Spectroscopic characterization and cytotoxicity assessment towards human colon cancer cell lines of acylated cycloartane glycosides from Astragalus boeticus L.

Article abstract of DOI:10.3390/molecules24091725

In several European countries, especially in Sweden, the seeds of the species Astragalus boeticus L. were widely used as coffee substitutes during the 19th century. Nonetheless, data regarding the phytochemistry and the pharmacological properties of this species are currently extremely limited. Conversely, other species belonging to the Astragalus genus have already been extensively investigated, as they were used for millennia for treating various diseases, including cancer. The current work was addressed to characterize cycloartane glycosides from A. boeticus, and to evaluate their cytotoxicity towards human colorectal cancer (CRC) cell lines. The isolation of the metabolites was performed by using different chromatographic techniques, while their chemical structures were elucidated by nuclear magnetic resonance (NMR) (1D and 2D techniques) and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. The cytotoxic assessment was performed in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in Caco-2, HT-29 and HCT-116 CRC cells. As a result, the targeted phytochemical study of A. boeticus enabled the isolation of three new cycloartane glycosides, 6-O-acetyl-3-O-(4-O-malonyl)-β-d-xylopyranosylcycloastragenol (1), 3-O-(4-O-malonyl)-β-d-xylopyranosylcycloastragenol (2), 6-O-acetyl-25-O-β-d-glucopyranosyl- 3-O-β-d-xylopyranosylcycloastragenol (3) along with two known compounds, 6-O-acetyl-3-O-β-d-xylopyranosylcycloastragenol (4) and 3-O-β-d-xylopyranosylcycloastragenol (5). Importantly, this work demonstrated that the acetylated cycloartane glycosides 1 and 4 might preferentially inhibit cell growth in the CRC cell model resistant to epidermal growth factor receptor (EGFR) inhibitors.

Full text of DOI:10.3390/molecules24091725

molecules
Article
Spectroscopic Characterization and Cytotoxicity
Assessment towards Human Colon Cancer Cell Lines
of Acylated Cycloartane Glycosides from
Astragalus boeticus L.
Vittoria Graziani 1 , Assunta Esposito 1 , Monica Scognamiglio 2,*, Angela Chambery 1,
Rosita Russo 1, Fortunato Ciardiello 3, Teresa Troiani 3, Nicoletta Potenza 1
,
Antonio Fiorentino 1,4,
*
and Brigida D’Abrosca 1,4
1
Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche (DiSTABiF), Università degli
Studi della Campania “Luigi Vanvitelli”, via Vivaldi 43, I-81100 Caserta, Italy;
vittoria.graziani@unicampania.it (V.G.); assunta.esposito@unicampania.it (A.E.);
angela.chambery@unicampania.it (A.C.); rosita.russo@unicampania.it (R.R.);
nicoletta.potenza@unicampania.it (N.P.); brigida.dabrosca@unicampania.it (B.D.A.)
Department of Biochemistry, Max Planck Institute for Chemical Ecology-Beutenberg Campus,
Hans-Knöll-Straße, 8 D-07745 Jena, Germany
Dipartimento di Medicina di Precisione, Università degli Studi della Campania “Luigi Vanvitelli” - Via
Pansini, 5, 80131 Napoli, Italy; fortunato.ciardiello@unicampania.it (F.C.);
teresa.troiani@unicampania.it (T.T.)
2
3
4
Dipartimento di Biotecnologia Marina, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy
Correspondence: mscognamiglio@ice.mpg.de (M.S.); antonio.fiorentino@unicampania.it (A.F.);
Tel.: +49-(0)-3641-5701609 (M.S.); +39-(0)-823-274576 (A.F.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 10 April 2019; Accepted: 1 May 2019; Published: 3 May 2019
Abstract: In several European countries, especially in Sweden, the seeds of the species
Astragalus boeticus L. were widely used as coffee substitutes during the 19th century. Nonetheless,
data regarding the phytochemistry and the pharmacological properties of this species are
currently extremely limited. Conversely, other species belonging to the Astragalus genus have
already been extensively investigated, as they were used for millennia for treating various
diseases, including cancer. The current work was addressed to characterize cycloartane
glycosides from A. boeticus, and to evaluate their cytotoxicity towards human colorectal
cancer (CRC) cell lines. The isolation of the metabolites was performed by using different
chromatographic techniques, while their chemical structures were elucidated by nuclear
magnetic resonance (NMR) (1D and 2D techniques) and electrospray-ionization quadrupole
time-of-flight (ESI-QTOF) mass spectrometry. The cytotoxic assessment was performed in vitro by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in Caco-2, HT-29 and
HCT-116 CRC cells. As a result, the targeted phytochemical study of A. boeticus enabled the isolation
of three new cycloartane glycosides, 6-O-acetyl-3-O-(4-O-malonyl)-
), 3-O-(4-O-malonyl)- -d-xylopyranosylcycloastragenol ( ), 6-O-acetyl-25-O-
3-O- -d-xylopyranosylcycloastragenol along with two known
6-O-acetyl-3-O- -d-xylopyranosylcycloastragenol ( ) and 3-O- -d-xylopyranosylcycloastragenol
). Importantly, this work demonstrated that the acetylated cycloartane glycosides and might
β
-d-xylopyranosylcycloastragenol
-d-glucopyranosyl-
compounds,
(
1
β
2
β
β
(3)
β
4
β
(
5
1
4
preferentially inhibit cell growth in the CRC cell model resistant to epidermal growth factor receptor
(EGFR) inhibitors.
Keywords: Astragalus boeticus L.; spectroscopic analysis; cytotoxic activity; human colon cancer cell
lines; acetylated astragalosides; Fabaceae
Molecules 2019, 24, 1725; doi:10.3390/molecules24091725
www.mdpi.com/journal/molecules

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1. Introduction
Astragalus genus is the largest in the Fabaceae family and it is widely distributed throughout the cool,
temperate, semiarid and arid regions of the world [ ]. Astragalus boeticus L. is a Steno-Mediterranean
1
species, which has represented an important cultivation in several countries of Europe, as its seeds have
been widely used as coffee substitutes in times of poverty and coffee prohibition. In Sweden, during
the 19th century, the monarchy introduced an extensive cultivation of the aforementioned species to
produce the so-called Swedish coffee. After the beginning of the 20th century, its cultivation declined,
and it was replaced by other substitutes [2]. In addition to this information, available literature data
describing the phytochemistry and the bioactivities of A. boeticus are currently extremely limited.
On the contrary, a plethora of works regarding other species of the same genus exists. The Astragalus
species were employed as forage for animals, albeit many species were found to be toxic, and responsible
for causing locoism in cattle [
considered medicinal plants of great importance, as these have been successfully used to cure a
broad range of ailments [ ]. In the Traditional Chinese Medicine “Astragali radix” (dried roots of
3,4]. In both folk and modern medicine, several Astragalus spp. were
5
Astragalus membranaceus Bunge and other Astragalus spp.) was a very well-known drug for its immune
stimulant, hepato-protective, anti-diabetic, analgesic, expectorant and sedative properties [6].
Previous works investigated the chemical profile of Astragalus spp. in order to identify the
active principles responsible for the bioactivity of the plant’s crude extracts. Results from these
studies described imidazoline alkaloids, nitro toxins and selenium derivatives as toxic compounds,
while polysaccharides, phenols and saponins as biologically active constituents [6]. Astragalus saponins
include both oleanane and cycloartane-type glycosides, yet the former occur far less in nature, thus the
Astragalus genus was especially employed as an ideal source to find cycloartane saponins [7].
These compounds were the most extensively studied secondary metabolites from Astragalus,
as they exhibited a wide range of biological and pharmacological properties. Indeed, these molecules
were found to exert immunomodulatory, anti-cancer, anti-fungal, hepato-, kidney-, neuro- and
vascular-protective activities [7–12]. So far, the most well-characterized biological effects were those
related to their immune stimulant properties, which made these compounds ideal vaccine adjuvant
candidates [13]. Alongside the capacity to modulate key immunity pathways, recent evidence
supported the effectiveness of Astragalus saponins as anti-tumor compounds and/or as adjuvants in
combination with orthodox chemotherapeutic agents [14,15].
The anti-cancer activities of these compounds have been evaluated towards a wide range of
human malignancies, and a large part of these works evidenced the effectiveness of Astragalus saponins
against gastric and colorectal cancers [16]. Consistent with this, our group recently demonstrated the
anti-proliferative effects of A. boeticus in human colorectal cancer (CRC) cells [17].
Colorectal cancer is one of the most frequently-diagnosed malignant diseases in Europe, and one
of the leading causes of cancer-related deaths worldwide [18]. Even if the outcome of patients with
metastatic colorectal cancer (mCRC) has clearly improved during the last years, the current therapies
are still not entirely efficient. Nowadays, resistance to both chemotherapy and molecularly-targeted
therapies represents a major problem for setting up effective treatment. The EGFR, which was found
overexpressed in 60% to 80% of colorectal cancers, is a transmembrane tyrosine kinase receptor that,
once activated, triggers two main signaling pathways. These include the RAS-RAF-MAPK axis, which is
mainly involved in cell proliferation, and the PI3KPTEN-AKT pathway, which is especially involved
in cell survival and motility [19]. Thus, EGFR inhibitors, such as Cetuximab and Panitumumab, have
been developed to block specifically the abnormal activation of those pathways in wild-type KRAS
CRC patients [20].
In this study, we aimed at providing a detailed chemical characterization of cycloartane glycosides
from A. boeticus, and at assessing their anti-proliferative activity towards human colorectal cancer cells
endowed with diverse mutation profiles and drug sensitiveness. The ultimate goal of this research is
to contribute to the search for new effective agents against refractory CRCs. As a result, we isolated

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and characterized five cycloartane glycosides (
1–
5), identifying compound
4
as a strong inhibitor of
proliferation in CRC cell models resistant to anti-EGFR therapies.
2. Results and Discussion
2.1. Structural Elucidation of Cycloartane Glycosides from Astragalus boeticus L.
A crude hydro-alcoholic extract of A. boeticus leaves was partitioned between EtOAc and H2O.
The purification process, which was performed by using different chromatographic techniques, enabled
the isolation of compounds 1, 2, 4 from the organic phase, while we also obtained 3 and 5 from the
aqueous fraction (Figure 1). The structures of these metabolites were elucidated through a combination
of NMR spectroscopy (1D and 2D techniques) and ESI-QTOF mass spectrometry.
Figure 1. Structures of compounds 1–5.
Compound
mass spectrum. In fact, the 13C NMR displayed 40 signals, which were identified using the HSQC
experiment as eight methyls (—CH3), eleven methylenes (=CH2), eleven methines (=CH ), and ten
1 showed a molecular formula C40H62O13 on the basis of the NMR data and ESI-QTOF
−
quaternary carbons. The ESI-QTOF spectrum displayed the sodiated adduct of the quasimolecular
ion at m/z 773.49, and a strong peak at m/z 687.46, which indicated the easy loss of an 86 Da fragment.
In the 1H NMR spectrum (Table 1), two methylene protons at δH 0.40 and δH 0.61 (δC 30.1), along with
six singlet methyls at δH 0.98, 1.01, 1.05, 1.13, 1.22, 1.26, and 1.27 allowed compound
1 to be identified
as a cycloartane triterpene. The doublet at δH 4.32 (δC 105.9), as well as other protons that resonated in
the range between 3.18 and 4.85 ppm, suggested the presence of a sugar unit. Meanwhile, a methyl
singlet at δH 1.99 supported the presence of an acetate group in the molecule. The above-mentioned
methylene signals (δH 0.40 and δH 0.61) were assigned to H-19 protons; these, in the CIGAR-HMBC
experiment (Figure 2), showed cross peaks with the C-9 (δC 21.8), C-10 (δC 29.6), C-1 (δC 32.8) C-11
(δC 26.8), C-5 (δC 51.2) and C-8 (δC 46.7). In the same experiment, the H-5 proton correlated with the
C-1 methylene, the C-4 quaternary carbon at δC 42.8, and with two carbinols at δC 89.1 and δC 72.0.
Thanks to the long-range heterocorrelations between the H-6 proton (δH 4.75) and the C-4, C-5, and C-8
carbons, it was feasible to assign the first carbinol to the C-3 methine, and then the second to the
C-6 carbon. Moreover, the H-6 proton also had cross peaks with the carbonyl at δC 171.7, which in
turn correlated with the methyl at δH 1.99. These data allowed the acetoxy (CH3-COO-) group to
be located at position 6. On the other hand, the H-3 proton correlated with the anomeric carbon

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at δC 105.9, suggesting that the glycosylation site was located on the hydroxyl at the C-3 carbon.
Furthermore, the C-4 carbon showed correlations with the methyls at δH 0.98 and δH 1.05, values that
were consequently assigned to the H-29 and H-28 protons, respectively. In addition, the methyls at
δH 1.27 (δC 21.4) and 1.01 (δC 20.3) were attributed to the H-18 and H-30 protons, respectively, on the
basis of the long range heterocorrelations between the C-18 carbon with the proton at δH 2.38 (H-17),
which in turn correlated with the H-21 methyl at δH 1.22 (δC 28.4).
In the COSY experiment, the H-17 proton had cross peaks with a proton geminal to oxygen at
δ
H 4.65 (H-16), which homocorrelated with the methylene protons at δH 1.87 and δH 1.45 (H-15),
suggesting the presence of another hydroxyl at the C-16 carbon. Consequently, the remaining methyls
at δH 1.13 and δH 1.26 were attributed to the H-26 and H-27 protons, respectively. In the CIGAR-HMBC
experiment, these latter protons displayed heterocorrelations with a quaternary carbinol carbon at
δC 71.2 (C-25), and with a carbinol methine at δC 82.6, which was bound to a proton at δH 3.76.
This latter signal evidenced heterocorrelations with the diasterotopic protons at δH 2.02 and δH 1.73,
while homocorrelations were with the methylene protons at δH 2.62 and 1.87 (δC 35.5). All these data
supported the presence of a tetrahydrofuran moiety formed by an oxygen bridge among the C-20 and
C-24 carbons of the side chain. Besides the ester carbonyl of the acetate group, two additional carbonyls
at δC 170.6 and 171.7 were also evident in the 13C NMR spectrum. In the CIGAR-HMBC experiment,
both of them revealed cross peaks with the methylene at δH 3.69 (δH 51.8), while the carbon at δC 170.6
was further correlated with a proton at δC 4.72. These data were in agreement with the presence of a
malonyl that was bound to the saccharide unit. In the HSQCTOXY experiment, the anomeric carbon at
δC 105.9 showed cross peaks with the signals at δC 75.4, 75.2, 73.6 and δC 63.3. These carbons in the
HSQC experiment correlated with the methines at δH 3.26, 3.57, 4.72 and to the methylene protons at
δH 3.96 and 3.28, respectively. The H2BC experiment displayed the following correlations: Starting
from the anomeric proton at δC 4.32 (H-10)
→
75.4 (C-20)
→
3.57 (H30)
→
73.6 (C-40)
→
3.96/3.28 (H-50);
63.3 (C-50).
starting from the anomeric carbon at δC 105.9 (C-10)
→
3.26 (H-20)
→
75.2 (C-30)
→
4.72 (H-40)
→
These data further supported the presence of a pentose that was bound to a malonyl group at the
C-40 carbon.
Figure 2. Selected H–H and H–C long range correlations of compound
1 evidenced in COSY and
HMBC, respectively.
The stereostructure of the molecule was assigned on the basis of the nOe observed in the
NOESY experiment (Figure 3). The sugar was identified as xylose by the GC-MS analysis of the
acetylated alditol, which was obtained from the hydrolysis, reduction and acetylation of compound
1.
The coupling constant value of the anomeric proton allowed a configuration for the anomeric carbon
β

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to be determined. The absolute configuration of the sugar was assigned by GC-MS, after a reaction of
hydrolyzed saponins with l-cisteine methyl ester and acetylation [21].
Figure 3. Key nOe correlation of compound
1 (top); NOESY experiment of compound 1 in
CD3OD (bottom).
The nOe evidenced in the NOESY experiments (Figure 3) allowed the configuration at the C-6
and C-16 carbons to be assigned. Based on these data, compound was unequivocally identified as
6-O-acetyl-3-O-(4-O-malonyl)-β-d-xylopyranosylcycloastragenol (Figures S1–S9).
Compound showed the molecular formula C38H60O12 on the basis of the NMR data, and the
1
2
presence of a quasimolecular peak at m/z 731.41 in the ESI-QTOF MS spectrum. The loss of an 86 Da
fragment was demonstrated by the peak at m/z 645.39, indicating the presence of a malonyl moiety also
in this molecule.
The 1H NMR spectrum showed the H-19 protons at δH 0.37 and 0.53, and seven singlet methyls at δH
0.96 (H-30), 0.98 (H-28), 1.15 (H-26), 1.24 (H-27), 1.26 (H-29 and H-21). The absence of the characteristic

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signal associated with the methyl group of the acetate moiety demonstrated that compound
2 was
the deacetylated form of compound
1, and all the NMR data confirmed this hypothesis. In addition,
the C3-sugar was characterized as xylose, and the malonyl was positioned to the C-40 carbon of
this saccharide moiety. Based on these data, compound
2 was unequivocally identified as 3-O-β-d
(4-O-malonyl)xylopyranosylcycloastragenol.
Compound 3 showed the molecular formula C43H70O15, calculated on the basis of its spectro-scopic
features. In the 1H-NMR spectrum, the signals of the aglycone, and those belonging to the
C3-xylopyranose, were in good agreement with the previous metabolite. However, a further anomeric
doublet at δC 4.51 suggested the presence of a second sugar. The heterocorrelation between this
anomeric signal and the C-25 carbon at δH 79.9 allowed the second site of glycosylation to be located at
position 25. The HSQCTOCSY experiment revealed the presence of an additional spin system due to
this sugar, in which the anomeric proton (δC 4.51) correlated with the carbons at
77.5 and 62.7. These data were in agreement with the presence of a glucopyranose, a hypothesis that was
confirmed by GC-MS analysis. Moreover, the coupling constant value indicated a configuration for the
δc 99.6, 75.0, 78.2, 71.2,
β
anomeric carbon, while the D-series was established by GC-MS after the reaction of hydrolyzed saponins
with L-cisteine methyl ester and acetylation. Based on these data, compound 3 was unequivocally
identified as 6-O-acetyl-25-O-β-d-glucopyranosyl-3-O-β-d-xylo-pyranosylcycloastragenol.
Compound
data. Of interest, this compound was also obtained by the mild acidic hydrolysis of compound
when this compound was dissolved in water, after one day at room temperature, it was quantitatively
converted in
by loss of the C40-malonyl. All the NMR data confirmed this hypothesis, and allowed
the identification of compound 4 as 6-O-acetyl-3-O-β-d-xylopyranosylcycloastragenol [22].
4
showed a molecular formula C37H60O10, calculated on the basis of its spectroscopic
1
. In fact,
4
Compound
5 showed a molecular formula C35H58O9, calculated on the basis of its spectroscopic
data. The 1H-NMR and 13C-NMR signals of the aglycone and those belonging to the C3-xylopyranose
were superimposable with the previous metabolites. Conversely, the lack of the singlet peak at δH 1.99
proved the absence of the C6-acetyl. All of these data enabled the identification of compound
5 as
3-O-β-d-xylopyranosylcycloastragenol [23].
2.2. Cytotoxicity of Cycloartane Glycosides from Astragalus boeticus Against Human Colorectal Cancer Cells
The cytotoxic activity of the isolated compounds was assessed on three
(1–5)
human colorectal cancer cell lines (Caco-2, HT-29 and HCT-116), using MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) tetrazolium salt colorimetric
assay (Figure 4). Results from these experiments demonstrated that cell proliferation was reduced by
the treatment with compounds
significant effect. To our knowledge, no mechanistic studies regarding the proliferation reducing-effect
of compounds and are available in literature. Yet, several previous works shed the light on the
anticancer activity of other cycloartane glycosides (most of them isolated from A. membranaceus),
which act by inducing apoptosis and modulating crucial cellular signaling pathways [24 27].
1 and 4 in a dose-dependent manner, while 2, 3 and 5 did not exert any
1
4
–
Importantly, a recent investigation pointed out that certain semisynthetic cycloastragenol derivatives
impair inflammation-carcinogenesis by regulating the NF-KB signaling pathway [15].
Consistently, compound
was described as a potentially anti-inflammatory molecule, because it was found to exert an inhibitory
activity on the nitric oxide production in macrophages [21]. Recently, our group identified compound
as a metabolite responsible for the cytotoxicity of the A. boeticus extract. Here, this species underwent a
further phytochemical investigation to understand whether analogues of compound exert cytotoxicity
were the active
4, which has already been purified from the leaf extract of A. membranaceus,
4
4
as well. As it was already anticipated above, we demonstrated that compounds 1 and 4
cycloartane glycosides isolated from A. boeticus.
In an attempt to further validate our findings, we compared the cytotoxicity of
(inactive compound) with astragaloside IV (AS-IV), a commercially-available cycloartane
glycoside extensively recognized as one of the main active components of several Astragalus spp.
4 (active compound)
and
3

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Results from these experiments confirmed the notable cytotoxicity of compound
found for AS-IV, at least in our experimental conditions (Figure S10).
4, while no effect was
From a structural point of view, these insights proved that the C3-xylopyranosyl, along with
the C6-acetoxy group and the C25-free hydroxyl function, were essential structural requirements for
the cytotoxic activity of these triterpenoids. As already discussed, when compound
solutions, such as a cellular environment, it converted into compound by losing the C4’ malonyl.
Thereby, it was demonstrated that compound was the real bioactive structure. Compounds
and AS-IV present the C3-xylopyranosyl, while none of them has either the C6-acetoxy group or
1 was in water
4
4
2, 3,
5
the C25-free hydroxyl function. In accordance with previous investigations, acylation of the C-3
and C-6 secondary alcohols of diverse cycloartane derivatives resulted in a higher cytotoxic activity.
This evidence led researchers to hypotheses that acylated cycloartane glycosides could lose the acyl
substituents to modify proteins that play a key role in cellular signaling, whose deregulation is involved
in carcinogenesis. Specifically, the acyl groups can be covalently attached to the amino acid side chains
regulating the protein functions and impairing cancer progression [15].
In our study, a detailed analysis of the cytotoxic effect unveiled a differential response of Caco-2,
HT-29 and HCT-116 to the treatment with compound 4. These insights could be interpreted by
describing the human colon cancer cells employed in our experimental setting. Caco-2 cell line was the
wild type for the KRAS, NRAS, BRAF, PIK3CA genes; thus representing an ideal cellular model to
study metastatic CRCs sensitive to anti-EGFR agents. By contrast, HT-29 and HCT-116 harbored a
BRAF and some KRAS/PIK3CA mutations, respectively—therefore identifying metastatic CRCs with
intrinsic resistance to the anti-EGFR treatments [28,29].
Herein, the in vitro cytotoxic screening revealed as compound
4 was clearly more effective in
treating HT-29 (3 µM) than HCT-116 (40 µM) and Caco-2 (50 µM) cell lines (Figure 4).
In clinics, the BRAF mutation status is a strong indicator of a very poor prognosis for mCRC
patients; indeed, they showed a worse outcome for the therapies compared with those whose tumors
were wild type [30–32]. Of interest, the inhibition of BRAF oncoprotein by the small-molecule drug
PLX4032 (Vemurafenib) is highly effective in the treatment of melanoma [33], whilst metastatic CRC
patients associated with the same mutation showed a very limited response to this drug [34]. In fact,
Vemurafenib treatment induces EGFR feedback activation, causing continued proliferation in the
presence of BRAF (V600E) inhibition. Unlike CRCs, melanomas express low levels of EGFR, and thus,
they are not subject to this kind of feedback activation [35].

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Figure 4. Cytotoxic activity of compounds 1–5 towards Caco-2, HT-29 and HCT-116 human colon
cancer cell lines.
3. Materials and Methods
3.1. General Experimental Procedures
Analytical TLC was performed on Merck Kieselgel (Darmstadt, Germany) 60 F254 or RP-8
F254 plates with a 0.2 mm film thickness. Spots were visualized by UV light, or by spraying with
H2SO4/AcOH/H2O (1:20:4), and then heating at 120 ◦C for 5 min. Preparative TLC was performed on
Merck Kieselgel 60 F254 plates with a 0.5 or 1.0 mm film thickness.
Column chromatography (CC) was performed on Fluka (Seelze, Germany) Amberlite XAD-4
and XAD-7, on Pharmacia (Stockholm, Sweden) Sephadex LH-20, on Merck Kieselgel 60 (70–240
mesh), or on Baker (Deventer, Netherlands) RP-8. Nuclear magnetic resonance (NMR) spectra were
recorded at 300 (1H) and 75 MHz (13C) on a Varian Mercury 300 FT-NMR spectrometer in CD3OD

Molecules 2019, 24, 1725
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◦
or pyridine-d5 solutions at 25 C. Chemical shifts are reported in
δ
(ppm), and referenced to the
residual solvent signal; J (coupling constant) are given in Hz. Standard pulse sequences and phase
cycling from Varian library were used for 1H, 13C, DEPT, DQF-COSY, COSY, TOCSY, NOESY, HSQC,
H2BC, HMBC and CIGAR–HMBC experiments. 1H NMR spectra were acquired over a spectral
window from 14 to
−
2 ppm, with 1.0 s relaxation delay, 1.70 s acquisition time (AQ), and 90◦ pulse
1
width = 13.8 µs. The initial matrix was zero-filled to 64 K. 13C NMR spectra were recorded in H
broadband decoupling mode, over a spectral window from 235 to
−
15 ppm, 1.5 s relaxation delay,
90◦ pulse width = 9.50 µs and AQ = 0.9 s. The number of scans for both 1H and 13C NMR experiments
,
were chosen, depending on the concentration of the samples. With regards to the homonuclear and
heteronuclear 2D-NMR experiments, the data points, number of scans and increments were adjusted
according to the sample concentrations. Correlation spectroscopy (COSY) and double quantum filtered
COSY (DQF-COSY) spectra were recorded with gradient-enhanced sequence at spectral widths of
3000 Hz in both f2 and f1 domains; the relaxation delays were of 1.0 s. The total correlation spectroscopy
(TOCSY) experiments were performed in the phase-sensitive mode with a mixing time of 90 ms.
The spectral width was 3000 Hz. Nuclear Overhauser effect spectroscopy (NOESY) experiments
were performed in the phase-sensitive mode. The mixing time was 500 ms, and the spectral width
was 3000 Hz. For all the homonuclear experiments, the initial matrix of 512
×
512 data points was
zero-filled to give a final matrix of 1 k 1 k points. Proton-detected heteronuclear correlations
×
were also measured. Heteronuclear single-quantum coherence (HSQC) experiments (optimized for
1J(H,C) = 140 Hz) were performed in the phase sensitive mode with field gradient. The spectral
width was 12,000 Hz in f1 (13C) and 3000 Hz in f2 (1H) and 1.0 s of relaxation delay; the matrix of
1 k × 1 k data points was zero-filled to give a final matrix of 2 k
×
2 k points. Heteronuclear 2 bond
correlation (H2BC) spectra were obtained with T = 30.0 ms, and a relaxation delay of 1.0 s; the third
order low-pass filter was set for 130 < 1J(C,H) < 165 Hz. A heteronuclear multiple bond coherence
(HMBC) experiment (optimized for 1J(H,C) = 8 Hz) was performed in the absolute value mode with
field gradient; typically, 1H–13C gHMBC were acquired with spectral width of 18,000 Hz in f1 (13C)
and 3000 Hz in f2 (1H) and 1.0 s of relaxation delay; the matrix of 1 k × 1 k data points was zero-filled
to give a final matrix of 4 k × 4 k points. Constant time inverse-detected gradient accordion rescaled
heteronuclear multiple bond correlation spectroscopy (CIGAR–HMBC) spectra (8 > nJ(H,C) > 5) were
acquired with the same spectral width used for HMBC. Heteronuclear single quantum coherence-total
correlation spectroscopy (HSQC-TOCSY) experiments were optimized for nJ(H,C) = 8 Hz, with a
mixing time of 90 ms. For accurate mass measurements, the purified compounds were analyzed by an
electrospray hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF),
fitted with a Z-spray electrospray ion source (Waters S.p.A.). All analyses were carried out in positive
ion mode. The capillary source voltage and the cone voltage were set at 3500 V and 35 V, respectively.
The source temperature was kept at 80 ◦C, and nitrogen was used as a drying gas (flow rate about 50 l/h).
The time-of-flight analyzer of the mass spectrometer was externally calibrated, with GFP from m/z
50–1600. Accurate mass data were collected by directly infusing samples (1.5 pmol/
µL in CH3CN/H2O,
1:1) into the system at a flow rate of 15 L/min. The acquisition and processing of data were performed
µ
with the MassLynx 4.1 software (Waters S.p.A., Manchester, UK). GC-MS analyses were carried out
using an HP 6890 GC instrument (Zebron ZB-5MS column, He flow 1.0 mL/min), coupled with a
5973 N mass spectrometer, equipped with an electron ionization source (EIMS), and operating with
an electron energy of 70 eV. Full-scan mass spectra were collected between 0 and 600 amu at 2 scan/s.
The MS was operated in the electron impact (EI) ionization mode, with an electron energy of 70 eV.
The ion source and quadrupole temperatures were maintained at 230 and 150 ◦C, respectively. For the
analyses of the acetylated alditols, the column head pressure was set at 7.41 p.s.i.
AS-IV was purchased from Shanghai Tauto Biotech Co., LTD. (Shanghai, China).

Molecules 2019, 24, 1725
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3.2. GC–MS Analysis of the Sugar Moieties
The GC-MS analysis of the sugar moieties has been previously described by Scognamiglio et al. [31].
Briefly, each metabolite (0.5 mg) was subjected to an acid hydrolysis with 2 N TFA (150
L) at 120 ◦C
for 1 h, obtaining the sugar moiety. This was dried under N2 flow, and reduced by adding MeOH
(150 L) and NaBH4 (1.0 mg). The solution was incubated at room temperature for 1 h and then dried
under N2 flow after treatment with glacial AcOH and MeOH. The obtained alditol was acetylated
µ
µ
by using anhydrous pyridine (200
µ
L) and Ac2O (200
µ
L). This mixture was incubated for 20 min
◦
at 120 C. Then, 500
µL of H2O was added, and the product was extracted with CH2Cl2 (500
µL)
following centrifugation at 3500 rpm for 5 min. The organic phase was dried under N2 flow, dissolved
in CH2Cl2 (500 µL) and analyzed by GC-MS. Temperature conditions were as follows: Injector port at
250 ◦C; the initial oven temperature was 160 ◦C for 50 s, then linearly increased to 200 ◦C at 10 ◦C/min.
A further linear increase at 2.5 C/min was performed to 300 ◦C, and held for 40 min. Sample solutions
were injected using the split mode.
3.3. Determination of Absolute Configuration of Monosaccharides of Compound 1 and 5
◦
Compound
The reaction mixture was then dried under N2 flow and dissolved in dry pyridine (100
pyridine solution of l-cysteine methyl ester hydrochloride (0.06 mol/L) were added to pyridine solutions
of the hydrolyzed compounds and and pure d-xylose, and l-xylose (0.04 mol/L). These mixtures
were warmed at 60 ◦C for 1 h, afterwards, acetic anhydride (150 L) was added at 120 ◦C for 20 min.
The products were dried under N2, dissolved in 500 L of H2O, and extracted with CH2Cl2 (500 L)
following centrifugation at 3500 rpm for 5 min. The organic phase was dried under N2 flow, dissolved
1
and
5
(2 mg each) were hydrolyzed with 2 N TFA (250
µL) at 120 C for 1 h.
µL). 100 µL of
1
5
µ
µ
µ
in CH2Cl2 (500 µL), and analyzed by GC-MS. Temperature conditions were as follows: Injector port at
250 ◦C; initial oven temperature 45 ◦C, then increased linearly to 300 ◦C at 20 ◦C/min, and then held
for 25 min. Sample solutions were injected using the split mode. The retention times were: d-xylose
13.89 min, l-xylose 14.17 min.
3.4. Plant Material
Leaf samples of A. boeticus were collected at vegetative state, in April 2014 in “Castel Volturno
Nature Reserve” (40◦57.5870N, 14◦00.1050E; southern Italy). Samples were harvested, frozen in liquid
nitrogen and lyophilized. A Voucher specimen CE000016 has been deposited at the Herbarium of the
Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche of Universit
à degli Studi
della Campania “Luigi Vanvitelli”.
3.5. Extraction and Isolation of Compound 1–5
Dried leaves (24.0 g) of A. boeticus were powdered, and underwent three cycles of an
ultrasound-assisted extraction with an MeOH/H2O (1:1) solution (720 mL) [36], finally obtaining a
crude extract (7.1 g). This was dissolved in H2O and separated by liquid–liquid extraction, by using
EtOAc as our extracting solvent. As a result, an organic and a water fraction were obtained. The former
(1g) was chromatographed by SiO2 CC, and eluted using a solution with an increasing degree of
polarity (CHCl3, Me2CO/CHCl3, MeOH/CHCl3). Thus, 21 fractions have been collected. Of these,
number 13 was chromatographed by C18 CC, and eluted with H2O/MeOH (3:2) to give compound
1
(18.6 mg) and 2 (7.1 mg). Meanwhile, number 12 was purified by Flash-CC eluting with MeOH/ CHCl3
(3:100) to obtain compound 4 (28 mg). On the other hand, the water fraction was chromatographed by
XAD-4 (20–50 mesh; Fluka) and XAD-7 (20–50 mesh; Fluka) CC, obtaining an alcoholic eluate (900 mg)
that was in turn purified by Sephadex LH-20. Subsequently, 17 fractions were given. Of these, fraction
13 was purified through RP-18 CC by using H2O/MeOH (4:1) as the eluting system, consequently
obtaining 30 fractions. One of these was chromatographed by TLC (0,5 mm), and eluted with the
organic phase of CHCl3/MeOH/H2O (13:7:2) to obtain compound
3 (11.8 mg). Finally, fraction 11 was

Molecules 2019, 24, 1725
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chromatographed by TLC (0.5 mm) and eluted with the organic phase of CHCl3/MeOH/H2O (13:7:2)
to give compound 5 (4.6 mg).
25
Compound
1
: 6-O-acetyl-3-O-(4-O-malonyl)-
β
-d-xylopyranosylcycloastragenol.
[α
]
= +22.8
D
(c = 14.97 × 10−3, MeOH). 1H NMR (CD3OD) and 13C NMR (CD3OD) see Table 1; ESI/Q-TOF: m/z 773.49
[M + Na]+ (calcd.773.41 Da for C40H62O13Na);
25
Compound
2
: 3-O-(4-O-malonyl)-
β
-d- xylopyranosylcycloastragenol. [
α
]
D
= +9.70 (c = 2.06
×
10−3
,
MeOH). 1H NMR (CD3OD) and 13C NMR (CD3OD) see Table 1; ESI/Q-TOF: m/z 731.41 [M+Na]+ (calcd.
731.40 Da for C38H60O12Na);
Compound
3
:
6-O-acetyl-25-O-
×
β
-d-glucopyranosyl-3-O-
β
-d-xylopyranosylcycloastragenol.
[α]D25 = +13.7 (c= 6.06
10−3, MeOH). H NMR (CD3OD) and 13C NMR (CD3OD) see Table 1;
1
ESI/Q-TOF: m/z 849.46 [M + Na]+ (calcd.849.43 Da for C43H70O15Na);
25
Compound
4
:
6-acetyl-3-O-(4-O-malonyl)-
β
-d-xylopyranosylcycloastragenol.
[α
]
= +7.65
D
(c = 5.1 × 10−3, MeOH/H2O, 2:1) [22];
25
Compound
1:1) [23].
5: 3-O-
β-D-xylopyranosylcycloastragenol. [
α
]
D
=
−
340.1 (c = 3.74
×
10−3) MeOH/H2O,

Molecules 2019, 24, 1725
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Table 1. 1D and 2D nuclear magnetic resonance (NMR) data of compound 1–3 in CD3OD.
1
2
3
Position
δC
Type
δ
(J in Hz)
HMBCa
δC,
Type
δ
(J in Hz)
HMBC a
δC,
Type
δ (J in Hz)
H
HMBC a
H
H
1.85 s
1.67 s
2, 10
2, 10
1.84 s
1.65 s
2, 10
2, 10
1
32.8
CH2
32.3
CH2
32.8
CH2
1.29 s
1.98 ov
1.64 ov
3, 4
3, 4
1.88 ov
1.69 ov
3, 4
3, 4
2
30.3
CH2
30.0
CH2
30.2
CH2
1.99 ov
3, 4
1xyl
28, 29, 1xyl
28, 29, 1xyl
3
4
89.1
42.8
CH
C
3.21 d (J = 1.8)
89.1
42.0
CH
C
3.29 ov
89.1
42.8
CH
C
3.21 ov
1, 4, 7, 10, 28,
29
1, 4, 7, 10, 28,
29
1, 4, 7, 10, 28,
29
5
51.2
CH
1.71 s
53.9
CH
1.38 s
51.3
CH
1.72 s
6
7
72.0
34.0
CH
4.75 ov
1.56 ov
4, 5, 7, 8, 1Ac
5, 6
68.5
33.4
CH
3.48 ov
1.46 ov
4, 5, 7, 8
5, 6
72.1
34.1
CH
4.75 ov
4, 5, 1ac
CH2
CH2
CH2
1.57
5, 6, 8, 9, 14
6, 9, 10, 13, 14,
19, 30
6, 9, 10, 13, 14,
19, 30
6, 10, 13, 14,
15, 19
8
46.7
CH
1.95 s
48.0
CH
1.46 s
46.1
CH
1.94 s
9
21.8
29.6
26.8
C
C
21.4
29.2
26.4
C
C
21.6
29.2
26.4
C
C
10
11
CH2
1.97 m
8, 12, 19
CH2
1.97 s
8, 12, 19
CH2
1.97 s
12
1.64 ov
1.56 ov
14, 18
14, 18
1.54 ov
1.37 ov
14, 18
14, 18
1.64 ov
1.56 ov
12
34.1
CH2
33.4
CH2
34.1
CH2
13
14
47.0
46.4
C
C
44.7
47.0
C
C
46.9
46.6
C
C
8, 13, 14, 17,
30
13, 14, 30
1.87 d (J = 8.0)
1.39 d (J = 8.0)
8, 13, 17, 30
8, 13, 17, 30
1.98 d (J = 4.4)
1.32 d (J = 6.8)
8, 13, 17, 30
8, 13, 17, 30
1.86 d (J = 6.0)
1.42 d (J = 6.4)
15
46.2
CH2
46.4
CH2
46.0
CH2
16
17
18
74.5
58.9
21.4
CH
CH
4.65 m
2.37 d (J = 7.8)
1.27 s
13, 14, 15
73.9
58.2
21.4
CH
CH
4.64 m
2.37 d (J = 7.7)
1.23 s
13, 14, 15
74.3
58.7
21.7
CH
CH
4.65 m
2.38 d (J=8.0)
1.27 s
13, 14, 15
13, 14, 16, 20,
21, 22
13, 14, 16, 20,
21, 22
13, 14, 16, 20,
21, 22
CH3
12, 15, 17
CH3
12, 15, 17
CH3
12, 15, 17
1, 5, 8, 9, 10
1, 5, 8, 9, 10,
11
1, 5, 8, 9, 10
1, 5, 8, 9, 10,
11
1, 5, 8, 9, 10,11
1, 5, 8, 9, 10,
11
0.61 s
0.40 s
0.53 s
0.37 s
0.61 s
0.40 s
19
30.1
CH2
31.4
CH2
30.2
CH2
20
21
88.3
28.4
C
-
88.2
28.1
C
-
88.3
28.0
C
-
CH3
1.22 s
17, 20, 22
CH3
1.25 s
17, 20, 22
CH3
1.24 s
2.62 ov
1.87 ov
17, 20, 21
17, 20, 21
17, 20, 21
17, 20, 21
2.54 ov
1.84
17
17
22
35.5
CH2
34.9
CH2
1.69 ov
35.6
CH2
2.02 ov
1.73
20, 24, 25
17, 24, 25
20, 24, 25
17, 24, 25
2.16 ov
1.72
24, 25
24, 25
23
24
26.8
82.6
CH2
CH
26.4
81.4
CH2
CH
2.02 ov
3.80 m
26.8
83.0
CH2
CH
1glc
3.76 m
25
25
3.82 m

Molecules 2019, 24, 1725
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Table 1. Cont.
1
2
3
Position
25
δC
71.2
26.6
27.6
27.2
16.6
20.3
105.9
75.4
75.2
Type
C
δ
(J in Hz)
-
HMBCa
δC,
71.2
26.5
27.1
16.1
27.1
20.9
105.9
75.4
75.2
Type
C
δ
(J in Hz)
-
HMBC a
δC,
79.9
23.2
25.3
16.5
27.3
20.2
107.4
75.5
78.0
Type
C
δ
H
(J in Hz)
-
HMBC a
H
H
26
CH3
CH3
CH3
CH3
CH3
CH
1.13 s
1.26 s
1.05 s
0.98 s
1.01 s
17, 24, 25
24, 25, 26
3, 4, 5, 29
3, 4, 5, 28
9, 14, 15
3, 5xyl
CH3
CH3
CH3
CH3
CH3
CH
1.15 s
1.24 s
0.98 s
1.25 s
0.96 s
17, 24, 25
24, 25, 26
3, 4, 5, 29
3, 4, 5, 28
9, 14, 15
3, 5xyl
CH3
CH3
CH3
CH3
CH3
CH
1.22 s
1.38 s
0.98 s
1.04 s
0.99 s
20, 23, 24, 25
23, 24, 26
3, 4, 5, 28
3, 4, 5, 29
14, 15
27
28
29
30
1xyl
2xyl
3xyl
4.32 d (J = 7.4)
3.26 ov
4.43 d (J = 7.4)
3.26 ov
4.26 d (J = 7.0)
3.19 ov
3
4xyl
4xyl
CH
CH
CH
4xyl, 5xyl
4xyl, 5xyl
CH
3.57 ov
CH
3.57 ov
CH
3.29 ov
2xyl, 3xyl, 5xyl
2mal
,
2xyl, 3xyl, 5xyl
2mal
,
4xyl
5xyl
73.6
63.3
CH
4.72 m
73.6
63.3
CH
4.72 m
71.2
66.7
CH
3.46 m
1xyl, 3xyl, 4xyl
1xyl, 3xyl, 4xyl
1xyl, 3xyl, 4xyl
1xyl, 3xyl, 4xyl
3.28 ov
3.96 ov
3.28 ov
3.96 ov
3.18 ov
3.82 ov
CH2
CH2
CH2
1glc
2glc
3glc
4glc
5glc
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
99.6
75.0
78.2
71.2
77.5
CH
CH
CH
CH
CH
4.51 d (J = 6.6)
3.18 ov
25
5glc
1glc, 5glc
6glc
3.32 ov
3.31 ov
1glc, 3glc
3.24 ov
4glc
4glc
3.65 ov
3.79 ov
6glc
-
-
-
-
-
62.7
CH2
1ac
2ac
172.2
22.2
C
CH3
C
-
-
-
172.2
C
1.99 s
1ac
-
-
21.8
CH3
1.99 s
1ac
1mal
2 mal
3 mal
170.6
51.8
-
3.69 s
-
170.6
51.8
171.7
C
CH2
C
-
3.69 s
-
-
-
-
-
-
-
CH2
C
1mal, 3mal
1mal, 3mal
171.7
a
HMBC correlations, optimized for 6 Hz, are from proton(s) stated to the indicated carbon; b obscured; d = doublet, m = multiplet, ov = overlapped, s = singlet, t = triplet.

Molecules 2019, 24, 1725
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3.6. Cell Lines
The human HCT-116, HT-29, Caco-2 colorectal cancer cell lines were obtained from the American
Type Culture Collection (ATCC) (Manassas, VA). HCT-116, HT-29 cancer cells were cultured in
RPMI 1640 medium (Lonza, Cologne, Germany) supplemented with 10% fetal bovine serum, 2 mM
l-glutamine, 50 U/mL penicillin and 100
µg/mL streptomycin (Lonza, Cologne, Germany). The Caco-2
cell line was cultured in DMEM medium (Lonza, Cologne, Germany), supplemented with 10% fetal
bovine serum, 2 mM l-glutamine, 1% non-essential amino acid, 50 U/mL penicillin and 100 µg/mL
streptomycin (Lonza, Cologne, Germany).
3.7. Proliferation Assay
The cell proliferation assay was performed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay. Briefly, cells in logarithmic growth phase were plated in 96-well
plates and incubated for 24 h before exposure to increasing doses of DMSO-diluted compounds (20,
40, 60, and 80
line, a lower range of doses (2, 4, 6, and 10
of 1 mg/mL (MTT) were mixed with 200 L of medium and added to the well. 1 h after incubation
at 37 ◦C, the medium was removed, and the purple formazan crystals produced in the viable cells
were solubilized in 100 L of dimethyl sulfoxide, and quantitated by measurement of absorbance at
570 nm with a plate reader. Results were reported as mean s.d. of % of cell growth, with respect
µM). For compounds
1
and
4
that exerted a strong cytotoxic effect in the HT-29 cell
µM) was also investigated. 48 h after treatment, 50 µL
µ
µ
±
to the control from six replicate analyses. The control was represented by a 0.08% DMSO treatment,
which corresponded to the higher amount of DMSO used for the tests.
4. Conclusions
A plethora of previous investigation regarding the phytochemistry and the bioactive components
of diverse species belonging to the Astragalus genus are available in literature. However, to our
knowledge, there are no data regarding the phytochemical constituents of A. boeticus. In this study,
we focused on the cycloartane derivatives present in A. boeticus, because of its putative cytotoxic activity.
Specifically, the targeted phytochemical study of A. boeticus led to the isolation of five
cycloartane-type glycosides (
first time. The cytotoxic activity of compounds
proliferation-reducing effect of compound in the HT-29 human colon cancer cell line, which was
1–
5
); of these,
1,
2
and
3
were isolated and characterized for the
1–5 was evaluated in vitro, disclosing a strong
4
employed as a preclinical model to study refractory mCRCs that are resistant to anti-EGFR therapies,
as well as other chemotherapeutic drugs currently used in the clinical setting. Our results therefore
provide a small molecule scaffold that might be potentially important for the development of new
agents effective against refractory mCRCs.
Indeed, these insights pave the way to further investigations aimed to figure out whether
compound
4 acts on CRC cell models resistant to EGFR inhibitors with high selectivity, and if so,
to elucidate the mechanism by which the anti-proliferative activity occurs.
On the other hand, as several limitations are intrinsically associated with our in vitro experimental
system, future experiments shall also be addressed to evaluate the pharmacokinetic properties, and the
bioavailability of the active molecule in animal models.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/9/1725/s1,
1
Figure S1: H NMR spectrum of compound
1
. Figure S2: 13C NMR spectrum of compound
1
. Figure S3: COSY
. Figure S5: CIGAR-HMBC spectrum of
. Figure S7: HSQCTOCSY spectrum of compound
. Figure S9: ESI TOF MS/MS spectrum of compound . Figure S10:
cytotoxicity of AS-IV in comparison with compound 3 and 4.
spectrum of compound
compound . Figure S6: H2BC spectrum of compound
Figure S8: ESI QTOF spectrum of compound
1. Figure S4: HSQC spectrum of compound 1
1
1
1.
1
1
Author Contributions: V.G. and M.S. designed the experiments, discussed and interpreted the results. A.E. selected
and identified the plant species. V.G. and M.S. carried out the experiments. A.C. and R.R. carried out the M.S.
analyses. V.G. and M.S. wrote the manuscript. A.E., B.D.A. and F.C. analyzed the experimental data. A.E.,

Molecules 2019, 24, 1725
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B.D.A. interpreted the results. T.T., N.P., A.F. designed the experiments, discussed and interpreted the results and
reviewed the manuscript. All authors revised the manuscript.
Funding: This work was funded by Regione Campania, “Technology Platform per la Lotta alle Patologie
Oncologiche” Grant: iCURE (CUP B21C17000030007–SURF17061BP000000008).
Conflicts of Interest: The authors declare no conflict of interest.
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