Saponins from the roots of Chenopodium bonus-henricus L.

Article abstract of DOI:10.1080/14786419.2018.1483928

Two new glycosides of phytolaccagenin and 2β-hydroxyoleanoic acid, namely bonushenricoside A (3) and bonushenricoside B (5) together with four known saponins, respectively compounds 3-O-L-α-arabinopyranosyl-bayogenin-28-O-β-glucopyranosyl ester (1), 3-O-β-glucuronopyranosyl-2β-hydroxygypsogenin-28-O-β-glucopyranosyl ester (2), 3-O-β-glucuronopyranosyl-bayogenin-28-O-β-glucopyranosyl ester (4) and 3-O-β-glucuronopyranosyl-medicagenic acid-28-β-xylopyranosyl(1→4)-α-rhamnopyranosyl(1→2)-α-arabinopyranosyl ester (6) were isolated from the roots of Chenopodium bonus-henricus L. The structures of the compounds were determined by means of spectroscopic methods (1D and 2D NMR, IR and HRMS). The MeOH extract and compounds were tested for cytotoxic activity on five leukemic cell lines (HL-60, SKW-3, Jurkat E6-1, BV-173 and K-562). In addition, the ability of metanolic extract and saponins to modulate the interleukin-2 production in PHA/PMA stimulated Jurkat E6-1 cells was investigated as well.

Full text of DOI:10.1080/14786419.2018.1483928

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Saponins from the roots of Chenopodium bonus-
henricus L.
Zlatina Kokanova-Nedialkova, Paraskev T. Nedialkov & Georgi Momekov
To cite this article: Zlatina Kokanova-Nedialkova, Paraskev T. Nedialkov & Georgi Momekov
(2018): Saponins from the roots of Chenopodium bonus-henricus L., Natural Product Research,
DOI: 10.1080/14786419.2018.1483928
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2018.1483928
Saponins from the roots of Chenopodium bonus-henricus L.
Zlatina Kokanova-Nedialkovaa #, Paraskev T. Nedialkova # and Georgi Momekovb
aFaculty of Pharmacy, department of Pharmacognosy, Medical university of sofia, sofia, Bulgaria; bFaculty of
Pharmacy, department of Pharmacology, Pharmacotherapy andtoxicology, Medical university of sofia, sofia,
Bulgaria
ARTICLE HISTORY
received 4 March 2018
accepted 29 May 2018
ABSTRACT
Two new glycosides of phytolaccagenin and 2β-hydroxyoleanoic
acid, namely bonushenricoside A (3) and bonushenricoside B (5)
together with four known saponins, respectively compounds 3-O-L-α-
arabinopyranosyl-bayogenin-28-O-β-glucopyranosyl ester (1), 3-O-β-
glucuronopyranosyl-2β-hydroxygypsogenin-28-O-β-glucopyranosyl
ester (2), 3-O-β-glucuronopyranosyl-bayogenin-28-O-β-glucopyranosyl
ester (4) and 3-O-β-glucuronopyranosyl-medicagenic acid-28-β-
xylopyranosyl(1→4)-α-rhamnopyranosyl(1→2)-α-arabinopyranosyl
ester (6) were isolated from the roots of Chenopodium bonus-henricus L.
The structures of the compounds were determined by means of
spectroscopic methods (1D and 2D NMR, IR and HRMS). The MeOH
extract and compounds were tested for cytotoxic activity on five
leukemic cell lines (HL-60, SKW-3, Jurkat E6-1, BV-173 and K-562). In
addition, the ability of metanolic extract and saponins to modulate
the interleukin-2 production in PHA/PMA stimulated Jurkat E6-1 cells
was investigated as well.
KEYWORDS
Chenopodium bonus-henricus
l.; triterpene saponins;
cytotoxicity; interleukin-2
CONTACT Zlatina Kokanova-Nedialkova
pharmfac.mu-sofia.bg
#these authors were co-first author.
zlatina.kokanova@pharmfac.mu-sofia.bg; Paraskev t. Nedialkov
pnedialkov@
the supplemental data for this article is available online at https://doi.org/10.1080/14786419.2018.1483928.
© 2018 Informa uK limited, trading as taylor & Francis Group

2
Z. KOKANOVA-NEDIALKOVA ET AL.
1. Introduction
The genus Chenopodium (Amaranthaceae) includes about 150 species native mainly to sub-
tropical and temperate regions (Uotila and Tan 1997). The most widely recognised
Chenopodium species for their medicinal and nutrient properties are C. ambrosioides and C.
quinoa (Podolak et al. 2016; Shah and Khan 2017). Chenopodium bonus-henricus L. is a per-
ennial herbaceous plant and is spread in the mountainous regions of Bulgaria (Grozeva
2011). Its leaves and flowering tops are used as a vegetable in the same manner as spinach
in some European countries (Cheschmedzhiev et al. 1999; Toensmeier 2007). In Bulgarian
folk medicine the roots are known as‘chuven’and have been applied externally to treat skin
inflammations, wounds and boils. The infusion of the drug has been also used as a mild
laxative. In Bulgarian food industry the aqueous extract of the roots has been employed in
production of halva (Kokanova-Nedialkova et al. 2015). Recently phytochemical investigation
of the aerial parts of C. bonus-henricus L. led to the isolation of nine flavonoid glycosides of
patuletin, spinacetin and 6-methoxykaempferol (Kokanova-Nedialkova et al. 2017). From
the roots of title plant the ecdysteroids 20-hydroxyecdysone and polypodine B (Báthory et
al. 1982) as well as the flavonol glycosides 6-methoxykaempferol 3-O-[β-apiofuranosyl(1→2)]-
β-glucopyranosyl(1→6)-β-glucopyranoside, spinacetin 3-O-[β-apiofuranosyl(1→2)]-β-
glucopyranosyl(1→6)-β-glucopyranoside
and
spinacetin
3-O-gentiobioside
(Kokanova-Nedialkova et al. 2015) have been isolated. The flavonoids isolated from title plant
have exerted hepatoprotective and antioxidant activities comparable to those of silymarin
and silybine in in vitro model of CCl4 induced liver damage. They also possessed DPPH and
ABTS radical-scavenging activity and significantly inhibited the lipid peroxidation in a linoleic
acid system by the ferric thiocyanate method (Kokanova-Nedialkova et al. 2015, 2017;
Kokanova-Nedialkova and Nedialkov 2017). The present study deals with the isolation and
structural elucidation of two new and four known triterpene saponins. The cytotoxicity of
the MeOH extract and isolated compounds were evaluated on HL-60, SKW-3, Jurkat E6-1,
BV-173 and K-562 tumor cell lines. In addition, the ability of the metanolic extract and sap-
onins to modulate the interleukin-2 production in PHA/PMA stimulated Jurkat E6-1 cells
was investigated as well.
2. Results and discussion
An extensive chromatographic procedure of the combined MeOH and aq. MeOH extracts
from the roots of C. bonus-henricus led to the isolation and structural identification of six
saponins 1–6 (Figure 1). The structures of the compounds were elucidated by means of
spectral (MS, IR and NMR) methods and hydrolysis. The signals in the 1H and 13C NMR spectra
were unambiguously assigned using 2D NMR techniques. The known compounds were
identified as 3-O-α-arabinopyranosyl-bayogenin-28-O-β-glucopyranosyl ester 1, 3-O-β-
glucuronopyranosyl-2β-hydroxygypsogenin-28-O-β-glucopyranosyl ester 2, 3-O-β-
glucuronopyranosyl-bayogenin-28-O-β-glucopyranosylester4and3-O-β-glucuronopyranosyl-
medicagenic acid-28-β-xylopyranosyl(1→4)-α-rhamnopyranosyl(1→2)-α-arabinopyranosyl
ester 6, respectively by comparing their spectroscopic data with those reported in the liter-
ature. Compound 1 was initially found in Medicago arabica (Bialy et al. 2004). Compounds
2 and 4 have been previously isolated respectively from Spinacia oleracea (Mithöfer et al.
1999) and Meliosma lanceolata (Abe et al. 1996), respectively while 6 was found in Medicago
sativa (Oleszek et al. 1990).

NATURAL PRODUCT RESEARCH
3
Figure 1. structures of saponins 1–6, isolated from the roots of C. bonus-henricus l.
Compound 3 was obtained as optically active colourless crystals with m.p. 226–227 °C
and [ꢀ]2D0 +73.8 (c 0.100, MeOH). The IR spectrum showed absorption bands for hydroxyl
groups (3368 cm−1), C–H bonds (2923 cm−1), an ester group (1718 cm−1), carbonyl (1654 cm−1)
and C–O bonds (1050 cm−1). The molecular formula of 3 was established as C42H66O16 by
means of HR-ESI-MS, showing a formyl adduct ion [M + HCOO]− at m/z 871.4330.
The 13C NMR spectrum (Table S1) displayed 42 carbon resonances with two carbonyl
carbons appeared at δC 176.4 (C-28) and 177.3 (C-30) and two olefin carbon resonances
located at δC 124.1 (C-12) and 144.1 (C-13). Three resonances for carbons bearing oxygen
were observed at δC 71.3 (C-2), 83.0 (C-3) and 65.6 (C-23) in addition to one methoxy and 11
oxygenated carbon resonances of one hexose and one pentose sugars. HSQC spectrum
displayed signals corresponding to carbons, classified into 6 methyls, 13 methylenes, 14
methines and 9 quaternary carbons. The 1H NMR spectrum (Table S1) revealed the presence
of five tertiary methyl singlets at δH 1.33 (H-24), 1.60 (H-25), 1.17 (H-26), 1.23 (H-27), 1.18
1
(H-29), an O-methyl proton at δH 3.60 and an olefinic triplet δH 5.58 (H-12). The H NMR
spectrum also showed two hydroxymethine signals at δH 4.80 (H-2) and 4.34 (H-3) correlated
by HSQC with the carbon signals at δC 71.3 and 83.0. The β-orientation of the hydroxyls at
C-2 and C-3 was deduced from the coupling constant (J = 3.8 Hz) of H-2 and H-3. The HMBC
correlations of δH 1.33 (H-24) to the signal due to a hydroxymethyl carbon at δC 65.6 (C-23)
was used to place a hydroxymethyl group at C-4. Detailed analysis of the NMR spectra
pointed out phytolaccagenin (2β,3β,23‐trihydroxyolean‐12‐en‐28,30‐dioic acid 30‐methyl
ester) as aglycone (Spengel and Schaffner 1993; He et al. 2011). The 1H NMR spectrum showed
two anomeric sugar proton signals at δH 5.03 (7.7 Hz) and 6.31 (8.2 Hz) indicating a α- and
β-orientation, respectively of the glycosidic linkage. According to the proton and the carbon
resonances the sugar units were identified as α-arabinopyranose and β-glucopyranose (Bialy
et al. 2004). The HMBC correlations of anomeric proton signal δH 5.03 (H-1ꞌ) of arabinose

4
Z. KOKANOVA-NEDIALKOVA ET AL.
moiety with the carbon signal at δC 83.0 (C-3) indicated the attachment of arabinose at C-3
of the aglycone. The presence of a second monosaccharide moiety at C-28 was deduced
from the HMBC interactions of anomeric proton of glucose δH 6.31 (H-1″) with the signal of
δC 176.4 (C-28). The presence of L-arabinose and D-glucose was authenticated by alkaline
and acid hydrolysis and synthesis of their tolylthiocarbamoyl-thiazolidine derivatives (Tanaka
et al. 2007; Nedialkov et al. 2012). Thus, the structure of 3 was established as 2β,3β,23‐trihy-
droxyolean‐12‐en‐28,30‐dioic acid 30‐methyl ester 3-O-α-L-arabinopyranosyl-28-O-β-D-
glucopyranoside, named Bonushenricoside A. This compound is structurally similar to
esculentoside S that was isolated from Phytolacca acinosa (Spengel and Schaffner 1993).
Compound 5 was obtained as a colourless amorphous powder with[ꢀ]2D0: +17.9 (c 0.100,
MeOH). The IR spectrum indicated the presence of hydroxyl groups (3383 cm−1), C–H bonds
(2937 cm−1), an ester group (1729 cm−1), carbonyl (1653 cm−1) and C–O bonds (1063 cm−1).
The molecular formula of 5 was determined as C42H66O15 by means of HR-ESI-MS, showing
a [M − H]− ion at m/z 809.4328. The 1H NMR spectrum (Table S1) showed two hydroxymethine
signals at δH 4.73 (H-2) and 3.43 (H-3) correlated by HSQC with the carbon signals at δC 71.1
and 90.0. The β-orientation of the hydroxyls at C-2 and C-3 was deduced from the coupling
1
constant (J = 3.6 Hz) of H-3. The H NMR of 5 also revealed the presence of seven tertiary
methyl singlets at δH 1.36 (H-23), 1.43 (H-24), 1.51 (H-25), 1.16 (H-26), 1.30 (H-27), 0.92 (H-29),
0.89 (H-30) and an olefinic proton signal resonating at δH 5.43 (H-12). The last correlated in
the HSQC spectrum with a carbon signal at δC 123.5 (C-12) and together with the signal of
a quaternary carbon at δC 144.6 (C-13) indicated a typical ∆12 pentacyclic triterpene derivative
(De Tommasi et al. 1998). The 13C NMR spectrum (Table S1) displayed 42 carbon resonances
with one carbonyl carbon appeared at δC 176.9 (C-28), two olefin carbon resonances located
at δC 123.5 (C-12) and 144.6 (C-13) and two resonances for carbons bearing oxygen at δC
71.1 (C-2), 90.0 (C-3). The NMR spectra clearly pointed out 2β,3β-dihydroxyolean-12-en-28-
1
oic acid as the aglycone. In addition, the H NMR spectrum showed two anomeric sugar
proton signals at δH 5.12 (7.7 Hz) and 6.36 (8.0 Hz) indicating a β-orientation of the glycosidic
linkage of glucose and glucuronic acid. The former signal gave a cross-peak with C-3 (δC
107.4) of the aglycone, while the latter correlated with the signal of C-28 (δC 96.3). The pres-
ence of D-glucuronic acid and D-glucose was authenticated by alkaline and enzymatic
hydrolysis and synthesis of their tolylthiocarbamoyl-thiazolidine derivatives (Tanaka et al.
2007; Nedialkov et al. 2012). Thus, the structure of 5 was established as 2β,3β-dihydroxyolean-
12-en-28-oic acid 3-O-β-D-glucuronopyranosyl-28-O-β-D-glucopyranoside, named
Bonushenricoside B.
The methanolic extract of the plant, as well as compounds 1–6 were tested for cytotoxic
activity in a panel of human leukemic cell lines, representative for some important types of
human haematological malignancies, namely HL-60 (acute promyelocyte leukemia), SKW-3
and Jurkat E6-1 (T-cell leukemias), BV-173 and K-562 (chronic myeloid leukemias). The extract
displayed concentration-dependent cytotoxicity with IC50 values ranging 124.5–258 μg/ml.
The juxtaposition of the MTT-bioassay data unambiguously indicated that the aforementioned
cytotoxicity of the extract resides chiefly in compounds 1, 5 and 6, whereas the other tested
agents inhibited the growth of cultured malignant cells at substantially higher concentrations
with IC50 values close to or ever exceeding 500 μM (Table S2). Further, we sought to assess
the ability of the tested compounds and the total methanolic extract to modulate the
mitogen-induced production of IL-2 in Jurkat E6-1 (a well-established model of humanT-cell
activation) following sub-cytotoxic exposure. As evident from the data presented in Table

NATURAL PRODUCT RESEARCH
5
S3, the tested saponins stimulated the PHA/PMA-triggered release of IL-2 in Jurkat E6-1
human T cells by approximately 30–60% vs. the effects of the solvent alone. In this
experimental system compound 1 was the most potent of the series. Interleukine-2 is a
lymphokine with a well-appreciated role in the immune system. It stimulates clonal expansion,
proliferation and differentiation of native T-cells, activates the T-cells and natural killer-
mediated cytotoxicity, and thus is involved in immune responses (Hoyer et al. 2008; Olejniczak
and Kasprzak 2008).
3. Experimental
3.1. General experimental procedures
General experimental procedures are in the Supplementary material.
3.2. Plant material
The roots of Chenopodium bonus-henricus L. were collected from Beglica, Western Rhodopes,
Bulgaria in September 2011 and were identified by P. T. Nedialkov. A voucher specimen (No.
SOM-Co-169848) was deposited at the National Herbarium, Bulgarian Academy of Sciences,
Sofia, Bulgaria.
3.3. Extraction and isolation
The roots of C. bonus-henricus were dried in the shade and a powdered plant material (295 g)
was extracted subsequently with MeOH (3 L), 80% aq. MeOH (5 L) and H2O (1 L) by ultrason-
ic-assisted extraction. After filtration, the extracts were combined and the solvent was evap-
orated under reduced pressure to give 149 g white-yellow residue. The resulting residue was
dissolved in 200 mL H2O and then subjected to CC over Diaion HP-20 (45 × 4 cm) with H2O
(2 L) and 90% aq. MeOH (4 L) as eluants to obtain water and aq. methanol fractions. The
MeOH fraction (56 g) was purified using silica gel, MCI gel, RP-18 to afford pure pure (1)
(53 mg), (2) (64 mg), (3) (86 mg), (4) (41 mg). (5) (127 mg) and (6) (145 mg) (See a supple-
mentary material).
3.3.1. Bonushenricoside A (3) (3-O-α-L-arabinopyranosyl-phytolaccagenin-28-O-β-D-
glucopyranosyl ester)
White crystals, m.p. 226–227 °C; [ꢀ]D20 +73.8 (c 0.100, MeOH); IR (neat) υmax: 3368, 2923, 1718,
1654, 1050 cm−1; HR-ESI-MS: found m/z 871.4330 [M + HCOO]−, calcd. for C43H67O18 m/z 871.4322;
1H NMR (pyridine-d6, 600 MHz): (Table S1); 13C NMR (pyridine-d6, 150 MHz): (Table S1).
3.3.2. Bonushenricoside B (5) (3-O-β-D-glucuronopyranosyl-2β-hydroxyoleanoic
acid-28-O-β-D-glucopyranosyl ester)
White amorphous powder; [ꢀ]2D0: +17.9 (c 0.100, MeOH); IR (neat) υ max: 3383, 2937, 1729,
1653, 1063 cm−1; HR-ESI-MS: found m/z 809.4328 [M − H]−, calcd. for C42H65O15 m/z 809.4329;
1H NMR (pyridine-d6, 600 MHz): (Table S1); 13C NMR (pyridine-d6, 150 MHz): (Table S1).

6
Z. KOKANOVA-NEDIALKOVA ET AL.
3.4. Alkaline, acid and enzymatic hydrolysis
Compounds 3 and 5 (each 10 mg) were separately refluxed with 2 mL 5% aq. KOH for 2 h.
The reaction mixtures were filtered through Amberlite IRC-86 resin and washed with H2O.
The eluate was filtered through a Diaion HP-20SS column and subsequently eluted with H2O
and MeOH. The water portions were evaporated to dryness and analyzed for sugars by HPLC
according the method ofTanaka et al. (2007) with some modifications (Nedialkov et al. 2012).
The occurrence of D-glucose (tR value of the tolylthiocarbamoyl-thiazolidine derivative was
18.7 min) was found in both water residues. The methanol fraction from alkaline hydrolysis
of 3 was refluxed with 4 mL of a mixture of 0.57 M H2SO4-EtOH (1:1) for 2 h. The reaction
mixture was filtered through Amberlite IRC-86 resin, washed with H2O and then filtered
through Diaion HP-20SS, followed by subsequent elution with H2O and MeOH. The water
eluate showed presence of L-arabinose with tR of its tolylthiocarbamoyl-thiazolidine deriv-
ative at 21.2 min. While the methanol fraction from alkaline hydrolysis of 5 was dissolved in
1 mL water and 50,000 units of β-glucuronidase were added. The mixture was left for 48 h
at room temperature then was filtered through Diaion HP-20SS, followed by subsequent
elution with H2O and MeOH. The water portion was evaporated to dryness and analyzed for
sugars by HPLC. The presence of D-glucuronic acid with tR of its tolylthiocarbamoyl-thiazo-
lidine derivative at 19.6 min was established.
3.5. Cell lines and cytotoxicity assay
The cell lines HL-60 (acute myeloid leukemia), BV-173 (chronic myeloid leukemia), K-562
(chronic myeloid leukemia) and SKW-3 (T-cell leukemia) were purchased form DSMZ
(Germany), and Jurkat E6-1 (clone of the Jurkat-FHCRC cell line, a derivative of the Jurkat
T-cell leukemia cell line) was supplied by ATCC (USA). Cells were cultured routinely in a con-
trolled environment: 37 °C in a 5% CO2 humidified atmosphere and were maintained in
RPMI-1640 medium, supplemented with 2 mM L-glutamine and 10% fetal calf serum. The
cellular viability was assessed using the MTT-dye reduction assay, as described by Mosmann,
with slight modifications (Mosmann 1983; Konstantinov et al. 1999). Cisplatin was used as
a positive control. In addition, IC50 values were derived from the concentration-response
curves.
3.6. Modulation of IL-2 production assay
The IL-2 release study was conducted using Jurkat cells (E6-1 clone), a leukemic T-cell line
known to produce IL-2 upon mitogen stimulation (Gillis and Watson 1980; Manger et al.
1986; McIntyre et al. 1994). Jurkat E6-1 cells were seeded in 24 well microplates (106 cells/
well), in triplicate, stimulated with 20 ng/mL 12-O-tetradecanoylphorbol 13-acetate (PMA)
and 10 μg/mL phytohemaglutinin (PHA) for IL-2 release and incubated either in the presence
of test compounds 1–6 (at 25 μM) or the solvent DMSO for 24 h. The cultured supernatants
were obtained and the concentration of the released IL-2 was directly determined by a
commercially available ELISA kit (Diaclone, USA), according to the instructions of the man-
ufacturer and quantified as ng/mL using a freshly prepared standard curve. The results were
evaluated as percentage stimulation and interpreted as previously described (Yeşilada et al.

NATURAL PRODUCT RESEARCH
7
1997, 2001). Stimulation between 70 and 100% was considered high, between 40 and 69%
moderate, and between 20 and 39% low. Isoprinosine was used as a positive control.
4. Conclusion
Phytochemical investigation of the roots of C. bonus-henricus led to the isolation and struc-
tural elucidation of two new glycosides of phytolaccagenin and 2β-hydroxyoleanoic acid,
namely bonushenricoside A and bonushenricoside B together with four known saponins.
The MeOH extract and compounds were tested for cytotoxicity on five leukemic cell lines
(HL-60, SKW-3, Jurkat E6-1, BV-173 and K-562) and their IC50 values showed moderate or
marginal activity. In addition, the methanolic extract and saponins showed moderate stim-
ulatory effects on interleukin-2 production in PHA/PMA stimulated Jurkat E6-1 cells.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Medicinal Science Council at Medical University of Sofia [grant number
11/2012].
ORCID
Zlatina Kokanova-Nedialkova
http://orcid.org/ 0000-0002-1553-6428
Paraskev T. Nedialkov
Georgi Momekov
http://orcid.org/0000-0001-5640-6120
http://orcid.org/0000-0003-2841-7089
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