Verbascoside, synthetic derivatives and other glycosides from Argentinian native plant species as potential antitumoral agents

Article abstract of DOI:10.1080/14786419.2019.1710701

A phytochemical study was performed on three native plant species from the central-western zone of Argentina: Buddleja cordobensis Grisebach, Baccharis salicina Torr. & A. Gray and Nepeta cataria L. We could obtain verbascoside (1) from B. cordobensis. From N. cataria, we could obtain 1, 5, 9-epi-deoxyloganic acid (2) L. Finally, we could isolate 2-β-(L-rhamnopyranosyl)-3-angeloyloxy-15-acetyloxy-7,13(14)-E-dien-ent-labdane (3) and 2-β-(L-rhamnopyranosyl)-3-α-angeloyloxy-15-hydroxy-7,13(14)-E-dien-ent-labdane (4) from B. salicina. Moreover, three derivatives from 1, and one semi-synthetic derivative from 2, were prepared. PCR reaction was used to analyse the activity against DNA polymerase and cell culture to determine cytotoxicity and antitumoral activity. Verbascoside (1) was strongly active in the nanomolar scale (IC₅₀ = 356 nM) against DNA polymerization. Moreover, verbascoside was also strongly active in the nanomolar scale against human melanoma cell line (IC₅₀ = 256 nM) and human colorectal cell line (IC₅₀ = 320 nM). Furthermore, derivatives 6 and 7 were cytotoxic against both cancer cell lines.

Full text of DOI:10.1080/14786419.2019.1710701

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Verbascoside, synthetic derivatives and other
glycosides from Argentinian native plant species
as potential antitumoral agents
Hugo A. Garro, Ezequiel Bruna-Haupt, Valeria Cianchino, Florencia Malizia,
Silvina Favier, Mauricio Menacho-Márquez, Diego Cifuente, Claudio O.
Fernández & Carlos R. Pungitore
To cite this article: Hugo A. Garro, Ezequiel Bruna-Haupt, Valeria Cianchino, Florencia
Malizia, Silvina Favier, Mauricio Menacho-Márquez, Diego Cifuente, Claudio O. Fernández
& Carlos R. Pungitore (2020): Verbascoside, synthetic derivatives and other glycosides from
Argentinian native plant species as potential antitumoral agents, Natural Product Research, DOI:
10.1080/14786419.2019.1710701
To link to this article: https://doi.org/10.1080/14786419.2019.1710701
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1710701
SHORT COMMUNICATION
Verbascoside, synthetic derivatives and other glycosides
from Argentinian native plant species as potential
antitumoral agents
Hugo A. Garroa,b, Ezequiel Bruna-Haupta, Valeria Cianchinoa,c, Florencia Maliziab,
a,c
b
a
ꢀ
Silvina Favier , Mauricio Menacho-Marquez , Diego Cifuente , Claudio O.
b
a
ꢀ
Fernandez and Carlos R. Pungitore
b
aꢀ
ꢀ
ꢀ
Area de Quımica Organica, INTEQUI-CONICET-UNSL, San Luis, Argentina; Max Planck Laboratory for
Structural Biology, Chemistry and Molecular Biophysics of Rosario (MPLbioR, UNR-MPIbpC), and
ꢀ
Instituto de Investigaciones para el Descubrimiento de Farmacos de Rosario (IIDEFAR, UNR-CONICET),
cꢀ
ꢀ
Universidad Nacional de Rosario, Rosario, Argentina; Area de Farmacotecnia, Facultad de Quımica,
ꢀ
Bioquımica y Farmacia, Universidad Nacional de San Luis, San Luis, Argentina
ABSTRACT
ARTICLE HISTORY
Received 7 August 2019
Accepted 26 December 2019
A phytochemical study was performed on three native plant spe-
cies from the central-western zone of Argentina: Buddleja cordo-
bensis Grisebach, Baccharis salicina Torr. & A. Gray and Nepeta
cataria L. We could obtain verbascoside (1) from B. cordobensis.
From N. cataria, we could obtain 1, 5, 9-epi-deoxyloganic acid (2)
L. Finally, we could isolate 2-b-(L-rhamnopyranosyl)-3-angeloyloxy-
15-acetyloxy-7,13(14)-E-dien-ent-labdane (3) and 2-b-(L-rhamno-
pyranosyl)-3-a-angeloyloxy-15-hydroxy-7,13(14)-E-dien-ent-labdane
(4) from B. salicina. Moreover, three derivatives from 1, and one
semi-synthetic derivative from 2, were prepared. PCR reaction was
used to analyse the activity against DNA polymerase and cell cul-
ture to determine cytotoxicity and antitumoral activity.
Verbascoside (1) was strongly active in the nanomolar scale (IC50
¼ 356nM) against DNA polymerization. Moreover, verbascoside
was also strongly active in the nanomolar scale against human
melanoma cell line (IC50 ¼ 256nM) and human colorectal cell line
(IC50 ¼ 320nM). Furthermore, derivatives 6 and 7 were cytotoxic
against both cancer cell lines.
KEYWORDS
Glycosides; DNA
polymerase; Cancer cell
lines; Antitumoral agents;
Natural Products
CONTACT Hugo A. Garro
hgarro@unsl.edu.ar; Carlos R. Pungitore
carlos.pungitore@conicet.gov.ar
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1710701.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
H. A. GARRO ET AL.
1. Introduction
DNA polymerases are enzymes directly implicated in the genetic material synthesis
and replication. The inhibition of these proteins locks DNA synthesis and could affect
the unlimited replicative potential of malignant cells, and therefore antitumoral activity
(Pungitore 2014; Garro et al. 2015). Natural products may act as precursors or tem-
plates for pharmaceutical drugs development (Newman and Cragg 2007; Kabera
et al. 2014).
In continuation of our drug discovery schedule of antitumoral agents from
Argentinean medicinal herbs, our aim here was to elucidate the potential target of
action of verbascoside and related semi-synthetic derivatives, and other natural glyco-
sides, as possible polymerase inhibitors. To carry this out, we performed a phytochem-
ical study on three native plant species from Argentina: Buddleja cordobensis
Grisebach, Baccharis salicina Torr. & A. Gray and Nepeta cataria L.
2. Results and discussion
The iridoids present in the Genus Buddleja, are derivatives of aucubin that include
aucubin itself and conjugates with groups derivatives from shikimato as p-methoxycin-
namoylaucubin. Another large group of iridoids informed contains catalpol as a
fundamental structure; for example, catalpol, methylcatalpol, catalposide and
p-methoxycinnamoylcatalpol have been isolated from Buddleja globose Lam. Finally, a
third type of iridoids, which contain the ajugol skeleton, have been obtained from the
aerial parts from Buddleja japonica Hemsl., such as vanillylajugol, feruloylajugol and
buddlejoside (Pungitore et al. 2004; Khan et al. 2019). In previous work, we could
obtain different iridoids from Buddleja cordobensis Grisebach (Pungitore et al. 2004;
Garro et al. 2015) and a little account of per-O-acetylverbascoside from acetylated frac-
tions, but we could not isolate free verbascoside. In this new phytochemical study, B.

NATURAL PRODUCT RESEARCH
3
cordobensis Grisebach was found to be rich in glycosides, in the TLC analysis, showing
the major products in the methanolic extract. Using chromatographic methods from
the methanolic fractions from aerial parts of Buddleja cordobensis Grisebach we
obtained as amorphous brown powders three majority products. We used Si gel col-
umn chromatography using mixtures of n-chloroform/methanol in increasing polarities
to obtain Catalpol (726 mg), 6-epi-catalpol (533 mg) and verbascoside (1.050 gr). In our
knowledge, verbascoside was isolate for the first time in Buddleja cordobensis
Grisebach. Compound 1 (verbascoside) was identified (Figure S1) by spectroscopic
studies and using bibliographical data (Grisebach 1874; Gomez-Aguirre et al. 2012) as
1
a brown amorphous solid. The H NMR spectrum (200 MHz, MeOD) exhibited charac-
teristic signals arising from an ester of (E)-caffeic acid and 3,4-dihydroxyphenethyl alco-
hol moiety together with the signals for two trans-olefinic protons as AB system, dH
7.58 and 6.26 (JAB ¼ 16.0 Hz), a benzylic methylene proton dH 2.76 (t, 2 H, J ¼ 7.0 Hz).
Additionally, typical proton resonances for methyl group of rhamnose were observed
at dH 1.08 as doublet (d, 3 H, J ¼ 6.8 Hz). The 13C NMR data of compound 1 confirmed
the diglycosidic nature exhibiting two anomeric carbon resonances at dC 104.2 (b-glu-
cose) and 103.1 (a-rhamnose) and HSQC experiments confirm their corresponding
anomeric proton resonances at dH 5.18 (a-rhamnose) and 4.36 (b-glucose). All proton
1
1
1
and carbon resonances were assigned by H, H-COSY, H-13C HSQC and HMBC experi-
ments. The molecular formula of compound 1 was determined as C29H36O15 by the
spectroscopic data and ESI-MS: m/z 625.21 [M þ Hþ].
To increase the number of compounds for biological assays we prepare chemical
derivatives using verbascoside as substrate. The first transformation of verbascoside (1)
was started using acetone in acidic medium (Figure S1) to obtain an acetonide deriva-
tive (5) with 92% yield. The NMR spectra exhibited characteristic signals arising from a
1
typical 2,2-dimethyl-dioxolane moiety. The H NMR spectrum exhibited six new hydro-
gens at higher field between 1.3–1.5 ppm, and the 13C NMR spectrum showed a new
ketal carbon at 110.25 ppm (quaternary carbon) and signals at 26.79 and 28.55 ppm
attributed to two geminal methyl groups. It was assumed that only one cyclic ketal
protected regio- selectively positions 2ꢀand 3ꢀof L-rhamnose, since only these positions
have cis configuration in the rhamnosyl moiety. Moreover, the adjacent anomeric sig-
nal appeared now at 99.48 ppm instead of 103.11 ppm, and HMBC spectrum showed
correlations between sugar alcohol base hydrogen at 3.76 ppm with ketal Carbon at
110.25 ppm. Furthermore, with derivative 5 as substrate, a dimethyl derivative (6) was
obtained using Me2SO4 as alkylating agent, without affecting the 2,2-dimethyl-dioxo-
1
lane moiety previously incorporated with 79% yield (Figure S1). The H NMR spectrum
exhibited signals arising from two methyl groups, showing six new Hydrogens at dΗ
3.84 and prominent signals at 57.0 and 59.0 ppm in 13C spectra. Besides, methylation
of hydroxyl groups was carried out in a regio-selective manner. It seems that the phen-
olic hydroxyl groups were preferentially alkylated compared to sugar alcohols. One
methyl group on caffeoyl moiety was assumed to be positioned at the C-3ꢀ on the
basis of the deshielding of the H-7ꢀand H-8ꢀof a,b-unsaturated carbonyl, and the HMBC
cross-peak observed between O-CH3 Hydrogens and the aromatic Carbons (d 13C in
146.0–158.0). Instead, the methyl group on phenylethanoid moiety was assumed to be
positioned at the C-4. Furthermore, the subtle deshielding of aglycone Hydrogens at

4
H. A. GARRO ET AL.
position seven (b) and the HMBC correlation from methyl to aromatic Carbons suggest
another methyl attached at position three of phenylethanoid moiety.
In the last step, we produced compound 7 using verbascoside (1) as substrate and
ethyl bromide. We generated a regio-alkylation of the primary hydroxyl from glucose
(Figure S2). The selection of this position against phenolic and secondary hydroxyls
may be steric (ethyl is larger than methyl). The ethyl group was assumed to be
attached to the hydroxymethylene of the glucose unit based on the migration of the
1
signal attributed to C-5ꢀ. Furthermore, in the H NMR spectrum a new signal appeared
for two Hydrogens (dd, 2 H, J ¼ 8.0; 6.4 Hz) at 4.07 ppm and another signal at higher
field (t, 3 H, J ¼ 3.6 Hz) for three Hydrogens at 1.28 ppm.
The Nepeta genus phytochemistry has two main kinds of compounds. The first is
the nepetolactone chemotype, and the second is the 1,8-cineole and/or linalool che-
motype (Salehi et al. 2018). From Nepeta cataria L. we could isolate the iridoid 1, 5, 9-
epi-deoxyloganic acid, also called b–nepetanudoside (2). Compound 2 was identified
by spectroscopic studies and using bibliographical data (Murai et al. 1984). The 1H
NMR spectrum of compound 2 exhibited typical absorptions of iridoid glycosides.
Signals at dH 7.41 and 5.31 should be assigned to H-3 and H-1, respectively. H-1
appeared as a doublet (d, J ¼ 3.9 Hz) and H-3 as a singlet. Also, a doublet at dH 1.06
(d, J ¼ 6.9 Hz) corresponds to H-10. The 13C NMR spectra of 2 showed two absorptions
in the region 100-105 ppm. The signal at dC 100.9 and 104.1 correspond to C-1 and C-
1’, respectively. Then, using DMAP as a catalyst, the per-O-acetylated derivative (8) was
obtained as the sole product, 64% yield (Figure S3).
The most widespread secondary metabolites present in the genus Buddleja are cler-
odane and labdane diterpenoids, as well as triterpenoids of the oleanane series.
Moreover, kaurane diterpenoids, cinnamic acid esters, coumarin derivatives and flavo-
noids are frequent and bioactive (Abad et al. 2005). For example, hexane extracts from
Baccharis salicina Torr. & A. Gray were active against mice edema (Cifuente et al.
2001a). We could isolate from Baccharis salicina Torr. & A. Gray two ent-labdane
diterpene glycosides: 2-b-(L-rhamnopyranosyl)-3-a-angeloyloxy-15-acetyloxy-7,13(14)-E-
dien-ent-labdane (3) and 2-b-(L-rhamnopyranosyl)-3-a-angeloyloxy-15-hydroxy-7,13(14)-
E-dien-ent-labdane (4) (Figure S4). Interestingly, these products showed a L-rhamnosyl
moiety like verbascoside (Cifuente et al. 2001b).
Finally, we used all products to evaluate their potential antitumoral therapeutic
properties against Taq DNA polymerase, as possible DNA replication inhibitors.
Compound 1 showed a remarkable IC50 value of 356.20 1.15 nM, while compounds
4-7 had IC50 values equal to 104.83 0.94, 4.22 0.08, 3.08 0.29 and 3.96 0.77 lM,
respectively (Table S1 and Figure S7).
In order to determine the putative antitumoral activity of the compounds, their
effect on cell proliferation was evaluated in two different cancer cell lines. Two semi-
synthetic compounds (6 and 7), showed a significant reduction of cell proliferation in
the two tumor cell lines tested (Figure S9). Moreover, by the use of MTT assays, we
observed that these compounds effectively decreased cancer cell proliferation in vitro
in a dose dependent manner (Figure S6 and S5). Indeed, the IC50 values obtained for
the cell lines tested fit into the micromolar range (Table S2). Verbascoside (1), seemed
to be more effective than the others, affecting cell proliferation at lower doses in the

NATURAL PRODUCT RESEARCH
5
Table 1. Selectivity index.
Compound
Cell Line
7
6
1
A375 (melanoma)
0.8772
0.568
1.28159
It was made comparing the activities of compounds 1, 6 and 7 in A375 melanoma cell line with normal MDCK cells.
nanomolar range (Figure S9) (human melanoma cancer cell line IC50 ¼ 256 nM and
human colorectal cancer cell line IC50 ¼ 320 nM). Interestingly, the three compounds
(1, 6 and 7), showed polymerase inhibition and cytotoxic activity (Table S2 and S1).
The results obtained revealed that, except for compounds 2, 3 and 8, all the
assayed products were active below IC50 values of 125.0 lM in the polymerase inhib-
ition. Furthermore, the activity of compound 1 is very powerful, throwing inhibition
values to the nanomolar scale (Table S1 and Figure S7a). The activity of compound 1
cannot be improved with chemical transformations; however, it was possible to obtain
three derivatives with excellent activity. It is clear that the lack of free hydroxyl groups
in verbascoside derivatives and the increase of the hydrophobicity is an important rea-
son for inactivity or activity decrease. For glycosides 3 and 4, the lack of acetyl moiety
is clearly a determinant for activity. Compound 4 has a free hydroxyl group, showing
activity at 104.83 lM against DNA replication.
The analysis of the effect on tumor cells proliferation showed that some glycosides
have an important impact on cell growth (Figure S10), showing IC50 values at the
micromolar range (Table 1). For compound 4 there is some cytotoxicity with A375 cell
lines. Even more, verbascoside (1) exhibits a powerful activity in the nanomolar range
as DNA polymerase inhibitor (IC50 ¼ 356 nM) and cytotoxic agent (IC50 ¼ 256 nM).
Moreover, verbascoside has more activity against melanoma cancer cell lines than nor-
mal MDCK cells (Table 1), showing some selectivity. This finding makes verbascoside
to a possible candidate for further antitumoral development.
3. Conclusions
In this study, we have highlighted the pharmacological potential on the DNA replica-
tion by verbascoside, chemical derivatives from it, and 2-b-(L-rhamnopyranosyl)-3-
a-angeloyloxy-15-hydroxy-7,13(14)-E-dien-ent-labdane
against
DNA
polymerase.
Moreover, the antitumor effects at nanomolar level observed for the verbascoside
obtained from B. cordobensis suggest the development of new pharmacological stud-
ies in the future.
Acknowledgments
These subsidies are gratefully acknowledged. E. Bruna-Haupt thanks CONICET for a doctoral
grant. H. Garro thanks Max Planck Laboratory for Structural Biology, Chemistry and Molecular
ꢀ
Biophysics of Rosario for postdoctoral position. In addition, we want to thank to Dra. C. Garcıa
ꢀ
ꢀ
and Prof. V. Martın from IUBO (Spain) for their help in the use of HRMS; and Bioq. Joaquın
Morillas and Dr. G. Labadie for their help. We wish specially thank to Drs. L. Mascotti and M.
Juri-Ayub for DNA material gently provided. We appreciate language revision of the manuscript
by staff from the “Instituto de Lenguas, Universidad Nacional de San Luis”.

6
H. A. GARRO ET AL.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
ꢀ
This work was financially supported by UNSL (PROICO 02-2516), Agencia Nacional de Promocion
ꢀ
ꢀ
Cientıfica
y Tecnologica (PICT 2017-0785), CONICET (PIP 090-2015) and Alexander von
Humboldt Foundation.
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