Serjanic Acid Glycosides from Chenopodium hybridum L. with Good Cytotoxicity and Selectivity Profile against Several Panels of Human Cancer Cell Lines

Two triterpene saponins, including a novel serjanic acid derivative, were isolated from Chenopodium hybridum L. (Amaranthaceae) aerial parts. Their structures were elucidated by a combination of spectroscopic methods (MS, 1D and 2D NMR). Both compounds were evaluated for cytotoxicity and selectivity on skin, prostate, gastrointestinal, thyroid and lung cancer cells. Their effect was dose and time-dependent with varied potency, the highest against prostate PC3 and melanoma WM793, where IC50 was lower than the reference drug doxorubicin. Structure–activity relationship is briefly discussed.


Introduction
The search for natural compounds that would provide an effective medicine to combat cancer is an on-going effort of many scientists. The plant kingdom offers a wide reservoir of potential drug candidates that reveal significant activity in in vitro models of cancer cell lines. Triterpene saponins are one of the most extensively studied groups of natural metabolites that were reported to exert cytotoxicity via various mechanisms including apoptotic or non-apoptotic pathways [1]. These compounds are distributed in many plant families, including Amaranthaceae, which is a source of some commercially important species like Beta vulgaris or Chenopodium quinoa. The latter is a valued crop and food product for humans and animals. Saponins identified in various plants of the Amaranthaceae family represent diverse structural types, including mono-and bidesmosides of oleanolic acid and its hydroxy-derivatives, hederagenin, sophradiol, gipsogenin, saikogenins, phytolaccagenic, akebenoic, spergulagenic and serjanic acids [2,3]. While serjanic acid is an aglycone typical of the Phytolacca genus (Phytolaccaceae), compounds with this genin are seldom reported in plant sources and their distribution seems to be rather scattered across families. Serjanic acid glycosides have been identified to date in Diploclisia galucescens (Bl.) Diels (Menispermaceae) [4], Dendrobangia boliviana Rusby (Cardiopteridaceae) [5] and Phytolacca such as P. isocandra L'Hérit (Phytolaccaceae) [6]. As far as the Chenopodium genus is concerned, this sapogenin was reported solely in two species, Chenopodium quinoa Willd. [3,7] and Ch. berlandieri spp. [8].
In the search for bioactive compounds from the Chenopodium genus we have investigated Ch. hybridum L., commonly known as maple-leaved goosefoot. The plant is an annual herb, 0.4-2 m tall, distributed across temperate regions of Asia, North America and Europe, where it was used in folk medicine as a painkiller [9]. Previous phytochemical investigations of Ch. hybridum were focused on flavonoids and phenolic acids [9][10][11]. Moreover, anti-oxidative, anti-inflammatory, anti-hyaluronidase and anti-proliferative properties of plant extracts were reported [10,11].
As triterpene saponins are commonly found throughout the Chenopodium genus this study was centered on isolation and structure elucidation of previously undescribed compounds from this group in maple-leaved goosefoot. As a result, two serjanic acid derivatives were reported in this plant species for the first time, namely 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl serjanic acid 28-O-β-D-glucopyranosyl ester and 3-O-α-Larabinopyranosyl serjanic acid 28-O-β-D-glucopyranosyl ester [7]. The former is a novel compound, the structure of which was fully characterized based on detailed analysis of spectral data, including two-dimensional Nuclear Magnetic Resonance (2D NMR) experiments and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). Taking into account data on cytotoxic activity of triterpene saponins as well as reports on such activity observed for extracts obtained from Ch. hybridum, both serjanic acid glycosides were further evaluated for their cytotoxic activity and selectivity towards human cancer cell lines, grouped into four panels (skin, prostate, gastrointestinal, thyroid) as well as lung cancer.

Results and Discussion
Fruits, leaves, flowers and roots of Ch. hybridum L. were extracted sequentially with chloroform and methanol. Preliminary TLC comparison of methanolic extracts revealed the presence of saponins with similar chromatographic patterns in extracts from the aerial parts. Therefore, in order to isolate saponins, extract from Ch. hybridum aerial parts was prepared and fractionated by a combination of medium pressure liquid chromatography (MPLC) and open column chromatography (CC) on silica gel to afford two compounds, which gave a positive Liebermann-Burchard test and were isolated with purity of over 95% as confirmed by liquid chromatography-mass spectrometry (LC-MS).
Compound 1 (Figure 1) was obtained as a white powder. Its molecular formula was determined from the HR-ESI-MS spectrum, which gave a peak at m/z 963.4929 [M + Na] + (calculated for C 48 H 76 O 18 (Table 1) and Heteronuclear Single Quantum Coherence (HSQC) spectra allowed us to identify signals that confirmed an oleane-12-ene type for the aglycone, namely six methyl groups at δ 28.7 (C-23), 17.3 (C-24), 16.0 (C-25), 17.8 (C-26), 26.3 (C-27), 28.8 (C-29), a hydroxyl at C-3 (δ 89.2) and olefinic carbon signals of the double bond at δ 123.6 (C-12) and 144.3 (C-13). Two carbonyl carbons at δ 176.6 and 177.4 were assigned to C-28 and C-30, respectively, based on Heteronuclear Multiple Bond Correlation (HMBC) correlations ( Figure 2) between H-18 and C-28, and H-19, H-21, Me-29, and the proton of a methoxy group (δ 3.51) and C-30. The above data were in good agreement with serjanic acid [4][5][6][7].  The 1 H NMR spectrum of 1 (Table 1) (Table 1) and Heteronuclear Single Quantum Coherence (HSQC) spectra allowed us to identify signals that confirmed an oleane-12-ene type for the aglycone, namely six methyl groups at δ 28.7 (C-23), 17.3 (C-24), 16.0 (C-25), 17.8 (C-26), 26.3 (C-27), 28.8 (C-29), a hydroxyl at C-3 (δ 89.2) and olefinic carbon signals of the double bond at δ 123.6 (C-12) and 144.3 (C-13). Two carbonyl carbons at δ 176.6 and 177.4 were assigned to C-28 and C-30, respectively, based on Heteronuclear Multiple Bond Correlation (HMBC) correlations ( Figure 2) between H-18 and C-28, and H-19, H-21, Me-29, and the proton of a methoxy group (δ 3.51) and C-30. The above data were in good agreement with serjanic acid [4][5][6][7].  The linkage position of the sugar chains to the aglycone and interglycosidic linkages were established from 2D NMR spectra. The downfield shift values of C-3 (δ 89.2) and C-28 (δ 176.6) suggested that compound 1 is a 3,28-bidesmoside. The 1 H NMR spectrum showed three doublets of anomeric protons at δ 4.75 (J = 6.9 Hz), 6.08 (J = 7.9 Hz) and 6.46 (J = 1.6 Hz) that corresponded to the carbon signals at δ 107.7, 95.4 and 101.9 as revealed by the HSQC experiment. Sugars were identified based on the results from acid hydrolysis of compound 1 on a TLC plate together with mass spectra fragmentation, coupling constants values and detailed analysis of 1 H-1 H 2D COSY (Correlation Spectroscopy), TOCSY (Total Correlation Spectroscopy), 1 H-13 C HMBC, H2BC (Heteronuclear two-bond Correlation) and F 2 -coupled HSQC [12] as α-arabinopyranose, β-glucopyranose and α-rhamnopyranose, respectively. The attachment of the glucose moiety at C-28 was deduced from the upfield shift of the anomeric carbon (δ 95.4) and HMBC correlation between anomeric H-1 (δ 6.08) and the carbon signal at δ 176.6 (C-28). The second sugar chain was assigned at C-3 based on HMBC correlation between the anomeric proton of arabinose at δ 4.75 and carbon signal at δ 89.2. The 3β-configuration was confirmed by the T-ROESY (Transverse Rotating frame Overhauser Enhancement Spectroscopy) experiment in which cross peaks between H-3α, H-5α and proton signal of Me-23 were seen (Figure 2). The linkage of the terminal rhamnose unit was established from the HMBC correlation between its anomeric proton at 6.46 and C-2 of α-arabinose (δ 73.1). Consequently, the structure of saponin 1 was concluded to be a 18+H] + ions indicated the loss of a hexose, a pentose and dehydration, respectively. Likewise, the key 2D NMR correlations indicated that only two monosaccharide units were attached to serjanic acid, namely glucose at C-28 and arabinose at C-3. Comparison of the spectroscopic evidence with literature data confirmed that compound 2 is identical with 3-O-α-L-arabinopyranosyl serjanic acid 28-Oβ-D-glucopyranosyl ester, isolated previously from Ch. quinoa Willd. [7]. This saponin was also reported in the grains of Ch. berlandieri spp. based on the results obtained from HPLC coupled with an evaporative light scattering detector (ELSD) where peaks were identified according to their similarity with Ch. quinoa compounds [8]. Nevertheless, it is noteworthy that both serjanic acid bidesmosides are not common within the Chenopodium genus, and Ch. hybridum as their novel source represents plant species typical of the European flora as opposed to Ch. quinoa and Ch. berlandieri which are distributed mainly in Central and South America. This may be an important factor that could contribute to observations on inter-species relationships within the Chenopodium genus derived from genetic studies. A phylogeny-based classification of Chenopodium sensu lato [13] has revealed that while Ch. quinoa and Ch. berlandieri belong to the same clade (Chenopodium), Ch. hybridum was included into Ch. murale clade, which was given a new name-Chenopodiastrum. Nevertheless, both Chenopodium and Chenopodiastrum are classified to the Atripliceae tribe.
Based on literature reports confirming cytotoxic activity of crude methanolic extracts from Ch. hybridum [9,10] cytotoxicity of isolated compounds 1 and 2 was evaluated by the LDH assay on several human cancer and normal cell lines (Figure 3). Data expressed as IC 50 is summarized in Table 2. Bearing in mind that phenotypic heterogeneity of tumor cells is critical to their resistance to therapy and treatment failures, cytotoxicity experiment was designed in such a way to mimic the complex nature of a tumor. Therefore, the assay included cancer cells of different origin (skin, prostate, gastrointestinal, thyroid, lung), malignancy and metastatic potential (cells from primary tumor, cells derived from metastatic sites) and, in order to verify the selectivity of the tested substances, the corresponding normal cells.
Both serjanic acid glycosides were found to act selectively with no effect observed on normal PNT2, Nthy-ori 3-1 and HaCaT cells. Their cytotoxicity towards cancer cells was varied and depended on a given tumor type. It is interesting to note that both saponins were more potent than the reference compound (doxorubicin) against primary melanoma WM793 and prostate carcinoma derived from metastatic site PC3. In all cases cytotoxic activity of 1 and 2 was dose and time-dependent. Literature data indicate that bidesmosidic saponins of plant origin, oleanane derivatives especially, are generally less potent than monodesmosides with a free carboxylic group at C-17 (C-28) [14]. On the other hand, several studies revealed that the activity of oleanane-type saponins is affected not only by the functional groups in sapogenin but also by the number and type of monosaccharide units in the sugar part [1]. Numerous reports confirmed that a rhamnose unit in the sugar chain attached at C-3 is a structural feature that enhances cytotoxicity of triterpene saponins [15]. Moreover, a sugar chain that incorporates a monosaccharide unit composed of α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl located at C-3 is also considered to be a key factor for cytotoxic activity of oleanane type saponins [14,16]. Such a unique disaccharide sequence located at C-3 of serjanic acid may be responsible for the observed activity of compound 1 towards cells grouped in the skin panel (HTB-140, A375, WM793) where the activity of compound 1 was higher as compared to 2, which is devoid of the terminal rhamnose unit. However, this structure-activity correlation is not evident in other types of cancer cells. Compound 2 was found to be more effective towards gastrointestinal Molecules 2021, 26, 4915 6 of 11 cells (Caco-2, HepG2, HT29) than compound 1. Cytotoxicity data available in the literature have shown that compound 2, as well as other serjanic acid bidesmosidic saponins isolated from Ch. quinoa were inactive towards HeLa cells [7].
Molecules 2021, 26, x FOR PEER REVIEW 6 of 12 malignancy and metastatic potential (cells from primary tumor, cells derived from metastatic sites) and, in order to verify the selectivity of the tested substances, the corresponding normal cells.  A lack of influence on the viability of normal prostate cells and skin keratinocytes observed in our study is also noteworthy. In another experiment, other serjanic acid bidesmosidic saponins with glucuronic acid and rhamnose in the sugar chain attached at C-3 were found to be cytotoxic towards human skin fibroblasts with IC 50 5.6-6.4 µM [5]. Taken together, data from our experiment show that the structure of saponin influences the cytotoxicity and selectivity, but the overall effect depends mainly on the susceptibility of cells which represent a given tumor type.
Chemical shifts (δ) are given in ppm. Coupling constants are reported in Hz. High resolution mass spectra were obtained on a high-resolution quadrupole time-of-flight mass spectrometer (HR/Q-TOF/MS, Impact II HD, Bruker Daltonik GmbH, Bremen, Germany) operated in the negative and positive electrospray ionization mode with mass scan range set from m/z 375 to m/z 2200, using capillary voltage of 3.0 and 4.5 kV, respectively; nitrogen (N 2 ) was used as nebulizer and drying gas at 2.0 bar and 6.0 L min −1 , respectively; dry temperature was set to 200 • C. The MS/MS spectra were acquired using variable collision energy in the range from 10 to 100 eV. Internal calibration of the instrument was accomplished using 10 mM sodium formate solution introduced to the ion source via a 20 µL loop at the beginning of each analysis. Data were collected and processed by the Data Analysis 4.4 software (Bruker Daltonik GmbH, Bremen, Germany).
LC-MS/MS analysis was performed on UPLC/MS Waters ACQUITY TQD (Waters Corporation, Milford, MA, USA) apparatus with gradient elution (95% to 0% of eluent A; eluent A: water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid (0.1%, v/v)). Chromatographic separations were carried out using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column; 2.1 × 100 mm, and 1.7 µm particle size, equipped with Acquity UPLC BEH C18 VanGuard pre-column; 2.1 × 5 mm, and 1.7 µm particle size. Chromatograms were recorded using Waters eλ PDA detector. Spectra were analyzed in 200-700 nm range with 1.2 nm resolution and sampling rate 20 points/s. MS detection settings of Waters TQD mass spectrometer were as follows: source temperature 150 • C, desolvation temperature 350 • C, desolvation gas flow rate 600 L h −1 , cone gas flow 100 L h −1 , capillary potential 3.00 kV and cone potential 30 V. Nitrogen was used for both nebulizing and drying gas. The data were obtained in a scan mode ranging from 50 to 1000 m/z in 0.5 s time intervals.
Open column chromatography and MPLC was carried out on silica gel 230-400 mesh (Sigma-Aldrich, Darmstadt, Germany), thin-layer chromatography (TLC) was performed on pre-coated silica gel 60 plates (0.25 mm Merck), preparative thin-layer chromatography (pTLC) was carried out on commercial glass Silica Gel G (500 µ) ANALTECH. Spraying reagents for saponins: 25% solution of H 2 SO 4 in methanol and heating at 120 • C on a TLC plate heater III (CAMAG, Muttenz, Switzerland) for 3 min.; for sugars: aniline phthalate reagent and heating at 100 • C for 5 min on a TLC plate heater III (CAMAG, Muttenz, Switzerland). Cytotoxicity assay was performed using a Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA) equipped with Gen 5 software. All reagents used were of analytical grade. Reference standards of authentic sugars were from (Sigma-Aldrich, Germany).

Plant Material
Chenopodium hybridum L. was collected from natural stands in Cracow, Poland (50.06262, 19.92971). Botanical identification of the species was carried out by a botanist from the Department of Pharmacognosy UJCM. A voucher specimen (No. KFg/ChHbr-2019) is deposited at the Department of Pharmacognosy, Pharmaceutical Faculty, Medical College, Jagiellonian University, Cracow, Poland.
Plant material was air-dried under steady, controlled conditions (at room temperature, in the dark) and divided into aerial part and roots. Aerial parts of the herb were separated into leaves, flowers, fruits and stems. Plant material was stored in airtight containers.

Extraction and Isolation
Dried plant material (aerial parts) (leaves 10 g, fruits 10 g, flowers 10 g, stems 10 g, roots 10 g) of Ch. hybridum were ground to a fine powder using a laboratory mill (BOSCHMKM6003, BSHGmbH, Munich, Germany) and extracted successively with chloroform (plant material/solvent ratio-DSR 1:10 w/v, 2× for 2 h and methanol, 3× for 3 h, DSR 1:10) on a boiling water bath under reflux. Evaporation of methanol under reduced pressure on a rotary evaporator gave dark brown crude extracts from leaves (65 mg), fruits (125 mg), flowers (45 mg) and stems (38 mg), which were subjected to TLC (silica gel, CHCl 3 -MeOH-H 2 O (23:12:2 v/v), 25% H 2 SO 4 in MeOH + heating). Preliminary TLC analysis revealed a similar chromatographic pattern in all morphotic aerial parts. In order to isolate saponins, extracts from a larger sample of aerial parts of Ch. hybridum (200 g) was prepared in an analogous manner as described above. The MeOH extract was concentrated in vacuo to yield 14.8 g residue. Extract portions (3.0 g) were chromatographed by MPLC on silica gel (MPLC column 150 × 40 mm, flow rate 10 mL/min) using isocratic solvent system CHCl 3 /MeOH/H 2 O (23:12:2) to give 7 fractions (I-VII) which were further purified by repeated column chromatography (CC column: 80 × 12 mm) under the same conditions and preparative thin layer chromatography in CHCl 3 /MeOH/H 2 O (27:10:2) to yield compound 1 (17 mg from fr. V) and compound 2 (23 mg from fr. III).