (CD3OD): dC 163.2 (s, C-3), 142.1 (s, C-10),
127.1 (s, C-9), 125.1 (d, C-7), 124.2 (d, C-6), 119.8 (d, C-8), 116.8 (s, C-
5), 103.9. (d, C-1'), 96.5 (d, C-2), 78.5 (d, C-3'), 77.9 (d, C-5'), 74.9 (d,
C-2'), 71.1 (d, C-4'). 62.6 (t, C-6').
Blepharigenin (4): Colorless powder; UV max MeOH/H2O 208, 250,
278, 2852 (sh) nm; HR ESI-MS m/z 210.0139 [M+2Na-H]+ (calcd for
C8H6NO3Na2, 210.0143), m/z 164.0352 [M-H]- (calcd for C8H6NO3,
164.0348); 1H NMR (CD3OD): dH 7.00-6.97 (m, 3H, H-6, H-7 and H-8),
6.94-6.92 (m, 1H, H-5), 5.52 (s, 1H, H-2); 13C NMR (CD3OD): dC 165.3
(s, C-3), 142.5 (s, C-10), 127.5 (s, C-9), 124.9 (d, C-7), 123.7 (d, C-6),
118.8 (d, C-8), 116.8 (d, C-5), 92.0 (d, C-2).
4-Pinoresinol D-apio-b-D-furanosyl-(1!2)-b-D-glucopyranoside
(5): Colorless powder; UV max MeOH/H2O 228, 278 nm; HR ESI-MS m/z
675.2260 [M+Na]+ (calcd for C31H40O15Na 675.2264), m/z 1327.4608
[2M+Na]+ (calcd for C62H80O30Na, 1327.4631); m/z 651.2293 [M-H]-
(calcd for C31H39O15, 651.2289); 1H NMR (CD3OD): dH 7.10 (d, 1H, J =
8.4 Hz, H-5), 7.01 (d, 1H, J = 1.7 Hz, H-2), 6.95 (d, 1H, 1.7 Hz, H-2'),
6.90 (dd, 1H, J = 8.5, 1.8 Hz, H-6), 6.82 (dd, 1, J = 8.2, 1.7 Hz, H-6'), 6.77
(d, 1H, J = 8.2 Hz, H-5'), 5.54 (d, 1H, J = 1Hz, H-1'''), 4.98 (d, 1H, J = 7.9
Fig. 2. Isolated compounds from W. religiosa: 3-Indole D-apio-b-D-furanosyl-(1!6)-b-D-glucopyranoside (1), benzouracil (2), blepharin (3), blepharigenin (4), 4-pinoresinol D-apio-
b-D-furanosyl-(1!2)-b-D-glucopyranoside (5) and from W. pubescens: tryptanthrin (6) and kelampayoside A (7). Atomic labelling of compounds is indicated and follows respective
publications in general (see section 3.1). Numbering of NMR spectroscopic data in section 2.6 is in accordance to this numbering.
245
South African Journal of Botany 137 (2021) 242 248
Hz, H-1''), 4.76 (d, 1H, J = 4.2 Hz, H-7), 4.71 (d, 1H, J = 4.3 Hz, H-7'),
4.25 (m, 2H, H-9a and H-9'a), 4.17 (d, 1H, J = 9.8 Hz, H-4'''a), 3.97 (d,
1H, J = 1.1 Hz, H-2'''), 3.86 (m, 3H, H-6''a; H-9b and H-9'b), 3.85 (s, 6H,
2x OMe), 3.74 (d, 2H, J = 9.8 Hz, H-4'''b), 3.71 (m, 1H, H-2''), 3.67 (m,
1H, H-6''b), 3.59 (m, 1H, H-4''), 3.56 (d, 1H, J = 11.6 Hz, H-5'''a), 3.51
(d, 1H, J = 11.6 Hz, H-5'''b), 3.40 (m, 1H, H-3''), 3.39 (m, 1H, H-5''),
3.14 (m, 1H, H-8'), 3.14 (m, 1H, H-8); 13C NMR (CD3OD): dC 150.9 (s,
C-3), 149.1 (s, C-3'), 147.5 (s, C-4), 147.4 (s, C-4'), 137.0 (s, C-1), 133.8
(s, C-1'), 120.1 (d, C-6'), 119.5 (d, C-6), 117.2 (d, C-5), 116.1 (d, C-5'),
111.4 (d, C-2), 111.0 (d, C-2'), 110.3 (d, C-1'''), 101.0 (d, C-1'), 87.5 (d,
C-7'), 87.2 (d, C-7), 80.8 (d, C-3'''), 78.8 (d, C-4''), 78.1 (d, C-5''), 77.9
(d, C-2'''), 77.5 (d, C-2''), 72.7 (t, C-9), 72.7 (t, C-9'), 71.4 (d, C-3''), 66.2
(t, C-5'''), 62.5 (t, C-6''), 56.4 (q, OMe (2x)), 55.5 (d, C-8 and C-8').
Tryptanthrin (6): Colorless powder, (UV max MeOH/H2O 206, 226,
252, 280, 314, 332, 390 nm, HR ESI-MS m/z 249.0659 [M+H]+ (calcd
for C15H9N2O2 249.0664); m/z 271.0479 [M+Na]+ (calcd for
C15H8N2O2Na 271.0484), m/z 519.1064 [2M+Na]+ (calcd for
C30H16N4O4Na, 519.1070); 1H NMR (CDCl3): dH 8.64 (d, 1H, J = 8.2 Hz,
H-10), 8.45 (dd, 1H, J = 8.0, 1.4 Hz, H-1), 8.04 (d, 1H, J = 8.0 Hz, H-4),
7.92 (d, 1H, J = 7.5 Hz, H-7), 7.86 (ddd, 1H, J = 8.5, 6.9, 1.5 Hz, H-3),
7.80 (ddd, 1H, J = 8.6, 7.2, 1.3 Hz, H-9), 7.68 (ddd, 1H, J = 8.2, 7.0, 1.2
Hz, H-2), 7.43 (ddd, 1H, J = 7.5, 7.5, 0.9 Hz, H-8); 13C NMR (CDCl3): dC
182.6 (s, C-6), 158.1 (s, C-12), 146.6 (s, C-4a), 146.4 (s, C-10a), 144.3
(s, C-5a), 138.3 (d, C-9), 135.1 (d, C-3), 130.8 (d, C-4), 130.3 (d, C-2),
127.6 (d, C-1), 127.2 (d, C-8), 125.4 (d, C-7), 123.7 (s, C-12a), 121.9 (s,
C-6a), 118.0 (d, C-10).
Kelampayoside A (7): Colorless powder, (UV max MeOH/H2O 204,
224 (sh), 270 nm, HR ESI-MS m/z 501.1572 [M+Na]+ (calcd for
C20H30O13Na 501.1584); 1H NMR (CD3OD): dH 6.46 (s, 2H, H-2 and H-
6), 4.96 (d, 1H, J = 2.7 Hz, H-1''), 4.80 (d, 1H, J = 7.5 Hz, H-1'), 4.05 (m,
1H, H-6'a), 3.95 (d, 1H, J = 9.7 Hz, H-4''a), 3.88 (d, 1H, J = 2.7 Hz, H-2''),
3.82 (s, 6H, 3-OCH3 and 5-OCH3), 3.74 (d, 1H, J = 9.7 Hz, H-4''b), 3.71
F. Traxler, N. Iamprasertkun, A.M. Tschigg et al.
(s, 3H, 4-OCH3), 3.59 (m, 1H, H-6'b), 3.58 (m, 1H, H-5'), 3.57 (m, 1H,
H-5''a), 3.55 (d, 1H, J = 1.9 Hz, H-5''b), 3.43 (m, 1H, H-3'), 3.42 (m, 1H,
H-2'), 3.32 (m, 1H, H-4'); 13C NMR (CD3OD): dC 156.0 (s, C-4), 154.8 (s,
C-3 and C-5), 134.6 (s, C-1), 110.9 (d, C-1''), 103.2 (d, C-1'), 96.3 (d, C-
2 and C-6), 80.5 (s, C-3''), 77.9 (d, C-3'), 77.9 (d, C-2''), 77.0 (d, C-5'),
74.9 (t, C-4''), 74.9 (d, C-2'), 71.6 (d, C-4'), 68.8 (t, C-6') 65.4 (d, C-5''),
62.2 (q, 4-OCH3), 56.7 (q, 3-OCH3 and 5-OCH3).
3. Results and discussion
3.1. Structure elucidation
Structure elucidation of the isolated compounds was performed
with data from 1D (1H, 13C) and 2D (COSY, TOCSY, NOESY, HSQC,
HMBC) NMR spectroscopic measurements as well as from HRESIMS
measurements. All compounds had been described earlier in
Fig. 3. A: HPLC profiles of organs and the latex of W. religiosa. B: HPLC profile of the stem bark extract of W. pubescens. Numbering of the compounds is in accordance to Figure 2. The
samples were injected at a concentration of 5 mg/mL for W. religiosa and 10 mg/mL for W. pubescens. The wavelength of detection was set at 230 nm.
246
South African Journal of Botany 137 (2021) 242 248
literature and their structures were verified by comparison of physi-
cal data to reported literature values. Compounds were identified as
indoxyl 3-O-b-D-apiofuranosyl-(1!6)-b-D-glucopyranoside (1)
(Sahakitpichan et al., 2018), benzouracil (2) (Castro et al., 2008), ble-
pharin (3) (Sahakitpichan et al., 2018), blepharingenin (4)
(Sahakitpichan et al., 2018), pinoresinol-apiosyl(1!2)-glucoside (5)
(Abe and Yamauchi, 1989), tryptanthrin (6) (Garcellano et al., 2019)
and kelampayoside A (7) (Kitagawa et al., 1996). The structural for-
mulae are given in Fig. 2 and NMR shifts are listed in Section 2.6. The
NMR spectra of the isolated compounds are depicted in the Supple-
mentary material (Figs. S1 S54).
In contrast to the general notion that Apocynaceae are rich sour-
ces of various complex tryptamine-derived alkaloids (Dey et al.,
2017), the so far studied Wrightia species exhibit a different special-
ized metabolite profile with some unique features. Thus, indole-
derived compounds generated from anthranilic acid
F. Traxler, N. Iamprasertkun, A.M. Tschigg et al.
(Muruganandam and Bhattacharya, 2000), are accumulated in
Wrightia species (Sahakitpichan et al., 2018), whereas no tryptamine-
derived alkaloids could be detected, neither by HPLC coupled with
UV diode array detection nor by forming orange colored spots on TLC
plates after spraying with Dragendorff’ reagent. The present work
revealed, that W. religiosa accumulates 3-indole D-apio-b-D-furano-
syl-(1!6)-b-D-glucopyranoside (1) as major compound in all organs
(Fig. 3). Its changes of the color on TLC plates from colorless to blue
after detection with Dragendorff’ reagent indicating an oxidation to a
compound structurally related to indigo. Furthermore, benzouracil
(2) could be isolated from the leaf extract as a minor compound. The
lignan derivative 4-pinoresinol D-apio-b-D-furanosyl-(1!2)-b-D-glu-
copyranoside (5) (Abe and Yamauchi, 1989) Abe and Yamauchi, 1989
was shown to be present in the stem bark extract. Chromatographic
separation of the methanolic root bark extract yielded the benzoxazi-
noid glucoside blepharin (3). This compound was recently isolated
from leaves and twigs of W. religiosa collected in Nonthaburi province
in Thailand (Sahakitpichan et al., 2018), but we could not confirm its
presence in the leaves.
Interestingly, its aglycone blepharigenin (4) (Chatterjee et al.,
1990) could successfully be obtained from the latex. This compound
might have resulted from hydrolysis during the time span between
collection and extraction, as blepharin (3) is known to be the storage
form, from which the aglycone is liberated upon tissue disruption
(Pratt et al., 1995). A conceivable reason could be hydrolysis of 3 by
glucosidases present in lacticifers. Presumably, such enzymes are
also spatially segregated in the lacticifers from the latex otherwise
hydrolysis under uncontrolled conditions would occur which may
lead to damage of the plant tissue. Generally, the role of latex in this
context remains unclear, but as 4 is probably toxic also for the plant,
compartmentation in safe compartments such as lacticifers is an
option to avoid self-intoxication. So far, W. tinctoria has been studied
for the presence of serine proteases in latex, due to its pharmaceutical
importance (Tomar et al., 2008), but no other compounds were speci-
fied. How far biotic and/or abiotic factors contributed to the detected
pattern of compounds remains open since only a limited number of
plant individuals growing under uncontrolled conditions were ana-
lyzed.
From stem bark of W. pubescens, the strong fluorescent indole-
derived compound tryptanthrin (6) and the phenolic apioglucoside
kelampayoside A (7) have been isolated. The well-known tryptan-
thrin (6) has earlier been isolated from various plant species, and has
been found in some microorganisms, too (Honda and Tabata, 1979;
Honda et al., 1979; 1980). A possible precursor in its biosynthesis is
the indole S,O-bisdesmoside, which also leads to the structurally
related compounds indirubin and isatin (Yoshikawa et al., 1998).
Kelampayoside A (7) was described firstly from Anthocephalus chi-
nensis (Lam.) Rich. ex Walp. (Rubiaceae) (Kitagawa et al., 1996). Since
then it has also been described from Nauclea pobeguinii (Hua ex
Pobe g.) Merr. (Rubiaceae) (Xu et al., 2012) and Ilex macropoda Miq.
(Aquifoliaceae) (Fuchino et al., 1997), but to best of our knowledge,
not detected in species belonging to the Apocynaceae yet.
3.2. Radical scavenging activities
The performed DPPH assay revealed in general low radical scav-
enging activities of all tested extracts as well as the isolated com-
pounds. Concerning the crude extracts of W. religiosa, the highest
exhibited free radical scavenging activity showed the crude extract
from stem bark with an EC50 of 272 mg/mL. The two other extracts
showed higher EC50 values with 474 mg/mL for the root bark and
299 mg/mL for the leaves, respectively. The EC50 values of compounds
1, 3 and 4 ranged from 200 to 276 mg/mL (Table 1). These values sug-
gest that the antioxidant activity is rather caused by kaempferol and
other phenolic compounds, which have been identified in W. religiosa
by Sahakitpichan et al. (2018) than by the compounds described in
247
South African Journal of Botany 137 (2021) 242 248
Table 1
Results of the DPPH assay of the isolated com-
pounds except 5. Ascorbic acid was used for com-
parison. The EC50 values are given in mg/mL.
Compounds EC50
1 200.3
2 >1000
3 232.0
4 275.2
6 >1000
7 35.7
Ascorbic acid 5.2
the present work. The stem bark extract of W. pubescens showed an
even much higher EC50 value of 2.8 mg/mL, which suggests the
absence of compounds possessing radical scavenger activities in
higher quantities. Only kelampayoside A (7), isolated from this plant
species, showed an EC50 value of 35.7 mg/mL (Table 1) but due to its
low conc. in the stem bark (0.8 mg from 120 g plant material) no sig-
nificant contribution of this compound to the antioxidative proper-
ties of the stem bark extract can be expected.
3.3. Feeding assay of extracts
Two concentrations, 1.8 and 4.5 mg/g, of the methanolic crude
extract from leaves, stem bark and root bark of W. religiosa, were sub-
jected to feeding bioassays on larvae of S. littoralis over 96 h to
observe an eventual growth inhibition of neonate larvae. The alkaloid
nicotine served as positive control (tested in a range of 28 mg/g and
2.7 mg/g; EC50 790 mg/g). However, no anti-feedant effect was
observed in the tested concentrations (data not shown). Not even
extracts containing the bitter tasting blepharin (3) (Lal, 1936; Pratt
et al., 1995); could exert an anti-feedant effect on this polyphagous
organism. Apparently, also the indole-derived compounds are less
effective as compared to the more complex tryptamine-derived alka-
loid glucosides, which are capable of protein cross-linking and pre-
cipitation after cleavage of the glucose moiety (Guirimand et al.,
2010).
4. Phylogenetic and ecological aspects
Expression of the indole biosynthetic pathway, starting from
anthranilic acid, and leading to indigo-related structures (1 and 6), to
benzouracil (2), and to benzoxazinoid compounds (3 and 4) appears
to be a characteristic feature of some Wrightia species
(Garcellano et al., 2019; Sahakitpichan et al., 2018). To date, none of
the more complex tryptamine-derived alkaloids could be detected in
these species (e.g. Muruganandam and Bhattacharya, 2000). The for-
mation of indole-derived compounds in this genus may be inter-
preted as a basal character state, which coincides with the
phylogenetic position in the tribe Wrightieae of Apocycnaceae
(Livshultz et al., 2007; Fishbein et al., 2018). Within Wrightia, the
accumulation of indigo-related compounds suggests a close relation-
ship of W. religiosa to W. tinctora. Limited phytochemical data prevent
further conclusions in this respect.
Finally, it should be mentioned that benzoxacinoids, being more
widespread in higher plants, frequently serve the role as phytoalex-
ins and defense compounds (Frey et al., 2009; Wouters et al., 2016).
Their role in the studied species cannot be assessed with certainty.
Similarly, a phytoalexin role was suggested for tryptanthrin (6),
detected in leaves of abiotically elicited Isatis indigotica Fortune, but
not in untreated plants (Pedras et al., 2019). In addition, this com-
pound exhibited pronounced antifungal activity (Pedras et al., 2019).
As for Wrightia species, its indole-derived compounds might be
involved in other biotic interactions than those tested here. They nei-
ther appear to serve as good antioxidants, nor are they effective
F. Traxler, N. Iamprasertkun, A.M. Tschigg et al.
insect deterrents. They might be good antifungal compounds though,
which needs to be evaluated in future studies.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors thank Dr. Natthawadi Wongthet and Wanitcha
Muangrom, MSc. for sampling of W. pubescens. We gratefully
acknowledge Susanne Felsinger and Peter Unteregger (Institute of
Organic Chemistry, University of Vienna) for recording the NMR and
mass spectra, respectively. We are grateful to Development and Pro-
motion of Science and Technology Talents Project (DPST) and Kaset-
sart University Research and Development Institute (KURDI) for
financial support of N. Iamprasertkun. We further acknowledge the
effort and comments of the reviewers.
Supplementary materials
Supplementary material associated with this article can be found,
in the online version, at doi:10.1016/j.sajb.2020.10.019.
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