New knowledge about old drugs; a cardenolide type glycoside with cytotoxic effect and unusual secondary metabolites from Digitalis grandiflora Miller

Article abstract of DOI:10.1016/j.fitote.2019.02.001

Phytochemical investigation of the aerial parts of Digitalis grandiflora Miller (Plantaginaceae) led to the isolation of an undescribed cardenolide type glycoside digigrandifloroside (1) along with five known compounds, rengyoside A (2), rengyoside B (3), cleroindicin A (4), salidroside (5), and cornoside (6), from its aqueous fraction of methanolic extract. Structures of the isolated compounds were determined by means of spectroscopic techniques. 1–6 were isolated for the first time from D. grandiflora. 2 and 3 are being reported for the first time from Digitalis genus and Plantaginaceae family with this study. This is the second report for occurrence of 4 from a Digitalis species. Cytotoxic activity of the aqueous fraction was also tested against HEp-2 (Human larynx epidermoid carcinoma) and HepG2 (Human hepatocellular carcinoma) cancer cell lines and L929 (Mouse fibroblast cell) non-cancerous cell line. Aqueous fraction showed stronger cytotoxicity on HEp-2 cells than HepG2. Therefore, the cytotoxic activity of 1, 2, 4, and 6 were tested against HEp-2 and L929 cell lines. 3 and 5 couldn't be tested due to their insufficient amount. 1 showed the highest cytotoxicity against HEp-2 cells with IC₅₀ value 10.1 μM when compared with the positive control, etoposide and 2–6 (IC₅₀ of etoposide; 39.5 μM).

Full text of DOI:10.1016/j.fitote.2019.02.001

Fitoterapia134(2019)73–80
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
New knowledge about old drugs; a cardenolide type glycoside with cytotoxic
effect and unusual secondary metabolites from Digitalis grandiflora Miller
Vahap Murat Kutluaya,⁎, Toshiaki Makinob, Makoto Inouec, Iclal Saracoglua
a Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, 06100, Sihhiye, Ankara, Turkey
b Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
c Laboratory of Medicinal Resources, School of Pharmacy, Aichi Gakuin University, 464-8650 Nagoya, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Digitalis species
Plantaginaceae
cardenolide glycosides
digigrandifloroside
antiproliferative activity
Phytochemical investigation of the aerial parts of Digitalis grandiflora Miller (Plantaginaceae) led to the isolation
of an undescribed cardenolide type glycoside digigrandifloroside (1) along with five known compounds, re-
ngyoside A (2), rengyoside B (3), cleroindicin A (4), salidroside (5), and cornoside (6), from its aqueous fraction
of methanolic extract. Structures of the isolated compounds were determined by means of spectroscopic tech-
niques. 1–6 were isolated for the first time from D. grandiflora. 2 and 3 are being reported for the first time from
Digitalis genus and Plantaginaceae family with this study. This is the second report for occurrence of 4 from a
Digitalis species. Cytotoxic activity of the aqueous fraction was also tested against HEp-2 (Human larynx epi-
dermoid carcinoma) and HepG2 (Human hepatocellular carcinoma) cancer cell lines and L929 (Mouse fibroblast
cell) non-cancerous cell line. Aqueous fraction showed stronger cytotoxicity on HEp-2 cells than HepG2.
Therefore, the cytotoxic activity of 1, 2, 4, and 6 were tested against HEp-2 and L929 cell lines. 3 and 5 couldn't
be tested due to their insufficient amount. 1 showed the highest cytotoxicity against HEp-2 cells with IC50 value
10.1 μM when compared with the positive control, etoposide and 2–6 (IC50 of etoposide; 39.5 μM).
1. Introduction
Recent studies showed the role of cardioactive glycosides in cancer
prevention and anticancer agents [22]. It was shown that Na-K ATPase
Plants have been used as folk medicines for a long time in history for
the therapy of a wide range health problems. Evaluation of anticancer
agents used in therapy, from 1940s to 2011, have shown that from 175
small molecular compounds approved 85 of them are natural products
or derived from natural sources [23].
Although, there is no traditional use of genus Digitalis L., its first
known use as a medicine was in 18th century by Withering. Nearly
more than a half-century Digitalis species and Digitalis-derived cardi-
oactive glycosides have been investigated for their effects in heart
failure. Their toxicities and therapeutic effects were identified in de-
tailed [18].
In the flora of Turkey, the genus Digitalis is presented by 9 species, 5
of which are endemic [5]. Digitalis species was formerly a member of
Scrophulariaceae family, but after phylogenetic and chemotaxonomic
studies it has been moved to Plantaginaceae. Cardioactive glycosides,
phenylethanoid glycosides, flavonoids, and anthraquinones were re-
ported from the genus Digitalis so far [16–18,27].
pump α-1 subunit can play a role in proliferation and migration in
cancer cells. The results of the study indicated that in cancer cells there
became an over expression of α-1 subunit compared to normal cells. So,
targeting this subunit could be an option in cancer therapy [22]. Some
other reports also suggested different mechanisms for the cytotoxicity.
Apoptosis, autophagy and immunologic cell death were also reported
for cardioactive glycosides [30]. There are several reports about in vitro
cytotoxic activity of cardioactive glycosides against different human
cancer cell lines [27,28]. Fujino et al. [7] pointed out the selectivity
between cancer cells and non-cancerous cells in their report. A cardi-
oactive glycoside glucodigifucoside showed selective high cytotoxicity
against ACHN (human renal adenocarcinoma cell line) when compared
with HK-2 (normal human renal proximal tubule-derived cell line) [7].
Digitalis species also reported to have emetic, antibacterial, anti-
viral, antifungal, antiinflammatory and antioxidant activities
[3,11,21,24,25,33].
The purpose of our study is the isolation of secondary metabolites
with cytotoxic activity from the aerial part of D. grandiflora. D. grand-
iflora is an annual or perennial herb growing wildly in north-west
Cardioactive glycosides show their effect on myocardial cells by
binding to Na-K ATPase pump to inhibit this ion transport enzyme [15].
⁎ Corresponding author.
E-mail address: muratkutluay@hacettepe.edu.tr (V.M. Kutluay).
https://doi.org/10.1016/j.fitote.2019.02.001
Received 6 December 2018; Received in revised form 2 February 2019; Accepted 3 February 2019
Availableonline06February2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

V.M. Kutluay, et al.
Fitoterapia134(2019)73–80
Turkey and Balkan region. There are only a few studies about the
phytochemical content of the plant so far. Therefore, to clarify the
chemical content, phytochemical studies were planned to perform.
Bioactivity tests were also carried out according to the recent research
interest on Digitalis species and cardenolide type glycosides. In this
study, cytotoxicity was tested against both cancer (HEp-2, HepG2) and
non-cancerous (L929) cell lines to determine the selectivity. This se-
lectivity is important for the compounds to become an anticancer drug
candidate.
chromatography and eluted with H2O and continued by a gradient
system with increasing ratios of MeOH from 25 to 100% MeOH to yield
five different fractions (Fr. A, 21.3 g; Fr. B, 1.8 g; Fr. C, 0.50 g; Fr. D,
0.20 g; Fr. E, 0.10 g). Further chromatographic methods applied on Fr.
A, which is the most cytotoxic fraction among the all fractions, to yield
1–6. Fr.A was dissolved in H2O and liquid-liquid extraction was per-
formed using n-BuOH. 1 g of n-BuOH fraction (total 8.2 g) was sub-
jected to silica gel column chromatography with a stepwise gradient
system of CHCl3: MeOH: H2O (100:0:0–50:50:5). Isolation studies on
subfractions yield to 1–6.
2. Experimental
1 and 5 were isolated from the silicagel column subfraction ob-
tained by CHCl3: MeOH: H2O (75:25:2.5). Subfraction (65 mg) was
applied to vacum liquid chromatography and eluted with MeOH: H2O.
5 (6.5 mg) was eluted with 16% MeOH, 1 (9.4 mg) was eluted with 45%
MeOH.
2 were isolated from the silicagel column subfraction obtained by
CHCl3: MeOH: H2O (65:35:3.5). Subfraction (109 mg) was applied to
vacum liquid chromatography and eluted with MeOH: H2O. 2 (8.7 mg)
was eluted with 8% MeOH.
3 and 6 were isolated from the silicagel column subfraction ob-
tained by CHCl3: MeOH: H2O (70:30:3). Subfraction (140 mg) was ap-
plied to vacum liquid chromatography and eluted with MeOH: H2O.
Elution with 10% MeOH 3 (5.8 mg) and 6 (7 mg) were isolated.
4 was isolated from the silicagel column subfraction obtained by
CHCl3: MeOH: H2O (80:20:2). Subfraction (169 mg) was applied to
sephadex column chromatography and then applied to vacum liquid
chromatography and eluted with MeOH: H2O. 4 (99.2 mg) was eluted
with 5% MeOH.
2.1. General experimental procedures
Optical rotations were recorded on a JASCO P-2100 polarimeter.
HR-ESIMS data were obtained from Bruker Daltonics APEX III. 1H-NMR
(500 MHz), 13C-NMR (125 MHz) and 2D-NMR spectra of the com-
pounds were measured in CD3OD with an Agilent Varian VNS500
spectrometer. Chemical shifts (ppm) were referenced to the residual
solvent peaks (δH 3.31 and δC 49.0). Tetramethylsilane was used as
internal standard.
2.2. Chemicals/reagents and cell culture material
Column chromatography separations were carried out with poly-
amide (Polyamide 6, 50–160 μm; obtained from Sigma-Aldrich; St.
Louis, MI, USA), silica gel (Kieselgel 60, 70–230 mesh, 0.063–0.2 μm,
from Merck; Darmstadt, Germany) and RP silica gel (LiChroprep C18,
40–63 μm, Merck). D-glucose and L-rhamnose were obtained from
Sigma-Aldrich. Lanatoside C was purchased from Fluka AG. TLC plates
Silica gel 60 F254, RP18 F254 and all solvents were obtained from Merck.
Etoposide was purchased from Deva Holding A.S. (Istanbul, Turkey).
Fetal bovine serum (FBS), minimal essential medium with Earle's salts
(MEM-Earle) and antibiotics (penicillin and streptomycin) were ob-
tained from Biochrom AG (Berlin, Germany). Trypsin/EDTA solution
was purchased from Merck. Dulbecco's Modified Eagle's Medium
(DMEM), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide] and sodium dodecyl sulphate was purchased from Sigma (St
Louis, MI, USA). HEp-2 (Human larynx epidermoid carcinoma) cell line
was obtained from Riken Bioresource Center Cell Bank (Tokyo, Japan).
HepG2 (Human hepatocelular carcinoma) cells was obtained from
Health Science Research Resources Bank (Osaka, Japan). L929 (Mouse
fibroblast cell) cell line was purchased from Biota Lab (Istanbul,
Turkey).
2.5. Cytotoxic activity
HEp-2, HepG2 and L929 cells were used for cytotoxic activity tests.
Cells were seeded in a 96-well plate at a density of 1 × 105cells/mL for
HEp-2, 4 × 105 cells/mL for HepG2 and 8 × 104 cells/mL for L929
cells. MEM's Earle medium for HEp-2 and L929 cells and Dulbecco's
Modified Eagle's Medium (DMEM) for HepG2 cell were used.
Cells were cultured in respective culture media supplemented with
10% FBS, 1% penicillin-streptomycin solution in a humidified 5% CO2
in air at 37 °C for 24 h. Cells were treated with different concentrations
of samples for the next 48 h. After incubation, the cells were washed
and replaced by fresh medium. 10 μL of MTT solution (5 mg/mL in PBS)
was added and incubated for 4 h. After that, 100 μL of 10% SDS (sodium
dodecyl sulphate) was added to each well to dissolve formazan crystals.
The absorbance was measured at 570/620 nm using microplate reader.
Results were given as percentage of inhibitory effect treated cells to
untreated cells that served as control [29]. Three independent test re-
sults were considered, averages and standard deviations were calcu-
lated and shown in the figures.
2.3. Plant material
Digitalis grandiflora Miller was collected from Kırklareli, Turkey in
July, 2013. Identification of the plant was done by Dr. Z. Ceren Arituluk
(Hacettepe University Faculty of Pharmacy Department of
Pharmaceutical Botany, Ankara, Turkey). Voucher specimen was de-
posited in Hacettepe University Faculty of Pharmacy Herbarium
(HUEF13007).
2.6. Statistical analysis
Results of MTT experiments are expressed as mean
standard
2.4. Extraction and isolation
deviation. The statistical significance was determined by one- way
ANOVA post hoc Dunnett's test by using IBM SPSS Statistic 23 software.
The IC50 values were computed by GraphPad Prism 6.0 software
(GraphPad Software Inc., San Diego, CA, USA).
Aerial parts of D. grandiflora was dried in appropriate conditions.
Dried plant material was powdered, 290 g out of it was weighed and
macerated in 5 L MeOH overnight, then extracted for 8 h at 40 °C. This
procedure was repeated three more times. The extracts were combined
and dried by evaporating under vacuum at 40 °C. The methanol extract
was lyophilized to yield 37 g dry weight. The lyophilized extract was
dissolved in 400 mL of water and partitioned with petroleum ether to
discard lipophylic fractions. Aquoeus extracts (26 g) was used for cy-
totoxic activity tests and isolation studies.
2.7. Digigrandifloroside (1) (12-epidigoxigenin 3-O-β-D-glucopyranosyl
(1 → 4) β-D-digitoxopyranoside)
White amorphous powder; [α]2D2 + 7.0 (c 1.0, MeOH); 1H and 13C
NMR data, see Tables 1–3; HR-ESIMS: 705.34919 m/z [M + Na]+
(calculated for C35H54NaO13 705.34621).
The aquoeus extract (25 g) was subjected to polyamide column
74

V.M. Kutluay, et al.
Fitoterapia134(2019)73–80
Table 1
Table 2
1H (500 MHz) and 13C (125 MHz) NMR data and HMBC correlations of 1 in
13C NMR (125 Hz) data of isolated compounds 2–6 in CD3OD.
CD3OD.
2
3
4
5
6
Position Multiplicity δC ppm δH ppm
J (Hz)
HMBC (H → C)
Aglycon
δC
δC
δC
δC
δC
Aglycon
1.38–1.52b
1.59–1.70b
4.02 brs
1.29b
C-2, C-5, C-10
1
2
3
4
5
6
7
8
9
α
β
70.5
36.2
31.3
70.8
31.3
36.0
70.9
38.5
38.4
215.5
38.4
38.5
130.7
130.9
116.1
156.7
116.1
130.9
69.3
1
2
3
4
CH2
CH2
CH
31.3
27.5
74.5
31.0
59.1
45.6
70.8
36.0
31.3
70.8
31.3
36.0
154.5
127.9
187.9
127.9
154.4
C-1
C-5
C-5
CH2
1.44–1.52b
1.65b
5
6
CH
CH2
38.0
27.8
C-4, C-7
C-4, C-5, C-8
1.26b
1.90b
7
CH2
22.5
1.32b
C-5
C-8
C-11, C-14
C-14
1.77b
67.0
43.0
67.4
42.5
71.6
36.3
65.7
41.0
8
9
10
11
12
CH
CH
C
CH2
CH
42.8
30.2a
36.0
30.3a
76.5
1.63b
2.12 m
(10.0)
Glucose
1″
2′
3′
4′
5′
6′
104.4
75.1
78.1
71.6
78.0
62.8
104.9
75.6
78.7
72.2
78.5
63.3
104.3
75.1
78.1
72.1
77.9
62.7
104.3
75.0
78.1
71.6
77.9
62.7
1.55b
3.59 brs
C-8
C-9, C-11, C-14, C-
18
13
14
15
C
C
CH2
54.6
86.6
35.0
1.65b
2.27 m
1.82
C-13, C-14, C-17
C-16
16
CH2
30.2a
(min) of the derivatized sugar standards were as follows: D-glucose
(14.23), D-digitoxose (13.59). D-glucose and D-digitoxose were de-
tected in compound 1 by comparison with retention times of standard
sugars.
2.06 m
3.39b
0.83 s
C-14
C-22
17
18
CH
CH3
46.7
17.7
C-12, C-13, C-14, C-
17
C-5, C-10,
19
20
21
CH3
C
CH2
24.2
179.6
75.7
0.94 s
4.94 d
4.98 d
5.90 s
(18.5)
(18.5)
C-22
3. Results and discussion
22
23
CH
C=O
117.7
177.3
C-17, C-21
3.1. Structure elucidation of compounds
Digitoxose
1′
2′
Isolation studies carried out on aqueous fraction of methanolic ex-
tract of D. grandiflora afforded 6 pure compounds (1–6), one of which
was a new compound (1). Their chemical structures are shown in Fig. 1.
1, was obtained as a white amorphous powder, with the molecular
formula C35H54O13 was established by HR-ESIMS m/z 705.34919
[M + Na]+ in association with 35 carbon signals observed at 13C NMR
data. The 13C NMR and DEPT spectra revealed 35 carbon signals in-
cluding three methyl (δC 17.7, 18.6, 24.2; CH3), eleven methylene (δC
22.5, 27.5, 27.8, 30.2, 30.3, 31.0, 31.3, 35.0, 39.1, 62.3, 75.7; CH2),
sixteen methine (δC 30.2, 38.0, 42.8, 46.7, 68.6, 69.6, 71.1, 74.5, 75.1,
76.5, 77.7, 77.9, 84.3, 96.8, 105.8, 117.7; CH), and five quaternary (δC
36.0, 54.6, 86.6, 177.3, 179.6; C) carbons (Table 1). The detailed ex-
amination of HR-ESI-mass (MS), 1H, 13C, COSY, HMQC, HMBC, and
NOESY NMR spectra suggested this substance should be a cardenolide
type glycoside. All 1H and 13C NMR data and HMBC correlations were
given in Table 1. 1H NMR signals of α,β-unsaturated γ-lactone [δ 4.98
(1H, brd, J = 18.5 Hz, H-21b), 4.94 (1H, brd, J = 18.5 Hz, H-21a)
oxymethylene signals, and 5.90 (1H, brs, H-22) olefinic signal] have
been seen at 1H NMR spectrum. At 13C NMR spectrum the signals
corresponding to these protons [δ 117.7, CH, (C-22) and 75.7, CH2, (C-
21)] with C signals [δ 177.3, CO, (C-23) and 179.6 C, (C-20)] supported
the presence of a lactone ring in the structure. Two methyl signals at δ
0.83 (3H, s, H-18) and δ 0.94 (3H, s, H-19) and an oxygen bearing
quaternary carbon (δ 86.6, C-14) was determined. Signals of methyl
and methylene protons between δH 1.30–2.30 ppm were evaluated to-
gether with the data given above. Oxymethylene signals at δH 4.02 (brs,
H-3) and 3.59 (brs, H-12) ppm were also detected. The 13C signals
corresponding to these protons were determined using HMQC spectra.
Fragments were generated by means of 1H-1H COSY spectrum and they
were coupled up by using long range correlations observed in HMBC
spectrum. According to these findings, the aglycone of 1 was considered
to possess similar chemical structure with digoxigenin. Therefore, 1H
and 13C NMR spectral data was compared with the data of digoxigenin
[9,20,26] and it was recognized that the data of 1 showed discrepancy
CH
CH2
96.8
39.1
4.92 brd
1.74b
(8.0)
C-3, C-2′, C-3′
C-1′
C-4′
C-1′
1.95b
3′
CH
68.6
4.30 ddd
(3.0/3.0/
3.0)
4′
5′
6′
CH
CH
CH3
84.3
69.6
18.6
3.28b
3.87
1.30 d
C-3′, C-1″
C-4’
(6.5)
Glucose
1″
2″
3″
4″
CH
CH
CH
CH
CH
CH2
105.8
75.1
77.9
71.1
77.7
62.3
4.38 d
3.23 dd
3.34b
(8.0)
(8.0/8.0)
C-4′, C-2″
C-3″
C-1″
3.34b
C-3″, C-5″
5″
6″
3.28b
3.70 dd
3.82 dd
(12.0/5.0)
(12.0/5.0)
C-4″
a
These signals are exchangeable.
Signal coupling patterns are unclear due to overlapping.
b
2.8. Acid hydrolysis and configuration determination of sugars
Compound 1 (2 mg) was dissolved in 1 mL of methanol and hy-
drolyzed with 2 mL of 12% HCl at 80 °C. The mixture was extracted
with 2 mL of chloroform twice. The aqueous layer was dried under
vacuum. Residue was dissolved in 100 μL pyridine and 100 μL of N-
methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was added for
derivatisation. D-glucose was used as sugar standard. D-digitoxose was
obtained by hydrolyzation of the known compound lanatoside C. The
same acid hydrolysis procedure was applied to lanatoside C. Sugar
standards were also dissolved in 100 μL pyridine and 100 μL of MSTFA
was added for silylation. Samples were analyzed by GC/MS analysis.
Agilent 7890b Gas Chromatograph was used with HP-5 ms column
(30μmx250μmx0.25 μm). The injection volume was 1 μL. Samples were
applied to the column held initially at 40 °C for 1 min and then in-
creased to 260 °C with a 15 °C/min heating ramp. The retention times
75

V.M. Kutluay, et al.
Fitoterapia134(2019)73–80
Table 3
1H NMR (500 MHz) data of isolated compounds 2–6 in CD3OD.
2
3
4
5
6
Aglyco
δΗ ppm
J (Hz)
δΗ ppm
J (Hz)
δΗ ppm
3.74 t
1.70a
J (Hz)
δΗ ppm
7.06 d
6.69 d
J (Hz)
(8.5)
(8.5)
δΗ ppm
7.02 d
6.12dd
J (Hz)
2
3
1.41 t
1.72 m
1.66 m
(12.5)
2.03 m
1.85 m
2.68 m
2.20 d
(7.0)
(10.0)
(14.5)
(14.5)
(10.0/2.5)
4
5
3.52 m
1.66 m
2.68 m
2.20 d
2.03 m
1.85 m
1.43 td
1.73a
(12.0/4.0)
(12.0/4.0)
6.69 d
7.06 d
(8.5)
(8.5)
6.12dd
7.02 d
(10.0/2.5)
(10.0)
6
1.41 t
1.72 m
(12.5)
(7.5)
1.64a
7
8
9
α
3.52 m
1.64a
1.43 td 1.73a
4.08 m
3.71 m
1.77 t
4.13 m
3.77 m
1.90 t
4.03 m
3.70 m
2.83 m
3.99 m
3.62 m
2.05 t
β
(7.0)
(6.5)
Glucose
1′
2′
3′
4′
5′
6′
4.25 d
(7.5)
(7.5/8.5)
4.28 d
3.16 dd
3.35a
(7.5)
(7.5/9.0)
4.29 d
3.18 dd
3.35 t
(8.0)
(8.5/8.0)
(8.5)
4.22 d
3.14 dd
3.34 t
3.28 dd
3.26 m
3.85 brd
3.66 dd
(7.5)
(7.5/9.0)
(9.0)
3.15 dd
a
3.34
3.27
a
3.32a
3.30a
(7.5/9.0)
3.26 m
3.86 dd
3.65 d
3.31a
3.88 brd
3.66 brd
3.28 m
3.86 brd
3.66 dd
(11.5)
(11.5/5.0)
(12.0)
(12.0)
(12.0)
(12.0/5.0)
(12)
(12/5.5)
a
Signal patterns are unclear due to overlapping.
from the literature. The 13C signal belongs to C-18 methyl group (δ
17.7, CH3) at C-13 of 1 shifted downfield approximately 8 ppm and
signal of C-13 (δ 54.6, C) shifted downfield approx. 3 ppm [20,26]
(Table 1). The relative configuration of these groups was clarified by
the correlations observed in the NOESY spectrum. The key NOESY
correlations were shown in Fig. 2. The NOESY correlations were ob-
served between H-12 with H-18 in the spectrum. In addition, there was
no correlation observed between H-17 and H-18. These findings sug-
gested that the C-12 hydroxyl group should be attached to the skeleton
in α configuration which differs from digoxigenin. The 13C NMR data of
12- α hydroxyl and 12- β hydroxyl digoxigenin given in the literature
were compared with the data of compound 1 [9]. The chemical shifts
observed for C and D rings confirmed that hydroxyl group at C-12 is in α
configuration. 13C NMR spectra was compared with the literature data
and A/B and C/D connections were found as cis and B/C was trans. With
all the data mentioned above showed that aglycone was a 12 epimer of
digoxigenin.
Anomeric proton signals at δ 4.92 (H, brd, J = 8.0 Hz, H-1′) and
4.38 (H, d, J = 8.0 Hz, H-1″) at 1H NMR spectrum indicated the pre-
sence of a sugar moiety with two sugar unites. The coupling constants
of these anomeric signals explained that both sugar units are in β-
configuration. The signals: a methyl group at δ 1.30 (3H, d, J = 6.5 Hz,
H-6′); seven oxymethines at δ 3.23 (H, dd, J = 8.0, 8.0 Hz, H-2″), 3.28
(H, m, H-4′), 3.28 (H, m, H-5″), 3.34 (H, m, H-3″), 3.34 (H, m, H-4″),
3.87 (H, m, H-5′), 4.30 (H, ddd, J = 3.0, 3.0, 3.0 Hz, H-3′); a methylene
at δ 1.74 (H, m, H-2’a) and 1.95 (H, m, H-2’b), an oxymethylene at δ
3.70 (H, dd, J = 12.0, 5.0 Hz, H-6″a) and 3.82 (H, dd, J = 12.0, 2.0 Hz,
H-6″b). The sugar moiety was deduced as digitoxose and glucose using
1H-1H COSY spectrum and carbon signals were identified using HMQC
correlations. The signals at δH 1.74–1.95 ppm (H-2′) and δH 1.30 ppm
(3H, d, J = 6.5 Hz, H-6′) showed the 2,6 dideoxyose structure of digi-
toxose as the first sugar. The long range correlations observed between
H-1″ of glucose (δH 4.38, d) and C-4′ of digitoxose (δC 84.3) in the
HMBC spectrum indicated that the second sugar unite was attached at
the 4th position of the first sugar unite (Fig. 3, Table 1). Combining the
HMBC and COSY spectra the sugar moiety was identified as β- gluco-
pyranosil (1 → 4) β- digitoxopyranoside. HMBC correlation between H-
1′ and C-3, and the occurrence of H-3 oxymethine signal at δ 4.02 as a
broad singlet explained that the sugar unit connected to the aglycone at
C-3 in β-orientation. Configuration of the sugars were determined as D-
glucose and D-digitoxose by GC/MS analysis. Therefore, the structure of
1 was determined to be 12-epidigoxigenin 3-O- β-D-glucopyranosyl
(1 → 4) β-D-digitoxopyranoside and named as digigrandifloroside.
There is no literature data that the presence of 12-epidigoxigenin as a
naturally occuring aglycone in the cardenolide type glycosides so far.
Synthetic derivatives of 12-epidigoxigenin were reported in the litera-
ture [9]. Therefore, we have suggested this unusual aglycone was iso-
lated from nature for the first time with this study.
The known compounds were characterized as rengyoside A (2),
rengyoside B (3) [31], cleroindicin A (4) [32], salidroside (5) and
cornoside (6) [19] comparing their NMR and physical data with pub-
lished data in the literature. 1H and 13C NMR data of the known com-
pounds were given in Tables 2 and 3. Rengyosides A and B are being
reported for the first time from Digitalis species and Plantaginaceae
family with this study. This is the second report for the isolation of
cleroindicin A from Digitalis species, the first report was from D. trojana
[17].
3.2. Cytotoxic activity
The aqueous fraction of D. grandiflora methanolic extract was tested
for its cytotoxicity against Hep-2, HepG2 cancer cells and L929 non-
cancerous cells. Extract showed higher cytotoxicity with an IC50 value
of 63.7 μg/mL on Hep2 cells when compared to HepG2 cells with an
IC50 value of 514 μg/mL (Table 4). This difference showed that the
extract has selectivity between cancer cell lines. Therefore, Hep2 cells
were selected for the cytotoxicity tests of the isolated compounds. Cy-
totoxic activity of the extract was found to be lower on L929 cell line
with an IC50 value of 215 μg/mL (Table 4). The selectivity was also
observed between cancer and non-cancerous cell lines.
Digigrandifloroside (1), rengyoside A (2), cleroindicin A (4), and
cornoside (6) were evaluated for their cytotoxic activity against HEp-2
cancer cell line and L929 noncancerous cell line. Etoposide was used as
positive control as it is a semisynthetic derivative of a nature originated
compound, podophyllotoxin. IC50 values for rengyoside A (2), cler-
oindicin A (4) and cornoside (6) on HEp-2 cell line were found to be
76

V.M. Kutluay, et al.
Fitoterapia134(2019)73–80
Fig. 1. Structures of the isolated compounds from D. grandiflora
382, 2.56 × 103, 2.03 × 103 μM respectively. These compounds
showed concentration dependent low cytotoxicity against selected
cancer cell line. Their cytotoxicity on L929 cells were almost two folds
lower than they had on HEp-2 cells. IC50 values for rengyoside A (2)
and cornoside (6) on L929 cell line were found to be 1.20 × 103 and
3.77 × 103 μM, respectively (Table 4).
The new compound digigrandifloroside (1) showed concentration
dependent high cytotoxicity against HEp-2 cells which is significant
when compared with the positive control etoposide (Fig. 4, Table 4).
Digigrandifloroside has an IC50 value of 10.1 μM on HEp-2 cells and
IC50 value of 12.9 μM on L929 cells (Table 4). In comparison with an
anticancer agent etoposide, digigrandifloroside showed higher poten-
tial on HEp-2 cell line. In previous reports cardenolide type glycosides
were tested on several cell lines. Digoxin was tested against brain SH-
SY5Y, brain SK-N-AS, breast MDA-MB-231, breast MDA-MB-435, liver
Hep3B, prostate PC3, prostate PPC-1, and transformed human B-lym-
phocytes P493-Myc cancer cells [4]. SH-SY5Y and SK-N-AS neuro-
blastoma cell lines were found to be the most sensitive among the tested
77

V.M. Kutluay, et al.
Fitoterapia134(2019)73–80
Fig. 2. The key NOESY correlations of compound 1
Fig. 3. The key HMBC correlations of compound 1
digigrandifloroside when compared with other cardenolide glycosides.
When the cytotoxicity results evaluated in detailed, it could be seen
that digigrandifloroside showed biphasic effect, both cytotoxic and
cytostatic (antiproliferative), depends on concentration, on L929 cell
line (Figs. 5 and 6). A dramatical decrease in cell viability (cytotoxic
effect) observed till 5 μg/mL concentration corresponding to 7.33 μM
(cell viability; 52.51%). Cytostatic (antiproliferative) effect was ob-
served with the increase of digigrandifloroside concentration. Even at
100 and 200 μg/mL concentrations corresonding to 146 and 293 μM of
digigrandifloroside, viability of L929 cells were found as 41.0 and
36.4% respectively (Fig. 5). However, the viability % of HEp-2 cells at
100 and 200 μg/mL concentrations corresponding to 146 and 293 μM of
digigrandifloroside, were found as 1.23 and 1.09% respectively (Fig. 4).
These results showed that the cytostatic activity can't be seen on Hep-2
cells, as high cytotoxicity have been detected. Although the IC50 values
of digigrandifloroside were found to be similar on Hep-2 and L929 cells,
it has a type of activity selectivity between cancer and noncancerous
cells (Fig. 6).
Table 4
IC50 values of extract and compounds against HEp-2, HepG2 and L929 cell lines
Extract/Compounds
IC50 values
Cell lines
Hep-2
HepG2
L929
D. grandiflora extract
Digigrandifloroside (1)
Rengyoside A (2)
Cleroindicin A (4)
Cornoside (6)
63.7 μg/mL
10.1 μM
514 μg/mL
nd.a
215 μg/mL
12.9 μM
382 μM
nd.a
1.20 × 103 μM
2.58 × 103 μM
3.77 × 103 μM
45.2 μM
2.56 × 103 μM
2.03 × 103 μM
39.5 μM
nd.a
nd.a
a
Etoposide
nd
a
Not determined.
cell lines with the IC50 values of 34 and 22 ng/mL. The clinical studies
performed on digoxin at prostate cancer is in Phase II level [1]. Lana-
toside C was tested against U87 glioblastoma cells and the IC50 value
was calculated as 1 μg/mL [2].
Even there is no other study reported on HEp-2 cancer cells treated
with cardenolide glycoside so far, digigrandifloroside showed lower
cytotoxicity than digoxin and some other cardenolide glycosides against
cancer cells. In this study, IC50 of digigrandifloroside was found as
10.1 μM corresponding to 6.9 μg/mL. The epimerisation at C-12 posi-
tion could be responsible from the lower cytotoxic activity of
4. Conclusion
Studies on aqueous fraction of methanolic extract of D. grandiflora
afforded 1 new total 6 compounds. All compounds were isolated for the
first time from D. grandiflora. Among the isolated compounds, rengyo-
sides
A (2) and B (3) were reported in Digitalis genus and
78

V.M. Kutluay, et al.
Fitoterapia134(2019)73–80
Fig. 4. Screening cytotoxic activity of digigrandifloroside and etoposide on HEp-2 cell line using MTT method. Results are given as inhibition %, and expressed as
mean
S.D. (n = 3). ⁎p < 0.05 as compared to positive control etoposide.
Fig. 5. Screening cytotoxic activity of digigrandifloroside and etoposide on L929 cell line using MTT method. Results are given as inhibition %, and expressed as
mean
S.D. (n = 3). ⁎p < 0.05 as compared to positive control etoposide.
Structure of a new cardenolide glycoside named digigrandifloroside
(1) has been identified by advanced spectral analysis. Cytotoxic activity
tests performed on the extract and the isolated compounds.
Digigrandifloroside showed strong cytotoxicity on Hep-2 cancer cell
line with the IC50 value of 10.1 μM when compared with anticancer
agent etoposide used as a positive control. Digigrandifloroside also
showed selectivity between cancer and noncancerous cell lines. The
antiproliferative (cytostatic) effect was seen against noncancerous cells.
This selectivity is valuable for the future studies to evaluate the struc-
ture activity relationship of the epimerisation in C-12 of the cardenolide
glycosides.
Acknowledgement
This work was supported by Hacettepe University Scientific
Research Project Coordination Unit (grant number TDK-2016-12687).
Vahap Murat Kutluay was supported by two research grants from
The Scientific and Technological Research Council of Turkey (grant
numbers TUBITAK 2214/A, 2211).
Authors are thankful for the Kan'ichiro Ishiuchi from Department of
Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya
City University (Japan) for operating the NMR and HR-ESIMS analysis
and discussion of the digigrandifloroside's structure.
Fig. 6. Comparison of cytotoxic activity of digigrandifloroside on HEp-2 and
L929 cell lines. Results are given as cell viability %, and expressed as
mean
S.D. (n = 3).
Plantaginaceae family for the first time. In previous studies, rengyoside
A was isolated only from Oleaceae (Forsythia sp.) and Bignoniacae
(Millingtonia sp.) families, and rengyoside B was isolated from Oleaceae
(Forsythia sp.), Bignoniacae (Incarvillea, Markhamia, Millingtonia,
Santisukia, Tecoma sp.) and Lamiaceae (Clerodendrum sp.) families
[8,10,12–14,31,34]. This is the second time for cleroindicin A (4) being
reported from Digitalis species, the first report was from D. trojana [17].
Previous studies on rengyoside A and B showed that these structures
can be afforded by biogenesis like transformation starting from sali-
droside [6]. Salidroside firstly transform to cornoside, and then re-
ngyosides A and B occur respectively. While salidroside is distributed
widely in different plant families, the distribution of rengyosides A and
B is restricted. In the future studies, this type of compounds with
cleroindicin structure might be studied in detailed with the view of
chemotaxonomy of the related families.
Conflicts of interest
The authors declare that there is no conflict of interest.
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