Chlorinated iridoid glucosides from Veronica longifolia and their antioxidant activity

Article abstract of DOI:10.1021/np100366k

From Veronica longifolia were isolated three chlorinated iridoid glucosides, namely, asystasioside E (6) and its 6-O-esters 6a and 6b, named longifoliosides A and B, respectively. The structures of 6a and 6b were proved by analysis of their spectroscopic data and by conversion to the catalpol ester verproside (5a) or to catalpol (5), respectively. The configuration of the previously known vanilloyl analogue, urphoside B, was shown to be the 6β-epimer (6c) of the structure originally reported. Longifoliosides A (6a) and B (6b) were found to exhibit radical-scavenging activity against nitric oxide, superoxide, and 2,2-diphenyl-1-picrylhydrazyl radicals.

Full text of DOI:10.1021/np100366k

J. Nat. Prod. 2010, 73, 1593–1596
1593
Chlorinated Iridoid Glucosides from Veronica longifolia and Their Antioxidant Activity
Søren R. Jensen,*,† Charlotte H. Gotfredsen,† U. Sebnem Harput,‡ and Iclal Saracoglu‡
Department of Chemistry, The Technical UniVersity of Denmark, DK-2800, Lyngby, Denmark, and Department of Pharmacognosy, Faculty of
Pharmacy, Hacettepe UniVersity, TR-06100, Ankara, Turkey
ReceiVed June 1, 2010
From Veronica longifolia were isolated three chlorinated iridoid glucosides, namely, asystasioside E (6) and its 6-O-
esters 6a and 6b, named longifoliosides A and B, respectively. The structures of 6a and 6b were proved by analysis of
their spectroscopic data and by conversion to the catalpol ester verproside (5a) or to catalpol (5), respectively. The
configuration of the previously known vanilloyl analogue, urphoside B, was shown to be the 6ꢀ-epimer (6c) of the
structure originally reported. Longifoliosides A (6a) and B (6b) were found to exhibit radical-scavenging activity against
nitric oxide, superoxide, and 2,2-diphenyl-1-picrylhydrazyl radicals.
Plants of the genus Veronica (Speedwell, Plantaginaceae) have
traditionally been considered members of the family Scrophulari-
aceae; however, the genus has recently been transferred to the
Plantaginaceae on the basis of DNA sequence investigations.1,2
With regard to chemistry, Veronica is generally characterized by
its content of the iridoid glucosides aucubin (4) and catalpol (5) as
well as a variety of esters of the latter.3-5 As a part of an earlier
project, one of us has previously investigated V. longifolia L,5
finding the iridoid glucosides 4 and 5 as well as the 6-O-catalpol
esters verproside (5a), verminoside (5b), and catalposide (5c).
Material of V. longifolia was briefly boiled with ethanol, and
after extraction, the water-soluble part of the extract was subjected
to reversed-phase column chromatography. Mannitol (1) was the
main carbohydrate in the sugar fraction. The iridoid glucosides
isolated comprised the common compounds 8-epiloganic acid (2),
gardoside (3), aucubin (4), catalpol (5), and two 6-O-esters of the
latter (5a and 5c), as well as the chloro-containing asystasioside E
(6) and two compounds (6a and 6b) that were apparently esters
of 6.
Compound 6a was isolated as a colorless glass, [R]25 -102.
D
The molecular formula was C22H27ClO13 as deduced from the
quasimolecular 3:1 ion cluster obtained by LC-HRESIMS (observed
m/z 557.1015 [M + Na]+ and its 37Cl isotope m/z 559.1034 [M +
2 + Na]+). The NMR spectroscopic data (Table 1) could be
assigned using 1D and 2D techniques. The 13C NMR spectrum of
6a exhibited the expected 22 signals, of which six could be assigned
to a ꢀ-glucopyranosyl group and seven to a 3,4-dihydroxybenzoyl
moiety, when comparing with the spectrum of verproside (5a). The
remaining 10 signals were surmised as belonging to an iridoid
aglucon, and since it also contained a chlorine atom, this was
compared to the data of asystasioside E (6) (Table 1). The chemical
shifts were very similar, except for the C-6 signal (δ 85.0) of 6a,
which showed a downfield shift of 2.1 ppm, while the C-5 and
C-7 signals were found 1.1 and 2.1 ppm upfield, respectively. This
indicated that 6a is the 6-O-(3,4-dihydroxybenzoyl) ester of 6. The
1H NMR spectrum of 6a (Table 1) was consistent with this,
including the expected acylation shifts (data not shown). The HMBC
spectrum allowed the carbons of attachment between the aglucon
and the peripheral moieties to be discerned. Thus, the position of
the ester was confirmed by a correlation between H-6 (δH 5.05)
and the carbonyl of the acyl group (δC 167.7), and that of the
glucopyranosyl moiety by a correlation between H-1′ of the glucosyl
moiety (δH 4.65) and C-1 of the aglucon (δC 92.8). This compound
has been accorded the trivial name longifolioside A.
Longifolioside B (6b) was similarly isolated as a colorless glass,
D
[R]25 -84. Again, the molecular formula, C24H29ClO13, was
deduced from the quasi-molecular 3:1 ion cluster obtained by LC-
HRESIMS (observed m/z 583.1166 [M + Na]+ and its 37Cl isotope
m/z 585.1151 [M + 2 + Na]+). As for compound 6a above, the
NMR spectroscopic data (Table 1) could be assigned using 1D and
2D techniques. The 13C NMR spectrum of 6b contained 24 signals,
of which six as above could be attributed to a ꢀ-glucopyranosyl
group and nine to a caffeoyl group, by comparison with the
* To whom correspondence should be addressed. Tel: +45 45252103.
Fax: +45 45933968. E-mail: srj@kemi.dtu.dk.
† Technical University of Denmark.
‡ Hacettepe University.
10.1021/np100366k  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/31/2010

1594 Journal of Natural Products, 2010, Vol. 73, No. 9
Notes
Table 1. 1H (500 MHz) and 13C (75 MHz) NMR Spectroscopic Data for Longifoliosides A (6a) and B (6b) and Urphoside B in
CD3OD
longifolioside A (6a)
δH (J in Hz)
longifolioside B (6b)
δH (J in Hz)
6
urphoside B (6c)a
δH (J in Hz)
position
δC
δC
δC
δC
Agluc
1
3
4
5
6
7
8
9
92.8
141.0
105.6
37.0
85.0
69.6
80.8
48.9
63.5
5.69, d (3.5)
92.8
141.0
105.6
37.1
84.7
69.6
80.8
48.9
63.5
5.68, d (3.6)
6.29, mb
5.22, dd (6.2, 3.3)
2.84, m
4.99, dd (5.1, 7.4)
4.29, d (7.4)
93.3
140.8
106.1
38.1
82.9
71.7
80.1
ca. 49
63.5
92.9
141.2
105.7
36.8
85.3
69.7
81.0
48.5
63.6
5.70, d (3.7)
6.30, dd (6.2, 1.8)
5.25, dd (6.2, 3.3)
2.88, m
5.05, dd (5.0, 7.4)
4.35, d (7.4)
6.31, dd (6.3, 2.1)
5.26, dd (6.3, 3.5)
2.90, m
5.10, dd (4.9, 7.3)
4.20, d (7.3)
2.62, dd (10.5, 3.5)
4.00, d (11.8)
2.60, dd (10.5, 3.6)
3.99, d (11.8)
2.63, dd (10.5, 3.7)
4.10, d (11.6)
10
3.82, d (11.8)
3.81, d (11.8)
4.83, d (11.6)
Glc
1′
99.5
74.7
77.8
71.6
78.1
62.8
4.65, d (7.9)
99.5
74.7
77.9
71.6
78.1
62.8
4.65, d (7.9)
99.7
74.7
77.9
71.7
78.1
62.9
99.6
74.8
78.0
71.7
78.2
62.9
4.66, d (7.9)
3.20, t (9.1)
2′
3.19, dd (9.2, 7,9)
3.36, t-like (9.2)
3.28, mb
3.19, dd (9.2, 7.9)
3.36, t-like (9.2)
3.28, mb
3′
3.36, t (9.3)
4′
3.28, mb
5′
3.29, mb
3.29, mb
3.28, mb
6′
3.87, br d (11.5)
3.67, dd (11.5, 5.1)
3.87, br d (11.2)
3.66, dd (11.2, 4.5)
3.83, dd (12.0, 2.1)
3.67, dd (12.0, 6.0)
Aroyl
1′′
2′′
3′′
4′′
5′′
6′′
R
122.0
117.5
146.2
152.0
115.9
123.9
127.5
115.2
147.7
149.8
116.5
123.2
114.4
146.8
168.6
122.7
113.7
148.8
153.3
116.1
125.6
7.45 (m)
7.05, d (1.9)
6.81 (d-like 8.8)
7.45 (m)
6.77, d (8.2)
6.96, dd (8.2, 1.9)
6.30, d (15.9)
7.59, d (15.9)
ꢀ
CO
167.7
167.6
a Data from ref 6 in this paper; the coupling constant J5,6 was given as 12.2 Hz due to a misreading. b Signal obscured.
analogous data for verminoside (5b). The 10 signals not accounted
being due to a spinning sideband from the solvent peak. The NOE
correlations observed between H-6 and H-7 and between H-7 and
H-9 in 6c are also visible in the spectra of 6a (see Supporting
Information); the proximity of H-6 and H-7 despite their trans-
disposition must thus be due to the conformation of the five-
membered ring; therefore, this is an additional example that extra
care should be taken when determining the structure of iridoid
glucosides using mainly NOE correlations. Assuming that com-
pounds 6a and 6b are both 7,8-trans-chlorohydrins, a simple way
to prove the structures would be a transformation to the known 5a
and 5b, respectively, by treatment with sodium hydroxide.13,14 Thus,
a sample of 6b was dissolved in D2O in a NMR tube and an excess
of NaOD in D2O was added, with the changes being followed by
1H NMR spectroscopy. After 1 h the 1H NMR signal (δH 5.45) of
6b had diminished by 40%, and after 24 h a complete conversion
to catalpol (5) had taken place. Similarly, a sample of 6a was
transformed to 5a within 24 h, with saponification evidently being
much slower in this case. It can be concluded that the two
longifoliosides (6a and 6b) and consequently also urphoside B (6c)
must be 6-O-esters of asystasioside E.
The difference in iridoid content from the two different sources
of V. longifolia is not readily explained, but it could be ascribed
either to a difference in age or to geographical variation. In the
first case,5 the plant was collected in July from old growth of an
outdoor plant of unrecorded origin in The Botanical Garden of The
University of Copenhagen. In the present case, seeds obtained from
the Botanic Garden of the University of Hohenheim, Stuttgart,
Germany, were sown in a greenhouse in April and harvested when
the plants flowered in September. Unfortunately, both samples are
of unknown origin, but since the natural distribution of the species
is most of Europe,15 the possibility of geographical variation cannot
be dismissed.
for were almost superimposable on those of the aglucon of
1
compound 6a. Except for the ester groups, the H NMR data for
6b and 6a were almost identical (Table 1), including the measured
coupling constants. Again, the HMBC spectrum allowed the relative
positions of the individual moieties to be pinpointed. Thus, the
position of the acyl group was confirmed by a correlation between
H-6 (δH 4.99) and the carbonyl of the acyl group (δC 168.6) and
that of the glucopyranosyl group by a cross-peak between H-1′ (δH
4.65) and C-1 of the aglucon (δC 92.8). Longifolioside B therefore
has the structure shown as 6b.
Having elucidated these structures, the data were compared
(Table 1) with two similar compounds reported in the literature,
namely, a 6R-O-vanilloyl ester named urphoside B (assigned
structure 7; [R]23D -122) isolated from Veronica pectinata L. var.
glandulosa Riek ex. M.A.6 and a compound named 6-O-p-
hydroxybenzoyl asystasioside E (6d; [R]26 -71), isolated from
D
“Catalpae fructus” from a Catalpa species (Bignoniaceae).7 Of these
two compounds, the former had been assigned the 6R-configuration
by analysis of coupling constants and a NOE correlation between
H-6 and H-7 as well as another between H-7 and H-9, which
indicated the cis-disposition of these three atoms. Compound 6d
has been assigned with a 6ꢀ-substituent, solely using the NOE
technique. In view of the similarity of the NMR data for the four
compounds (Table 1), which were almost identical except for the
signals arising from the differing acyl moieties, and since the
specific optical rotations were similar, it was assumed that they all
had the same relative configuration. It has been demonstrated8,9
previously that the configuration of a 6-O-substituent in iridoid
glucosides has considerable effects (1-3 ppm) on the resultant
chemical shifts of C-1, C-3, and C-4. Furthermore, it has been
shown more recently that determination of the stereochemistry of
iridoids using NOE correlations can be uncertain.10-12 By rein-
spection of the original 1H NMR spectrum of urphoside B we could
conclude that the measured coupling constants for J5,6 (12.5 and
7.3 Hz) instead should be 4.9 and 7.3 Hz, respectively, the confusion
Iridoid glucosides containing a chlorine atom have been found
previously in the plant family Plantaginaceae. Besides 6c, the
chlorohydrin linarioside has been reported from Linaria japonica
Miq.16 together with the corresponding epoxide anthirrinoside, while

Notes
Journal of Natural Products, 2010, Vol. 73, No. 9 1595
Longifolioside B (6b). Chromatography on a Merck HiBar column
Table 2. Antioxidative Activities of Compounds 6a and 6b as
Expressed by 50% Inhibition Concentrations (IC50, µg/mL) in
Vitro
(250-25) packed with LiChrosorb RP-18 gave the pure compound as a
1
colorless, amorphous solid: [R]25 -84 (c 0.3; MeOH); H and 13C
D
NMR, Table 1; LC-HR ESIMS m/z 583.1166 [M + Na]+ (calcd for
compound
DPPH
SO
NO
C24H2935ClNaO13, 583.1194).
6a
6b
27
19
<10
<10
<10
92
199
377
68
149
285
238
96
>1000
Treatment with Sodium Hydroxide. Sodium (5 mg) was dissolved
in D2O (0.5 mL), and an excess was added to NMR tubes (5 mm)
containing 6a and 6b dissolved in D2O. The reactions were monitored
by recording NMR spectra at 300 MHz.
DPPH Radical-Scavenging Effect. The DPPH radical-scavenging
effect of compounds 6a and 6b was assessed spectroscopically by the
decoloration of a methanol solution of 2,2-diphenyl-1-picrylhydrazyl
(DPPH). A MeOH solution (100 µL) of the compounds at various
concentrations was added to a DPPH-MeOH (80 µg/mL) solution,
and the absorbance of the remaining DPPH was measured at 520 nm
after 30 min. The radical-scavenging activity was determined by
comparing the absorbance with that of blank (100%) containing only
DPPH and solvent.24,25
Superoxide Radical-Scavenging Effect by an Alkaline DMSO
Method. The method of Kunchandy and Rao26 was used for the
detection of superoxide radical-scavenging activity with slight modi-
fication. To the reaction mixture containing 10 µL of NBT (1 mg/mL
solution in DMSO) and 30 µL of the extract or standard compounds
dissolved in DMSO was added 100 µL of alkaline DMSO (1 mL of
DMSO containing, 5 mM NaOH in 0.1 mL of water) to give a final
volume of 140 µL, and the absorbance was measured at 560 nm using
a microplate reader.26,27
Nitric Oxide Scavenging Effect. In order to determine NO radical-
scavenging activity of the compounds, 60 µL of 10 mM sodium
nitroprusside, dissolved in phosphate-buffered saline (PBS), was added
to 60 µL of serial diluted sample. After incubation under light at room
temperature for 150 min, an equal volume of Griess reagent (1%
sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5%
H3PO4) was added into each well in order to measure the nitrite content.
After 10 min, the chromophore was formed, and an absorbance at 577
nm was measured in a microplate reader.24,28
BHAa
quercetin
AAb
13
a Butylated hydroxyanisole. b Ascorbic acid.
asystasioside E (6) and baldaccioside, the 10-O-cinnamoyl ester
of 6, have been reported from Wulfenia baldaccii Degen.17 As it
is evident also in the present investigation, the iridoid chlorohydrins
are consistently found together with the corresponding epoxides,18
and the latter are therefore likely to be their precursors. It is,
however, a possibility that the esters could be formed from 6 in
parallel with the formation of the catalpol esters from 5.
The two new compounds longifoliosides A (6a) and B (6b) were
tested for radical-scavenging activity against nitric oxide (NO),
superoxide (SO), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radi-
cals. Iridoid glucosides are known for their low radical-scavenging
activity.19 However, the tested compounds showed radical-scaveng-
ing activity comparable to that of the standard compounds butylated
hydroxyanisole (BHA), quercetin, and ascorbic acid (Table 2).
These results show that the dihydroxybenzoyl and caffeoyl substitu-
tion of the iridoid glycosides increase the radical-scavenging
activities of the compounds.
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 polarimeter. 13C NMR spectra were recorded
1
at 25 °C on a Varian Mercury-300 instrument. 1D H and 2D DQF-
COSY, gHSQC, gHMBC, and NOESY NMR spectra were recorded
similarly on a Varian Unity Inova 500 MHz spectrometer. Spectra were
recorded in CD3OD, and the chemical shifts are given as δ values with
reference to the solvent peaks (δH 3.31 or δC 49.0), respectively. The
mixing time used in the NOESY spectra was 600 ms. LC-HRESIMS
was performed on an Agilent HP 1100 liquid chromatograph equipped
with a BDS-C18 reversed-phase column running a water-acetonitrile
(50 ppm TFA in water) gradient. The LC was coupled to a LCT of a
TOF MS (Micromass, Manchester, UK) operated in the positive
electrospray ion mode using 5-leucine-enkephalin as lock mass.
Chromatography was performed on a Merck Lobar RP-18 column (size
B) eluting with H2O-MeOH mixtures (1:0 to 1:1). Compounds are
listed in order of elution. The amount of mannitol (1) was estimated
from the 13C NMR spectrum of the crude sugar fraction. The known
compounds isolated were identified by comparison with their published
NMR data: mannitol (1),20 iridoids 2-6,21 verproside (5a),22 vermi-
noside (5b).23
Plant Material. Seeds of Veronica longifolia were obtained from
the Botanic Garden of the University of Hohenheim, Stuttgart, Germany.
The plants were grown in a greenhouse at the experimental field of the
Botanical Garden of The University of Copenhagen in Tåstrup, being
sown in April and harvested when flowering in September 2008. The
voucher (IOK-1/2008) was verified by Prof. Dirk Albach and deposited
in the Herbarium of Johannes Gutenberg-Universita¨t Mainz, Germany.
Extraction and Isolation. Fresh plant material (100 g) was blended
in ethanol (500 mL) and the extract taken to dryness. The concentrated
extract was partitioned in Et2O-H2O. The aqueous phase was taken to
dryness (5.3 g) and an aliquot (1.0 g) separated by preparative reversed-
phase chromatography to give a sugar fraction (490 mg) consisting
mainly of mannitol (1); gardoside (3, 50 mg); 8-epiloganic acid (2, 40
mg); a 2:1 mixture of catalpol and asystasioside E (5 and 6, 50 mg);
aucubin (4, 25 mg); verproside (5a, 80 mg); longifolioside A (6a, 40
mg); a mixture of unidentified compounds (60 mg); verminoside (5b,
60 mg); and longifolioside B (6b, 15 mg).
Acknowledgment. We thank the staff of The Botanic Garden, The
University of Copenhagen, for growing the plant material, Dr. K. F.
Nielsen, BioCentrum, DTU, for recording the mass spectra, and Prof.
D. Albach, Carl von Ossietzky-Universita¨t Oldenburg, for verifying
the plant material.
Supporting Information Available: NMR spectra (1H, 13C) of
longifoliosides A (6a) and B (6b) are available free of charge via the
Internet at http://pubs.acs.org.
References and Notes
(1) Olmstead, R. G.; DePamphilis, C. W.; Wolfe, A. D.; Young, N. D.;
Elisons, W. J.; Reeves, P. A. Am. J. Bot. 2001, 88, 348–361.
(2) Albach, D. C.; Meudt, H. M.; Oxelman, B. Am. J. Bot. 2005, 92, 297–
315.
(3) Grayer-Barkmeijer, R. J. Biochem. Syst. Ecol. 1978, 6, 131–137.
(4) Taskova, R. M.; Albach, D. C.; Grayer, R. J. Plant Biol. 2004, 6,
673–682.
(5) Jensen, S. R.; Albach, D. C.; Ohno, T.; Grayer, R. J. Biochem. Syst.
Ecol. 2005, 33, 1031–1047.
(6) Harput, U. S.; Nagatsu, A.; Ogihara, Y.; Saracoglu, I. Z. Naturforsch.
2003, C58, 481–484.
(7) Machida, K.; Ogawa, M.; Kikuchi, M. Chem. Pharm. Bull. 1998, 46,
1056–1057.
(8) Damtoft, S.; Jensen, S. R.; Nielsen, B. J. Phytochemistry 1981, 20,
2717–2732.
(9) Bianco, A.; Passacantili, P.; Polidori, G. Gazz. Chim. Ital. 1983, 113,
829–834.
(10) Franzyk, H.; Jensen, S. R.; Stermitz, F. R. Phytochemistry 1998, 49,
2025–2030.
(11) Franzyk, H.; Jensen, S. R.; Thale, Z.; Olsen, C. E. J. Nat. Prod. 1999,
62, 275–278.
(12) Jensen, S. R.; Cal1s, I.; Gotfredsen, C. H.; Søtofte, I. J. Nat. Prod.
2007, 70, 29–32.
(13) Demuth, H.; Jensen, S. R.; Nielsen, B. J. Phytochemistry 1989, 28,
3361–3364.
(14) Sudo, H.; Ide, T.; Otsuka, H.; Hirata, E.; Takushi, A.; Takeda, Y.
Longifolioside A (6a). Repeated chromatography gave the pure
compound as a colorless, amorphous solid: [R]25D -102 (c 0.4; MeOH);
1H and 13C NMR, Table 1; LC-HR ESIMS m/z 557.1015 [M + Na]+
(calcd for C22H2735ClNaO13, 557.1038).
Phytochemistry 1997, 46, 1231–1236.
(15) Flora Europea (http://193.62.154.38/FE/fe.html).

1596 Journal of Natural Products, 2010, Vol. 73, No. 9
Notes
(16) Kitagawa, I.; Tanai, T.; Akita, K.; Yosioka, I. Chem. Pharm. Bull.
1973, 21, 1978–1987.
(17) Taskova, R. M.; Gotfredsen, C. H.; Jensen, S. R. Phytochemisty 2005,
66, 1440–1447.
(18) Jensen, S. R.; Nielsen, B. J. Phytochemistry 1989, 28, 811–816.
(19) Pacifico, S.; D’Abrosca, B.; Pascarella, M. T.; Letizia, M.; Uzzo,
P.; Piscopo, V.; Fiorentino, A. Bioorg. Med. Chem. 2009, 17, 6173–
6179.
(24) Harput, U. S.; Saracoglu, I.; Inoue, M.; Ogihara, Y. Biol. Pharm. Bull.
2002, 25, 483–486.
(25) Kostadinova, E. P.; Alipieva, K. I.; Kokubun, T.; Taskova, R. M.;
Handjieva, N. V. Phytochemistry 2007, 68, 1321–1326.
(26) Kunchandy, E.; Rao, M. N. A. Int. J. Pharm. 1990, 58, 237–240.
(27) Srinisavan, R.; Chandrasekar, M. J. N.; Nanjan, M. J.; Suresh, B. J.
Ethnopharmacol. 2007, 113, 284–291.
(28) Hensley, K.; Mou, S.; Pye, Q. N. In Methods in Pharmacology and
Toxicology: Methods in Biological OxidatiVe Stress; Hensley, K.,
Floyd, R. A., Eds.; Humana Press Inc.: Totowa, NJ, 2003; pp
185-193.
(20) Bock, K.; Pedersen, C. AdV. Carbohydr. Chem. Biochem. 1983, 41,
27–66.
(21) Rønsted, N.; Go¨bel, E.; Franzyk, H.; Jensen, S. R.; Olsen, C. E.
Phytochemistry 2000, 55, 337–348.
(22) Afifi-Yazar, F. U.; Sticher, O. HelV. Chim. Acta 1980, 63, 1905–1907.
(23) Sticher, O.; Afifi-Yazar, F. U. HelV. Chim. Acta 1979, 62, 535–539.
NP100366K