Antiproliferative Cardenolides from the Aerial Parts of Pergularia

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Antiproliferative Cardenolides from the Aerial Parts of Pergularia tomentosa Seyed Hamzeh Hosseini,†,# Milena Masullo,‡,# Antonietta Cerulli,‡ Stefania Martucciello,§ Mahdi Ayyari,⊥ Cosimo Pizza,‡ and Sonia Piacente*,‡ †

Department of Plant Biology, University of Jiroft, Jiroft, Iran Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Salerno, Italy § Dipartimento di Chimica e Biologia, Università degli Studi di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Salerno, Italy ⊥ Department of Horticultural Science, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

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‡

S Supporting Information *

ABSTRACT: The LC-MS analysis of the MeOH extract of the aerial parts of Pergularia tomentosa led to the isolation of 23 compounds, of which the structures were elucidated unambiguously by NMR spectroscopic data analysis. Three new doubly linked cardenolides (4, 13, 14) along with several known cardenolides (1−3, 5, 7, 8, 15−23) and flavonol glycosides (6, 9−12) were identified. LC-HRESIMS analysis, in the negative-ionization mode, showed the absence of flavonoids in a methanol extract of the roots of P. tomentosa. On the basis of the antiproliferative activity reported for cardenolides, the isolated compounds were tested for their ability to decrease the cell viability of five different human cancer cell lines, PC3, HeLa, Calu-1, MCF-7, and U251MG, exhibiting IC50 values ranging from 0.2 to 8.0 μM. Moreover, an S-phase entry assay was performed to investigate if the compounds could affect the cell cycle progression of PC3 prostate carcinoma cells. The results obtained demonstrated that the compounds 4, 7, and 14 at 1 μM considerably reduced the number of cells in the S-phase. sides from the plant genera Digitalis and Strophanthus contain sugar units linked to OH-3β of the steroidal aglycone (single link), doubly linked cardiac glycosides occurring in plants of the Asclepiadaceae contain a single sugar linked to the 2α- and 3β-positions of the aglycone by hemiketal and acetal functions, respectively, generating a “dioxanoid” structure.6,9 The sugar unit is represented by 4,6-dideoxyhexosulose and its modified form, 4-deoxyhexosulose.5 The former sugar unit has been reported thus far in ghalakinoside and its derivatives,1,4,5 which are cardenolides found only in P. tomentosa. Previously, our group carried out a phytochemical investigation of the roots of P. tomentosa, leading to the isolation and characterization of several doubly linked cardiac glycosides. These compounds were found to exert proapoptotic effects on Kaposi sarcoma cells and exhibited marked cytotoxic activity as well as the ability to inhibit Na+/K+-ATPase activity.4,5 As a part of ongoing research on new bioactive compounds from P. tomentosa and on the basis of the interesting biological activity shown by cardenolides isolated from the roots, attention was focused on the aerial parts of this plant with the aim of investigating the occurrence of cardiac glycosides with potential cytotoxic activity. Therefore, the LC-MS profile

Pergularia tomentosa L. is a milkweed tropical plant, belonging to the subfamily Asclepiadaceae of the Apocynaceae and grows to close to 50 cm high. It is a native species of the Middle East and North Africa.1 Different parts of P. tomentosa, including the aerial parts and roots, are used in traditional medicine for the treatment of several diseases, such as bronchitis, tuberculosis, diabetes mellitus, constipation, and skin diseases, and also have molluscicidal activity.2 Previous investigations on P. tomentosa have led to the isolation of cardiac glycosides such as desglucouzarin, coroglaucigenin, and uzarigenin in the leaves1,3 and uzarigenin, ghalakinoside, calactin, 6′-hydroxycalactin, 6′hydroxy-16α-acetoxycalactin, 16α-hydroxycalactin, 12′-dehydroxyghalakinoside, 3-O-β-glucopyranosiylcalactin, and 6′dehydroxyghalakinoside in the roots.4,5 Structurally, cardiac glycosides are compounds with a steroidal nucleus having a lactone moiety at position C-17, containing also a sugar unit at position C-3. The nature of the lactone ring at position C-17 defines the compound class, i.e., cardenolides (with an unsaturated butyrolactone ring) and bufadienolides (with an α-pyrone ring).6,7 In cardiac glycosides produced by plants from the milkweed family Asclepiadaceae (such as calactin and ghalakinoside), the A/B rings are transfused, resulting in rather flat structures, with a markedly more potent binding to the Na+/K+-ATPase pump (particularly to the Na+/K+-ATPase α1 subunits).5,8 Whereas cardiac glyco© XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 30, 2018

A

DOI: 10.1021/acs.jnatprod.8b00630 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

ion [(M + HCOOH) − H]− at m/z 595, and compounds 20 and 21 showed a deprotonated molecular ion [(M + HCOOH) − H] − at m/z 557. In order to define unambiguously the molecular structure of 1−23 and to discriminate among structural isomers or stereoisomers, the first step was to perform an LC-HRESIMS analysis of naturally occurring standards, previously isolated from the roots of P. tomentosa.4,5 In this manner, ghalakinoside (1), 6′-hydroxycalactin (2), 12-dehydroxyghalakinoside (5), 6′-dehydroxyghalakinoside (15), 3-O-β-glucopyranosiylcalactin (16), and calactin (21) could be identified. Nevertheless, using this approach compounds 7, 13, and 14, structural isomers of 6′hydroxycalactin (2), could not be identified. Compounds 6 and 9−12 showed in the LC-HRESIMS principal precursor [M − H]− ions at m/z 463, 447, 447, 463, and 477, respectively, with fragmentation patterns characterized by the occurrence of a neutral loss of 162 amu, typical of a hexose sugar unit linked to a flavonol aglycone. The aerial parts of P. tomentosa were extracted with methanol, and the dried extract obtained was fractionated over Sephadex LH-20. The fractions generated were chromatographed by RP-HPLC to yield three new cardenolides (4, 13, and 14), along with 15 known cardenolides (1−3, 5, 7, 8, and 15−23) and five known flavonoids (6 and 9−12). Compound 4 was obtained as an amorphous, white solid, and its molecular formula of C29H40O11 was deduced by HRESIMS analysis (m/z 563.2476 [M − H]−, calcd for C29H39O11, 563.2492). Its IR spectrum exhibited absorption bands for hydroxy (3438 cm−1), carbonyl (1750 cm−1), and olefinic (1635 cm−1) groups. The 1H NMR spectrum of 4 showed the typical features of a cardenolide glycoside including signals corresponding to a butenolactone ring at δ 5.93 (1H, t, J = 1.6 Hz), 5.01 (1H, dd, J = 18.2, 1.6 Hz), and 4.95 (1H, overlapped).10,11 Further signals of a formyl group

of the MeOH extract of the aerial parts of P. tomentosa was performed to guide the isolation of 23 compounds, among which the structures of three new cardiac glycosides (4, 13, 14) were elucidated unambiguously by spectroscopic data analysis. The LC-HRESIMS analysis of the methanol extract of the roots was also performed. In order to investigate their potential anticancer activity, cardenolides isolated from the aerial parts of P. tomentosa were tested against five human cancer cell lines: PC3 (prostate carcinoma cells), HeLa (cervical carcinoma cells), Calu-1 (epithelial lung cancer cells), MCF-7 (breast cancer cells), and U251MG (human glioma cells). A further study aimed at investigating the cardenolides 4, 7, 13, and 14 in the inhibition of S-phase entry in PC3 cells was performed.

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RESULTS AND DISCUSSION A preliminary metabolite fingerprint of the methanol extract of P. tomentosa aerial parts was obtained by LC-HRESIMS analysis in the negative-ion mode. The LC-MS profile (Figure S1, Supporting Information, panel A) showed 23 main peaks, corresponding to cardenolides (1−5, 7, 8, 13−23) and flavonol glycosides (6, 9−12). Experiments using the “datadependent scan” mode, in which the MS software selects precursor ions corresponding to the most intense peaks in the LC-MS chromatogram, were carried out. Some of the main peaks were attributed tentatively according to the accurate masses, characteristic fragmentation patterns, and retention times and by comparison with literature data on P. tomentosa and the “KNApSAcK” database. As reported in Table S1 (Supporting Information), showing the retention times, molecular formula, and MS/MS values of compounds 1−23, compounds 2, 7, 13, and 14 showed a deprotonated molecular ion [(M + HCOOH) − H]− at m/z 593, compounds 5 and 15 showed a deprotonated molecular B

DOI: 10.1021/acs.jnatprod.8b00630 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR Spectroscopic Data (600 MHz, CD3OD) for Compounds 4, 13, and 14a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′

4 37.9 70.3 73.0 33.1 47.9 28.4 27.9 41.7 49.3 53.7 33.2 75.2 56.9 86.0 32.5 27.6 45.9 9.5 209.7 178.8 75.4 117.4 177.1 96.9 92.5 74.0 33.6 74.0 65.7

2.42 dd (12.5, 4.7); 1.20 t (12.5) 3.89 m 4.03 m 1.57 m 1.16 m 2.12, 1.94 m 2.22 m 1.80 m 1.80 m 1.69 m 3.31 m

1.90, 1.54 m 2.19, 1.71 m 3.33, dd (5.7, 3.1) 0.86 s 10.10 s 5.01 dd (18.2, 1.6); 4.95 overlapped 5.93 t (1.6) 4.53 s 3.64 dd (12.0, 4.6) 1.76, 1.67 m 3.60 m 3.61 (2H) m

13 37.3 70.1 73.0 34.3 44.3 28.6 28.6 43.5 49.3 53.7 22.8 40.2 51.1 85.5 32.7 27.6 51.8 15.7 208.7 175.0 75.4 117.5 177.3 97.0 92.8 74.0 34.0 74.0 65.6

2.49 dd (12.5, 4.7); 1.16 t (12.5) 3.89 m 4.01 m 1.66, 1.45 m 1.58 m 2.05, 1.68 m 2.07 m 1.62 m 1.45 m 1.74, 1.32 m 1.64, 1.45 m

2.11, 1.72 m 2.19, 1.86 m 2.85 dd (5.2, 3.1) 0.85 s 10.06 s 5.03 dd (18.2, 1.6); 4.95 overlapped 5.92 t (1.6) 4.50 s 3.61 dd (12.0, 4.6) 1.74, 1.66 m 3.64 m 3.60 (2H) m

14 37.3 70.2 72.8 34.2 42.9 28.3 28.4 43.5 46.4 53.7 28.2 74.7 56.5 85.7 28.4 28.3 46.1 9.2 209.5 175.4 75.4 117.5 177.7 95.4 91.2 71.7 37.9 67.7 20.4

2.42 dd (12.5, 4.7); 1.16 t (12.5) 3.88 m 3.98 dt (10.4, 3.4) 1.69, 1.45 m 1.58 m 2.05, 1.67 m 2.06 m 1.60 m 1.46 m 1.75, 1.29 m 3.38 m

2.13, 1.74 m 2.24 dd (12.6, 2.1); 1.93 m 3.34, m 0.75 s 10.06 s 4.97 dd (18.2, 1.6); 4.93 overlapped 5.92 t (1.6) 4.73 s 3.64 t (3.1) 1.71, 1.67 m 4.10 m 1.22 d (6.3)

a

Assignments were confirmed by HSQC and HMBC experiments.

at δ 3.33 (1H, dd, J = 5.7, 3.1 Hz), in agreement with data published for 17β-cardenolides.5 The β-configuration of the hydroxy group at C-12 was deduced from the ROESY correlations between H-12 (δ 3.31) and H-9 (δ 1.80) and H-17 (δ 3.33) and by the absence of correlations between H12/Me-18 and H-12/CHO-19. The J coupling constant value of H-3′ (δ 3.64, dd, J = 12.0, 4.6 Hz) indicated an axial position and thus a β-orientation of H-3′. On the basis of 1DTOCSY, DQF-COSY, HSQC, and HMBC experiments, the occurrence of a 2,3-doubly linked 4-deoxyhexosulose unit was deduced.13 From this evidence, the structure of compound 4 was established as the new compound 12β,6′-dihydroxycalotropin. The HRESIMS of compound 13 (m/z 547.2558 [M − H]−, calcd for C29H39O10, 547.2543) and the 13C NMR data supported a molecular formula of C29H40O10. The 1H NMR spectrum of 13 showed signals corresponding to a butenolactone ring at δ 5.92 (1H, t, J = 1.6 Hz), 5.03 (1H, dd, J = 18.2, 1.6 Hz), and 4.95 (1H, overlapped), to a formyl group proton at δ 10.06 (1H, s), to a tertiary methyl at δ 0.85 (3H, s), along with signals ascribable to secondary alcohol functions at δ 4.01 (1H, m) and 3.89 (1H, m). The 1H and 13C NMR spectra of 13 were almost superimposable to those of 4 except for the absence of a secondary alcoholic function on the aglycone moiety. The analysis of HSQC, HMBC, and COSY experiments confirmed that compound 13 in comparison with 4 lacked the C-12 hydroxy group. Thus, the structure of 13 was established as the new compound 6′-hydroxycalotropin.

proton at δ 10.10 (1H, s), a tertiary methyl at δ 0.86 (3H, s), and signals ascribable to secondary alcoholic functions at δ 4.03 (1H, m), 3.89 (1H, m), and 3.31 (1H, m) were evident.5 Moreover, the proton spectrum showed a signal corresponding to an anomeric proton of a doubly linked sugar unit at δ 4.53 (s). The 13C NMR spectrum of 4 showed 29 carbon signals, of which 23 could be assigned to the aglycone moiety and six to a sugar portion. The 13C NMR chemical shifts of all the hydrogenated carbons were assigned unambiguously from the HSQC spectrum. In particular, the analysis of the 13C NMR spectrum on the basis of the HSQC correlations clearly confirmed in the aglycone moiety the occurrence of a formyl group carbon (δ 209.7) along with signals typical of a butenolactone ring: a carbonyl group (δ 177.1), an olefinic quaternary function (δ 178.8), an olefinic methine (δ 117.4), and a primary alcoholic function (δ 75.4). The 1H and 13C NMR chemical shifts of compound 4 were almost superimposable on those of the known compound calotropin (20),12 except for a signal centered at δ 3.60 (2H, m) corresponding to a CH2OH group at C-5′, replacing the methyl signal at δ 1.15 (3H, d). In addition, a further signal at δ 3.31 (m) was observed, ascribable to a secondary alcohol function. HMBC correlations of the proton value at δ 3.31 with the carbon resonances at δ 9.5 (Me-18), 56.9 (C-13), and 86.0 (C-14) supported the location of the hydroxy group at C12. The relative configurations of C-12, C-17, and C-3′ were established from the analysis of 1H NMR coupling constants and ROESY correlations. The relative configuration of H-17 was assigned as α, based on the coupling constants of the signal C

DOI: 10.1021/acs.jnatprod.8b00630 J. Nat. Prod. XXXX, XXX, XXX−XXX

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trans fused and containing a single sugar in a unique “dioxanoid” attachment. Therefore, doubly linked cardenolides (1, 2, 4, 5, 7, 13−16, and 19−21) along with the remaining cardenolides (3, 8, 17, 18, 22, and 23) isolated from the aerial parts of P. tomentosa were tested in an in vitro growth inhibitory assay (an MTT colorimetric assay), including five different human cancer cell lines, PC3 (prostate carcinoma cells), HeLa (cervical carcinoma cells), Calu-1 (epithelial lung cancer cells), MCF-7 (breast cancer cells), and U251MG (human glioma cells). The PC3 and MCF-7 cancer cell lines were used in a previous investigation.5 Herein, additional different cell lines have been selected to investigate if the antiproliferative activity is dependent on cell type. As shown in Table 2, all the cardenolides (1−5, 7, 8, 13−23) from P.

The negative-ion HRESIMS of 14 showed a deprotonated ion [M − H]− at m/z 547.2535 (calcd for C29H39O10, 547.254), which, in combination with the 13C NMR data, supported a molecular formula of C29H40O10. The 1H NMR spectrum of 14 showed the typical signals of a butenolactone ring at δ 5.92 (1H, t, J = 1.6 Hz), 4.97 (1H, dd, J = 18.2, 1.6 Hz), and 4.93 (1H, overlapped), along with a formyl group proton at δ 10.06 (1H, s), a methyl group signal at δ 0.75 (3H, s), and proton signals adjacent to secondary hydroxy groups at δ 3.98 (1H, dt, J = 10.4, 3.4 Hz), 3.88 (1H, m), and 3.38 (1H, m). The 1H NMR spectrum of compound 14 showed a resonance at δ 4.73 (s) in the saccharide region, typical of a doubly linked sugar unit, and a signal at δ 1.22 (3H, d, J = 6.3 Hz) ascribable to a secondary methyl group (Table 1). On the basis of 1D-TOCSY, DQF-COSY, HSQC, and HMBC experiments, the occurrence of a 4,6-dideoxyhexosulose unit was identified. In particular, the 1H NMR spectral data of 14 were similar to those of calactin (21), with the exception of an additional signal at δ 3.38 (1H, overlapped), located at C-12, as shown by the HMBC correlations between the proton signals at δ 3.38 with the carbon resonances at δ 9.2 (Me-18), 56.5 (C-13), and 85.7 (C-14). The relative configurations of C-3′, C-12, and C-17 were established by analysis of 1H NMR coupling constants and ROESY correlations as reported for compound 4. A difference with 4 was observed for the J coupling constant value of H-3′ (δ 3.64, t, J = 3.1 Hz), indicating an equatorial position and thus an α-orientation of H-3′. Thus, the structure of 14 was established as the new compound 12β-hydroxycalactin. The other cardiac glycosides isolated were identified as the known compounds 12β-hydroxycoroglaucigenin (3),14 16αhydroxycalotropin (7),15,16 glucocoroglaucigenin (8),17 desglucouzarin (17),3 coroglaucigenin (18),3 16α-acetoxycalotropin (19),12 6′-O-feruloyl-desglucouzarin (22),18 and uzarigenin (23).3 Also, the known flavonoids 6 and 9−12 were identified as quercetin 3-O-β-D-galactopyranoside, kaempferol3-O-β-D-galactopyranoside, kaempferol-3-O-β-D-glucopyranoside,19 quercetin-3-O-β-D-glucopyranoside,20 and isorhamnetin-3-O-β-D-glucopyranoside,19 respectively. With the aim of investigating the secondary metabolite composition of roots, the same LC-HRESIMS conditions used for the aerial parts were applied to the analysis of P. tomentosa root methanol extract, obtaining the profile shown in Figure S1 (Supporting Information, panel B). Analysis of this profile demonstrated the absence of flavonoids 6 and 9−12 and the occurrence of the doubly linked cardenolides 6′-hydroxy-16αacetoxycalactin (24) and 16α-hydroxycalactin (25) (Table S1, Supporting Information). This investigation highlights that the aerial parts of P. tomentosa represent a source of doubly linked cardenolides. Chemically, they can be considered as belonging to two groups: the calactin derivatives (1, 2, 5, 14−16, and 21) and the calotropin derivatives (4, 7, 13, 19, and 20), differing in the configuration at C-3′. The doubly linked cardenolides have been found in the Asclepiadaceae family, particularly in the genera Asclepias, Calotropis, and Pergularia. This structural feature is rarely found in the cardenolides from other families.21 Our previous investigations on the doubly linked cardenolides revealed that these compounds cause apoptotic cell death of Kaposi sarcoma cells4 and display marked cytotoxic activity against cancer cell lines, due to their ability to inhibit Na+/K+ATPase activity.5 These activities are ascribable to their structural features, with the A/B rings of the steroidal skeleton

Table 2. Antiproliferative Activity for Cancer Cell Lines Induced by Cardenolides from Pergularia tomentosaa compound

PC3

HeLa

MCF-7

CaLu-1

U-251MG

1 2 3 4 5 7 8 13 14 15 16 17 18 19 20 21 22 23

4.2 8.9 8.4 0.9 3.0 0.6 1.0 3.2 0.3 0.6 1.0 0.3 0.4 4.2 2.1 3.7 0.3 0.3

0.9 0.3 0.9 2.0 0.3 2.3 0.3 0.3 2.5 7.0 0.6 0.6 4.2 0.5 0.6 0.2 4.5 3.0

0.6 2.0 2.5 2.8 0.2 7.0 8.0 1.8 6.0 5.0 0.2 6.0 7.0 0.4 0.2 0.2 0.2 6.0

0.7 3.7 1.3 2.0 0.6 4.3 6.0 1.4 7.0 6.0 0.3 6.5 5.8 0.7 0.6 0.5 6.0 8.0

5.2 5.5 5.0 6.0 1.2 1.5 6.0 5.0 5.3 8.0 4.4 8.0 5.0 6.5 1.0 0.9 5.8 6.0

a

Data presented as IC50 values (μM). The SEM values are not reported for the sake of clarity. IC50 (nM) values of docetaxel (used as reference compound): 0.6, 0.3, 0.5, 0.4, 0.7 against PC3, HeLa, MCF7, CaLu-1, and U-251MG cancer cell lines, respectively. Additionally, it should be noted that the highest SEM value calculated was less than 10% of its mean value.

tomentosa were found to possess antiproliferative activity. All compounds tested were able to reduce cell viability, with IC50 values ranging from 0.2 to 8.9 μM, depending on cell type. The U251MG cell line was less sensitive to the isolated compounds (IC50 ranging from 0.9 to 8.0 μM). Compounds 4, 7, 14, 15, and 18 showed more potent effects against the PC3 cell line than in the HeLa, MCF7, and Calu cell lines. Literature reports document both the proapoptotic action of cardiac glycosides at μM concentrations and proautophagic effects if used at nM concentrations.5 Therefore, with the aim to further characterize the properties of the doubly linked cardenolides 4, 7, 13, and 14 isolated from P. tomentosa, an S-phase entry assay was carried out to verify whether these compounds could affect cell cycle progression in PC3 cells. The S-phase entry in vitro assay is used commonly in the detection of proliferating cells by measuring the transition G0 → S of the cell cycle as a percentage of cells that incorporate bromodeoxyuridine (BrdU) in G0 synchronized cells. The results obtained demonstrated that the treatment of cells for 24 h with D

DOI: 10.1021/acs.jnatprod.8b00630 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Effect of compounds 4, 7, 13, and 14 on BrdU incorporation in PC3 cells. (A) Microscopic visualization, ×40 with oil, of representative Hoechst-stained (blue) and BrdU-stained (red) nuclei of PC3 cells treated with 1 μM of compounds for 24 h. (B) Quantification of BrdU incorporation by cells cultured for 24 h in the presence of 1 μM of compounds 4, 7, 13, and 14. *p < 0.05 vs control cells treated with DMSO. three times). After filtration and evaporation of the solvent to dryness in vacuo, 10 g of a crude MeOH extract was obtained. Of this, 3 g was fractionated using a Sephadex LH-20 (Pharmacia) column (100 × 5 cm), with MeOH as mobile phase, affording 53 fractions (8 mL), as monitored by TLC. Fractions 10−15 (930.0 mg) were subjected to n-butanol−water partition to remove free sugars, and the final butanol fractions (250.0 mg) were analyzed by an RP-HPLC-UV system.19 The elution gradient was obtained using water with 0.1% formic acid as eluent A and acetonitrile with 0.1% formic acid as B at a flow rate of 2.0 mL/ min. In particular, for fractions 10−12, the HPLC gradient started at 10% B, after 10 min % B was at 30%, after 10 min it was set at 40%, holding it for 10 min, after 10 min % B was at 50%, after 14 min it was at 70%, and finally after 3 min at 100%, it was held for 10 min, to yield compounds 3 (2.3 mg, tR = 16.04 min), 13 (4.7 mg, tR = 21.52 min), 8 (3.0 mg, tR = 22.28 min), 16 (4.3 mg, tR = 29.61 min), and 17 (3.8 mg, tR = 32.05 min). For fractions 13−15 (817.5 mg), the HPLC gradient started at 10% B, after 10 min % B was at 30%, after 16 min, it was at 54%, after 7 min it was at 63%, and after 11 min it was at 100%, holding it for 10 min. In this way compounds 1 (4.7 mg, tR = 14.21 min), 4 (3.0 mg, tR = 14.30 min), 7 (7.5 mg, tR = 16.30 min), 5 (2.5 mg, tR = 17.04 min), 18 (2.2 mg, tR = 19.17 min), 19 (2.3 mg, tR = 23.25 min), 20 (6.5 mg, tR = 23.95 min), 21 (3.0 mg, tR = 25.67 min), 22 (2.6 mg, tR = 26.83 min), and 23 (2.7 mg, tR = 32.41 min) were obtained. Fractions 16 and 17 (112.7 mg) were chromatographed by reversed-phase-HPLC-IR23 with a Knauer Prep MSC18 column (300 × 8 mm i.d.), using MeOH−H2O (5:5) as mobile phase (flow rate 2.5 mL/min) to yield compounds 2 (3.0 mg, tR = 12.0 min) and 14 (8.5 mg, tR = 16.5 min). Fractions 26 and 27 corresponded to compound 10 (13.8 mg), and fraction 34 corresponded to compound 6 (36.9 mg). Finally, fractions 25/26, 27, 34/35, and 38/39 corresponded to compounds 9 (8.0 mg), 6 (36.9 mg), 11 (9.7 mg), and 12 (9.9 mg), respectively.

compounds 4, 7, and 14 at 1 μM considerably reduced the number of cells incorporating BrdU or those in the S-phase of the cell cycle (Figure 1).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations, IR measurements, and NMR experiments were performed as reported previously.22 HRESIMS were carried out by an LTQ Orbitrap XL mass spectrometer as reported previously.20 Plant Material. Fresh aerial parts and roots of P. tomentosa L. were collected in the growing stage from rangelands at Kahnouj, Iran, in April 2016 and identified by one of the authors (S.H.H.). The plant material was air-dried and stored at room temperature. A voucher specimen (no. 8644) was deposited in the Natural Resource Herbarium of the University of Tehran, Iran. LC-HRESIMS Analysis. The MeOH extracts of the aerial parts and roots of P. tomentosa were analyzed separately, using a liquid chromatograph coupled to electrospray ionization and high-resolution mass spectrometer (LC-HRESIMS), operating in the same conditions reported previously.20 Linear gradient elution was carried out by using water with 0.1% formic acid as eluent A and acetonitrile with 0.1% formic acid as B. The HPLC gradient started at 10% (B), after 25 min, % B was at 60%, and after a further 10 min it was at 100%, holding it at this percentage for 10 min, before returning back to the starting percentage. The autosampler was set to inject 2 μL of both extracts (0.5 mg/mL CH3CN−H2O, 2:1). The source voltage was 3.5 kV, the capillary voltage was −48 kV, the tube lens offset was −176.5 V, the auxiliary gas was set at 5 (arbitrary units), and the sheath gas was set at at 15 (arbitrary units). Extraction and Isolation. The aerial parts of P. tomentosa (200 g) were dried and extracted at room temperature using solvents of increasing polarity inclusive of hexane (1.2 L for 3 days, two times), CHCl3 (1.3 L for 3 days, two times), and MeOH (1.3 L for 3 days, E

DOI: 10.1021/acs.jnatprod.8b00630 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

12β,6′-Dihydroxycalotropin (4): amorphous powder; [α]25D −15.5 (c 0.11, MeOH); IR (KBr) νmax 3438, 1750, 1635 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 2; HRESIMS m/z 563.2476 [M − H]−, calcd for C29H39O11, 563.2492. 6′-Hydroxycalotropin (13): amorphous powder; [α]25D +12.2 (c 0.10, MeOH); IR (KBr) νmax 3435, 1750, 1635 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 2; HRESIMS m/z 547.2558 [M − H]−, calcd for C29H39O10, 547.2543. 12β-Hydroxycalactin (14): amorphous powder; [α]25D −6.2 (c 0.15, MeOH); IR (KBr) νmax 3430, 1750, 1636 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 2; HRESIMS 547.2535 [M − H]− (calcd for C29H39O10, 547.2543). Cancer Cell Lines. PC3 prostate carcinoma cells, HeLa cervical carcinoma cells, Calu-1 epithelial lung cancer cells, and MCF-7 breast cancer cells were obtained from Interlab Cell Line Collection (IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy). They were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% antibiotic mixture. The U251MG human glioma cell line was obtained from CLS CellLines Service GmbH (Eppelheim, Germany). Cells were cultured in EMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 1% sodium pyruvate, and 1% nonessential amino acids and maintained as reported.24 Cell Viability Assays. To assess cell viability in the presence of isolated compounds from P. tomentosa aerial parts, cells were seeded in flat-bottomed 96-well microwells at the density of 1.0 × 104 to 5.0 × 104/cm2 depending on cell type and cultured for 24 h; then treatments were carried out for 72 h. Cell viability was performed by MTT assay as described previously.24 IC50 values were calculated as described by Piacente et al.5 S-Phase Entry Assay in PC3 Prostate Carcinoma Cells. The BrdU incorporation study was performed as reported previously.24 BrdU incorporation of cells treated with 10% serum was used as positive control.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00630. LC-HRESIMS profiles of the methanol extracts of P. tomentosa aerial parts and roots, 1H NMR, HSQC, HMBC, COSY, and ROESY spectra for compounds 4, 13, and 14, and LC-MS table of compounds 1−23 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39 089969763. Fax: +39 089969602. E-mail: [email protected]. ORCID

Sonia Piacente: 0000-0002-4998-2311 Author Contributions #

S. H. Hosseini and M. Masullo contributed equally to this work. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.8b00630 J. Nat. Prod. XXXX, XXX, XXX−XXX