Antiproliferative Carvotacetones from Sphaeranthus africanus

Article Cite This: J. Nat. Prod. 2018, 81, 1829−1834

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Antiproliferative Carvotacetones from Sphaeranthus africanus Huyen T. Tran,† Eva-Maria Pferschy-Wenzig,† Nadine Kretschmer,† Olaf Kunert,‡ Loi Huynh,§ and Rudolf Bauer*,† †

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitaetsplatz 4, 8010 Graz, Austria Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Graz, Universitaetsplatz 1, 8010 Graz, Austria § Saigon Pharmaceutical Sciences and Technology Center (SAPHARCEN), University of Medicine and Pharmacy at Ho Chi Minh City, 41 Dinh Tien Hoang Street, District 1, Ho Chi Minh City, Vietnam Downloaded via KAOHSIUNG MEDICAL UNIV on August 24, 2018 at 07:52:13 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Five carvotacetone derivatives, including two known ones, 3,5-diangeloyloxy-7-hydroxycarvotacetone (1) and 3-angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (2), along with three new compounds, 3-angeloyloxy-5-[3″-chloro2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (3), 5angeloyloxy-7-hydroxy-3-tigloyloxycarvotacetone (4), and 3-angeloyloxy-5,7-dihydroxycarvotacetone (5), were isolated from the aerial parts of Sphaeranthus africanus collected in Vietnam. Bioassay-guided fractionation was monitored by the antiproliferative activity on CCRF-CEM human cancer cells. The structures of compounds 1−5 were determined on the basis of NMR spectroscopic and mass spectrometric data. Activities of compounds 1−5 were evaluated in vitro against the human cancer cell lines CCRF-CEM, MDA-MB-231, U-251, and HCT-116. All compounds exhibited significant antiproliferative activity against all four cancer cell lines. CCRF-CEM was most sensitive to the compounds, with IC50 values ranging from 0.6 to 1.5 μM. Compounds 3 and 4 possessed the highest activity, with IC50 values in the four cell lines ranging from 0.6 to 2.9 μM and 1.3 to 2.5 μM, respectively. These compounds also showed inhibitory activity toward the HEK293 human embryonic kidney cells with IC50 values ranging from 2.5 to 5.5 μM. This is the first time that antiproliferative activity of S. africanus has been reported, and 1−5 are the most cytotoxic carvotacetone derivatives reported so far.

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an antitussive and expectorant. The pounded leaves are applied externally to relieve pain and swelling.6 Phytochemical investigations have revealed that the major secondary metabolites of members of this genus are monoterpenes,7−10 sesquiterpenes,11 essential oils,12,13 flavonoids,14 and alkaloids.15 Chemical investigations of S. africanus are scant, and, hitherto, only two compounds and a mixture of carvotacetone derivatives, chrysosplenol D, squalene, spinasterol, and stigmasterol, from S. africanus were reported.16 There is no literature reporting on the cytotoxicity of the plant. In the current study, bioguided fractionation led to the isolation of potent antiproliferative constituents.

ancer is one of the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases in 2012 and 8.8 million cancer-related deaths in 2015, affecting populations in all countries and all regions of the world.1 Natural products have played a major role in cancer chemotherapy for over 50 years.2 A 2016 report of Newman and Cragg reviewed the time frame from around the 1940s to the end of 2014 and found that of the 175 small molecules approved, 131, or 75%, are other than “S” (synthetic), with 85, or 49%, actually being either natural products or directly derived therefrom.3 The genus Sphaeranthus, with ca. 40 species, is distributed mainly over the tropical areas of Africa, southern Asia, and Australia.4 Sphaeranthus af ricanus L. (syn. Sphaeranthus cochinchinensis Loureiro; S. microcephalus Willdenow; S. suberiflorus Hayata; family Asteraceae) occurs naturally in East Africa, Madagascar, southern Asia, and northern Australia.5 In Vietnam, it is known as “Cúc chân vịt” or as ́ S. africanus has been used in traditional medicine in “Bọ xit”. Vietnam to alleviate swelling and as a sedative. Pressed juice from the fresh leaves of S. africanus is used for mouth and throat washes to treat sore throat. The decoction is also used as © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The antiproliferative activity of crude extracts and chromatographic fractions of S. af ricanus was tested in CCRF-CEM (human acute lymphoblastic leukemia) cells. The n-hexane and CH2Cl2 extracts exhibited high activity (5 μg/mL). HPLC-MS analysis of the active extracts showed similar phytochemical Received: April 16, 2018 Published: August 3, 2018 1829

DOI: 10.1021/acs.jnatprod.8b00309 J. Nat. Prod. 2018, 81, 1829−1834

Journal of Natural Products

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profiles for the n-hexane and CH2Cl2 extracts, but a higher enrichment of constituents of medium polarity in the CH2Cl2 extract, prompting us to select this extract for chromatographic separation. From the chromatographic fractions showing cytotoxic activity, two known (1 and 2) and three new carvotacetone compounds (3, 4, and 5) were obtained. All pure compounds were also evaluated against MDA-MB-231 (human breast adenocarcinoma), U-251 (human glioblastoma astrocytoma), HCT-116 (human colon carcinoma), and HEK293 (human embryonic kidney) cells.

was recorded in DMSO-d6. The resonances of two hydroxy groups were observed, and both showed a set of HMBC correlations that allowed an unambiguous assignment of these two groups: The hydroxy proton at 5.15 ppm showed HMBC correlations to carbon resonances at 57.2 and 139.9 ppm, which placed this hydroxy group at C-7. The hydroxy proton at 5.70 ppm was correlated with a carbonyl resonance at 172.8 ppm and the two oxidized carbons at 77.1 and 62.5 ppm in the side chain. Hence, the second hydroxy group was assigned to C-2″ and, consequently, the chlorine atom to C-3″. Therefore, the new compound (3) is the hydrochlorinated derivative of compound 2, and its structure was assigned as 3-angeloyloxy-5[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone. From the HRESIMS data, a molecular formula of C20H28O6 was deduced for compound 4, indicating an isomer of compound 1. The 1H NMR spectrum of 4 showed the same number of methyl groups and olefinic protons as 1, and, in addition, these groups showed the same multiplicities. However, the proton and carbon resonance of two methyl groups and an olefinic methine group were different. This suggested the replacement of an angeloyl moiety with a tigloyl moiety in 4, which was confirmed by comparison with reported carbon shift values.17 HMBC correlation of the angeloyl methyl resonance at 1.91 ppm with the carbonyl carbon at 168 ppm and the correlation of H-5 (5.79 ppm) with the same carbonyl resonance placed the angeloyl moiety at C-5. Correspondingly, the HMBC correlations of the tigloyl methyl group at 1.88 ppm and H-3 (5.80 ppm) with the same carbonyl carbon at 168.7 ppm placed the tigloyl moiety at C-3. Hence, the structure of the new compound 4 was elucidated as 5-angeloyloxy-7-hydroxy-3-tigloyloxycarvotacetone. The negative mode HRESIMS data of compound 5 indicated a molecular formula of C15H22O5. The 1H NMR data, which showed only four methyl resonances and the mass difference in comparison to 1 or 4, indicated the absence of a pentanoate moiety. The assignment was done by comparing shift values: As the chemical shift value of H-5 changed from around 5.8 in compounds 1−4 to 4.5 ppm in 5, and the C-5 shift from 75 to 73.2 ppm, a free OH at C-5 is plausible, in particular because the proton and carbon chemical shift values at C-1 to C-3 did not change. The side chain was identified as an angeloyl moiety. Therefore, the structure of the new compound 5 was defined as 3-angeloyloxy-5,7-dihydroxycarvotacetone. For H-3 and H-5, compounds 1−5 showed the same multiplicity and similar J-values. These findings indicate that the relative configurations at C-3 and C-5 are the same in all compounds; that is, H-3 has an equatorial orientation, which leads to a (3R*)-configuration, while H-5 has an axial orientation, and thus a (5S*)-configuration. Compounds 1−5 were evaluated for their antiproliferative activity against CCRF-CEM, MDA-MB-231, U-251, and HCT116 cancer cells. The new carvotacetone (3), 3-angeloyloxy-5[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone, was found to be the most active in inhibiting the proliferation of CCRF-CEM and MDA-MB-231 cancer cells, with IC50 values of 0.6 and 1.5 μM, respectively. New compound 4, 5-angeloyloxy-7-hydroxy-3-tigloyloxycarvotacetone, was most potent toward U-251 and HCT-116 cancer cells, with IC50 values of 2.1 and 2.5 μM, while new compound 5, 3-angeloyloxy-5,7-dihydroxycarvotacetone, was less active against all four cell lines (Table 3). Thus, the acyl side chain at

Compounds 1 and 2 were identified on the basis of their NMR spectroscopic and mass spectrometric data as known carvotanacetone derivatives described by Ragasa et al. (2010),9 namely, 3,5-diangeloyloxy-7-hydroxycarvotacetone (1) and 3angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (2), respectively. From the HRESIMS data, C20H29O7Cl was deduced as the most likely molecular formula for compound 3. The isotopic pattern of the HR mass spectra indicated the presence of a chlorine atom in the molecule. In order to rule out that the presence of chlorine was due to in situ adduct formation in the ESI source, compound 3 was subjected to GC-MS analysis. The main ion in the GC-EI mass spectrum was derived from the angeloyl moiety. The m/z 416 molecular ion showed a similar isotope pattern to the one found by HRESIMS analysis. Therefore, a chlorine must be present in the C-5 ester moiety. The 1H NMR spectrum indicated a similar structure to that for compound 2, i.e., a carvotacetone oxidized at C-3, C-5, and C-7 with the presence of substituents at C-3 and C-5. Comparison of proton shift values with compound 2 revealed an angeloyl moiety, which was confirmed by comparison of the carbon shift values with NOE-based assignments by Bodensieck et al.17 HMBC correlations of H-3 (5.87 ppm) and H3-5′ (1.92 ppm) with the carbonyl carbon at 167.9 ppm indicated the presence of the angeloyl moiety at C-3. The chemical shift pattern of the second side chain indicated a 2-methylbutyrate moiety substituted with two electronegative groups at C-2″ and C-3″. The mass of the compound required that one of these substituents is a chlorine and the second a hydroxy group. However, with the data recorded in methanold4, there was no clear experimental evidence for the position of this substituent. Therefore, an NMR data set of compound 3 1830



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Table 1. 1H NMR Chemical Shifts of Compounds 1−5 in MeOH-d4 at 25 °Ca position 2 3 4 5 7 8 9 10 7-OH C-3 OR1 3′ 4′ 5′ C-5 OR2 3″ 4″ 5″

1 7.01, 5.74, 2.48, 5.82, 4.27, 2.05, 0.97, 1.03, 2.14,

2

d (6.0) dd (3.6, 6.0) ddd (3.6, 4.8, 12.0) d (12.0) d (14.4); 4.34, d (14.4) m d (7.2) d (6.6) brs

7.02, 5.74, 2.48, 5.82, 4.26, 2.05, 0.96, 1.02, 2.14,

d (6.0) dd (3.6, 6.0) ddd (3.6, 4.8, 12.6) d (12.6) d (14.4); 4.33, d (14.4) m d (7.2) d (6.6) brs

3

4

5

7.06, dt (5.8, 1.6) 5.87, dd (5.8, 3.5) 2.57, ddd (3.5, 4.5, 12.6) 5.82, d (12.6) 4.24/4.24, brs 2.18, m 0.99, d (7.0) 1.09, d (7.0)

7.06, dt (5.8, 1.8) 5.80, dd (5.8, 3.4) 2.56, ddd (3.4, 4.7, 12.7) 5.79, d (12.7) 4.23/4.23, brs 2.04, m 0.98, d (6.7) 1.04, d (6.7)

6.98, dt (5.7, 1.7) 5.76, dd (5.7, 3.0) 2.15, m 4.50, d (12.5) 4.25/4.25, brs 2.16, m 1.06, d (6.7) 1.10, d (6.7)

6.18, qq (7.0, 1.3) 1.97, dq (7.0, 1.3) 1.89, m

6.13, d (7.2) 2.01, d (7.2) 1.90, s

6.16, q (7.2) 2.00, d (7.2) 1.88, s

6.21, qq (7.3, 1.4) 2.01, dq (7.3, 1.3) 1.92, m

6.95, qq (7.1, 1.4) 1.86, m 1.88, m

6.14, q (7.2) 2.01, d (7.2) 1.94, s

3.10, q (4.8) 1.42, d (4.8) 1.62, s

4.43, q (6.6) 1.55, d (6.7) 1.53, s

6.20, qq (7.3, 1.3) 1.99, dq (7.3, 1.5) 1.90, m

a

J values (Hz) are given in parentheses. Assignments are based on 1H−1H, DQFCOSY, HSQC, and HMBC NMR spectra.

Table 2. 13C NMR Chemical Shifts of Compounds 1−5 in MeOH-d4 at 25 °Ca position

1

1 138.89, C 2 139.08, CH 3 66.82, CH 4 46.45, CH 5 73.04, CH 6 195.30, C 7 60.75, CH2 8 27.69, CH 9 19.56, CH3 10 19.87, CH3 C-3 OR1 1′ 166.70, C 2′ 126.80, C 3′ 140.35, CH 4′ 16.02, CH3 5′ 20.60, CH3 C-5 OR2 1″ 166.90, C 2″ 127.20, C 3″ 139.39, CH 4″ 15.80, CH3 5″ 20.52, CH3

2

3

4

5

139.00, C 139.06, CH 66.53, CH 46.23, CH 74.07, CH 194.19, C 60.54, CH2 27.51, CH 19.39, CH3 19.80, CH3




166.62, C 126.83, C 140.52, CH 16.01, CH3 20.60, CH3

167.9, C 128.6, C 140.7, CH 16.3, CH3 20.6, CH3

168.7, C 129.3, C 139.9, CH 14.5, CH3 12.2, CH3

168.2, C 128.6, C 140.1, CH 16.0, CH3 20.7, CH3

169.33, C 60.03, C 60.18, CH 13.94, CH3 19.36, CH3

174.8, C 67.7, C 63.0, CH 18.6, CH3 23.8, CH3

168.1, C 128.5, C 140.6, CH 16.1, CH3 20.7, CH3

C-5 of these compounds seems to be required for potent cytotoxicity. Particularly, when the acyl moiety at C-5 is removed as in compound 5, the resulting cytotoxicity is reduced 2- to 3-fold when compared with 3,5-diangeloyloxy-7hydroxycarvotacetone (1), 3-angeloyloxy-5-[2″,3″-epoxy-2″methylbutanoyloxy]-7-hydroxycarvotacetone (2), and 3-angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (3). All compounds were also tested against HEK-293 human embryonic kidney cells and showed IC50 values ranging from 2.5 to 5.5 μM. Table 3 indicates a lack of selective inhibitory activity toward adherent cancer cell lines; however, the IC50 values of 1−5 against leukemia cells were 2.3- to 3.6-fold lower than against nontumorigenic cells, suggesting some selectivity against this type of cancer cells. Carvotacetone is one of the major volatile constituents of certain species of genera belonging to the Asteraceae family, such as Blumea,18−21 Pulicaria,22−24 and Francoeuria.25 In essential oils of Pluchea26 and Vernonia27 species, it was identified as a dominating constituent. The general structure of carvotacetones isolated from members of the genus Sphaeranthus was initially elucidated by Bohlmann and Mungai in 1990.4 These derivatives were reported in five species of Sphaeranthus: for S. bullatus, 11 derivatives were identified,4,7 followed by Sphaeranthus confertifolius, S. suaveolens, and S. ukambensis with seven, five, and six constituents identified, respectively.8,10,28 For S. africanus, five carvotacetone derivatives with three new compounds and two known compounds were described in this report for the first time.

a

Measured at 100 MHz; J values (Hz) are given in parentheses. Assignment are based on HSQC and HMBC NMR spectra.

Table 3. IC50 (μM) Values of Antiproliferative Activity of Compounds 1−5 After 72 h of Treatmenta compound 1 2 3 4 5 vinblastine

HEK-293 2.5 3.0 2.8 3.0 5.5 2.7

± 0.1 ± 0.2 ± 0.2 ± 0.1 ± 0.5 × 10−2 ± 4.0 × 10−4

CCRF-CEM 0.8 1.2 0.6 1.3 1.5 9.4

± 0.03 ± 0.03 ± 0.1 ± 0.02 ± 0.04 × 10−3 ± 2.0 × 10−4

MDA-MB-231 1.8 2.0 1.5 2.0 6.5 3.1

± 0.1 ± 0.03 ± 0.1 ± 0.1 ± 0.1 × 10−2 ± 4.6 × 10−3

U-251 3.0 2.9 2.1 2.1 4.3 8.1

± 0.1 ± 0.03 ± 0.01 ± 0.02 ± 0.04 × 10−3 ± 1.0 × 10−3

HCT-116 3.9 2.6 2.9 2.5 9.5 8.7

± 0.1 ± 0.1 ± 0.04 ± 0.02 ± 0.2 × 10−3 ± 5.0 × 10−4

Results are expressed as means ± SEM of six independent experiments. IC50 values were determined using the four-parameter logistic curve and individual values of all independent experiments; CCRF-CEM = human acute lymphoblastic leukemia; MDA-MB-231 = human breast adenocarcinoma; HCT-116 = human colon carcinoma; U-251 = human glioblastoma astrocytoma; HEK-293 = human embryonic kidney cells.

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used between each run. The detection wavelength was 254 nm, and the column thermostat was set at 25 °C. The injection volume was 10 μL; the flow rate was 0.8 mL/min. Aluminum TLC plates coated with silica gel 60 F254 20 × 20 (Merck) were used for TLC and analyzed at 254 and 366 nm and daylight using a Camag Reprostar 3 TLC visualizer. Column chromatographic separations were performed using Sephadex LH-20 (Pharmacia Biotech AB, Stockholm, Sweden) and silica gel 60 (0.040−0.063 mm; Merck, VWR, Darmstadt, Germany) as stationary phases. Plant Material. The stems and leaves of S. africanus were harvested in the Tam Thành commune, Phú Ninh District, Quang Nam Province of Vietnam, in February 2016. The plant was identified by Dr. Vo Van Chi, former lecturer in the Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, Vietnam. The identity of the plant was confirmed using genomic analysis based on its ITS region by Prof. Dr. Günther Heubl, Faculty of Biology, Ludwig-Maximilians University, Munich, Germany. A voucher specimen (SA-VN-0216) has been deposited at the Institute of Pharmaceutical Sciences, Pharmacognosy Department, University of Graz. Extraction and Activity-Guided Compound Isolation. The air-dried and milled material containing leaves and stems (1.5 kg) of S. africanus was percolated with 20 L of 96% EtOH at room temperature. The solvent was removed under reduced pressure to yield 130 g of crude extract, which was suspended in 0.5 L of water and partitioned sequentially with n-hexane (200 mL × 6), CH2Cl2 (200 mL × 6), EtOAc (200 mL × 6), and then n-BuOH (200 mL × 6). Each of the combined organic layers was evaporated to dryness, affording n-hexane (SA-Hx, 6.0 g), CH2Cl2 (SA-DCM, 4.0 g), EtOAc (SA-Et, 7.5 g), and n-BuOH (SA-Bu, 10 g) extracts. The SA-Hx and SA-DCM extracts exhibited activity against the CCRF-CEM cell line at a concentration of 5 μg/mL. Since the minor compounds of the nhexane extract were also present in a higher amount in the dichloromethane (DCM) extract (verified by HPLC-MS experiments), accordingly, bioassay-guided fractionation of SA-DCM was performed to facilitate the isolation process. The SA-DCM extract (3 g) was chromatographed over a silica gel column (4 × 70 cm), eluted with gradient mixtures of n-hexane− EtOAc (100:1 → 50:50). The eluates were monitored by TLC analysis to afford 20 combined fractions (SA.D.1 to SA.D.20). Fraction SA.D.9 (n-hexane−EtOAc, 80:20; 80 mg) was separated by Sephadex LH-20 column chromatography (2.5 × 70 cm) using MeOH as mobile phase to obtain compound 1 (15 mg). Fraction SA.D.16 (n-hexane−EtOAc, 70:30; 50 mg) was applied to Sephadex LH-20 column chromatography (2.5 × 70 cm), eluted with MeOH, affording compound 2 (7 mg). Fraction SA.D.19 (n-hexane−EtOAc, 60:40; 110 mg) was rechromatographed on a silica gel column (1.5 × 30 cm), eluted with a gradient solvent system of n-hexane−EtOAc (90:10 → 50:50), to give four subfractions (SA.D.19.1 to SA.D.19.4). Compound 3 (6 mg) was obtained from SA.D.19.4. Fraction SA.D.12 (n-hexane−EtOAc, 80:20; 57 mg) was separated by silica gel column chromatography (1.5 × 30 cm), using n-hexane−EtOAc (90:10 → 50:50) as eluting solvent, to yield five subfractions (SA.D.12.1 to SA.D.12.5). Compound 4 (5 mg) was obtained from subfraction SA.D.12.4. Fraction SA.D.15 (n-hexane−EtOAc, 70:30; 45 mg) was fractionated by silica gel column chromatography (1.5 × 30 cm), with a gradient solvent system of n-hexane−EtOAc (95:5 → 50:50), to give six subfractions (SA.D.15.1 to SA.D.15.6). Subfraction SA.D.15.4 was purified by preparative HPTLC (silica gel 60 F251 10 × 20 cm, Merck), using n-hexane−EtOAc (70:30) as solvent, to afford compound 5 (3 mg). 3,5-Diangeloyloxy-7-hydroxycarvotacetone (1): C20H28O6, colorless oil; [α]25589 −102 (c 0.09, MeOH); UV (MeOH) λmax 215 nm; 1 H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz), see Tables 1 and 2, respectively; HRESIMS negative mode m/ z 363.1810 [M − H]−, 263.1286 [M − H − angelic acid]−, positive mode m/z 365.1965 [M + H]+, 387.1783 [M + Na]+, 265.1438 [M + H − angelic acid]+, 247.1333 [M + H − angelic acid − H2O]+. HRESIMS data were consistent with the chemical formula C20H28O6.

Among carvotacetone derivatives that have been identified from the genus Sphaeranthus, overlap can be observed between the four species S. bullatus, S. suaveolens, S. ukambensis, and S. confertifolius. 5α-Acetoxy-7-hydroxy-3β-tigloyloxycarvotacetone, the main constituent of S. suaveolens,10 was also isolated from S. bullatus. These species also share the more common compounds 3β-acetoxy-5α,7-dihydroxycarvotacetone and 3,7dihydroxy-5-tigloyloxycarvotacetone.4,7 3-Acetoxy-7-hydroxy5-tigloyloxycarvotacetone has been identified in three species, S. bullatus, S. confertifolius, and S. ukambensis.7,8,28 Moreover, 3α-acetoxy-7-hydroxy-5β-[4-hydroxytigloyloxy]carvotacetone, the major compound of S. ukambensis, was also reported from S. confertifolius.8,28 Interestingly, the carvotacetone derivatives isolated from S. africanus are exclusive to this species. Concerning biological effects, essential oils dominated by carvotacetone exhibited antimicrobial,23,26 insecticidal,26 antifungal,22 and antioxidant activity19 but only weak cytotoxic activity.23,27,29 Four carvotacetones from S. bullatus were evaluated for antiparasitic activity in vitro, and three of these were found to be cytotoxic.7 Four derivates from S. ukambensis were reported as inhibitors of the ubiquitin-proteasome pathway.8 Our results show that leukemia cells reacted more sensitively to these compounds than nontumorigenic cells. Therefore, carvotacetones may be interesting lead compounds in leukemia research.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Jasco P-2000 polarimeter (Gross-Umstadt, Germany). NMR data (1H, 13C, DQFCOSY, HSQC, and HMBC) were acquired on a Varian Unitylnova spectrometer (400 MHz) or a Bruker Avance spectrometer (700 MHz, equipped with a cryoprobe). Data sets of all compounds were recorded in MeOH-d4 (Eurisotop, Saint-Aubin Cedex, France), and the solvent resonance was set to 3.31 ppm in the proton spectra and 49.0 ppm in the carbon spectra. In addition, a set of NMR data of compound 3 was recorded in DMSO-d6 at 25 °C. For LC-DAD-ESI-HRMS experiments, a Thermo Scientific Dionex Ultimate 3000 UHPLC system equipped with a Thermo Q Exactive hybrid quadrupole orbitrap mass spectrometer was used. As a stationary phase, a Kinetex C18 column (1.7 μm, 2.1 × 10 mm, Phenomenex) was used. The mobile phase consisted of H2O (solvent A) and CH3CN (solvent B), using the following gradient: 0−15 min, 20−50% B in A; 15−30 min, 50−70% B in A; 30−43 min, 70−90% B in A; 45−45 min, 90−20% B in A; 46−55 min, re-equilibration. The flow rate was 0.5 mL/min, and the column temperature was 25 °C. The MS parameters were as follows: probe heater temperature: 400 °C; capillary temperature: 350 °C; sheath gas flow: 50 arbitrary units; auxiliary gas flow: 15 arbitrary units; spray voltage: 3.44 kV (ESI positive mode), −3 kV (ESI negative mode). GC-MS analyses were performed on an HP7890A GC System hyphenated with a 5975C VL MSD (Agilent Technologies, Waldbronn, Germany). An HP-5MS column of 0.25 mm inner diameter × 30 m length, film thickness 0.5 μm (Agilent Technologies, Waldbronn, Germany), was used as stationary phase, and helium was used as carrier gas. The injector and detector temperatures were 240 °C. The sample was injected in the splitless mode. The temperature program was as follows: 50 °C for 2 min; 50−300 °C at 6 °C/min; 300 °C for 10 min. HPLC analyses were carried out using an Agilent 1260 Infinity system equipped with an autosampler, DAD, and column thermostat. Separations were performed on a Grace Alltima C18 100A (250 × 4.6 mm i.d., 5 μm) column (Alltech Associates, Inc., USA). The mobile phase consisted of water (HPLC grade) (solvent A) and (solvent B), using MeCN (HPLC grade) with the following gradient: 0−15 min, linear gradient from 10% to 40% B; 15−30 min, linear gradient from 40% to 70% B; 30−40 min, linear gradient from 70% to 90% B; 40− 45 min, linear gradient from 90% to 95% B. A postrun column equilibration period consisting of 5 min at the starting conditions was 1832



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(0.5% DMSO). The final DMSO concentration of 0.5% did not affect the cells. Statistical Analysis. Nonlinear regression (with sigmoidal dose response) was used to calculate the IC50 values using SigmaPlot 13.0 (Systat Software Inc., Chicago, IL, USA). IC50 values were determined using the four-parameter logistic curve. At least eight concentrations of the test compound and two different cell passages, each tested in three independent wells, were used. Results are expressed as mean ± SEM.

1

H and 13C NMR data were in accordance with reported literature data.9 3-Angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7hydroxycarvotacetone (2): C20H28O7, colorless oil; [α]25589 −112 (c 0.08, MeOH); UV (MeOH) λmax 225 nm; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz), see Tables 1 and 2, respectively; HRESIMS negative mode m/z 379.1759 [M − H]−, 279.1338 [M − H − angelic acid]−, positive mode m/z 381.1914 [M + H]+, 403.1736 [M + Na]+. From 13C NMR and HRESIMS data, the molecular formula C20H28O7 was deduced. 1H and 13C NMR data were in accordance with reported literature data.9 3-Angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]7-hydroxycarvotacetone (3): C20H29ClO7, colorless oil; [α]25589 −109 (c 0.1, MeOH); UV (MeOH) λmax 230 nm; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz), see Tables 1 and 2, respectively; HRESIMS negative mode m/z 415.1530 [M − H]−, 451.1300 [M + Cl]−, 461.1585 [M + HCOO]−, 315.1006 [M − H − angelic acid]−, 279.1237 [M − H − angelic acid − HCl]−, positive mode m/z 439.1492 [M + Na]+, 265.1434 [M + H − 3chloro-2-hydroxy-2-methylbutanoic acid]+ (calcd for C20H29ClO7, 416.1601); GCMS (EI) 416 [M]+, 83 [angeloyl]+. 5-Angeloyloxy-7-hydroxy-3-tigloyloxycarvotacetone (4): C20H28O6, colorless oil; [α]25589 −102 (c 0.03, MeOH); UV (MeOH) λmax 227 nm; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz), see Tables 1 and 2, respectively; HRESIMS negative mode (very weak ionization) m/z 363.1815 [M − H]−, 263.1288 [M − H − angeloyl or tigloyl]−, positive mode m/z 387.1777 [M + Na]+, 365.1959 [M + H]+, 265.1435 [M + H − angeloyl or tigloyl]+ (calcd for C20H28O6, 364.1886). 3-Angeloyloxy-5,7-dihydroxycarvotacetone (5): C15H22O5, colorless oil; [α]25589 −57 (c 0.03, MeOH); UV (MeOH) λmax 227 nm; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz), see Tables 1 and 2, respectively; HRESIMS negative mode m/ z 281.1394 [M − H]−, 181.0869 [M − H − angeloyl]−, positive mode: no ionization (calcd for C15H22O5, 282.1467). Cell Culture. Human CCRF-CEM leukemia and MDA-MB-231 breast cancer cell lines were maintained in RPMI 1640 medium (Gibco, Invitrogen, Vienna, Austria), supplemented with 2 mM Lglutamine (Gibco), 10% fetal bovine serum (FBS, Gibco), 100 units/ mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco) (1% penicillin/streptomycin). Human U-251 glioblastoma and HCT-116 colon cancer cell lines were grown in high-glucose Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 4 mM L-glutamine, 10% FBS, and 1% penicillin/streptomycin. Human embryonic kidney HEK-293 cells were cultivated in DMEM:Ham’s F12 (1:1 mixture) supplemented with 2 mM L-glutamine, 10% FBS, and 1% penicillin/streptomycin. All cells were kept in a humidified 5% CO2 atmosphere at 37 °C and passaged at 90% confluence. XTT Viability Assay. The XTT viability assay was performed to determine the cytotoxicity of extracts, fractions, and isolated pure compounds. This assay is based on the cleavage of the tetrazolium salt XTT by mitochondrial dehydrogenases and provides an easy, safe, and nonradioactive method to determine cell growth and viability.30 It was done in accordance with the manufacturer’s protocol and as reported previously31 (Roche Diagnostics, Mannheim, Germany; cell proliferation kit II (XTT), cat. no 11 465 015 001). Briefly, 10 000 cells/well in the case of CCRF-CEM and HEK-293 cells and 5000 cells/wells for MDA-MB-231, HCT-116, and U-251 cell lines were seeded into 96-well plates (100 μL, flat bottom) and treated with various concentrations of the extracts, fractions, and pure compounds for 72 h. Adherent cell lines were grown overnight before the test compounds were added. After 72 h, a freshly prepared XTT solution (5 mL of XTT plus 100 μL of electron coupling reagent) was added and analyzed after another 1.5 h (adherent cells) or 4 h (CCRF-CEM cells), and cell viability was determined by photometric measurement using a Hidex Sense microplate reader (Hidex, Turku, Finland). Vinblastine served as positive control (0.01 μM). Sample Preparation. Extracts, fractions, and pure compounds were freshly dissolved in DMSO, subsequently diluted with medium, and used immediately. Control cells represent vehicle-treated cells

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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.8b00309. 1

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H and 13C NMR spectra of compounds 3 and 5; 1H NMR spectra of compound 4; HSQC spectra for compound 3−5 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +43 (0)316 380-8700. Fax: +43 (0)316 380-9860. Email: rudolf.bauer(at)uni-graz.at. ORCID

Huyen T. Tran: 0000-0003-1382-5020 Rudolf Bauer: 0000-0002-0057-5547 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Vo Van Chi, former lecturer in Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, Vietnam, for identification of the plant and Prof. Dr. Günther Heubl, Faculty of Biology, Ludwig-Maximilians University, Munich, Germany, for confirming the plant authenticity by DNA analysis. We thank Ass. Prof. Dr. Beate Rinner, Core Facility Alternative Biomodels & Preclinical Imaging, Medical University of Graz, Austria, for providing the HEK-293 cells.

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REFERENCES

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Antiproliferative Carvotacetones from Sphaeranthus africanus

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