Bioactive Isocedrenes from Perezia multiflora

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Chemicals
• General experimental procedures
• Plant material
• Extraction and isolation
• Antiparasitic activity
• Cytotoxicity evaluation on J774A.1 macrophages
• Cytotoxicity evaluation on human macrophages generated from THP-1 cells
• Contributorsʼ Statement
• References

Abstract

Four isocedrenes (1 – 4), including one new compound (2), were isolated from an ethanolic extract of the aerial parts of Perezia multiflora by bioactivity-guided fractionation. For compounds 1 and 3, a revised stereochemical assignment is proposed based on molecular modeling studies using DFT-NMR calculations. Antiparasitic activity of the four compounds was evaluated using an in vitro culture of Plasmodium falciparum and axenic amastigotes of Leishmania infantum. IC₅₀ values ranged from 0.81 to 16.1 µM (P. falciparum) and 0.16 to 2.03 µM (L. infantum). Toxicity was evaluated against J774A.1 mouse macrophages or human macrophages generated from THP-1 monocytic cells (IC₅₀ values ranging from 0.16 to 2.64 µM). Compound 4 exhibited weak selectivity against P. falciparum with a selectivity index (SI = CC₅₀/IC₅₀) of 3. No selectivity was observed for compounds 1 – 3 against both parasites.

Introduction

Perezia multiflora (Humb. & Bonpl.) Less. (Asteraceae) is a small herb growing in the highlands of the Andean mountains (3800 – 4500 m) in Peru, Bolivia, Chile, Argentina, Ecuador, and Colombia [1]. In Peru, the plant is sold as a fresh plant on Andean markets and is very popular against cough, bronchitis, and fever [2].

Isocedrenes are sesquiterpenes first described in P. multiflora and Proustia pyrifolia [3] by Bohlman, and then also in Trixis wrightii and Trixis inula [4]. Further studies have confirmed the occurrence of this group of sesquiterpenes in various genera, like Jungia [5], Moscharia [6], Pleocarphus [7], and Acourtia [8], all belonging to the tribe Nassauviineae, subfamily Mutisioidaea, in the Asteraceae family [9]. The name isocedrene was first proposed by Bohlman because of the similarity of this skeleton with α-cedrene, from which it differs by the shift of a methyl group from C-3 to C-5 [3]. However, a biogenetic relationship between α-cedrene and isocedrene is unlikely, and isocedrene is more probably formed from a cyperene precursor [9]. Therefore, some authors proposed to use the name trixane instead of isocedrene [10], [11], [12], [13].

In our hands, an ethanol extract from the aerial parts of P. multiflora displayed strong inhibitory activity against in vitro culture of the parasite Plasmodium falciparum (IC₅₀ = 4 µg/mL). Herein, we describe the bioactivity-guided isolation of 4 isocedrene sesquiterpenes (1 – 4) ([Fig. 1]), and the evaluation of their antiparasitic activity against P. falciparum and Leishmania infantum axenic amastigotes. To assess their selectivity, their cytotoxicity was determined on J774A.1 mouse macrophages or human macrophages generated from THP-1 cells. One isocedrene (2) is new, and we propose a stereochemical revision of the previously published structures of two of them (1 and 3) based on a careful examination of their NMR data and their comparison with computed ones.

Results and Discussion

Compounds 1 – 4 were isolated from the ethanol extract of the aerial parts of P. multiflora by bioactivity-guided fractionation.

Compound 1 was isolated as colorless oil. Its molecular formula was established as C₂₉H₃₈O₉ by positive HRESIMS (m/z 548.2856, [M + NH₄]⁺) corresponding to 11 indices of hydrogen deficiency. Careful examination of 2D NMR spectra enabled the determination of the planar structure and assign all ¹H and ¹³C NMR signals ([Fig. 2] and [Tables 1] and [2]). The two methyl singlets at δ _H 0.74 and 1.19 showed HMBC correlations with one another and carbon signals at δ _C 43.3 (C), 52.9 (CH), and 60.5 (CH), which are typical of a gem-dimethyl group, thereby allowing the assignment of C-11, C-2, and C-10. The ¹H-¹H COSY correlation between H-2 (δ _H 2.03, m) and the signal at δ _H 5.96 enabled the assignment of H-3. The position of the olefinic methine proton at δ _H 5.49 (q, J = 2.0 Hz, H-4) was deduced from the vicinal ¹H-¹H COSY correlation with H-3. Long-range ¹H-¹H COSY correlations of H-3 and H-4 with the signal at δ _H 6.92 (dd, J = 2.8, 2.0 Hz) allowed for the identification of H-15. This was confirmed by HMBC correlations between H-15, C-4, and C-5. Substitution of C-15 (CH, δ _C 87.5) by an acetoxy group was confirmed by the HMBC correlation between H-15 and a carbonyl at δ _C 169.5, and by the HMBC correlation of this latter signal with a methyl singlet at δ _H 1.59. Starting from H-10 (1.72, m), successive ¹H-¹H COSY correlations between signals at δ _H 1.32 (m), 2.07 (m), and δ _H 5.20 (ddd, 10.5, 8.9, 5.9) allowed their assignment as H-9a, H-9b, and H-8, respectively. The HMBC correlation of H-9b with a methine signal at δ _C 49.5 positioned C-7, and an ¹H-¹H COSY correlation between H-7 (δ _H 2.10) and a signal at δ _H 6.45 (d, J = 1.4 Hz) identified H-14. The HMBC correlation of H-14 with a carbonyl at δ _C 168.6 and the HMBC correlation of this carbonyl with a methyl singlet at δ _H 1.70 located an acetoxy substituent on C-14. The epoxy bridge between C-14 and C-15 was suggested by the chemical shifts of H-14 and H-15, typical of acetalic protons, and by the HMBC correlation between H-15 and C-14. The HMBC correlation between a quaternary carbon at δ _C 47.5 and H-4 (5.49, q, J = 2.0 Hz) and H-7 or H-9 (δ _H 2.09) indicated the central position of C-6 (δ _C 47.5). The isocedrene structure is completed by the assignment of H-1a (δ _H 1.81) based on its COSY correlation with H-2 (δ _H 2.03) and the HMBC correlation between C-1 (δ _C 44.5) and H-7 (δ _H 2.10). Signals typical of two angelate moieties were also observed in the 1D and 2D NMR spectra. HMBC correlations between the two carbonyl signals (δ _C 166.9 and 167.0) belonging to these angeloyl groups with H-3 and H-8 positioned the angelate substituents at C-3 and C-8. This planar structure was identical to that already described for 3-angeloyl-8-angeloyloxyproustianol by Bohlman and Zdero, who also reported ¹H NMR data comparable to those observed here (Table 21S, Supporting Information) [3]. However, the reported configuration was not compatible with our NMR data (coupling constants and NOESY correlations), which prompted us to propose a stereochemical revision. Bohlmanʼs structure was, in particular, not in agreement with the NOE effects observed between H-8 and H-14, and between H-8 and H-10. Only an 8-angeloyloxy and a 14-acetoxy substituent, both in α-orientation, could explain these NOE effects. A β-orientation for the 3-angeoyl moiety was confirmed by the observed NOE effect between H-3 and H-1b, and this allowed for discriminating between H-1a and H-1b. The NOE effects between H-15 and H-10 enabled the determination of the C-15 configuration. Finally, the NOE effects observed between H₃-12 and H-9a, and H₃-13 and H-10 allowed for discriminating between H₃-12 and H₃-13, and H-9a and H-9b ([Fig. 3]).

The proposed absolute configuration ([Fig. 1]) is in agreement with a biosynthetic origin from a (−)-cyperene skeleton. It is also in line with the absolute configuration of natural trixanolides determined by Mosherʼs method [13], [14], and by ECD analysis supported by quantum chemical calculations [12]. Furthermore, the ECD spectrum of compound 1 showed the same shape as that of previously reported trixanes [12]. Therefore, the structure of compound 1 was assigned as (2R,3R,6S,7S,8S,10S,14S,15R)-14,15-diacetoxy-3,8-diangeloyloxy-14,15-epoxy-4-isocedrene.

Compound 2 was isolated as a colorless oil. Its molecular formula was established as C₂₈H₃₈O₈ by positive HRESIMS (m/z 525.2457, [M + Na]⁺), which indicated 10 indices of hydrogen deficiency. The ¹H and ¹³C NMR data of 2 ([Tables 1] and [2]), as well as the 2D NMR spectra, were similar to those of 1, except for the disappearance of the signals of an acetoxy moiety and the appearance of signals of a methoxy group (δ _H 3.27, s, δ _C 55.5, CH₃). The latter was positioned at C-15 based on the HMBC correlation of the methoxy protons with the carbon at δ _C 96.5. This latter signal could be assigned to C-15 because of the ¹H-¹H COSY correlation between the proton at δ _H 4.96 (dd, J = 2.7, 1.9 Hz, H-15) and the proton at δ _H 5.99 (dt, J = 4.8, 2.5 Hz, H-3). NOESY correlations and coupling constants as well as the ECD spectrum were similar to those of 1. Therefore, compound 2 could be identified as (2R,3R,6S,7S,8S,10S,14S,15S)-14-acetoxy-3,8-diangeloyloxy-14,15-epoxy-15-methoxy-4-isocedrene. This is the first occurrence of this compound in the literature.

Compound 3 was isolated as a colorless oil. Its molecular formula was established as C₂₈H₃₈O₈ by positive HRESIMS (m/z 525.2458, [M + Na]⁺) and corresponded to 10 indices of hydrogen deficiency. The ¹H NMR data matched with those published for an isocedrene isolated from Perezia runcinata but again, the published structure was not consistent with our data, especially with the NOESY correlations [15].

Analysis of ¹H and ¹³C NMR data ([Tables 1] and [2]), as well as the 2D NMR spectra indicated that compound 3 was a diastereomer of compound 2. The most striking differences on the ¹H NMR spectra were the chemical shifts of H-1a, H-1b, and H-10 and the coupling constants of the H-14 and H-15. The strong difference between the coupling constant between H-14 and H-7 (1.7 Hz for compound 2 and 7.0 Hz for compound 3) prompted us to describe 3 as an epimer of 2 with an opposite configuration at C-14. However, this structure was not consistent with the strong NOE effect observed between H-14 and H-8. The isocedrene structure could present different conformations due to the conformational flexibility of the tetrahydropyrane ring, which influences the geometry around C-14 and C-15. For compounds 1 and 2, the data matched with a conformation where H-14 and H-15 were both pseudoequatorial. The greater stability of this conformation in a tetrahydropyrane ring substituted in position 2 by electron-acceptor substituents is a well-known stereoelectronic effect called “anomeric effect”. In this conformation, the electron-acceptor bonds (C-OCH₃ or C-OCOCH₃ in our case) are antiperiplanar to the tetrahydropyranose oxygen lone pair, maximizing orbital overlap [16]. For compound 3, NMR data could match with the structure of the epimer of 2 at C-15, but with a different conformation of the tetrahydropyrane ring, in which H-14 would be pseudoaxial and H-15 pseudoequatorial. Therefore, it seemed to us necessary to accurately determine the major conformers contributing to the structures of 1 – 3. Calculation of the NMR data corresponding to these conformers, and comparison with experimental spectra, should allow for the evaluation of the validity of the structures we proposed for the three compounds.

For each compound, the conformers were modeled by DFT using Gaussian 09 at the B3LYP/6 – 31+G(d,p) level in the gas phase. Frequencies calculations were performed on the optimized geometries at 298 K, showing all positive frequencies and allowing for the evaluation of the enthalpies of the minima.

¹³C and ¹H NMR chemical shift calculations were then performed using the GIAO NMR method with B3LYP/6 – 31+(d,p) and using the chloroform polarizable continuum model (PCM) on optimized geometries at the B3LYP/6 – 31+G(d,p) level in the gas phase. Isotropic shielding constants (σ) for the ¹³C and ¹H nuclei were transformed into chemical shifts (δ) using the linear regression procedure proposed by Tantillo [17]. The contribution of each conformer to the overall spectrum was based on Boltzmann conformational analysis.

Optimized geometries and calculated values on the major conformers of compounds 1 and 2 match with their experimental data, which were also recorded in CDCl₃. Detailed calculations and ¹H NMR simulated spectra of compounds 1 and 2 are consigned in Tables 1S – 6S and Figs. 1S – 2S, Supporting Information.

As for compound 3, the four isomers corresponding to the two possible configurations of C-14 and C-15 were considered and, for each structure, the two conformations (A and B) of the tetrahydropyrane ring were modeled ([Fig. 4]). Of note, the calculation of total energies for each conformer confirmed that iso1B is the preferred conformation for compound 2, with pseudoaxial electron-acceptor substituents at C-14 and C-15, which can be explained by the anomeric effect.

The strong NOE effect observed between H-14 and H-8 in 3 allowed us to retain only isomers where the distance between H-14 and H-8 was less than 3 Å. The experimental coupling constant between H-14 and H-7 was 7.0 Hz. The calculated values that best fit the experimental one are, respectively, 8.4 Hz for iso1A, 5.9 Hz for iso2A, and 8.1 Hz for iso4A, with the calculated values for the others isomers and conformers being quite smaller. Furthermore, the root mean square deviation (RMSD) on the calculated ¹³C chemical shifts has to be less than 2.94 with the chosen method [17]; calculations of the RMSD for the A and B conformations of each four isomers indicate, without any ambiguity, the matching of compound 3 with iso4A ([Table 3]). For this structure, where H-7 and H-14 are both pseudoaxial ([Fig. 5]), the NOE effect observed between them is puzzling. However, Tables 4S, 8S, and 20S, Supporting Information, show that under the conditions where the spectra were recorded, NOE effects could be observed between protons more than 3 Å apart, the calculated distance between H-7 and H-14 being 3.05 Å.

The summary of these DFT-NMR calculations and comparison with the experimental data are presented in the Supporting Information. Therefore, the structures proposed for 1, 2, and 3 ([Fig. 1]) are confirmed.

Compound 4 was isolated as a colorless oil. Its molecular formula was established as C₂₀H₂₄O₄ by positive HRAPCIMS [m/z 329.1742, [M + H]⁺] and showed nine indices of hydrogen deficiency. Analysis of ¹H and ¹³C NMR data ([Tables 1] and [2]) as well as the 2D NMR spectra allowed us to establish the structure of 4. This compound was first described by Bohlman and Zdero as the product of the saponification of a natural isocedrene isolated from P. pyrifolia [3], and ¹H NMR data matched with published ones. It is noteworthy that we could not find any MS signal of significant intensity corresponding to compound 4 in the LC-MS chromatogram of the crude extract recorded in APCI⁺ mode. This compound may therefore be an artefact resulting from the hydrolysis of 1, 2, or 3 during purification on silica gel.

Antiparasitic activity of compounds 1, 2, 3, and 4 was evaluated on P. falciparum and L. infantum axenic amastigotes following already published protocols. To assess the selectivity of their antiparasitic activity, their toxicity on J774A.1 mouse macrophages or human macrophages generated from the THP-1 monocytic cell line was evaluated ([Table 4]). Only compound 4 displayed a slightly better antiplasmodial activity than the methanol-soluble fraction (IC₅₀ = 0.81 µM or 0.26 µg/mL for compound 4; IC₅₀ = 0.8 µg/mL for the methanol-soluble fraction), suggesting that some other compounds contribute to the activity of the extract. Compound 4 was the only one to present some weak selectivity against P. falciparum, with a selectivity index (SI = CC₅₀/IC₅₀) of 3. Compounds 1 – 3 showed only moderate activity against P. falciparum and displayed no selectivity. These compounds displayed better activity on L. infantum axenic amastigotes, but in the same range as their toxicity on J774A.1 macrophages. Antileishmanial activity of a trixane derivative isolated from Trixis antimenorrhoea has already been reported on promastigotes of L. infantum and L. amazonensis [14].

In this work, we describe four isocedrenes isolated from P. multiflora, a highly valued herbal remedy in the Andean mountains. One of them, compound 2, is new. Compound 1 was already isolated from P. multiflora and compound 3 from P. runcinata, but careful examination of their NMR data, together with the calculation of stability of their conformers and prediction of their NMR spectra, allowed us to propose a stereochemical revision for both of them. This study highlights the influence of the substitution of the tetrahydropyranose ring of isocedrenes on their more stable conformation and therefore on their NMR spectra. Moreover, this is the first report of antiplasmodial and cytotoxic activity of isocedrene sesquiterpenes. Their antiparasitic activity is not selective enough, but it could be interesting to evaluate their cytotoxicity on cancerous cells lines. As isocedrenes appear here as potentially toxic molecules, it may be relevant to determine their concentration in traditional preparations of P. multiflora to assess the safety of use of this herbal remedy.

Materials and Methods

Chemicals

Ethanol (96%) was from Laboratoire ALKOFARMA E. I. R. L. Petroleum ether, cyclohexane, dichloromethane, ethyl acetate, and methanol were of analytical or HPLC grade and purchased from Fisher Scientific SAS. Merck silica gel 60 (15 – 40 µm) was used for medium-pressure column chromatography (MPLC). Sephadex LH-20 was purchased from Sigma-Aldrich. Analytical TLC was carried out on precoated silica gel plates (Kieselgel 60 F₂₅₄, 0.25 mm; Merck) with detection under UV 254 nm and after spraying with 1% vanillin/10% H₂SO₄ in ethanol.

General experimental procedures

MPLC was performed on a Büchi pump manager C-615 connected to a Büchi pump module C-601 with glass columns of various sizes. Optical rotations were determined with a JASCO P2000 digital polarimeter. CD spectra were recorded on a JASCO J-815, at 20 °C, with a measurement range of 190 – 280 nm, a data interval of 0.2 nm, a bandwidth of 1 nm, and a scanning speed of 20 nm/min, at a concentration of 7.75 ppm. IR spectra were recorded on a Perkin-Elmer Frontier FT-IR in ATR mode. The NMR spectra were recorded on a Bruker Avance 500 MHz instrument in C₆D₆ (δ _H 7.16 and δ _C 128.06) or a Bruker Avance 300 MHz instrument in CDCl₃ (δ _H 7.28 and δ _C 77.1). UHPLC/MS analysis was performed using a UHPLC Ultimate 3000 system (Dionex) controlled by Chromeleon Xpress 6.8 software (Dionex), coupled with an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). All spectra were acquired and processed using LCQ Xcalibur 3.0 software (Thermo Fisher Scientific).

Plant material

P. multiflora was purchased as a whole fresh plant in the Challwa market (town of Huaraz, Peru) in August 2017. A voucher specimen was deposited at the National Herbarium of the Museum of Natural History (Universidad Nacional Mayor de San Marcos, Lima, Peru, deposit N°026 – 2018-USM-MHN) where it was identified by botanists. Whole plants were dried at ambient temperature in a ventilated place away from the sun. Aerial parts and roots were separated, and the aerial parts were ground.

Extraction and isolation

The ground aerial parts (375 g) were extracted successively three times with 3.3 L of 96% EtOH at an ambient temperature for 12 h. After filtration, the solvent was evaporated under reduced pressure to give 53 g of extract (IC₅₀ = 4 µg/mL). This extract was partitioned between ethyl acetate (1 L) and water (1 L). A white precipitate was removed, and the ethyl acetate phase was separated. The solvent was removed under reduced pressure to give 20.4 g of ethyl acetate-soluble fraction. This fraction was further partitioned between petroleum ether (400 mL) and methanol containing 10% of water (400 mL). After removal of the solvents under reduced pressure, 7.9 g of the petroleum ether-soluble fraction (IC₅₀ = 3.1 µg/mL) and 10.3 g of the methanol-soluble fraction (IC₅₀ = 0.8 µg/mL) were obtained.

Isolation of compounds 1 – 3: A portion (5.0 g) of the methanol-soluble fraction was submitted to MPLC (23 cm × 4 cm, i. d., 65 g silica gel). The column was eluted with dichloromethane containing increasing amounts of methanol (500 mL 0%, 500 mL 1%, 500 mL 2%, 500 mL 5%, 500 mL 10%, and 300 mL 100%) and 25 mL fractions were collected. Based on TLC analysis, the eluted fractions were pooled into six fractions (F1 – F6), each of which was tested in the P. falciparum assay. Fraction 2 (546 mg) displayed the best activity against P. falciparum (IC₅₀ = 0.4 µg/mL). A portion of fraction 2 (520 mg) was submitted to MPLC on silica gel (23 cm × 3 cm i. d., 35 g). The column was eluted with petroleum ether containing increasing amounts of ethyl acetate (500 mL 10%, 600 mL 15%, 500 mL 25%), and 20 mL fractions were collected. Fractions were pooled according to their TLC profiles into eight fractions (F2-1–F2 – 8). F2 – 4 (110 mg) was submitted to CC on Sephadex LH-20 (20 cm × 1 cm i.d, 10 g) with methanol as the eluent. Two mL fractions were collected and pooled according to their TLC profile to give compound 1 (89 mg, 84% purity by ¹H NMR). F2-1 (18.5 mg) was treated with the same protocol to give compound 3 (10.7 mg, 81% purity by ¹H NMR). F2-2 (37.8 mg) was submitted to CC on Sephadex LH-20 (2 cm × 1 cm, i. d., 1 g) with methanol as the eluent. Finally, 0.5 mL fractions were collected and pooled according to their TLC profile to give compound 2 (26.3 mg, 72% purity by ¹H NMR).

Isolation of compound 4: Another portion (5.0 g) of the methanol-soluble fraction was submitted to MPLC as described above. Fraction pooling was slightly different and gave eight fractions (F1′-F8′). Fractions F2′ (281 mg) and F3′ (302 mg) displayed the best activity against P. falciparum (IC₅₀ values of 0.23 and 0.7 µg/mL, respectively). Fraction F2′ was submitted to MPLC on silica gel (26 cm × 2.5 cm i. d., 35 g). The column was eluted with cyclohexane containing increasing amounts of ethyl acetate (500 mL 10%, 600 mL 15%, 500 mL 25%, 350 mL 50%), and 20 mL fractions were collected. Fractions were pooled according to their TLC profiles into five fractions (F2-1′ – F2 – 5′). F2-2′ (76.3 mg) was further purified by CC on Sephadex LH-20 (20 cm × 2 cm i. d., 40 g) with methanol as the eluent. Finally, 2 mL fractions were collected and pooled according to their TLC profile to give compound 4 (23.7 mg, 84% purity by ¹H NMR).

Compound 1: colorless oil; R_f 0.40, silica gel 60 F254, petroleum ether/EtOAc (80 : 20); [α]_D ²⁰ + 22.5 (c 0.475, CH₂Cl₂), CD (CH₃CN) Δε _204.8 + 21.1, Δε _227.8 − 5.4; IR (ATR mode) ν _max 2958, 2928, 1751, 1713, 1647, 1459, 1368, 1228, 1154, 1109, 1020, 991, 941 cm⁻¹. ¹H and ¹³C NMR: see [Tables 1] and [2] for data in C₆D₆ and Table 21S, Supporting Information, for data in CDCl₃. HRESIMS (pos. ion mode) m/z 548.2856, [M + NH₄]⁺ (calcd. for C₂₉H₄₂NO₉ 548.2854).

Compound 2: colorless oil; R_f 0.60, silica gel 60 F254, petroleum ether/EtOAc (80 : 20); [α]_D ²⁰ + 3.0 (c 0.43, CH₂Cl₂), CD (CH₃CN) Δε _206.2 + 25.5, Δε _227.4 − 7.6; IR (ATR mode) ν _max 2957, 2925, 1758, 1712, 1647, 1457, 1365, 1229, 1152, 1085, 1040, 1013, 961 cm^{− 1 1}H and ¹³C NMR: see [Tables 1] and [2] for data in C₆D₆ and Table 21S, Supporting Information, for data in CDCl₃. HRESIMS (pos. ion mode) m/z 525.2457, [M + Na]⁺ (calcd. for C₂₈H₃₈NaO₈ 525.2459).

Compound 3: colorless oil; R_f 0.65, silica gel 60 F254, petroleum ether/EtOAc (80 : 20); [α]_D ²⁰ − 38.2 (c 0.75, CH₂Cl₂), CD (CH₃CN) Δε _204.4 + 25.3, Δε _224.8 − 7.9; IR (ATR mode) ν _max 2958, 2922, 1754, 1714, 1650, 1457, 1365, 1229, 1154, 1088, 1041, 977 cm⁻¹. ¹H and ¹³C NMR: see [Tables 1] and [2] for data in C₆D₆ and Table 21S, Supporting Information, for data in CDCl₃. HRESIMS (pos. ion mode) m/z 525.2458, [M + Na]⁺ (calcd. for C₂₈H₃₈NaO₈ 525.2459).

Compound 4: colorless oil; R_f 0.36, silica gel 60 F254, petroleum ether/EtOAc (80 : 20); ¹H and ¹³C NMR (C₆D₆): see [Tables 1] and [2]. HRAPCIMS (pos. ion mode) m/z 329.1742, [M + H]⁺ (calcd. for C₂₀H₂₅O₄ 329.1747).

Antiparasitic activity

Antiplasmodial and antileishmanial activities were assessed as previously described [18].

Cytotoxicity evaluation on J774A.1 macrophages

Cytotoxicity evaluation on the J774A.1 cell line (mouse macrophage cell line; Sigma-Aldrich) was performed using an MTT assay. Briefly, cells (5.10⁴ cells/mL) in 100 µL of complete medium [DMEM, high glucose supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin)] were seeded into each well of 96-well plates and incubated at 37 °C in a humidified 5% CO₂ with 95% air atmosphere. After a 24-h incubation, 100 µL of medium with various compound or extract concentrations and appropriate controls were added and the plates were incubated for 72 h at 37 °C. Each plate well was then microscope examined for detecting possible precipitate formation before the medium was aspirated from the wells. Next, 100 µL of MTT solution (0.5 mg/mL in DMEM) were then added to each well. Cells were incubated for 2 h at 37 °C. After that, the MTT solution was removed and DMSO (100 µL) was added to dissolve the resulting formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min and the absorbance was measured at 570 nm with a microplate spectrophotometer. DMSO or MeOH was used as a blank and doxorubicin (Sigma Aldrich) as a positive control. CC₅₀ values were calculated by nonlinear regression analysis processed on dose-response curves using TableCurve 2D V5 software.

Cytotoxicity evaluation on human macrophages generated from THP-1 cells

A cytotoxicity evaluation on human macrophages generated from THP-1 cells was done in two steps: first, the THP-1 cell line (human monocyte cell line) was incubated with PMA to generate macrophages, then a cytotoxicity evaluation was done by an MTT assay. Briefly, cells (0.77.10⁵ cells/mL) in 200 µL of complete medium [RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin)] + PMA (50 ng/mL) were seeded into each well of 96-well plates and incubated at 37 °C in a humidified 5% CO₂ with 95% air atmosphere. After a 96-h incubation, plates were rinsed 3 times with medium and 100 µL of medium were added. Finally, 100 µL of medium with various compound or extract concentrations and appropriate controls were added and the plates were incubated for 72 h at 37 °C. Further treatment of the plates was done as described above for the J774A.1 macrophages.

Contributorsʼ Statement

Conception and design of the work: V. Jullian, C. André-Barrès. Data collection: S. Bourgeade-Delmas, V. Jullian, C. André-Barrès. M. Trinel, J. Lucas, D. Castillo. Analysis and interpretation of the data: S. Bourgeade-Delmas, V. Jullian, C. André-Barrès, M. Trinel, J. Lucas, D. Castillo. Drafting the manuscript: S. Bourgeade-Delmas, V. Jullian, C. André-Barrès. M. Trinel, J. Lucas, D. Castillo. Critical revision of the manuscript: S. Bourgeade-Delmas, V. Jullian, C. André-Barrès, M. Trinel, J. Lucas, D. Castillo.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors thank the logistic support of the LMI-LaVi (UPCH-IRD, Lima, Peru) and their directors: Pr. Rosario Rojas (UPCH) and Dr. Michel Sauvain (IRD). The authors are grateful to Pierre Lavedan, Marc Vedrenne, and Caroline Toppan (NMR platform of the Institut de Chimie de Toulouse, ICT) for NMR analyses and to Dr. Stéphane Massou (NMR platform of the Institut de Chimie de Toulouse, ICT) for fruitful discussions and help with the redaction of the SI section. The authors thank Dr. Charles-Louis Serpentini (IMRCP, UMR 5623 CNRS-Université Toulouse 3) for the recording of CD spectra, and Dr. Céline Deraeve (LCC, CNRS, Toulouse) for the recording of IR spectra. The authors are indebted to the personnel of the Universidad Nacional Mayor de San Marcos, Lima, Peru (UNMSM) herbarium for plant determination and the Servicio Nacional Forestal y de Fauna Silvestre (SERFOR) for issuing the P. multiflora collecting and research permits (No 406-2016-SERFOR/DGGSPFFS and No 80-2017-SERFOR/DGGSPFFS).

• References

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Correspondence

Publication History

Received: 08 July 2020

Accepted after revision: 17 October 2020

Article published online:
23 November 2020

© 2020. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Missouri Botanical Garden. Tropicos.org. Perezia multiflora . Accessed August 25, 2020 at: https://www.tropicos.org/name/2701110
  PubMed
• 2 Mostacero-León J, Castillo-Picón F, Mejía-Coico FR, Gamarra-Torres OA, Charcape-Ravelo JM, Ramirez-Vargas RA. Plantas medicinales del Perú, 1 ed. Trujillo: Asamblea Nacional de Rectores; 2011
  Search in Google Scholar
• 3 Bohlmann F, Zdero C. Neue Sesquiterpene mit anomalem Kohlenstoffgerüst aus der Tribus Mutisieae . Chem Ber 1979; 112: 427-434
  CrossrefPubMedSearch in Google Scholar
• 4 Bohlmann F, Zdero C. Über eine neue Gruppe von Sesquiterpenlactonene aus der Gattung Trixis . Chem Ber 1979; 112: 435-444
  CrossrefPubMedSearch in Google Scholar
• 5 Bohlmann F, Zdero C, King RM, Robinson H. A tetracyclic sesquiterpene, further isocedrene, and guaiene derivatives from Jungia stuebelii . Phytochemistry 1983; 22: 1201-1206
  CrossrefPubMedSearch in Google Scholar
• 6 Singh P, Jakupovic J, Bohlmann F. Isocedrene derivatives and other sesquiterpenes from Moscharia pinnatifida . Phytochemistry 1985; 24: 1525-1529
  CrossrefPubMedSearch in Google Scholar
• 7 Zdero C, Bohlmann F, Niemeyer HM. Isocedrene and guaiane derivatives from Pleocarphus revolutus . J Nat Prod 1988; 51: 509-512
  CrossrefPubMedSearch in Google Scholar
• 8 Zdero C, Bohlmann F, Sanchez H, Dominguez XA. Isocedrene derivatives and other constituents from Acourtia nana . Phytochemistry 1991; 30: 2695-2697
  CrossrefPubMedSearch in Google Scholar
• 9 Zdero C, Bohlmann F, King RM, Robinson H. α-Isocedrene derivatives, 5-methyl coumarins and other constituents from the subtribe Nassauviinae of the Compositae. Phytochemistry 1986; 25: 2873-2882
  CrossrefPubMedSearch in Google Scholar
• 10 De Riscala EC, Catalan CAN, Sosa VE, Gutierrez AB, Herz W. Trixane derivatives from Trixis praestans . Phytochemistry 1988; 27: 2343-2346
  CrossrefPubMedSearch in Google Scholar
• 11 Ybarra MI, Catalan CAN, Diaz JG, Herz W. A cyperane and trixanes from Jungia polita . Phytochemistry 1992; 31: 3627-3629
  CrossrefPubMedSearch in Google Scholar
• 12 Azevedo L, Faqueti L, Kritsanida M, Efstathiou A, Smirlis D, Franchi GCJ, Genta-Jouve G, Michel S, Sandjo LP, Grougnet R, Biavatti MW. Three new trixane glycosides obtained from the leaves of Jungia sellowii less. using centrifugal partition chromatography. Beilstein J Org Chem 2016; 12: 674-683
  CrossrefPubMedSearch in Google Scholar
• 13 Kotowicz C, Hernandez LR, Cerda-Garcia-Rojas CM, Villecco MB, Catalan CAN, Joseph-Nathan P. Absolute configuration of trixanolides from Trixis pallida . J Nat Prod 2001; 64: 1326-1331
  CrossrefPubMedSearch in Google Scholar
• 14 Maldonadoa EM, Salamanca E, Gimenez A, Saavedra G, Sterner O. Antileishmanial metabolites from Trixis antimenorrhoea . Phytochem Lett 2014; 10: 281-286
  CrossrefPubMedSearch in Google Scholar
• 15 Zdero C, Bohlmann F, Solomon J, Dominguez XA. Further isocedrene derivatives and other constituents from Perezia species. Phytochemistry 1988; 27: 849-853
  CrossrefPubMedSearch in Google Scholar
• 16 Kirby AJ. Stereoelectronic Effects. Oxford: Oxford University Press; 1996
  CrossrefSearch in Google Scholar
• 17 Lodewyk MW, Siebert MR, Tantillo DJ. Computational prediction of 1H and 13C chemical shifts: a useful tool for natural product, mechanistic, and synthetic organic chemistry. Chem Rev 2012; 112: 1839-1862
  CrossrefPubMedSearch in Google Scholar
• 18 Castro I, Fabre N, Bourgeade-Delmas S, Saffon N, Gandini C, Sauvain M, Castillo D, Bourdy G, Jullian V. Structural characterization and anti-infective activity of 9, 10-seco-29-norcycloartane glycosides isolated from the flowers of the peruvian medicinal plant Cordia lutea . J Nat Prod 2019; 82: 3233-3241
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material