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Author(s): Stefano Dall’Acqua, Gregorio Peron, Sara Ferrari, Valentina Gandin, Massimo Bramucci, Luana Quassinti, Pavol Martonfi, and Filippo Maggi
Article title: Phytochemical investigations and antiproliferative secondary metabolites fromThymus alternans growing in Slovakia
Article no: IPHB_A_1291689 Enclosures: 1) Query sheet
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RESEARCH ARTICLE
Phytochemical investigations and antiproliferative secondary metabolites
from
Thymus alternans growing in Slovakia
Stefano Dall’Acquaa, Gregorio Perona, Sara Ferraria, Valentina Gandina, Massimo Bramuccib, Luana Quassintib, Pavol Martonfic and Filippo Maggib
a
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy;bSchool of Pharmacy, University of Camerino, Camerino, Italy;cDepartment of Botany, Institute of Biology and Ecology, P. J. Safarik University, Kosice, Slovakia
ABSTRACT
Context: Thymus alternans Klokov (Lamiaceae) is a neglected species of the genus Thymus (Sect. Serpyllum) endemic to Carpathian area, where it is used as a flavouring agent and for medicinal purposes. Objective: The aim of the work was to identify antiproliferative constituents from the flowering aerial parts of this plant.
Materials and methods:Thymus alternans extracts were analyzed by HPLC-MSnand subjected to exten-sive chromatographic separations. The isolated compounds (phenolics and triterpenes) were structurally elucidated by MS and 1D and 2D NMR experiments. Essential oil (EO) composition was determined by GC-FID and GC-MS. Six purified triterpenes and EO were assayed for in vitro antiproliferative activity against a panel of human cancer cells, namely, breast (MDA-MB 231), colon (HCT-15 and HCT116), lung (U1810), pancreatic (BxPC3), melanoma (A375) and cervical carcinoma (A431) cells.
Results: The structures of the isolated compounds were achieved on the basis of H-NMR and MS experiments. Luteolin-40-O-b-D-glucopyranoside (P1), chrysoeriol-7-O-b-D-glucopyranoside (P2),
chrysoeriol-5-O-b-D-glucopyranoside (P3), apigenin-7-O-b-D-glucopyranoside (P4), rosmarinic acid (P5), rosmarinic
acid-30-O-b-D-glucopyranoside (P6), caffeic acid-3-O-b-D-glucopyranoside (P7), 3a-hydroxy-urs-12,15-diene (T1),
a-amyrin (T2), b-amyrin (T3), isoursenol (T4), epitaraxerol (T5), and oleanolic acid (T6). GC-MS analysis revealed that the EO ofT. alternans was devoid of phenols and belonged to the nerolidol-chemotype, that is, typical of the Sect.Serpyllum. The six purified triterpenes (T1-T6) were active with IC50 ranging
from 0.5 to 5lM being comparable or better than those of reference compounds betulinic acid and cis-platin. The EO exhibited significant effects on A375, MDA-MB 231 and HCT116 cell lines with IC50 in the
range of 5–8 lg/mL.
Conclusion: The reported results suggest thatT. alternans can be considered as a good source of phyto-constituents with possible importance in the pharmaceutical field.
ARTICLE HISTORY Received 1 August 2016 Revised 17 January 2017 Accepted 1 February 2017 KEYWORDS Triterpene; flavonoids; essential oil; GC-MS; HPLC-MS Introduction
The genus Thymus L. (Lamiaceae) includes 215 species divided in 8 sections, with the evolutionary centres in the Mediterranean area from where it spreads to Europe, Asia, Northern America and Abyssinia, as well as to Greenland (Morales 2002). Many species of this genus are used throughout the world in the trad-itional medicine and are nowadays important species in food, cosmetics and pharmaceutics (Nabavi et al. 2015). For this rea-son, the evaluation of the phytochemical composition of different Thymus species can be considered fundamental for possible uses of the different plants in pharmaceutical, cosmetic and food products. A large number of studies were conducted especially on T. vulgaris L., a spice used in many regions of the world and one of the most important herbal drugs used as an antibacterial agent (Nabavi et al.2015). However, several species of the genus remain unexplored and deserve attention of scientists. Among them T. alternans Klokov is endemic to Carpathian regions and can be considered a neglected species of the genus Thymus
(Klokov 1960). Thymus alternans grows in dry meadows and hilly areas of Carpathian regions up to 1100 m above the sea level (Martonfi 1996; Nachychko 2014). This species belongs to the Sect. Serpyllum (Miller) Bentham, Subsect. Alternantes Klokov, and taxonomically is widely accepted by specialists of the genus Thymus, as well as in the IPNI (The International Plant Names Index, www.ipni.org) database. Actually, T. alternans is recognized as a well distinguished taxon, characterized by sympo-dial branching with terminal fertile branch and alelotrichous indument (Martonfi 1996). The taxon belongs to the group of tetraploid taxa with 2 n¼ 4 ¼ 56 (Martonfi & Martonfiova 1996) and is probably closely related to the species T. nummular-ius M. Bieb. and T. pseudonummularnummular-ius Klokov et Des.-Shost. from Caucasus.
Thymus alternans has been frequently employed in the folk medicine of the regions in which occurs, often together with T. pulegioides L., with which it shares the same habitat. Owing to the polymorphism occurring in many Thymus species, including 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128
CONTACTStefano Dall’Acqua stefano.dallacqua@unipd.it Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-bution, and reproduction in any medium, provided the original work is properly cited.
PHARMACEUTICAL BIOLOGY, 2017
T. serpyllum s.l., which makes their identification very problem-atic, spontaneous plants of T. alternans are often used together with those of T. pulegioides (both belonging to the sect. Serpyllum) as an ingredient of the drug called ‘Serpylli herba’, which is included in the European Pharmacopoeia (2008).
Continuing our research on secondary metabolites from unex-plored species of the genus Thymus growing in Slovakia (Maggi et al. 2014), the present work reports a comprehensive phyto-chemical analysis on polar phenolics, triterpenes, and volatile constituents obtained from the flowering aerial parts of T. alter-nans growing in Slovakia. Extracts obtained with solvents of increasing polarity were analyzed by HPLC-MSn and were fur-ther subjected to extensive chromatographic separations, leading to the isolation of 12 compounds that were fully characterized by 1D and 2D NMR. Furthermore, hydrodistilled essential oils (EOs) from different samples were obtained and the quali-quanti-tative compositions were carefully investigated by GC-FID and GC-MS. Finally, the purified triterpene constituents as well as the EO were assayed for in vitro cytotoxic activity on a panel of human cancer cell lines.
Materials and methods Plant material
Aerial parts of T. alternans were collected in the time of flower-ing on 16 June 2014 by Albert Rakai and Erik Ducar in three dif-ferent localities in Bukovske vrchy hills in eastern Slovakia (Figure 1). These localities are: 1. the village of Runina, path across the fields; 494’32.34” N; 2224’29.68” E; 572 m a.s.l. (vou-cher specimen KO 31164); 2. the village of Topol'a, near the bridge over the small river “Ulicka”; 493’11.46” N 2221’14.32” E; 398 m a. s. l. (KO 31165); 3. the village of Nova Sedlica, path across the forest; 493’14.95” N; 2231’8.13” E; 452 m a. s. l. (KO 31166). Plants were identified by the specialist of the genus Thymus, Pavol Martonfi, and voucher specimens are deposited in the KO herbarium (Herbarium of the Botanical Garden, P. J. Safarik University, Kosice, Slovakia). Plant samples were then stored in absence of light and at r.t. (ca. 22C) until com-pletely dry.
HPLC, HPLC-MS, flash chromatography and semi-preparative HPLC
Silica gel plates (cod. 5171 Merck) and silica gel (60 mesh) were acquired from Sigma (Milan, Italy). Solvents were acquired from Carlo Erba (Milan). Varian Intelliflash flash chromatograph was used for preparative chromatography. A Varian 920 liquid chromatograph was used for semi-preparative HPLC. ESI-MS measurements were performed on a Varian 500 MS ion trap spectrometer. HPLC-DAD analyses were performed on an Agilent 1100 chromatographic system with Diode Array (1100 series). HPLC-MS was performed using a Varian 212 binary chromatograph hyphenated with a Varian 500 MS ion trap spectrometer.
NMR analysis
1D and 2D NMR spectra were obtained on Bruker AVANCE 300 MHz and Bruker AVANCE III 400 MHz spectrometers, dis-solving the samples in deuterated methanol. 2D experiments, namely, COSY, HSQC-DEPT, HMBC, TOCSY and NOESY were performed on a Bruker AVANCE III 400 MHz spectrometer and were used for structure elucidation.
Analysis of nonvolatile constituents
Aerial parts of Thymus alternans (130 g dry material) collected in Nova Sedlica were chopped and extracted with n-hexane (100 mL) at room temperature for 10 min in an ultrasound bath (NE extract). Extraction was repeated twice yielding a brown residue. Solvent was removed and the residue was extracted with dichloromethane (100 mL) at room temperature for 10 min in an ultrasound bath, repeating the extraction twice (DM extract). Residual plant material was then extracted with methanol (100 mL) in an ultrasound bath, repeating the extraction twice (ME extract). Solvents were removed under vacuum, yielding a semi-solid dark-brown residue (NE 0.6%, DM 1.88%, ME 3.4%). Preliminary investigation on NE extract showed the presence of lipids, thus this fraction was discharged. DM extract (2.11 g) was applied to a pre-packed silica-gel column Supel Flash Cartridge, 80 g, 40–60 lm silica. Loaded column was eluted with cyclohex-ane (2 column volumes), then gradually adding acetone (up to 50%) to increase polarity of the eluent. Finally, the column was eluted with 100% methanol. Ninety-eight fractions (25 mL) were collected and pooled on the basis of their TLC chromatographic behaviour in nine different fractions: A (101.4 mg), B (27.4 mg), C (849.7 mg), D (80.2 mg), E (196 mg), F (94.7 mg), G (345.1 mg), H (397.2 mg), I (212.1 mg). The purification of frac-tions E, F, G, H by semi-preparative HPLC was performed using a PLRP-S 100 Å column (8lm) as stationary phase. HPLC condi-tions were as follows: 0.1% formic acid in water (A) and aceto-nitrile (B) were used as eluents; gradient elution started from 20% (B), then in 30 min to 80% (B); the flow rate was 1.5 mL/ min and the injection volume was 100lL. Monitored wavelength was 205 nm. Isolated compounds were T1 (22 mg), T2 (15 mg), T3 (12 mg), T4 (10 mg), T5 (10 mg) and T6 (25 mg); NMR assignments are reported in supplementary material.
ME extract (1.9 g) was charged on Supel Flash Cartridge, 120 g, 40–60 lm silica and eluted in isocratic mode with a 10:5:0.5 mixture of dichloromethane, methanol and water, respectively. Fifty fractions were collected (50 mL) and then pooled in 10 fractions on the basis of TLC behaviour. Fractions 4 (450 mg), 5 (300 mg) and 6 (350 mg) were then used for 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256
Figure 1. Distribution of the collection sites of T. alternans in Slovakia. 2 S. DALL’ACQUA ET AL.
semi-preparative HPLC. Semi-preparative reverse-phase HPLC was performed using an Agilent XDB C-18 (2.1 150 mm, 5 lm) stationary phase and eluting the column with a gradient of 1% formic acid in water (A) and acetonitrile (B), as follows: 0 min, 10% B; 30 min, 85% B; 35 min, 85% B. Flow rate was 4 mL/min; the monitored wavelengths were 254, 280 and 350 nm; the injec-tion volume was 100lL. The isolated compounds were P1 (35 mg), P2 (14 mg), P3 (15 mg), P4 (12 mg), P5 (18 mg), P6 (11 mg) and P7 (14 mg), NMR assignments are reported in Supplementary information.
Hydrodistillation
Dry aerial parts (187–257 g) were cut into small pieces, inserted into 6 L flasks filled with 3.5 L of deionized water and subjected to hydrodistillation using a Clevenger-type apparatus until no more oil was obtained. The oil yield was determined on a dry weight basis, by calculating the moisture content by leaving plant material in a stove at 105C for 8 h. The pure EO was stored in a sealed vial protected from light at20C before chemical ana-lysis and biological assays.
GC-FID and GC-MS analyses
Volatile components of T. alternans were analyzed on an Agilent 4890D instrument coupled to an ionization flame detector (FID). Separation was achieved on an HP-5 capillary column (5% phenylmethylpolysiloxane, 30 m, 0.32 mm i.d.; 0.25lm film thickness; J and W Scientific, Folsom, CA), using the following temperature program: 5 min at 60C, then 4C/min up to 220C, then 11C/min up to 280C, held for 15 min. Injector and transfer line temperatures were 280C; He was the mobile phase, with a flow rate of 1.8 mL/min; split ratio was 1:34. A standard mixture of aliphatic hydrocarbons (C8–C30; Sigma, Milan) diluted in n-hexane was used to deter-mine the retention indices (RIs) of peaks. Oil samples were diluted to 1:100 in n-hexane and 1lL of the solution was injected into GC system. Analyses were performed in triplicate and the mean values were reported. Data analysis was carried out by HP3398A GC Chemstation software (Hewlett Packard, Rev. A.01.01). Quantification of volatiles was performed by peak-area normalization considering the response factors at FID equal to 1. GC-MS analysis was carried out on an Agilent 6890N gas chromatograph equipped with a 5973N mass spec-trometer and using a HP-5 MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1lm film thickness; J & W Scientific, Folsom) capillary column. The temperature program was the same as that reported above. Injector and transfer line tempera-tures were 280C; He was used as the mobile phase, with a flow rate of 1 mL/min; split ratio was 1:50; acquisition mass range was 29–400 m/z. Mass spectra were acquired in electron-impact (EI) mode with an ionization voltage of 70 eV. Oil sam-ples were diluted to 1:100 in n-hexane, and 2lL of the solution was injected into GC-MS system. Data analysis was done by MSD ChemStation software (Agilent, Version G1701DA D.01.00). The major EO constituents were identified by com-parison with authentic standards available in the laboratory. Otherwise, the peak assignment was carried out by comparison of retention indices (RIs) and mass spectra (MS) with respect to those reported in commercial (Adams 2007; NIST 08 2008; FFNSC 2 2012) and home-made libraries.
Cell cultures
Human breast (MDA-MB 231), colon (HCT-15 and HCT116), lung (U1810) and pancreatic (BxPC3) carcinoma cell lines together with melanoma (A375) cells were obtained from American Type Culture Collection (ATCC, Rockville, MD). Human cervical carcinoma (A431) cells were kindly provided by Prof. F. Zunino (Division of Experimental Oncology B, Istituto Nazionale dei Tumori, Milan, Italy). Cell lines were maintained in the logarithmic phase at 37C in a 5% carbon dioxide atmos-phere using the following culture media, containing 10% fetal calf serum (Euroclone, Milan, Italy), antibiotics (100 units/mL penicillin and 100lg/mL streptomycin) and 2 mM L-glutamine: (i) RPMI-1640 medium (Euroclone) for HCT-15, A431, BxPC3, HCT116 and U1810 cells; (ii) DMEM (Sigma Chemical Co.) for A375 and MDA-MB 231 cells.
Cytotoxicity assays
The MTT assay (MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide) was used as a relative measure of cell viability. Briefly, (3-8) 103 cells/well, dependent upon the growth characteristics of the cell line, were seeded in 96-well microplates in growth medium. After 24 h, the medium was removed and replaced with fresh medium containing the com-pound to be studied at the appropriate concentration (0.1–30 lM for isolated compounds, 1–100 lg/mL for EO). Triplicate cultures were established for each treatment. After 72 h, each well was treated with 10lL of a 5 mg/mL MTT solution in phosphate-buf-fered saline (PBS) and, after 4 h of incubation, 100lL of a sodium dodecylsulfate (SDS) solution in 0.01 M HCl were added. After an overnight incubation, the extent of MTT reduction was quantified spectrophotometrically using a microplate reader by absorbance measurement at 540 nm. The mean absorbance for each drug dose was expressed as a percentage of the control, untreated, well absorbance and plotted vs. drug concentration. Cytotoxicity is expressed as the concentration of compound inhibiting cell growth by 50% (IC50). The IC50 values, the drug concentrations that decrease the mean absorbance at 570 nm to 50% of that of untreated control wells, were calculated using GraphPad Prism 4 (GraphPad Software, S. Diego, CA). The final value is the mean ± S.D. of at least three independent experi-ments performed in triplicate.
Results and discussion
LC-MSnand NMR analysis
The HPLC-MSn analysis of the methanolic extract from aerial parts of T. alternans allowed the identification of several phenolic constituents on the basis of pseudomolecular [M-H] ions and their relative fragmentation pathways, as summarized inTable 1.
Some of the identified constituents could not be assigned to a single compound due to the presence of possible different iso-mers and due to the non-availability of reference compounds. Thus, in order to establish correct structures, we decided to iso-late them. Chromatographic separations starting from flash chro-matography and then using semi-preparative HPLC allowed the isolation of seven phenolic derivatives whose structures were elu-cidated by 1D and 2D NMR and mass spectrometric analyses (Figure 2). The1H, HSQC and HMBC spectra of compound P1allowed the identification of luteolin, and the presence of a glycosidic residue that was assigned to a glucopyranoside unit. Glycosidation position was deduced from HMBC correlations 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 PHARMACEUTICAL BIOLOGY 3
observed from H-1glc (d 4.88) and C-40 (d 145.2) and from the NOESY correlation observed from H-1glcand H-30 (d 6.56); thus the compound P1was assigned to luteolin-40-O-b- D-glucopyrano-side. The compound P2 showed similar NMR data compared to P1except for the presence of a methoxy signals (d 3.95) and the glycosidation position that has been revealed by the HMBC cor-relation observed from the H-1glc and the C-7 (d 162.3) thus allowing to assign to compound P2 the structure of chrysoeriol-7-O-b-D-glucopyranoside (Agrawal & Bansal 1989). Compound P3 was assigned to chrysoeriol-5-O-b-D-glucopyranoside (Agrawal & Bansal 1989) on the basis of NOESY correlation
observed from H-1glc (d 6.70) and H-6 (d 6.96) and due to the HMBC correlation observed from H-1glc and C-5 (d 160.5). The compound P4 presented the apigenin as aglycone and the glyco-sidation position was revealed by the NOESY correlations observed from the H-1glc (d 5.10), H-6 and H-8 (d 6.82 and 6.51) as well as by the HMBC correlation observed between H-1glc and C-7 (d 158.6) allowing the assignment to apigenin-7-O-b-D-glucopyranoside (Agrawal & Bansal1989). The rosmarinic acid structure was assigned to P5 due to the presence of a trans double bond supported by a pair of doublet at d 7.51 and 6.26 (J¼ 15.85), as well as to signals ascribable to two 1,3,4 trisubsti-tuted aromatic rings and a 2-hydroxy-propanoic substituent. Compound P6 presented a similar NMR spectrum characterized by signals ascribable to a glucopyranosil moiety that resulted to be linked to the rosmarinic acid to position 30 due to NOESY correlation observed from the H-1glc (d 4.30) and H-20 (d 6.70) being the rosmarinic acid-30-O-b-D-glucopyranoside P6 (Claire et al. 2005). The structure of caffeic acid-3-O-b- D-glucopyrano-side was assigned to compound P7 and the glycosidation position was deduced by NOESY correlations from H-1glc (d 4.37) and H-2 (d 6.79) (Agrawal & Bansal1989).
The chromatographic separation from the lipophilic fraction yielded the isolation of six triterpenes (T1-T6) (Figure 3). The NMR spectra (H, HSQC, HMBC, COSY, NOESY) of the different compounds were similar and the analysis and comparison with the literature data allowed the characterization of the isolated compounds. The NMR spectrum of T1 is characterized by the presence of six tertiary methyl groups at d 0.68, 0.69, 0.76, 0.84, 1.01 and 1.25 and two secondary methyl signals 0.79 (3H, d, J¼ 6.5 Hz) and 0.92 (3H, d, J ¼ 6.3 Hz,), and a hydroxyl methyne signal atd 3.50 as well as three olefinic protons, two at d 5.11 and 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512
Table 1.Non-volatile compounds extracted from T. alternans, tentatively identi-fied on the basis of mass fragmentation spectra.
Identified compound [M-H]and fragments
Luteolin-3-O-glucopyranosidea 447, 285,175 Luteolin-7-O-glucopyranosidea 447, 285,175 Luteolin-7-O-rutinosidea 593, 285, 175 Chrysoeriol-hexoside 461, 446, 299, 284, 256 Methoxy luteolin-hexoside 461, 447, 285, 256, 175 Chrysoeriol-7-O-hexosyl-deoxyhexoside 923, 461, 285, 175
Rosmarinic acid hexoside 521, 367, 359, 191
Esculina 339, 177
Rosmarinic acida 359, 161
Caffeic acid-hexoside 341, 179, 89
30-O-(800-Z-Caffeoyl) rosmarinic acidb 537, 493, 359
Caffeic acid derivative 377, 341, 179
647, 601, 341, 179 3’-O-methyl- rosmarinic acidb 377, 161, 133, 105
Apigenin-7-O-glucopyranosidea 431, 269, 175
Rosmarinic acid derivative 493, 359, 161, 133
aStructure identity confirmed by injection of reference compound. b
The compound was assigned on the basis of literature data.
O R3O OR4 OR2 OR1 O OH O HO O HO HO OH O OH P1: R1=Glc, R2=R3=R4=H P2: R1=H, R2=Me, R3=Glc, R4=H P3: R1=H, R2=Me, R3=H, R4=Glc P7 OH HO O O OH O OH OR1 P5: R1=H P6: R1=Glc O O OH O OH O HO HO OH OH P4
Figure 2. Structures of the isolated phenolic derivatives. 4 S. DALL’ACQUA ET AL.
one at d 5.33. 13C-NMR shifts and heteronuclear correlations allowed the assignment of structure to 3a-hydroxy-urs-12,15-diene, that was previously reported by Gosh et al. (2013) from Croton bonplandianum Bail. T2 and T3 were assigned to the well-known a- and b-amyrin (that were also compared with authentic standard available in the lab). 1H-NMR spectrum of compound T4 presented six quaternary methyl groups and two secondary methyl groups as doublets at d 0.93 and 1.02, and a broad doublet at d 5.50 (assigned to position H-15). The data were in agreement with previously published for isoursenol (Zhang et al. 2008; Shan et al. 2015). Compound T5 was assigned to epitaraxerol characterized by the presence of eight quaternary methyl groups and the olefinic signal at d 5.50 assigned to position H-15 (Ahiahonu & Goodenowe 2007). Finally, compound T6 was assigned to oleanolic acid by com-parison with authentic standard.
Essential oil analysis
The composition of the EOs hydrodistilled from the flowering aerial parts of T. alternans is reported in Table 2. A total of 71 volatile constituents were identified in the three samples ana-lyzed, accounting for 91.0–93.2% of the total compositions. The EOs were mainly composed of terpenoids, notably oxygenated monoterpenes (35.3–39.1%), oxygenated sesquiterpenes (22.5–34.1%), monoterpene hydrocarbons (5.1–15.6%) and ses-quiterpene hydrocarbons (12.2–15.1%). The sesses-quiterpene alcohol (E)-nerolidol was the most abundant component in all three oils analyzed (15.8–31.4%). Geranial (6.8–7.7%), linalool (1.7–6.4%), geraniol (3.3–6.2%) and neral (4.9–5.4%) were the most repre-sentative compounds among oxygenated monoterpenes. Germacrene D (6.7–7.4%) and (E)-b-ocimene (2.6–7.0%) were the predominant compounds among sesquiterpene and monoter-pene hydrocarbons, respectively. The three EOs did not show relevant differences in qualitative composition each other. Only quantitative variation was noticed among them. For instance, the population from the village of Topol'a exhibited the highest levels of oxygenated monoterpenes (39.1%) and sesquiterpenes (34.1%), with the major amount of (E)-nerolidol (31.4%). On the other hand, the population from Nova Sedlica showed the highest lev-els of monoterpene hydrocarbons (15.6%) which were mainly represented by (E)-b-ocimene (7.0%), and minor amounts of oxygenated sesquiterpenes (22.5%), among which (E)-nerolidol attained the lowest level (15.8%).
The main volatile component of T. alternans, the tertiary alco-hol (E)-nerolidol, is one of the most important volatile compo-nents of kiwi (Actinidia chinensis), tea (especially Oolong tea), strawberry, grape, snapdragon and maize, and is approved and used as a food flavouring agent by the FDA. Nerolidol is involved in attracting pollinators and in defence against preda-tors (Green et al.2011). Interestingly, nerolidol is currently under study as a skin penetration enhancer for transdermal drugs. Furthermore, it was shown to inhibit carcinogenesis on azoxyme-thane-induced neoplasia in rats (Wattenberg 1991), and it was effective as an antioxidant in vivo (Nogueira Neto et al.2013), as an antimicrobial and anti-biofilm agent against Staphylococcus aureus (Lee et al. 2014) and Candida albicans (Curvelo et al. 2014), and as anti-parasitic versus Leishmania species (Arruda et al.2005) and acari (de Assis Lage et al.2015). Recently, neroli-dol was proven to exert antinociceptive activity mediated by the GABAergic system, and anti-inflammatory activity decreasing TNF-a and IL-1b proinflammatory cytokines (Fons^eca et al. 2016).
The present work constitutes the first investigation on volatile components of T. alternans. In all the populations considered in the present work, the phenolic compounds thymol and carvacrol, which are considered as the most frequent EO constituents in Thymus taxa, have not been found or have been detected in scarce amount (0.5%). This may be due to adaptation of Slovak populations to low temperatures. Accordingly, it has been reported that the production of phenolic compounds in Thymus species is higher in warmer and drier regions, while non-phenolic compounds usually accumulate in higher quantities in colder and damper areas (Keeler & Tu 1991). Therefore, the T. alternans populations investigated are to be included in the nerolidol-che-motype, as already reported for other species of the same genus such as T. serpyllum (Jamali et al. 2012), T. caucasicus (Jamzad et al. 2011), T. praecox (Vidic et al. 2010), T. atticus (Tzakou & Constantinidis 2005), T. pulegioides (Mockute & Bernotiene 2005) and T. zygioides (Baser et al. 1999). As regards the related T. roegneri, one paper reported on the EO composition of plants growing in Turkey (T€umen et al. 1997). In this case thymol (37.15%), p-cymene (12.94%) carvacrol (7.76%) andb-bisabolene (6.96%) were found as the major components, while (E)-neroli-dol was completely missing. The different chemical profile obtained for T. alternans may represent an additional evidence at the basis of the taxonomical differentiation of the two species.
Cytotoxic activity of isolated triterpenes and EO from thymus alternans on tumour cells
The cytotoxic activity of the six isolated triterpenes and EO iso-lated from plants collected near the village of Nova Sedlica was evaluated by the MTT assay. The triterpenes (T1-T6) were tested on a panel of five human tumour cell lines including examples of pancreatic (BxPC3), colorectal (HCT-15), cervical (A431), and lung (U1810) cancers as well as of melanoma (A375). For com-parison purposes, the cytotoxicity of cisplatin, one of the most widely used anticancer drugs, and of betulinic acid, a naturally occurring triterpene that has been shown to possess a promising anticancer activity (Chudzik et al. 2015), were evaluated under the same experimental conditions. IC50 values, calculated from the dose-survival curves obtained after 72 h of drug treatment, are shown inTable 3.
Cytotoxicity data showed that all the considered triterpenes are endowed with a considerable activity, which is greater than that exerted by the reference drug betulinic acid against all tested cancer cell lines. On the other hand, the antiproliferative activity of all isolated triterpenes was even higher than that of cisplatin on melanoma (A375, p< 0.05), colon cancer (HCT-15, p < 0.05) and pancreatic cancer (BxPC3, p< 0.05) cells. Among all isolated derivatives, T3 was the most effective, with a mean IC50 value of roughly 0.9lM, about 6.7 times lower than that calculated for cisplatin (mean IC50¼6.1 lM, p < 0.05). Similarly, the carboxy-lated derivative T6 exhibited a cytotoxicity profile comparable to that of T3, with an average IC50 value of about 1lM (p < 0.05). Notably, the cytotoxicity of T3 and T6 exceeded that of the met-allodrug by a factor of about 25 against HCT-15 colon cancer cells, which are barely chemosensitive to cisplatin. On average, T1, T2, T4 and T5 showed a quite similar pattern of response against all tested cell lines, eliciting average IC50 values of about 2lM, roughly three times lower than those obtained with the reference drugs (mean IC50 of 6.7 and 6.1lM for betulinic acid and cisplatin, respectively, p< 0.05).
Considering the similar pair of compounds T2/T3 (differenc-ing each other only in the structure of r(differenc-ing E), T3 showed higher 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 PHARMACEUTICAL BIOLOGY 5
641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768
Table 2.Composition of EOs obtained from three populations of Thymus alternans growing in Slovakia (NS – village of Nova Sedlica, RUN – village of Runina, POL– village of Topol'a. For details of localities see part Plant material).
N. Componenta RIb RI LIT.c Area%d IDe NS RUN POL 1 a-Thujene 916 924 0.1 trf RI, MS 2 a-Pinene 921 932 0.3 0.1 tr Std 3 Camphene 934 946 0.5 0.1 0.1 Std 4 Sabinene 959 969 tr tr tr RI, MS 5 b-Pinene 962 974 0.3 0.2 0.1 Std 6 1-Octen-3-one 968 972 0.2 0.1 tr RI, MS 7 1-Octen-3-ol 974 974 1.6 1.1 1.4 Std 8 3-Octanone 979 979 0.2 0.1 0.1 RI, MS 9 Myrcene 981 988 4.0 1.9 1.3 Std 10 3-Octanol 990 988 0.2 0.2 0.1 RI, MS 11 A-terpinene 1009 1014 0.4 0.3 0.1 RI, MS 12 P-cymene 1017 1020 0.3 0.3 0.1 Std 13 Limonene 1020 1024 0.4 0.3 0.2 Std 14 1,8-Cineole 1021 1026 0.7 0.3 0.4 Std 15 (Z)-b-ocimene 1032 1032 1.0 0.5 0.4 RI, MS 16 (E)-b-ocimene 1041 1044 7.0 4.3 2.6 RI, MS 17 c-Terpinene 1050 1054 1.0 1.0 0.2 Std
18 cis-Sabinene hydrate 1057 1065 2.4 2.3 0.7 RI, MS
19 Terpinolene 1079 1086 0.2 0.2 0.1 Std
20 trans-Sabinene hydrate 1089 1098 0.3 0.3 0.1 RI, MS
21 Linalool 1095 1095 2.7 1.7 6.4 Std
22 n-Undecane 1100 1100 0.2 0.1 Std
23 1-Octen-3-yl acetate 1110 1110 0.1 0.6 0.1 RI, MS
24 cis-p-Menth-2-en-1-ol 1113 1118 tr 0.2 0.1 RI,MS
25 allo-Ocimene 1124 1128 0.1 tr Std
26 Camphor 1132 1141 1.1 0.4 1.1 Std
27 trans-Chrysanthemal 1141 1153 0.2 0.3 0.2 RI, MS
28 Nerol oxide 1149 1154 tr 0.1 0.1 RI, MS
29 Isoborneol 1154 1155 1.1 0.5 1.0 Std 30 Terpinen-4-ol 1166 1174 1.8 1.8 1.0 Std 31 Isogeranial 1178 1174 0.4 0.4 0.4 RI, MS 32 a-Terpineol 1181 1186 0.5 0.3 0.5 Std 33 Nerol 1222 1227 1.9 4.9 3.1 Std 34 Citronellol 1225 1223 0.1 0.1 0.1 Std
35 Thymol, methyl ether 1229 1232 tr tr tr RI, MS
36 Neral 1234 1235 5.1 5.4 4.9 Std
37 Carvacrol, methyl ether 1236 1241 0.1 RI, MS
38 Geraniol 1251 1249 3.3 6.0 6.2 Std
39 Linalool acetate 1254 1254 2.9 tr 2.4 RI, MS
40 Geranial 1265 1264 6.8 7.7 7.2 Std 41 Isobornyl acetate 1276 1283 tr tr tr Std 42 Carvacrol 1299 1298 0.5 Std 43 d-Elemene 1325 1335 0.1 0.1 RI, MS 44 Neryl acetate 1360 1359 3.6 1.5 2.3 Std 45 b-Bourbonene 1369 1387 1.8 1.0 1.3 RI, MS
46 Geranyl acetate 1379 1379 0.3 0.9 1.1 RI, MS
47 (E)-caryophyllene 1402 1417 1.5 1.0 1.6 Std 48 b-Copaene 1413 1430 0.2 0.2 0.2 RI, MS 49 a-Humulene 1436 1454 0.2 0.4 0.2 Std 50 allo-Aromadendrene 1443 1458 0.7 0.1 0.1 RI, MS 51 cis-Cadina-1(6),4-diene 1449 1461 0.1 RI, MS 52 (E)-b-Farnesene 1450 1454 0.1 RI, MS 53 Germacrene D 1465 1484 6.7 7.4 7.4 RI, MS 54 Bicyclogermacrene 1480 1500 0.5 0.5 0.2 RI, MS 55 a-Muurolene 1487 1500 0.2 tr 0.1 RI, MS 56 b-Bisabolene 1498 1505 1.5 0.7 1.3 RI, MS 57 (E,E)-a-Farnesene 1501 1505 0.3 0.1 0.1 RI, MS 58 d-Cadinene 1510 1522 1.1 0.2 0.4 RI, MS 59 b-Sesquiphellandrene 1512 1521 0.2 0.4 tr RI, MS 60 a-Cadinene 1533 1537 0.1 tr RI, MS 61 Hedycaryol 1536 1546 2.9 3.6 1.4 RI, MS 62 (E)-Nerolidol 1559 1561 15.8 26.4 31.4 Std
63 Germacrene D-4-ol 1559 1574 tr tr tr RI, MS
64 Viridiflorol 1584 1590 0.2 Std 65 c-Eudesmol 1615 1630 0.4 0.1 0.2 RI, MS 66 epi-a-Cadinol 1626 1638 0.4 0.1 0.1 RI, MS 67 epi-a-Muurolol 1626 1640 0.4 0.3 0.2 RI, MS 68 b-Eudesmol 1631 1649 0.2 0.2 0.1 RI, MS 69 a-Eudesmol 1635 1652 0.3 0.3 0.2 RI, MS 70 a-Cadinol 1648 1652 1.8 0.8 0.6 RI, MS 71 n-Nonacosane 2900 2900 0.1 0.2 0.3 Std (continued) 6 S. DALL’ACQUA ET AL.
cytotoxic activity against all the tested cell lines compared to T2. Thus, minor changes in the structure of these compounds may induce moderate changes in the observed activity. On the other hand, considering the pair T4/T5, also these differing in the structure of ring E, T5 showed higher activity against A375, A431 and U1810 cell lines compared to T4. Therefore, a system-atic Structure Activity Relationship (SAR) study may be useful in order to assess notable key point in the basic structure bearing useful improvement of activity.
Concerning possible mechanism of action on tumour cells, betulinic acid is considered as a promising anticancer agent act-ing through activation of the mitochondrial pathway of apoptosis in cancer cells (Fulda & Kroemer2009). On the other hand, no data are available on the mode of action of T1-T6. For this 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 Table 2. Continued N. Componenta RIb RI LIT.c Area%d IDe NS RUN POL Total identified (%) 91.0 91.4 93.2 Oil yield (%) 0.2 0.2 0.1 Grouped compounds (%) Monoterpene hydrocarbons 15.6 9.2 5.1 Oxygenated monoterpenes 35.3 34.6 39.1 Sesquiterpene hydrocarbons 15.1 12.2 12.9 Oxygenated sesquiterpenes 22.5 32.1 34.1 Others 2.6 2.4 2.0 a
Compounds are in order of elution from a HP-5MS column. bRetention index on HP-5MS column.
c
Retention index taken from literature (Adams,2007; NIST08,2008; FFNSC2,2012).
dPercentage values are means of three determinations, with a RSD% for the main components below 10% in all cases. e
Identification methods: MS, by comparison with the mass libraries Wiley, Adams and NIST 08; RI, by comparison with RIs-containing libraries (Adams,2007; NIST08,2008; FFNSC2,2012); Std, by comparison with authentic standard.
f
tr, traces (mean value below 0.1%).
H H H H HO H H HO H H H H HO H H OH O T1 T2 T3 T4 T5 T6 H HO HO HO
Figure 3. Structures of the isolated triterpenes.
Table 3. In vitro cytotoxic activity of triterpenes isolated from aerial parts of T. alternans collected in Nova Sedlica, Slovakia. Cells (5–8 104mL 1) were treated for 72 h with increasing concentrations of tested compounds.
IC50(lM) ± S.D.
Compound A375 HCT-15 A431 BxPC3 U1810
T1 1.57 ± 0.72 0.88 ± 0.22 4.54 ± 1.06 0.52 ± 0.24 2.86 ± 0.83 T2 1.26 ± 0.12 1.46 ± 0.13 4.98 ± 1.14 1.84 ± 0.31 2.25 ± 1.05 T3 0.48 ± 0.67 0.67 ± 0.09 2.03 ± 0.98 0.42 ± 0.13 0.98 ± 0.21 T4 1.45 ± 0.56 1.69 ± 0.72 4.85 ± 1.32 1.42 ± 0.73 3.59 ± 1.31 T5 0.87 ± 0.32 2.42 ± 1.05 3.28 ± 1.09 1.84 ± 0.91 2.93 ± 1.13 T6 0.45 ± 0.10 0.54 ± 0.08 2.16 ± 0.83 0.19 ± 0.09 1.62 ± 0.85 Betulinic acid ND 6.85 ± 2.18 8.96 ± 2.83 4.28 ± 1.35 ND Cisplatin 2.06 ± 1.01 15.53 ± 2.48 1.96 ± 0.84 10.17 ± 1.65 0.76 ± 0.85 S.D.¼ standard deviation. ND ¼ not detected. Cell viability was evaluated by means of MTT test. IC50values were calculated by the dose–response curves by means of four parameter logistic model (p < 0.05).
reason, further studies on T. alternans triterpenes are needed in order to evaluate their possible role on apoptosis. It is worthy to underline that these compounds are well known for possessing moderate toxic effects when administered orally, thus are consid-ered as significant models for the development of new classes of antiproliferative compounds.
The cytotoxic effects of T. alternans EO were evaluated on A375, MDA-MB 231, and HCT116 human tumour cell lines. Tumour cells were treated with various concentrations (1–100 lg/mL) of EO for 72 h and then submitted to the MTT assay. As shown in Table 4, the EO showed notable growth inhibition effects on all human tumour cells in a dose-dependent manner (p< 0.05). The inhibition was remarkable with IC50 val-ues of 5.51, 5.96 and 8.45lg/mL on A375, MDA-MB 231 and HCT116, respectively. These IC50 values are commonly consid-ered as promising for anticancer drug discovery and develop-ment. In fact, according to the standards of the National Cancer Institute (NCI), a plant extract may be considered as active and potential source of anticancer drugs if its IC50 is lower than 20lg/mL (Cordell et al.1993). The observed cytotoxicity was not specific toward a cancer cell line and it cannot be attributed to a compound or few compounds as shown in the composition of EO. In order to compare the obtained results with literature we considered the previously published data on some of the main constituents. (E)-nerolidol, the main compound present in T. alternans EO, has been shown to be cytotoxic on renal cell adenocarcinoma ACHN, the hormone-dependent prostate carcin-oma LNCaP, human lung carcincarcin-oma A-549 and colon adenocar-cinoma DLD-1 (Loizzo et al. 2007; Sylvestre et al. 2007). The IC50 values of this compound determined by us on tumour cell lines were 2.92, 4.13 and 5.76lg/mL for A375, HCT116 and MDA-MB 231, respectively, but the differences in the various types of tumour cells and the possible variation in the assay protocol should be also considered. The antiproliferative activity of neral and geranial, the two isomers of citral, were tested in our previous study (Maggi et al. 2013), and IC50 values ranged from 5.73 to 11.3lg/mL on MDA-MB 231 and A375, respect-ively. Germacrene D has been reported to inhibit the growth of MDA-MB-231, MCF7, Hs 578T, PC-3, and Hep-G2 cell lines (Kuzma et al. 2009). Neryl acetate and nerol were also assayed and showed lower cytotoxic activity against the tested tumour cell lines (Table 4). No data are available in the literature regard-ing cytotoxic activity of (E)-b-ocimene. The concentrations of (E)-nerolidol (15.8%), (E)-b-ocimene (7%), geranial (6.8%),
germacrene D (6.7%), and neral (5.1%) cannot fully explain the observed cytotoxic activity. This means that other minor com-pounds contributed to the activity, taking into account that syn-ergism between constituents may increase the total antiproliferative activity exhibited by the EO. T. alternans EO presented an excellent inhibitory activity against human tumour cell lines with IC50 values close to those of the cisplatin (p< 0.05). These results suggest further investigations on the pos-sible mechanisms of action.
Conclusions
Phytochemical investigations on T. alternans allowed identifica-tion of the main phytoconstituents of the plants aerial parts, showing the presence of glycosylated flavonoids and rosmarinic acid, pentacyclic triterpenes and EO. Triterpenes and EO exhib-ited significant antiproliferative activity against a panel of tumour cell lines. In particular, the pentacyclic triterpenes showed improved activity compared to the reference compounds cisplatin and betulinic acid. (E)-nerolidol, the main volatile component of T. alternans EO, appeared to afford the major contribution to the cytotoxic activity of the EO. This work pointed out the great chemical diversity of T. alternans phytocomplex. In addition, the significant cytotoxic activity of some of the isolated triterpenes underlines the possible role of the plant as a potential source of bioactive compounds for therapeutical applications.
Acknowledgments
The authors acknowledge MIUR and the University of Camerino (FPI000044, Fondo di Ateneo per la Ricerca 2014-2015) for financial support.
Disclosure statement
The authors declare that they have no conflict of interest.
Funding
The authors acknowledge MIUR and the University of Camerino (FPI000044, Fondo di Ateneo per la Ricerca 2014-2015) for financial
support. Q1
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b
Compound A375c MDA-MB 231d HCT116e
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b
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