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Phytochemical and biological studies on Piptocarpha axillaris (Less.) Baker (Asteraceae)
Biochemical Systematics and Ecology 85 (2019) 24–30
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Phytochemical and biological studies on Piptocarpha axillaris (Less.) Baker (Asteraceae)
T
Isabela de Souza Pinto Pereiraa,∗, Maria Raquel Garcia Vegaa, Marcelo da Silva Mathiasa, Amaro Chaves Ramosa, Rodrigo Rodrigues de Oliveiraa, Marina Meirelles Paesb,c, Milton Masahiko Kanashirob a
Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto Lamego, 2000, Campos dos Goytacazes, Rio de Janeiro, Brazil Laboratório de Biologia de Reconhecer, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto Lamego, 2000, Campos dos Goytacazes, Rio de Janeiro, Brazil c Centro das Ciências Biológicas e da Saúde, Universidade Federal do Oeste da Bahia, Rua Professor José Seabra de Lemos, 316, Barreiras, Bahia, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Piptocarpha axillaris (less.) baker Cytotoxic activity UPLC-MS/MS HPCCC
The phytochemical investigation of Piptocarpha axillaris (Less.) Baker (Asteraceae) leaves resulted in the identification of 20 known compounds. Terpenes, steroids, flavonoids and phenolic acids were identified and isolated by column chromatography, high performance countercurrent chromatography (HPCCC), UPLC-MS/MS, 1D and 2D NMR, GC-MS and compared to literature data. Extract (hexane, dichloromethane, ethyl acetate) and 5hydroxy-3′,4′,7-trimethoxyflavanone (compound 10) cytotoxicity against the growth of two human tumor cell lines (histiocytic lymphoma U-937 and acute monocytic leukemia THP-1) and a human non-tumor cell line (PBMC) were assessed by the MTT method. The dichloromethane extract displayed low activity, but the most significant result against the U-937 cell line, with an IC50 63.82 ± 1.06 μg/mL and a good selectivity index (SI) of 5.51. All extracts tested in PBMC cells displayed high cytotoxicity. The cytotoxic evaluation of compound 10 displayed low activity against the U-937 and THP-1 cell lines. Twelve compounds comprising triterpenes, steroids, flavonoids, and phenolic acids are described herein for the first time for the Piptocarpha genus. In addition, this is the first phytochemical investigation and cytotoxic evaluation of Piptocarpha axillaris (Less.) Baker that displays chemotaxonomic significance.
1. Introduction Piptocarpha belongs to the Asteraceae family and is a neotropical genus comprising about 46 species distributed throughout southern and eastern Brazil and northern Argentina, the West Indies and Central America. Their habitat extends from temperate forests to the savannah (Robinson, 2002; Smith, 1981; Smith and Coile, 2007). Previous phytochemical studies have revealed the occurrence of sesquiterpenes, steroids, triterpenes and flavonoids in plants belonging to the Piptocarpha genus (Bohlmann et al., 1980; Castro and Warning, 1987; Cowall et al., 1981; Herz and Kulanthaivel, 1983), and several biological investigations have been carried out with Piptocarpha species extracts or isolated compounds. These include antimicrobial activity evaluations against Bacillis cereus and Candida albicans; cytotoxic assessments against 9 KB human nasopharynx carcinoma cells and P-388 lymphoid leukemia system; investigations concerning activity against human saliva α-amylases and larvicidal, molluscicidal, antihelminthic and ∗
leishmanicidal activities (Antinarelli et al., 2015; Cowall et al., 1981; Gilbert et al., 1972; Mendes et al., 1999; Rodrigues et al., 2006; Scio et al., 2012; Silva et al., 2009). Piptocarpha axillaris (Less.) Baker is a tree ranging from 3 to 15 m in height, with cylindrical brownish collared branches and alternate leaves. Several popular names have been attributed to this species, due to the internal oxidation that occurs when its wood is cut, characterized by a color change to almost black, such as: “canela-podre”, “vassourão” and “cambará”. Piptocarpha axillaris is found in the Atlantic Rainforest, in Southeastern and Southern Brazil, and is the most abundant species in the state of Paraná (Grokoviski et al., 2009). Reports on the use of this species as both ornamental and used as temporary fences are available, and their flowers are also known for being melliferous (Ribeiro et al., 2010). To the best of our knowledge, no phytochemical study has been previously performed concerning P. axillaris. However, plant extracts have been analyzed by LC-UV-HRMS by dereplication of major
Corresponding author. E-mail address: [email protected] (I.d.S.P. Pereira).
https://doi.org/10.1016/j.bse.2019.05.001 Received 27 February 2019; Received in revised form 26 April 2019; Accepted 4 May 2019 Available online 16 May 2019 0305-1978/ © 2019 Elsevier Ltd. All rights reserved.
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secondary metabolites, with the identification of phenolic acids and flavonoids (Gallon et al., 2018). One biological investigation is available regarding the antihelminthic activity of the essential oil present in wood from this species, where the oil was proven to be a Schistosoma mansoni cercaria skin penetration inhibitor. The authors attributed the activity to sesquiterpenes and diterpenes through literature data (Gilbert et al., 1972). In this context, this is the first phytochemical study and cytotoxic evaluation concerning Piptocarpha axillaris.
corresponding to compound 12. Fractions A6 (2.7 mg) and A7 (2.3 mg) displayed a phenolic profile when analyzed by TLC. They were subsequently evaluated by UPLC-MS/MS, allowing for the identification of compounds 13–20. Two milligrams of the samples (A6 and A7) were solubilized in 1.0 mL of a 5% acetonitrile solution in ultrapure water in 2.0 mL polypropylene microtubes, with the aid of an ultrasonic bath for 10 min. The samples were then centrifuged at 3,000 rpm for 10 min. The supernatants were filtered through a 0.45 μm membrane (TEDIA) and transferred to 1.5 mL glass vials.
2. Material and methods
2.3. General
2.1. Plant material
1
H and 13C NMR spectra, were recorded on a Bruker NMR, operating at 400 MHz for 1H and 100 MHz for 13C, and on a Bruker Ascend 500 operating at 500 MHz for 1H and 125 MHz for 13C. Samples were dissolved in appropriate deuterated solvents (d-chloroform or d-methanol) and TMS was used as the internal standard. High performance countercurrent chromatography (HPCCC) was performed using a Dynamic Extractions Spectrum with 142 mL multilayer coil columns (1.6 mm i.d.). The β-value ranges from 0.52 to 0.86 and revolution speed can be adjusted to up to 1600 rpm. The chromatographic system comprises two Knauer Smartline 100 V5010 pumps, a 5 mL manual injection valve and a Büchi C-660 fraction collector. The eluate was monitored by a Knauer V7604 UV absorbance detector. A GC-MS analysis was performed on a Shimadzu GCMS-QP5050A equipped with a DB5 column (30 m × 0.25 mm, 0.25 μm film thickness). The mass spectrometer was set to 280 °C and the electron impact (EI) energy to 70 eV. The initial oven temperature was set to 100 °C, and a 20 °C/min rate was used, until reaching 280 °C. The final temperature (280 °C) was held for 78 min (total run time 87 min). The injection port temperature was set at 280 °C and 1 μL of sample was injected. The UPLC-MS/MS analysis was performed on a Shimadzu LC-20A instrument (Kyoto, Japan) equipped with a degasser, binary pump, autosampler, temperature controlled column oven, and diode array detector (DAD) coupled to a high resolution mass spectrometer equipped with an electrospray ion source (ESI) and quadrupole time of flight mass analyzer (Q-TOF) (Bruker Daltonics, Bremen, Germany). The separation was performed on a C-18 analytical Zorbax column (1.7 μm, 2.1 × 75 mm, Phenomenex). The mobile phase consisted of ultra pure water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The following gradient elution program was determined after several optimization procedures: 0–1 min 5% B, 1–5 min 5–17% B, 5–42 min 17–22% B, 42–50 min 22–100% B, 50–55 min 100% B, 55–60 min from 100 to 5% B, 60–65 min 5% B. The flow rate was maintained at 0.25 mL min−1. The column temperature was set to 40 °C. The final 15 min of the method aimed to clean the system for the next injection, in sequence, rebalancing the column with the initial mobile phase composition. An injection volume of 50 μL was used. The UV/DAD detector was monitored at 345 nm (flavonoids and phenolic acids). Chromatograms were recorded and processed using the LC Solution v. 1.25 software (Shimadzu). The nebulizer gas consisted in high purity nitrogen (N2) produced online by a Peak Scientific NM32LA nitrogen generator. The analysis parameters were set using the negative ionization mode with spectra acquired over a mass range from m/z 50 to 1400. The optimum ESI-MS parameters were as follows: capillary voltage, +3.5 kV; drying gas temperature, 210 °C; drying gas flow, 10.0 L/min; nebulizing gas pressure, 72.5 psi; collision RF, 200 Vpp; transfer time 120 μs; and pre-pulse storage, 3 μs. Moreover, automatic MS/MS experiments were performed adjusting the collision-energy values as follows: m/z 500, 30 eV; m/z 1000, 50 eV; m/z 1400, 70 eV using nitrogen as collision gas. The MS data were processed using the Data Analysis 4.0 software (Bruker Daltonics, Bremen, Germany). External instrument calibration was performed using a Cole Palmer syringe pump (Vernon Hills, IL, USA) directly connected to the interface, eluting a sodium formate (NaCHO2) solution cluster containing 5 mM sodium hydroxide (NaOH) and 0.2% formic acid in
Piptocarpha axillaris leaves were collected at the União Biological Reserve (22°27′30″S; 42°02′15″O, Rio das Ostras, Atlantic Rainforest, Rio de Janeiro, Brazil) in March 2013. A voucher specimen (HUENF 9476) is deposited at the UENF Herbarium, at the Center of Biosciences and Biotechnology, Universidade Estadual do Norte Fluminense, Rio de Janeiro, Brazil. It was also archived and published at JABOT/JBRJ (http://huenf.jbrj.gov.br/v2/consulta.php), available for public consultation. 2.2. Extraction and isolation Piptocarpha axillaris dried and crushed leaves (390.489 g) were extracted by maceration at room temperature with solvents presenting different polarities. The extraction step was repeated weekly with 1–2 L of solvent. Subsequently, the liquid was filtered, and the solvent removed under vacuum to obtain a dried extract. The following solvents were used: hexane, for 8 weeks (20.0981 g - PH), CH2Cl2, for 7 weeks (3.9431 g - PD) and EtOAc, for 3 weeks (2.5593 g - PA). The PH extract (6.0981 g) was chromatographed on a silica gel column eluted with hexane/EtOAc, gradually increasing solvent polarity to obtain 9 fractions (H1–H9). Fraction H4 (781.8 mg) was washed with MeOH, resulting in a 1 + 2 mixture (50.5 mg). Fraction H5 (696.7 mg) was also washed with MeOH, resulting in the pure compound 4 (107.0 mg). The insoluble H4 fraction (726.8 mg) was chromatographed on a silica gel column eluted with hexane/EtOAc gradients, obtaining 8 fractions. Fraction H-4.2.2 (712.3 mg) was further purified by washing with EtOAc, obtaining H-4.2.2.1 (449.4 mg) and H4.2.2.2 (101.0 mg). Fraction H-4.2.2.1 was purified on a silica gel CC eluted with an isocratic system comprising hexane/ethyl ether 8.5:1.5 (v/v), resulting in a triterpene mixture 1 + 2 + 3 (39.4 mg). Fraction H-4.2.2.2 was chromatographed on a low pressure silica flash applying hexane/CHCl3 gradients to obtain compound 2 (12.3 mg). Fractions H5 (589.7 mg), H6 (257.8 mg), and H8 (769.1 mg) were chromatographed on silica gel CC with hexane/EtOAc gradients and yielding a 4 + 5 + 6 mixture (187.0 mg). Fraction H-8.7 was further purified by crystallization with CH2Cl2, yielding a steroid mixture 7 + 8 + 9 (41.8 mg). The PD extract (3.12 g) was chromatographed on a filtration column (using activated charcoal) under low pressure, with ethyl ether/ CH2Cl2/EtOAc/MeOH as the eluent, resulting in 4 fractions (PD2-E, PD2-D, PD2-A, PD2-M). Fraction PD2-D was chromatographed on a silica gel column eluted with CH2Cl2/MeOH, gradually increasing eluent polarity to obtain 13 fractions. Fraction PD2-D9 was purified by another silica gel CC eluted with hexane/CH2Cl2. Fraction PD2-D9.6 (11.5 mg) was further purified by crystallization with hexane, resulting in compound 10 (5.5 mg). The PA extract (0.7593 g) was submitted to HPCCC using a solvent system comprising Hexane:EtOAc:MeOH:H2O (5:8:5:8) (v/v). A total of 18 fractions were obtained (A1-A18). After a qualitative analysis performed on TLC, fraction A4 displayed a major yellow spot corresponding to compound 11 (4.0 mg). Fractions A15 (52.6 mg) and A16 (76.1 mg) were combined and submitted to a new HPCCC analysis using Hexane:EtOAc:MeOH:H2O (1:3:1:3) (v/v) as the solvent system. Fraction A15–16.7 (5.4 mg) presented a major yellow spot on TLC, 25
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water:isopropanol 1:1 (v/v). The LC/MS system was controlled by the HyStar 3.2 software (Bruker Daltonics, Bremen, Germany). Column chromatography (CC) separations were carried out on silica gel 60 (63–200 μm, Merck) and silica flash (230–400 mesh, Merck). TLC was performed on commercial plates (silica gel 60 F254, Merck). Spot visualization was performed in the UV range (254 and 365 nm) using vanillin sulfuric acid, followed by heating.
[4,5-dimethy-2-lthiazolyl-]2,5-diphenyl-2H-tetrazolium bromide), as described previously (Paes et al., 2015). After 48 h of incubation with the samples, 20 μL of MTT (5 mg/mL - Sigma) were added to each well, followed by incubation for 4 h at 37 °C for MTT metabolism. After this period, 150 μL of the supernatant were removed from each well and 100 μL of an HCl solution in isopropanol were then added for formazan crystal solubilization. Absorbance values were determined by spectrophotometry at 570 nm (EpochTM, BioTex® Instruments, Inc.). Each experiment was performed in triplicate. Doxorubicin (Sigma) was used as the positive control (Paes et al., 2015). The selectivity degree of the fractions was expressed by the selectivity index (SI), calculated by dividing the IC50 value for the noncancer PBMC by the IC50 value for the U937cancer cell line.
2.4. Compound identification All compounds are known and have been identified by comparison to literature data and libraries. Identification of compounds 1–10 was based: (i) on comparison with published 1D-2D NMR spectroscopy data (Balestrin et al., 2008; Carvalho et al., 1998; Chaturvedula and Prakash, 2012; Gangwal et al., 2010; Harbone and Mabry, 1982; Silva et al., 1998; Sobrinho et al., 1991); (ii) on comparison of their mass fragmentation similarities by GC-MS using NIST library versions 107, 05, 21 and literature data (Silva et al., 2016). Identification of compounds 11–12 was based on comparison with published 1D-2D NMR spectroscopy data (Alves et al., 2010; Junior et al., 2010). Identification of compounds 13–20 was based on comparison of their mass fragmentation similarities by UPLC-MS/MS, literature data, and the MassBank database (Bueno et al., 2016; Clifford et al., 2005; D'Urso et al., 2018; He et al., 2016; MassBank, 2018; Zheleva-Dimitrova et al., 2017).
2.6. Statistical analyses The cytotoxicity to each sample was expressed as IC50 and analyzed by a one-way ANOVA using the GraphPad Prism 5.0 software. Significant differences were set at p < 0.05. 3. Results and discussion 3.1. Isolation and structural elucidation The phytochemical investigation of Piptocarpha axillaris leaves allowed the identification and isolation of 12 known compounds: six triterpenes, β-amyrin acetate (1), lupeol acetate (2), α-amyrin acetate (3), lupeol (4), α-amyrin (5), β-amyrin (6), three steroids, stigmasterol (7), β-sitosterol (8), campesterol (9), and three flavonoids, 5-hydroxy3′,4′,7-trimethoxyflavanone (10), 4′,5-dihydroxy-7-methoxyflavone (11), kaempferol 3-O-β-D-(6″-O-E-p-coumaroyl)-glucopyranoside (trans-tiliroside) (12). Their structures were established on the basis of a spectral NMR analysis by comparison to literature data and a GC-MS analysis. Sesquiterpene lactones and flavonoids are considered chemotaxonomic markers for the Asteraceae family (Emerenciano et al., 2001; Seaman, 1982). Table 1 summarizes the compounds isolated from the Piptocarpha genus since the first study in 1980, as sesquiterpene lactones, steroids, sesquiterpenes, terpenes, and flavonoids. As no
2.5. Cytotoxic activity bioassay evaluation U-937 (histiocytic lymphoma) and THP-1 (acute monocytic leukemia) cells were obtained from the American Type Culture Collection (ATCC). Human non-tumor PBMC (peripheral blood mononuclear cells) were obtained from the venous blood of healthy volunteers as described elsewhere (Junior et al., 2010). The cells were cultured in DMEM-F12 (Gibco) medium containing 10% fetal bovine serum (Gibco) and gentamicin 20 μg/mL (Gibco). The culture was maintained at 37 °C with 5% CO2 and controlled humidity. Briefly, cells were incubated in 96well plates (1 × 106 cells/well) containing 100 μL and incubated at different extract/fractions/compound concentrations during 48 h cytotoxicity was determined by a colorimetric microassay using MTT (3Table 1 Compounds isolated from the Piptocarpha genus. Compounds
Species
References
germacrene D (s) lupeol acetate (t) lupeol (t)
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
Bohlmann et al. (1980) Bohlmann et al. (1980) Bohlmann et al. (1980) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Bohlmann et al. (1980) Bohlmann et al. (1980) Bohlmann et al. (1980) Cowall et al. (1981) Cowall et al. (1981) Cowall et al. (1981) Cowall et al. (1981) Cowall et al. (1981) Cowall et al. (1981) Castro and Warning (1987) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983)
α-amirin (t) β-amirin (t) sakuranetin (f) naringenin 7,4′-dimethyl ether (f) dimethyl eriodictyol (f) piptocarphin A (sl) piptocarphin B (sl) piptocarphin C (sl) piptocarphin D (sl) piptocarphin E (sl) piptocarphin F (sl) hirsutinolide (sl) 7,10-epoxycyclodeca (β) furan, 2-butenoic acid (sl) 7,10-epoxycyclodeca (β) furan, 2-propenoic acid (sl) sitosterol β-D-glucoside (st) stigmasterol β-D-glucoside (st) stigmasterol (st) β-sitosterol (st) stigmasterone (st) β-sitosterone (st)
oblonga oblonga oblonga opaca opaca opaca oblonga oblonga oblonga chontalensis chontalensis chontalensis chontalensis chontalensis chontalensis poeppigiana opaca opaca opaca opaca opaca opaca opaca opaca
(s) sesquiterpenes; (t) triterpenes; (f) flavonoids; (sl) sesquiterpene lactones; (st) steroids. 26
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Fig. 1. A6 and A7 fraction chromatograms obtained by UPLC-DAD-MS/MS at 345 nm.
phytochemical investigation has been reported for Piptocarpha axillaris species, the results obtained herein are new for Piptocarpha axillaris, with compounds 1, 3, 9, 10, 11 and 12 obtained from the Piptocarpha genus for the first time.
were identified as 3,5-dicaffeoylquinic acid (18) and 1,4-dicaffeoylquinic acid (19). Both acids lost caffeoyl units ([M-162-162]-), resulting in the ion m/z 191.06 relative to quinic acid, but the remainder of the fragmentation profiles of these acids are different. Baicalein-7-O-glucoronide (14) (m/z 445.0801), wogonoside (15) (m/z 459.0981) and isorhamnetin-3-O-glucoside (16) (m/z 477.1439) were also detected. Two tiliroside (m/z 593.13) isomers, compounds 20 and 12, with different retention times, were also detected. It is suggested that the more intense peak 11 is a trans-isomer, as it is thermodynamically stable than cis-isomer (Devi and Kumar, 2018). All compounds were identified by their relative change in fragments. Flavonoids are considered chemotaxonomic Asteraceae family markers, and the obtained results corroborate the observed chemical profile. The importance and efficiency of the analytical techniques used to identify the organic compounds are worth emphasizing. A previous investigation analyzed major secondary metabolites in Asteraceae species by LC-UV-HRMS and dereplication (Table 4), and identified 29 compounds in Piptocarpha genus species extracts and 12 compounds in P. axillaris, including as phenolic acids and flavonoids (Gallon et al., 2018). Compared to the UPLC-MS/MS analysis, compounds 13, 14, 15, 16, 19, 20 are described for the first time for the Piptocarpha genus.
3.2. UPLC-DAD-MS/MS analysis Two ethyl acetate extract fractions (A6 and A7) were analyzed by UPLC-DAD-MS/MS due the phenolic profile observed by TLC. The obtained chromatograms are displayed in Fig. 1. Compound identification was performed by comparing the fragmentations to the MassBank database and literature data (Tables 2 and 3). Fraction A6 presented a major peak at 13.8 min and was identified as baicalein-7-O-glucuronide (14) with m/z 445.0816 ([M-H]-). Its fragmentation profile is based on the loss of the glucuronide unit (−176 Da) for the formation of m/z 269.05 for aglycone baicalein. Some minor compounds were also detected, such as caffeic acid (13) (m/z 179.0355), characterized by the presence of m/z 135.04, due to loss of CO2 ([M-H-44]-), wogonoside (15) (m/z 459.0965) characterized by glucuronide and a CH3 unit loss (M-H-176-15]-), and isorhamnetin3-O-glycoside (16) (m/z 477.1439) characterized by the neutral loss of glucose for ion m/z 315.09 formation, relative to aglycone ([M-H-162]). Concerning fraction A7, the first peak at 6.2 min corresponds to 5-Ocaffeoylquinic acid (17), due to the presence of only one m/z 191.06 ion fragment, unlike 3-O-caffeoylquinic acid, which presents other fragments (Jaiswal et al., 2014). Two major peaks (12.4 min and 15.1 min)
3.3. Chemotaxonomic significance The most recent botanical classification used for the Piptocarpha genus is from the study carried out by (Smith and Coile, 2007), who used morphology, karyology, chemotaxonomy, phylogeny, evolution
Table 2 Compounds identified in the A6 fraction by UPLC-MS/MS. Peaks
RT (min)
[M-H]-
MS/MS (% relative abundance)
Compounds
Reference
1 2 3 4
6.0 13.8 27.1 36.8
179.0355 445.0816 459.0965 477.1439
135.04(100) 269.05(100) 459.09(100), 268.04(20) 315.09(100)
caffeic acid (13) putative baicalein-7-O-glucoronide (14) putative wogonoside (15) putative isorhamnetin-3-O-glucoside (16)
MassBank (2018) He et al. (2016) He et al. (2016) Bueno et al. (2016)
27
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Table 3 Compounds identified in the A7 fraction by UPLC-MS/MS. Peaks
RT (min)
[M-H]-
MS/MS (% relative abundance)
Compounds
Reference
5 6 7 8 9 10 11 12
6.2 12.4 13.8 15.1 27.2 30.3 32.8 36.8
353.0873 515.1257 445.0801 515.1260 459.0981 593.1340 593.1354 477.1411
191.06(100) 353.09(98), 191.06(100), 179.03(65), 161.02(4), 135.05(7) 269.05(100) 353.09(100), 191.06(13), 179.04(44), 173.04(55), 135.05(4) 459.09(100), 268.04(44) 593.13(100), 447.09(8), 285.04(66) 593.13(100), 447.09(7), 285.04(74) 315.09(100)
5-O-caffeoylquinic acid (17) 3,5-dicaffeoylquinic acid (18) putative baicalein-7-O-glucoronide (14) 1,4-dicaffeoylquinic acid (19) putative wogonoside (15) cis-tiliroside (20) trans-tiliroside (12) putative isorhamnetin-3-O-glucoside (16)
MassBank (2018) Zheleva-Dimitrova et al. (2017) He et al. (2016) Clifford et al. (2005) He et al. (2016) D'Urso et al. (2018) D'Urso et al. (2018) Bueno et al. (2016)
Table 4 Piptocarpha genus compounds identified by LC-MS (Gallon et al., 2018). Compounds
Species
3-O-caffeoylquinic acid (pa) 5-O-caffeoylquinic acid (pa) 4-O-caffeoylquinic acid (pa) 5-p-coumaroylquinic acid (pa) 5-O-feruloylquinic acid (pa) 3,5-di-O-caffeoylquinic acid (pa) 4,5-di-O-caffeoylquinic acid (pa) vicenin-2 (f) putative quercetin 3-O-rutinoside (f) vitexin (f) putative quercetin 3-O-glucoside (f) putative luteolin 7-O-glucuronide (f) putative kaempferol 3-O-rutinoside (f) putative 7,3′,5′-trihydroxy-4′-methoxy-3-O-glucosylflavone (f) putative apigenin 7-O-glucuronide (f) putative chrysoeriol 7-O-neohesperidoside (f) putative quercetin 3-O-(6-caffeoyl)-glucoside (f) putative chrysoeriol 7-O-glucuronide (f) putative isoorientin 3′-O-glucoside (f) quercetin (f) luteolin (f) putative acacetin 7-O-rutinoside (f) tiliroside (f) putative isorhamnetin 3-O-(6-p-coumaroyl) glucoside (f) apigenin (f) kaempferol (f) chrysoeriol (f) 3′,4′-dimethoxyluteolin (f) glaucolide B (sl)
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
axillaris, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii angustifolia, P. axillaris, P. macropoda, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii axillaris, P. macropoda, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii quadrangulares, P. sellowii axillaris angustifolia, P. axillaris, P. macropoda, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii angustifolia, P. axillaris, P. macropoda, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii macropoda macropoda, P. regnellii macropoda axillaris, P. macropoda, P. regnellii, P. rotundifolia, P. sellowii quadrangulares, P. rotundifolia quadrangulares regnellii, P. rotundifolia quadrangulares angustifolia, P. macropoda axillaris, P. regnellii, P. sellowii quadrangulares axillaris, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii axillaris, P. regnellii, P. rotundifolia, P. sellowii macropoda, P. quadrangulares macropoda macropoda, P. regnellii, P. rotundifolia, P. sellowii angustifolia, P. axillaris macropoda, P. quadrangulares axillaris, P. macropoda, P. regnellii, P. rotundifolia, P. sellowii angustifolia, P. quadrangulares angustifolia macropoda
(pa) phenolic acids; (f) flavonoids; (sl) sesquiterpene lactones.
and species distribution data. In this classification, Piptocarpha is divided into two subgenus (Piptocarpha and Hypericoides) and four sections (Piptocarpha, Oocephalus, Macrolepideae and Vanilosma). Tables 1 and 4 summarize compounds already isolated and identified in species belonging to the Piptocarpha genus. Table 5 displays the species represented by their respective sections and subgenera. It should be noted that P. paradoxa is no longer classified as a member of the Piptocarpha genus (Mathur and Gonzalez, 1982). Few species belonging to the Piptocarpha genus have been chemically studied, and no specific markers for this genus have been defined. According to phytochemical data available in the literature (Tables 1 and 4), up to now, these species show a chemical profile aimed at the production of, mostly, sesquiterpenes and flavonoids. An analysis of the available data indicates that these species are in evolutionary transition, which can be observed by a greater flavonoid production compared to flavones. In this context, another parameter that points to this evolution is the degree of protection of flavonoid hydroxyl groups, which are mostly protected by glycosylation and, in addition, some methylation and double protection (glycosylation and methylation) cases. No significant structural difference between the studied species belonging to the different sections is noted regarding the structures of isolated and identified flavonoids, except for P. oblonga, which presents
Table 5 Botanical classification of the assessed Piptocarpha species. Subgenus
Piptocarpha
Hypericoides
Sections
Piptocarpha
Oocephalus
Macrolepideae
Vanilosma
Species
P. rotundifolia P. opaca
P. poeppigiana
P. quadrangularis P. oblonga P. sellowii
P. axillaris P. regnellii P. angustifolia P. macropoda
Table 6 Cytotoxic activities expressed as IC50 (μg/mL) for extracts, and for doxorubicin (μM) against the U-937 histiocytic lymphoma cell line and non-tumor human PBMC. Extracts
U-937
PMBC
SI (selectivity index)
PH PD PA Doxorubicina
111.1 ± 1.11 63.82 ± 1.06 > 200.0 2.25 ± 1.03
> 400.0 352.00 ± 1.16 > 400.0 26.92 ± 1.21
> 3.6 5.51 > 2.0 11.96
PH-Hexane extract; PD-Dichloromethane extract; PA-Ethyl acetate extract. a Positive control. 28
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Alves do Nascimento, Tatiane Pereira de Souza, and Marcelo Trindade Nascimento for the collection and botanical identification of the species assessed herein.
Table 7 Cytotoxic activities expressed as IC50 (μM) for an isolated flavanone (10) against the U-937 histiocytic lymphoma and acute monocytic leukemia THP1 cell lines. Compounds
U-937
THP-1
Appendix A. Supplementary data
10 Doxorubicina
> 50.0 2.25 ± 1.03
> 50.0 13.73
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bse.2019.05.001.
a
Positive control.
References
flavanones protected by methylation. Thus, the results of the isolated and identified compounds corroborate the chemotaxonomic positioning of P. axillaris in the Vanilosma section, while also providing an important chemotaxonomy tool, with the description of 20 identified compounds.
Alves, H.S., Oliveira, G.E., Zoghbi, M.G., Chaves, M.C.O., 2010. Flavonóides de Piper carniconnectivum C.DC. Piperaceae. Brazilian J. Pharmacogn. 20, 160–164. https:// doi.org/10.1590/S0102-695X2010000200004. Antinarelli, L.M.R., Pinto, N.C., Scio, E., Coimbra, E.S., 2015. Antileishmanial activity of some brazilian plants, with particular reference to Casearia sylvestris. An. Acad. Bras. Cienc. 87, 733–742. https://doi.org/10.1590/0001-3765201520140288. Balestrin, L., Dias, J.F.G., Miguel, O.G., Dall'Stella, D.S.G., Miguel, M.D., 2008. Contribuição ao estudo fitoquímico de Dorstenia multiformis Miquel (Moraceae) com abordagem em atividade antioxidante. Brazilian J. Pharmacogn. 18, 230–235. https://doi.org/10.1590/S0102-695X2008000200016. Bohlmann, F., Zdero, C., King, R.M., Robinson, H., 1980. Seven guaianolides from the tribe Vernonieae. Phytochemistry 19, 2669–2673. https://doi.org/10.1016/S00319422(00)83941-2. Bueno, P.C.P., Passareli, F., Anhesine, N.B., Torres, R.B., Cavalheiro, A.J., 2016. Flavonoids from Casearia sylvestris swartz variety lingua (salicaceae). Biochem. Syst. Ecol. 68, 23–26. https://doi.org/10.1016/j.bse.2016.06.002. Carvalho, M.G., Velandia, J.R., Oliveira, L.F., Bezerra, F.B., 1998. Triterpenos isolados de Eschweilera longipes Miers (Lecythidaceae). Quim. Nova 21, 740–743. https://doi. org/10.1590/S0100-40421998000600014. Castro, V., Warning, U., 1987. Steiractinolides from Zexmenia virgulata and constituents from other Compositae from Costa Rica. Ing. y Cienc. Quim. 11, 5–6. Chaturvedula, V.S.P., Prakash, I., 2012. Isolation of Stigmasterol and β-Sitosterol from the dichloromethane extract of Rubus suavissimus. Int. Curr. Pharm. J. 1, 239–242. https://doi.org/10.3329/icpj.v1i9.11613. Clifford, M.N., Knight, S., Kuhnert, N., 2005. Discriminating between the six isomers of dicaffeoylquinic acid by LC-MSn. J. Agric. Food Chem. 53, 3821–3832. https://doi. org/10.1021/jf050046h. Cowall, P.L., Cassady, J.M., Chang, C., Kozlowski, J.F., 1981. Isolation and structure determination of Piptocarphins A-F, cytotoxic germacranolide lactones from Piptocarpha chontalensis. J. Org. Chem. 46, 1108–1114. https://doi.org/10.1021/ jo00319a014. D'Urso, G., Pizza, C., Piacente, S., Montoro, P., 2018. Combination of LC–MS based metabolomics and antioxidant activity for evaluation of bioactive compounds in Fragaria vesca leaves from Italy. J. Pharm. Biomed. Anal. 150, 233–240. https://doi.org/10. 1016/j.jpba.2017.12.005. Devi, S., Kumar, V., 2018. Comprehensive structural analysis of cis- and trans-tiliroside and quercetrin from Malvastrum coromandelianum and their antioxidant activities. Arab. J. Chem. https://doi.org/10.1016/j.arabjc.2018.01.009. Emerenciano, V.P., Militão, J.S.L.T., Campos, C.C., Romoff, P., Kaplan, M.A.C., Zambon, M., Brant, A.J.C., 2001. Flavonoids as chemotaxonomic markers for Asteraceae. Biochem. Syst. Ecol. 29, 947–957. https://doi.org/10.1016/S0305-1978(01) 00033-3. Gallon, M.E., Jaiyesimi, O.A., Gobbo-Neto, L., 2018. LC-UV-HRMS dereplication of secondary metabolites from Brazilian Vernonieae (Asteraceae) species supported through in-house database. Biochem. Syst. Ecol. 78, 5–16. https://doi.org/10.1016/j. bse.2018.03.002. Gangwal, A., Parmar, S.K., Sheth, N.R., 2010. Triterpenoid, flavonoids and sterols from Lagenaria siceraria fruit. Der Pharm. Lett. 2, 307–317. https://doi.org/http:// scholarsresearchlibrary.com/archive.html. Gilbert, B., Mors, W.B., Baker, P.M., Tomassini, T.C.B., Coulart, E.G., Holanda, J.C., Costa, J.A.R., Lopes, J.N.G., Filho, D.S., Sarti, S.J., Turco, A.M.T., Vichnewski, W., Lopes, J.L.C., Thames, A.W., Pellegrino, J., Katz, N., 1972. A atividade antelmíntica de óleos essenciais e de seus componentes químicos. Ann. Brazilian Acad. Sci. 44, 423–428. Grokoviski, L., Cervi, A.C., Tardivo, R.C., 2009. O gênero Piptocarpha R. Br.(Asteraceae: vernonieae) no estado do Paraná, brasil. Acta Bot. Bras. 23, 486–498. https://doi. org/10.1590/S0102-33062009000200020. Harbone, J.B., Mabry, T.J., 1982. The Flavonoids: Advances in Research, first ed. Chapman and Hall, London and New York, pp. 744. He, L., Zhang, Z., Lu, L., Liu, Y., Li, S., Wang, J., Song, Z., Yan, Z., Miao, J., 2016. Rapid identification and quantitative analysis of the chemical constituents in Scutellaria indica L. by UHPLC-QTOF-MS and UHPLC-MS/MS. J. Pharm. Biomed. Anal. 117, 125–139. https://doi.org/10.1016/j.jpba.2015.08.034. Herz, W., Kulanthaivel, P., 1983. Piptocarphol esters from Piptocarpha opaca. Phytochemistry 22, 1286–1287. https://doi.org/10.1016/0031-9422(83)80245-3. Jaiswal, R., Müller, H., Müller, A., Karar, M.G.E., Kuhnert, N., 2014. Identification and characterization of chlorogenic acids, chlorogenic acid glycosides and flavonoids from Lonicera henryi L. (Caprifoliaceae) leaves by LC-MSn. Phytochemistry 108, 252–263. https://doi.org/10.1016/j.phytochem.2014.08.023. Junior, H.M.S., Tirelli, A.A., Carvalho, H.W.P., Oliveira, D.F., Prado, N.R.T., Campos, V.P., 2010. Purificação do flavonóide trans-tilirosídeo do extrato metanólico das folhas de Gochnatia barrosii Cabrera (ASTERACEAE) e avaliação da sua atividade
3.4. Cytotoxic activity evaluation Extract cytotoxicity (hexane-PH, dichloromethane-PD, ethyl acetate-PA), and flavanone (5-hydroxy-3′,4′,7-trimethoxyflavanone) and the standard control doxorubicin activities against the growth of two human tumor cell lines (histiocytic lymphoma cell line U-937 and acute monocytic leukemia THP-1) and a non-tumor human cell line (Peripheral Blood Mononuclear Cells - PBMC) were evaluated using the MTT method. The PD extract displayed the best results against the U-937 cell line, with IC50 = 63.82 ± 1.06 μg/mL. All extracts were also tested for cytotoxicity against PBMC. High cytotoxicity was observed, with IC50 = 352.00 ± 1.16 μg/mL for PD and a good selectivity index (SI) of 5.51 (Table 6). A high SI value (> 2.0) indicated high specificity (Suffness and Pezzutto, 1991). Plants extracts are considered active when displaying IC50 ≤ 20.0 μg/mL (Suffness and Pezzutto, 1990). The IC50 value for the dichloromethane extract (PD) was more significant. The cytotoxic activity of the flavanone 5-hydroxy-3′,4′,7trimethoxyflavanone (10) isolated from this extract was evaluated against the U-937 and THP-1 cell lines (Table 7). According to the literature, a pure compound can be considered a potential antineoplastic when its IC50 value is of less than 10 μM (Suffness and Douros, 1982). This compound presented a mild value of IC50 > 50.0 μM for both cell lines. As a contribution for the micromolecular assessment of Atlantic Rainforest species, the present study describes 20 known compounds observed in Piptocarpha axillaris. These results corroborate the chemical profile observed in the literature data for the Asteraceae family, whose flavonoids and sesquiterpene lactones are considered chemotaxonomic markers. No phytochemical investigation has been previously reported for this species and an UPLC-MS/MS analysis verified the presence of phenolic acids and flavonoids. Thus, compounds 1, 3, 9, 10, 11, 12 were isolated, and compounds 13, 14, 15, 16, 19, 20, identified in the Piptocarpha genus for the first time. It is worth noting that the biological results presented herein for P. axillaris are new, while this study comprises the first cytotoxic activity evaluation concerning the histiocytic lymphoma U-937 and acute monocytic leukemia THP-1 cell lines for the flavonoid 5-hydroxy-3′,4′,7-trimethoxyflavanone (10). The dichloromethane extract displayed low activity, but the most significant result against the U-937 cell line, with an IC50 of 63.82 ± 1.06 μg/mL and a good selectivity index (SI) of 5.51. Acknowledgments The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for research fellowships (31033016013P0). Thanks are also due to the Universidade Federal Rural do Rio de Janeiro (UFRRJ) for the 1H (400 MHz) and 13C (100 MHz) NMR analysis. In addition, the authors also thank Aline 29
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I.d.S.P. Pereira, et al. nematicida. Cienc. E Agrotecnol 34, 1224–1231. https://doi.org/10.1590/S141370542010000500021. MassBank High Resolution Mass Spectral Database. accessed. https://massbank.eu/ MassBank, Accessed date: 12 September 2018. Mathur, S.B., Gonzalez, L., 1982. Identification of terpenoids from the leaves of Piptocarpha paradoxa and their biological activities. J. Nat. Prod. 45, 495–496. https://doi.org/10.1021/np50022a025. Mendes, N.M., Queiroz, R.O., Grandi, T.S., Anjos, A.M., Oliveira, A.B., Zani, C.L., 1999. Screening of Asteraceae (Compositae) plant extracts for molluscicidal activity. Mem. Inst. Oswaldo Cruz 94, 411–412. https://doi.org/10.1590/S007402761999000300023. Paes, M.M., Vega, M.R.G., Cortes, D., Cabedo, N., Kanashiro, M.M., 2015. Cytotoxic activities of acetogenins from annona dioica roots. Intermt. J. Sci. 4, 19–29. https://doi. org/10.18483/ijSci.875. Ribeiro, A.O., Silva, A.F., Castro, A.H.F., 2010. Identificação de espécies da família Asteraceae, revisão sobre usos e triagem fitoquímica do gênero Eremanthus da Reserva Boqueirão, Ingaí-MG. Rev. Bras. Plantas Med. 12, 456–465. https://doi.org/ 10.1590/S1516-05722010000400009. Robinson, H.E., 2002. Three new species of Piptocarpha (Asteraceae: vernonieae) from Ecuador and Peru. Novon 12, 393–398. https://doi.org/10.2307/3393087. Rodrigues, A.M.S., Paula, J.E., Degallier, N., Molez, J.F., Espíndola, L.S., 2006. Larvicidal activity of some cerrado plants extracts agains Aedes aegypti. J. Am. Mosq. Control Assoc. 22, 314–317. https://doi.org/10.2987/8756-971X. (2006)22[314]:LAOSCP] 2.0.CO;2. Scio, E., Mendes, R.F., Motta, E.V.S., Bellozi, P.M.Q., Aragão, D.M.O., Mello, J., Fabri, R.L., Moreira, J.R., Assis, I.V.L., Bouzada, M.L.M., 2012. Antimicrobial and antioxidant activities of some plant extracts. Phytochem. as Nutraceuticals - Glob. Approaches to Their Role Nutr. Heal 65–80. https://doi.org/10.5772/27308. Seaman, F.C., 1982. Sesquiterpene lactones as taxonomic characters in the Asteraceae. Bot. Rev. 48, 121–595. https://doi.org/10.1007/BF02919190. Silva, J.R.A., Rezende, C.M., Pinto, A.C., Pinheiro, M.L.B., Cordeiro, M.C., Tamborini, E.,
Young, C.M., Bolzani, V.S., 1998. Ésteres triterpênicos de Himatanthus sucuuba (Spruce) Woodson. Quim. Nova 21, 24–26. https://doi.org/10.1590/S010040421998000600005. Silva, C.A.M., Melo, R.O., Resck, I.S., Silveira, D., 2016. Triterpenes from Pouteria gardneri ( mart . & miq .) baehni extracts. J. Appl. Pharm. Sci. 6, 197–201. https://doi.org/10. 7324/JAPS.2016.601229. Silva, E.M., Valencia, A., Grossi-de-Sá, M.F., Rocha, T.L., Freire, É., Paula, J.E., Espindola, L.S., 2009. Inhibitory action of Cerrado plants against mammalian and insect αamylases. Pestic. Biochem. Physiol. 95, 141–146. https://doi.org/10.1016/j.pestbp. 2009.08.003. Smith, G.L., 1981. New taxa in Piptocarpha R . BR . (vernonieae: compositae). Ann. Mo. Bot. Gard. 68, 661–667. https://doi.org/10.2307/2398894. Smith, G.L., Coile, N.C., 2007. Piptocarpha (compositae: vernonieae). Flora Neotrop. 99, 1–94. https://www.jstor.org/stable/4393953. Sobrinho, D.C., Hauptli, M.B., Appolinario, E.V., Kollenz, C.L.M., Carvalho, M.G., BrazFilho, R., 1991. Triterpenoids isolated from Parahancornia amapa. J. Braz. Chem. Soc. 2, 15–20. https://doi.org/10.5935/0103-5053.19910004. Suffness, M., Douros, J., 1982. Current status of the NCl plant and animal product program. J. Nat. Prod. 45, 1–14. https://doi.org/10.1021/np50019a001. Suffness, M., Pezzutto, J.M., 1991. Assays for cytotoxicity and antitumor activity. In: Hostettmann, K. (Ed.), Methods in Plant Biochemistry. Academic Press, London, pp. 133. Suffness, M., Pezzutto, J.M., 1990. Assays related to cancer drug discovery. In: Hostettmann, K. (Ed.), Methods in Plant Biochemistry: Assays for Bioactivity. Academic Press, London, pp. 220. Zheleva-Dimitrova, D., Gevrenova, R., Zaharieva, M.M., Najdenski, H., Ruseva, S., Lozanov, V., Balabanova, V., Yagi, S., Momekov, G., Mitev, V., 2017. HPLC-UV and LC–MS analyses of acylquinic acids in geigeria alata (DC) oliv. & hiern. And their contribution to antioxidant and antimicrobial capacity. Phytochem. Anal. 28, 176–184. https://doi.org/10.1002/pca.2658.
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Phytochemical and biological studies on Piptocarpha axillaris (Less.) Baker (Asteraceae)
T
Isabela de Souza Pinto Pereiraa,∗, Maria Raquel Garcia Vegaa, Marcelo da Silva Mathiasa, Amaro Chaves Ramosa, Rodrigo Rodrigues de Oliveiraa, Marina Meirelles Paesb,c, Milton Masahiko Kanashirob a
Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto Lamego, 2000, Campos dos Goytacazes, Rio de Janeiro, Brazil Laboratório de Biologia de Reconhecer, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto Lamego, 2000, Campos dos Goytacazes, Rio de Janeiro, Brazil c Centro das Ciências Biológicas e da Saúde, Universidade Federal do Oeste da Bahia, Rua Professor José Seabra de Lemos, 316, Barreiras, Bahia, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Piptocarpha axillaris (less.) baker Cytotoxic activity UPLC-MS/MS HPCCC
The phytochemical investigation of Piptocarpha axillaris (Less.) Baker (Asteraceae) leaves resulted in the identification of 20 known compounds. Terpenes, steroids, flavonoids and phenolic acids were identified and isolated by column chromatography, high performance countercurrent chromatography (HPCCC), UPLC-MS/MS, 1D and 2D NMR, GC-MS and compared to literature data. Extract (hexane, dichloromethane, ethyl acetate) and 5hydroxy-3′,4′,7-trimethoxyflavanone (compound 10) cytotoxicity against the growth of two human tumor cell lines (histiocytic lymphoma U-937 and acute monocytic leukemia THP-1) and a human non-tumor cell line (PBMC) were assessed by the MTT method. The dichloromethane extract displayed low activity, but the most significant result against the U-937 cell line, with an IC50 63.82 ± 1.06 μg/mL and a good selectivity index (SI) of 5.51. All extracts tested in PBMC cells displayed high cytotoxicity. The cytotoxic evaluation of compound 10 displayed low activity against the U-937 and THP-1 cell lines. Twelve compounds comprising triterpenes, steroids, flavonoids, and phenolic acids are described herein for the first time for the Piptocarpha genus. In addition, this is the first phytochemical investigation and cytotoxic evaluation of Piptocarpha axillaris (Less.) Baker that displays chemotaxonomic significance.
1. Introduction Piptocarpha belongs to the Asteraceae family and is a neotropical genus comprising about 46 species distributed throughout southern and eastern Brazil and northern Argentina, the West Indies and Central America. Their habitat extends from temperate forests to the savannah (Robinson, 2002; Smith, 1981; Smith and Coile, 2007). Previous phytochemical studies have revealed the occurrence of sesquiterpenes, steroids, triterpenes and flavonoids in plants belonging to the Piptocarpha genus (Bohlmann et al., 1980; Castro and Warning, 1987; Cowall et al., 1981; Herz and Kulanthaivel, 1983), and several biological investigations have been carried out with Piptocarpha species extracts or isolated compounds. These include antimicrobial activity evaluations against Bacillis cereus and Candida albicans; cytotoxic assessments against 9 KB human nasopharynx carcinoma cells and P-388 lymphoid leukemia system; investigations concerning activity against human saliva α-amylases and larvicidal, molluscicidal, antihelminthic and ∗
leishmanicidal activities (Antinarelli et al., 2015; Cowall et al., 1981; Gilbert et al., 1972; Mendes et al., 1999; Rodrigues et al., 2006; Scio et al., 2012; Silva et al., 2009). Piptocarpha axillaris (Less.) Baker is a tree ranging from 3 to 15 m in height, with cylindrical brownish collared branches and alternate leaves. Several popular names have been attributed to this species, due to the internal oxidation that occurs when its wood is cut, characterized by a color change to almost black, such as: “canela-podre”, “vassourão” and “cambará”. Piptocarpha axillaris is found in the Atlantic Rainforest, in Southeastern and Southern Brazil, and is the most abundant species in the state of Paraná (Grokoviski et al., 2009). Reports on the use of this species as both ornamental and used as temporary fences are available, and their flowers are also known for being melliferous (Ribeiro et al., 2010). To the best of our knowledge, no phytochemical study has been previously performed concerning P. axillaris. However, plant extracts have been analyzed by LC-UV-HRMS by dereplication of major
Corresponding author. E-mail address: [email protected] (I.d.S.P. Pereira).
https://doi.org/10.1016/j.bse.2019.05.001 Received 27 February 2019; Received in revised form 26 April 2019; Accepted 4 May 2019 Available online 16 May 2019 0305-1978/ © 2019 Elsevier Ltd. All rights reserved.
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secondary metabolites, with the identification of phenolic acids and flavonoids (Gallon et al., 2018). One biological investigation is available regarding the antihelminthic activity of the essential oil present in wood from this species, where the oil was proven to be a Schistosoma mansoni cercaria skin penetration inhibitor. The authors attributed the activity to sesquiterpenes and diterpenes through literature data (Gilbert et al., 1972). In this context, this is the first phytochemical study and cytotoxic evaluation concerning Piptocarpha axillaris.
corresponding to compound 12. Fractions A6 (2.7 mg) and A7 (2.3 mg) displayed a phenolic profile when analyzed by TLC. They were subsequently evaluated by UPLC-MS/MS, allowing for the identification of compounds 13–20. Two milligrams of the samples (A6 and A7) were solubilized in 1.0 mL of a 5% acetonitrile solution in ultrapure water in 2.0 mL polypropylene microtubes, with the aid of an ultrasonic bath for 10 min. The samples were then centrifuged at 3,000 rpm for 10 min. The supernatants were filtered through a 0.45 μm membrane (TEDIA) and transferred to 1.5 mL glass vials.
2. Material and methods
2.3. General
2.1. Plant material
1
H and 13C NMR spectra, were recorded on a Bruker NMR, operating at 400 MHz for 1H and 100 MHz for 13C, and on a Bruker Ascend 500 operating at 500 MHz for 1H and 125 MHz for 13C. Samples were dissolved in appropriate deuterated solvents (d-chloroform or d-methanol) and TMS was used as the internal standard. High performance countercurrent chromatography (HPCCC) was performed using a Dynamic Extractions Spectrum with 142 mL multilayer coil columns (1.6 mm i.d.). The β-value ranges from 0.52 to 0.86 and revolution speed can be adjusted to up to 1600 rpm. The chromatographic system comprises two Knauer Smartline 100 V5010 pumps, a 5 mL manual injection valve and a Büchi C-660 fraction collector. The eluate was monitored by a Knauer V7604 UV absorbance detector. A GC-MS analysis was performed on a Shimadzu GCMS-QP5050A equipped with a DB5 column (30 m × 0.25 mm, 0.25 μm film thickness). The mass spectrometer was set to 280 °C and the electron impact (EI) energy to 70 eV. The initial oven temperature was set to 100 °C, and a 20 °C/min rate was used, until reaching 280 °C. The final temperature (280 °C) was held for 78 min (total run time 87 min). The injection port temperature was set at 280 °C and 1 μL of sample was injected. The UPLC-MS/MS analysis was performed on a Shimadzu LC-20A instrument (Kyoto, Japan) equipped with a degasser, binary pump, autosampler, temperature controlled column oven, and diode array detector (DAD) coupled to a high resolution mass spectrometer equipped with an electrospray ion source (ESI) and quadrupole time of flight mass analyzer (Q-TOF) (Bruker Daltonics, Bremen, Germany). The separation was performed on a C-18 analytical Zorbax column (1.7 μm, 2.1 × 75 mm, Phenomenex). The mobile phase consisted of ultra pure water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The following gradient elution program was determined after several optimization procedures: 0–1 min 5% B, 1–5 min 5–17% B, 5–42 min 17–22% B, 42–50 min 22–100% B, 50–55 min 100% B, 55–60 min from 100 to 5% B, 60–65 min 5% B. The flow rate was maintained at 0.25 mL min−1. The column temperature was set to 40 °C. The final 15 min of the method aimed to clean the system for the next injection, in sequence, rebalancing the column with the initial mobile phase composition. An injection volume of 50 μL was used. The UV/DAD detector was monitored at 345 nm (flavonoids and phenolic acids). Chromatograms were recorded and processed using the LC Solution v. 1.25 software (Shimadzu). The nebulizer gas consisted in high purity nitrogen (N2) produced online by a Peak Scientific NM32LA nitrogen generator. The analysis parameters were set using the negative ionization mode with spectra acquired over a mass range from m/z 50 to 1400. The optimum ESI-MS parameters were as follows: capillary voltage, +3.5 kV; drying gas temperature, 210 °C; drying gas flow, 10.0 L/min; nebulizing gas pressure, 72.5 psi; collision RF, 200 Vpp; transfer time 120 μs; and pre-pulse storage, 3 μs. Moreover, automatic MS/MS experiments were performed adjusting the collision-energy values as follows: m/z 500, 30 eV; m/z 1000, 50 eV; m/z 1400, 70 eV using nitrogen as collision gas. The MS data were processed using the Data Analysis 4.0 software (Bruker Daltonics, Bremen, Germany). External instrument calibration was performed using a Cole Palmer syringe pump (Vernon Hills, IL, USA) directly connected to the interface, eluting a sodium formate (NaCHO2) solution cluster containing 5 mM sodium hydroxide (NaOH) and 0.2% formic acid in
Piptocarpha axillaris leaves were collected at the União Biological Reserve (22°27′30″S; 42°02′15″O, Rio das Ostras, Atlantic Rainforest, Rio de Janeiro, Brazil) in March 2013. A voucher specimen (HUENF 9476) is deposited at the UENF Herbarium, at the Center of Biosciences and Biotechnology, Universidade Estadual do Norte Fluminense, Rio de Janeiro, Brazil. It was also archived and published at JABOT/JBRJ (http://huenf.jbrj.gov.br/v2/consulta.php), available for public consultation. 2.2. Extraction and isolation Piptocarpha axillaris dried and crushed leaves (390.489 g) were extracted by maceration at room temperature with solvents presenting different polarities. The extraction step was repeated weekly with 1–2 L of solvent. Subsequently, the liquid was filtered, and the solvent removed under vacuum to obtain a dried extract. The following solvents were used: hexane, for 8 weeks (20.0981 g - PH), CH2Cl2, for 7 weeks (3.9431 g - PD) and EtOAc, for 3 weeks (2.5593 g - PA). The PH extract (6.0981 g) was chromatographed on a silica gel column eluted with hexane/EtOAc, gradually increasing solvent polarity to obtain 9 fractions (H1–H9). Fraction H4 (781.8 mg) was washed with MeOH, resulting in a 1 + 2 mixture (50.5 mg). Fraction H5 (696.7 mg) was also washed with MeOH, resulting in the pure compound 4 (107.0 mg). The insoluble H4 fraction (726.8 mg) was chromatographed on a silica gel column eluted with hexane/EtOAc gradients, obtaining 8 fractions. Fraction H-4.2.2 (712.3 mg) was further purified by washing with EtOAc, obtaining H-4.2.2.1 (449.4 mg) and H4.2.2.2 (101.0 mg). Fraction H-4.2.2.1 was purified on a silica gel CC eluted with an isocratic system comprising hexane/ethyl ether 8.5:1.5 (v/v), resulting in a triterpene mixture 1 + 2 + 3 (39.4 mg). Fraction H-4.2.2.2 was chromatographed on a low pressure silica flash applying hexane/CHCl3 gradients to obtain compound 2 (12.3 mg). Fractions H5 (589.7 mg), H6 (257.8 mg), and H8 (769.1 mg) were chromatographed on silica gel CC with hexane/EtOAc gradients and yielding a 4 + 5 + 6 mixture (187.0 mg). Fraction H-8.7 was further purified by crystallization with CH2Cl2, yielding a steroid mixture 7 + 8 + 9 (41.8 mg). The PD extract (3.12 g) was chromatographed on a filtration column (using activated charcoal) under low pressure, with ethyl ether/ CH2Cl2/EtOAc/MeOH as the eluent, resulting in 4 fractions (PD2-E, PD2-D, PD2-A, PD2-M). Fraction PD2-D was chromatographed on a silica gel column eluted with CH2Cl2/MeOH, gradually increasing eluent polarity to obtain 13 fractions. Fraction PD2-D9 was purified by another silica gel CC eluted with hexane/CH2Cl2. Fraction PD2-D9.6 (11.5 mg) was further purified by crystallization with hexane, resulting in compound 10 (5.5 mg). The PA extract (0.7593 g) was submitted to HPCCC using a solvent system comprising Hexane:EtOAc:MeOH:H2O (5:8:5:8) (v/v). A total of 18 fractions were obtained (A1-A18). After a qualitative analysis performed on TLC, fraction A4 displayed a major yellow spot corresponding to compound 11 (4.0 mg). Fractions A15 (52.6 mg) and A16 (76.1 mg) were combined and submitted to a new HPCCC analysis using Hexane:EtOAc:MeOH:H2O (1:3:1:3) (v/v) as the solvent system. Fraction A15–16.7 (5.4 mg) presented a major yellow spot on TLC, 25
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water:isopropanol 1:1 (v/v). The LC/MS system was controlled by the HyStar 3.2 software (Bruker Daltonics, Bremen, Germany). Column chromatography (CC) separations were carried out on silica gel 60 (63–200 μm, Merck) and silica flash (230–400 mesh, Merck). TLC was performed on commercial plates (silica gel 60 F254, Merck). Spot visualization was performed in the UV range (254 and 365 nm) using vanillin sulfuric acid, followed by heating.
[4,5-dimethy-2-lthiazolyl-]2,5-diphenyl-2H-tetrazolium bromide), as described previously (Paes et al., 2015). After 48 h of incubation with the samples, 20 μL of MTT (5 mg/mL - Sigma) were added to each well, followed by incubation for 4 h at 37 °C for MTT metabolism. After this period, 150 μL of the supernatant were removed from each well and 100 μL of an HCl solution in isopropanol were then added for formazan crystal solubilization. Absorbance values were determined by spectrophotometry at 570 nm (EpochTM, BioTex® Instruments, Inc.). Each experiment was performed in triplicate. Doxorubicin (Sigma) was used as the positive control (Paes et al., 2015). The selectivity degree of the fractions was expressed by the selectivity index (SI), calculated by dividing the IC50 value for the noncancer PBMC by the IC50 value for the U937cancer cell line.
2.4. Compound identification All compounds are known and have been identified by comparison to literature data and libraries. Identification of compounds 1–10 was based: (i) on comparison with published 1D-2D NMR spectroscopy data (Balestrin et al., 2008; Carvalho et al., 1998; Chaturvedula and Prakash, 2012; Gangwal et al., 2010; Harbone and Mabry, 1982; Silva et al., 1998; Sobrinho et al., 1991); (ii) on comparison of their mass fragmentation similarities by GC-MS using NIST library versions 107, 05, 21 and literature data (Silva et al., 2016). Identification of compounds 11–12 was based on comparison with published 1D-2D NMR spectroscopy data (Alves et al., 2010; Junior et al., 2010). Identification of compounds 13–20 was based on comparison of their mass fragmentation similarities by UPLC-MS/MS, literature data, and the MassBank database (Bueno et al., 2016; Clifford et al., 2005; D'Urso et al., 2018; He et al., 2016; MassBank, 2018; Zheleva-Dimitrova et al., 2017).
2.6. Statistical analyses The cytotoxicity to each sample was expressed as IC50 and analyzed by a one-way ANOVA using the GraphPad Prism 5.0 software. Significant differences were set at p < 0.05. 3. Results and discussion 3.1. Isolation and structural elucidation The phytochemical investigation of Piptocarpha axillaris leaves allowed the identification and isolation of 12 known compounds: six triterpenes, β-amyrin acetate (1), lupeol acetate (2), α-amyrin acetate (3), lupeol (4), α-amyrin (5), β-amyrin (6), three steroids, stigmasterol (7), β-sitosterol (8), campesterol (9), and three flavonoids, 5-hydroxy3′,4′,7-trimethoxyflavanone (10), 4′,5-dihydroxy-7-methoxyflavone (11), kaempferol 3-O-β-D-(6″-O-E-p-coumaroyl)-glucopyranoside (trans-tiliroside) (12). Their structures were established on the basis of a spectral NMR analysis by comparison to literature data and a GC-MS analysis. Sesquiterpene lactones and flavonoids are considered chemotaxonomic markers for the Asteraceae family (Emerenciano et al., 2001; Seaman, 1982). Table 1 summarizes the compounds isolated from the Piptocarpha genus since the first study in 1980, as sesquiterpene lactones, steroids, sesquiterpenes, terpenes, and flavonoids. As no
2.5. Cytotoxic activity bioassay evaluation U-937 (histiocytic lymphoma) and THP-1 (acute monocytic leukemia) cells were obtained from the American Type Culture Collection (ATCC). Human non-tumor PBMC (peripheral blood mononuclear cells) were obtained from the venous blood of healthy volunteers as described elsewhere (Junior et al., 2010). The cells were cultured in DMEM-F12 (Gibco) medium containing 10% fetal bovine serum (Gibco) and gentamicin 20 μg/mL (Gibco). The culture was maintained at 37 °C with 5% CO2 and controlled humidity. Briefly, cells were incubated in 96well plates (1 × 106 cells/well) containing 100 μL and incubated at different extract/fractions/compound concentrations during 48 h cytotoxicity was determined by a colorimetric microassay using MTT (3Table 1 Compounds isolated from the Piptocarpha genus. Compounds
Species
References
germacrene D (s) lupeol acetate (t) lupeol (t)
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
Bohlmann et al. (1980) Bohlmann et al. (1980) Bohlmann et al. (1980) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Bohlmann et al. (1980) Bohlmann et al. (1980) Bohlmann et al. (1980) Cowall et al. (1981) Cowall et al. (1981) Cowall et al. (1981) Cowall et al. (1981) Cowall et al. (1981) Cowall et al. (1981) Castro and Warning (1987) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983) Herz and Kulanthaivel (1983)
α-amirin (t) β-amirin (t) sakuranetin (f) naringenin 7,4′-dimethyl ether (f) dimethyl eriodictyol (f) piptocarphin A (sl) piptocarphin B (sl) piptocarphin C (sl) piptocarphin D (sl) piptocarphin E (sl) piptocarphin F (sl) hirsutinolide (sl) 7,10-epoxycyclodeca (β) furan, 2-butenoic acid (sl) 7,10-epoxycyclodeca (β) furan, 2-propenoic acid (sl) sitosterol β-D-glucoside (st) stigmasterol β-D-glucoside (st) stigmasterol (st) β-sitosterol (st) stigmasterone (st) β-sitosterone (st)
oblonga oblonga oblonga opaca opaca opaca oblonga oblonga oblonga chontalensis chontalensis chontalensis chontalensis chontalensis chontalensis poeppigiana opaca opaca opaca opaca opaca opaca opaca opaca
(s) sesquiterpenes; (t) triterpenes; (f) flavonoids; (sl) sesquiterpene lactones; (st) steroids. 26
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Fig. 1. A6 and A7 fraction chromatograms obtained by UPLC-DAD-MS/MS at 345 nm.
phytochemical investigation has been reported for Piptocarpha axillaris species, the results obtained herein are new for Piptocarpha axillaris, with compounds 1, 3, 9, 10, 11 and 12 obtained from the Piptocarpha genus for the first time.
were identified as 3,5-dicaffeoylquinic acid (18) and 1,4-dicaffeoylquinic acid (19). Both acids lost caffeoyl units ([M-162-162]-), resulting in the ion m/z 191.06 relative to quinic acid, but the remainder of the fragmentation profiles of these acids are different. Baicalein-7-O-glucoronide (14) (m/z 445.0801), wogonoside (15) (m/z 459.0981) and isorhamnetin-3-O-glucoside (16) (m/z 477.1439) were also detected. Two tiliroside (m/z 593.13) isomers, compounds 20 and 12, with different retention times, were also detected. It is suggested that the more intense peak 11 is a trans-isomer, as it is thermodynamically stable than cis-isomer (Devi and Kumar, 2018). All compounds were identified by their relative change in fragments. Flavonoids are considered chemotaxonomic Asteraceae family markers, and the obtained results corroborate the observed chemical profile. The importance and efficiency of the analytical techniques used to identify the organic compounds are worth emphasizing. A previous investigation analyzed major secondary metabolites in Asteraceae species by LC-UV-HRMS and dereplication (Table 4), and identified 29 compounds in Piptocarpha genus species extracts and 12 compounds in P. axillaris, including as phenolic acids and flavonoids (Gallon et al., 2018). Compared to the UPLC-MS/MS analysis, compounds 13, 14, 15, 16, 19, 20 are described for the first time for the Piptocarpha genus.
3.2. UPLC-DAD-MS/MS analysis Two ethyl acetate extract fractions (A6 and A7) were analyzed by UPLC-DAD-MS/MS due the phenolic profile observed by TLC. The obtained chromatograms are displayed in Fig. 1. Compound identification was performed by comparing the fragmentations to the MassBank database and literature data (Tables 2 and 3). Fraction A6 presented a major peak at 13.8 min and was identified as baicalein-7-O-glucuronide (14) with m/z 445.0816 ([M-H]-). Its fragmentation profile is based on the loss of the glucuronide unit (−176 Da) for the formation of m/z 269.05 for aglycone baicalein. Some minor compounds were also detected, such as caffeic acid (13) (m/z 179.0355), characterized by the presence of m/z 135.04, due to loss of CO2 ([M-H-44]-), wogonoside (15) (m/z 459.0965) characterized by glucuronide and a CH3 unit loss (M-H-176-15]-), and isorhamnetin3-O-glycoside (16) (m/z 477.1439) characterized by the neutral loss of glucose for ion m/z 315.09 formation, relative to aglycone ([M-H-162]). Concerning fraction A7, the first peak at 6.2 min corresponds to 5-Ocaffeoylquinic acid (17), due to the presence of only one m/z 191.06 ion fragment, unlike 3-O-caffeoylquinic acid, which presents other fragments (Jaiswal et al., 2014). Two major peaks (12.4 min and 15.1 min)
3.3. Chemotaxonomic significance The most recent botanical classification used for the Piptocarpha genus is from the study carried out by (Smith and Coile, 2007), who used morphology, karyology, chemotaxonomy, phylogeny, evolution
Table 2 Compounds identified in the A6 fraction by UPLC-MS/MS. Peaks
RT (min)
[M-H]-
MS/MS (% relative abundance)
Compounds
Reference
1 2 3 4
6.0 13.8 27.1 36.8
179.0355 445.0816 459.0965 477.1439
135.04(100) 269.05(100) 459.09(100), 268.04(20) 315.09(100)
caffeic acid (13) putative baicalein-7-O-glucoronide (14) putative wogonoside (15) putative isorhamnetin-3-O-glucoside (16)
MassBank (2018) He et al. (2016) He et al. (2016) Bueno et al. (2016)
27
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Table 3 Compounds identified in the A7 fraction by UPLC-MS/MS. Peaks
RT (min)
[M-H]-
MS/MS (% relative abundance)
Compounds
Reference
5 6 7 8 9 10 11 12
6.2 12.4 13.8 15.1 27.2 30.3 32.8 36.8
353.0873 515.1257 445.0801 515.1260 459.0981 593.1340 593.1354 477.1411
191.06(100) 353.09(98), 191.06(100), 179.03(65), 161.02(4), 135.05(7) 269.05(100) 353.09(100), 191.06(13), 179.04(44), 173.04(55), 135.05(4) 459.09(100), 268.04(44) 593.13(100), 447.09(8), 285.04(66) 593.13(100), 447.09(7), 285.04(74) 315.09(100)
5-O-caffeoylquinic acid (17) 3,5-dicaffeoylquinic acid (18) putative baicalein-7-O-glucoronide (14) 1,4-dicaffeoylquinic acid (19) putative wogonoside (15) cis-tiliroside (20) trans-tiliroside (12) putative isorhamnetin-3-O-glucoside (16)
MassBank (2018) Zheleva-Dimitrova et al. (2017) He et al. (2016) Clifford et al. (2005) He et al. (2016) D'Urso et al. (2018) D'Urso et al. (2018) Bueno et al. (2016)
Table 4 Piptocarpha genus compounds identified by LC-MS (Gallon et al., 2018). Compounds
Species
3-O-caffeoylquinic acid (pa) 5-O-caffeoylquinic acid (pa) 4-O-caffeoylquinic acid (pa) 5-p-coumaroylquinic acid (pa) 5-O-feruloylquinic acid (pa) 3,5-di-O-caffeoylquinic acid (pa) 4,5-di-O-caffeoylquinic acid (pa) vicenin-2 (f) putative quercetin 3-O-rutinoside (f) vitexin (f) putative quercetin 3-O-glucoside (f) putative luteolin 7-O-glucuronide (f) putative kaempferol 3-O-rutinoside (f) putative 7,3′,5′-trihydroxy-4′-methoxy-3-O-glucosylflavone (f) putative apigenin 7-O-glucuronide (f) putative chrysoeriol 7-O-neohesperidoside (f) putative quercetin 3-O-(6-caffeoyl)-glucoside (f) putative chrysoeriol 7-O-glucuronide (f) putative isoorientin 3′-O-glucoside (f) quercetin (f) luteolin (f) putative acacetin 7-O-rutinoside (f) tiliroside (f) putative isorhamnetin 3-O-(6-p-coumaroyl) glucoside (f) apigenin (f) kaempferol (f) chrysoeriol (f) 3′,4′-dimethoxyluteolin (f) glaucolide B (sl)
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
axillaris, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii angustifolia, P. axillaris, P. macropoda, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii axillaris, P. macropoda, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii quadrangulares, P. sellowii axillaris angustifolia, P. axillaris, P. macropoda, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii angustifolia, P. axillaris, P. macropoda, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii macropoda macropoda, P. regnellii macropoda axillaris, P. macropoda, P. regnellii, P. rotundifolia, P. sellowii quadrangulares, P. rotundifolia quadrangulares regnellii, P. rotundifolia quadrangulares angustifolia, P. macropoda axillaris, P. regnellii, P. sellowii quadrangulares axillaris, P. quadrangulares, P. regnellii, P. rotundifolia, P. sellowii axillaris, P. regnellii, P. rotundifolia, P. sellowii macropoda, P. quadrangulares macropoda macropoda, P. regnellii, P. rotundifolia, P. sellowii angustifolia, P. axillaris macropoda, P. quadrangulares axillaris, P. macropoda, P. regnellii, P. rotundifolia, P. sellowii angustifolia, P. quadrangulares angustifolia macropoda
(pa) phenolic acids; (f) flavonoids; (sl) sesquiterpene lactones.
and species distribution data. In this classification, Piptocarpha is divided into two subgenus (Piptocarpha and Hypericoides) and four sections (Piptocarpha, Oocephalus, Macrolepideae and Vanilosma). Tables 1 and 4 summarize compounds already isolated and identified in species belonging to the Piptocarpha genus. Table 5 displays the species represented by their respective sections and subgenera. It should be noted that P. paradoxa is no longer classified as a member of the Piptocarpha genus (Mathur and Gonzalez, 1982). Few species belonging to the Piptocarpha genus have been chemically studied, and no specific markers for this genus have been defined. According to phytochemical data available in the literature (Tables 1 and 4), up to now, these species show a chemical profile aimed at the production of, mostly, sesquiterpenes and flavonoids. An analysis of the available data indicates that these species are in evolutionary transition, which can be observed by a greater flavonoid production compared to flavones. In this context, another parameter that points to this evolution is the degree of protection of flavonoid hydroxyl groups, which are mostly protected by glycosylation and, in addition, some methylation and double protection (glycosylation and methylation) cases. No significant structural difference between the studied species belonging to the different sections is noted regarding the structures of isolated and identified flavonoids, except for P. oblonga, which presents
Table 5 Botanical classification of the assessed Piptocarpha species. Subgenus
Piptocarpha
Hypericoides
Sections
Piptocarpha
Oocephalus
Macrolepideae
Vanilosma
Species
P. rotundifolia P. opaca
P. poeppigiana
P. quadrangularis P. oblonga P. sellowii
P. axillaris P. regnellii P. angustifolia P. macropoda
Table 6 Cytotoxic activities expressed as IC50 (μg/mL) for extracts, and for doxorubicin (μM) against the U-937 histiocytic lymphoma cell line and non-tumor human PBMC. Extracts
U-937
PMBC
SI (selectivity index)
PH PD PA Doxorubicina
111.1 ± 1.11 63.82 ± 1.06 > 200.0 2.25 ± 1.03
> 400.0 352.00 ± 1.16 > 400.0 26.92 ± 1.21
> 3.6 5.51 > 2.0 11.96
PH-Hexane extract; PD-Dichloromethane extract; PA-Ethyl acetate extract. a Positive control. 28
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Alves do Nascimento, Tatiane Pereira de Souza, and Marcelo Trindade Nascimento for the collection and botanical identification of the species assessed herein.
Table 7 Cytotoxic activities expressed as IC50 (μM) for an isolated flavanone (10) against the U-937 histiocytic lymphoma and acute monocytic leukemia THP1 cell lines. Compounds
U-937
THP-1
Appendix A. Supplementary data
10 Doxorubicina
> 50.0 2.25 ± 1.03
> 50.0 13.73
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bse.2019.05.001.
a
Positive control.
References
flavanones protected by methylation. Thus, the results of the isolated and identified compounds corroborate the chemotaxonomic positioning of P. axillaris in the Vanilosma section, while also providing an important chemotaxonomy tool, with the description of 20 identified compounds.
Alves, H.S., Oliveira, G.E., Zoghbi, M.G., Chaves, M.C.O., 2010. Flavonóides de Piper carniconnectivum C.DC. Piperaceae. Brazilian J. Pharmacogn. 20, 160–164. https:// doi.org/10.1590/S0102-695X2010000200004. Antinarelli, L.M.R., Pinto, N.C., Scio, E., Coimbra, E.S., 2015. Antileishmanial activity of some brazilian plants, with particular reference to Casearia sylvestris. An. Acad. Bras. Cienc. 87, 733–742. https://doi.org/10.1590/0001-3765201520140288. Balestrin, L., Dias, J.F.G., Miguel, O.G., Dall'Stella, D.S.G., Miguel, M.D., 2008. Contribuição ao estudo fitoquímico de Dorstenia multiformis Miquel (Moraceae) com abordagem em atividade antioxidante. Brazilian J. Pharmacogn. 18, 230–235. https://doi.org/10.1590/S0102-695X2008000200016. Bohlmann, F., Zdero, C., King, R.M., Robinson, H., 1980. Seven guaianolides from the tribe Vernonieae. Phytochemistry 19, 2669–2673. https://doi.org/10.1016/S00319422(00)83941-2. Bueno, P.C.P., Passareli, F., Anhesine, N.B., Torres, R.B., Cavalheiro, A.J., 2016. Flavonoids from Casearia sylvestris swartz variety lingua (salicaceae). Biochem. Syst. Ecol. 68, 23–26. https://doi.org/10.1016/j.bse.2016.06.002. Carvalho, M.G., Velandia, J.R., Oliveira, L.F., Bezerra, F.B., 1998. Triterpenos isolados de Eschweilera longipes Miers (Lecythidaceae). Quim. Nova 21, 740–743. https://doi. org/10.1590/S0100-40421998000600014. Castro, V., Warning, U., 1987. Steiractinolides from Zexmenia virgulata and constituents from other Compositae from Costa Rica. Ing. y Cienc. Quim. 11, 5–6. Chaturvedula, V.S.P., Prakash, I., 2012. Isolation of Stigmasterol and β-Sitosterol from the dichloromethane extract of Rubus suavissimus. Int. Curr. Pharm. J. 1, 239–242. https://doi.org/10.3329/icpj.v1i9.11613. Clifford, M.N., Knight, S., Kuhnert, N., 2005. Discriminating between the six isomers of dicaffeoylquinic acid by LC-MSn. J. Agric. Food Chem. 53, 3821–3832. https://doi. org/10.1021/jf050046h. Cowall, P.L., Cassady, J.M., Chang, C., Kozlowski, J.F., 1981. Isolation and structure determination of Piptocarphins A-F, cytotoxic germacranolide lactones from Piptocarpha chontalensis. J. Org. Chem. 46, 1108–1114. https://doi.org/10.1021/ jo00319a014. D'Urso, G., Pizza, C., Piacente, S., Montoro, P., 2018. Combination of LC–MS based metabolomics and antioxidant activity for evaluation of bioactive compounds in Fragaria vesca leaves from Italy. J. Pharm. Biomed. Anal. 150, 233–240. https://doi.org/10. 1016/j.jpba.2017.12.005. Devi, S., Kumar, V., 2018. Comprehensive structural analysis of cis- and trans-tiliroside and quercetrin from Malvastrum coromandelianum and their antioxidant activities. Arab. J. Chem. https://doi.org/10.1016/j.arabjc.2018.01.009. Emerenciano, V.P., Militão, J.S.L.T., Campos, C.C., Romoff, P., Kaplan, M.A.C., Zambon, M., Brant, A.J.C., 2001. Flavonoids as chemotaxonomic markers for Asteraceae. Biochem. Syst. Ecol. 29, 947–957. https://doi.org/10.1016/S0305-1978(01) 00033-3. Gallon, M.E., Jaiyesimi, O.A., Gobbo-Neto, L., 2018. LC-UV-HRMS dereplication of secondary metabolites from Brazilian Vernonieae (Asteraceae) species supported through in-house database. Biochem. Syst. Ecol. 78, 5–16. https://doi.org/10.1016/j. bse.2018.03.002. Gangwal, A., Parmar, S.K., Sheth, N.R., 2010. Triterpenoid, flavonoids and sterols from Lagenaria siceraria fruit. Der Pharm. Lett. 2, 307–317. https://doi.org/http:// scholarsresearchlibrary.com/archive.html. Gilbert, B., Mors, W.B., Baker, P.M., Tomassini, T.C.B., Coulart, E.G., Holanda, J.C., Costa, J.A.R., Lopes, J.N.G., Filho, D.S., Sarti, S.J., Turco, A.M.T., Vichnewski, W., Lopes, J.L.C., Thames, A.W., Pellegrino, J., Katz, N., 1972. A atividade antelmíntica de óleos essenciais e de seus componentes químicos. Ann. Brazilian Acad. Sci. 44, 423–428. Grokoviski, L., Cervi, A.C., Tardivo, R.C., 2009. O gênero Piptocarpha R. Br.(Asteraceae: vernonieae) no estado do Paraná, brasil. Acta Bot. Bras. 23, 486–498. https://doi. org/10.1590/S0102-33062009000200020. Harbone, J.B., Mabry, T.J., 1982. The Flavonoids: Advances in Research, first ed. Chapman and Hall, London and New York, pp. 744. He, L., Zhang, Z., Lu, L., Liu, Y., Li, S., Wang, J., Song, Z., Yan, Z., Miao, J., 2016. Rapid identification and quantitative analysis of the chemical constituents in Scutellaria indica L. by UHPLC-QTOF-MS and UHPLC-MS/MS. J. Pharm. Biomed. Anal. 117, 125–139. https://doi.org/10.1016/j.jpba.2015.08.034. Herz, W., Kulanthaivel, P., 1983. Piptocarphol esters from Piptocarpha opaca. Phytochemistry 22, 1286–1287. https://doi.org/10.1016/0031-9422(83)80245-3. Jaiswal, R., Müller, H., Müller, A., Karar, M.G.E., Kuhnert, N., 2014. Identification and characterization of chlorogenic acids, chlorogenic acid glycosides and flavonoids from Lonicera henryi L. (Caprifoliaceae) leaves by LC-MSn. Phytochemistry 108, 252–263. https://doi.org/10.1016/j.phytochem.2014.08.023. Junior, H.M.S., Tirelli, A.A., Carvalho, H.W.P., Oliveira, D.F., Prado, N.R.T., Campos, V.P., 2010. Purificação do flavonóide trans-tilirosídeo do extrato metanólico das folhas de Gochnatia barrosii Cabrera (ASTERACEAE) e avaliação da sua atividade
3.4. Cytotoxic activity evaluation Extract cytotoxicity (hexane-PH, dichloromethane-PD, ethyl acetate-PA), and flavanone (5-hydroxy-3′,4′,7-trimethoxyflavanone) and the standard control doxorubicin activities against the growth of two human tumor cell lines (histiocytic lymphoma cell line U-937 and acute monocytic leukemia THP-1) and a non-tumor human cell line (Peripheral Blood Mononuclear Cells - PBMC) were evaluated using the MTT method. The PD extract displayed the best results against the U-937 cell line, with IC50 = 63.82 ± 1.06 μg/mL. All extracts were also tested for cytotoxicity against PBMC. High cytotoxicity was observed, with IC50 = 352.00 ± 1.16 μg/mL for PD and a good selectivity index (SI) of 5.51 (Table 6). A high SI value (> 2.0) indicated high specificity (Suffness and Pezzutto, 1991). Plants extracts are considered active when displaying IC50 ≤ 20.0 μg/mL (Suffness and Pezzutto, 1990). The IC50 value for the dichloromethane extract (PD) was more significant. The cytotoxic activity of the flavanone 5-hydroxy-3′,4′,7trimethoxyflavanone (10) isolated from this extract was evaluated against the U-937 and THP-1 cell lines (Table 7). According to the literature, a pure compound can be considered a potential antineoplastic when its IC50 value is of less than 10 μM (Suffness and Douros, 1982). This compound presented a mild value of IC50 > 50.0 μM for both cell lines. As a contribution for the micromolecular assessment of Atlantic Rainforest species, the present study describes 20 known compounds observed in Piptocarpha axillaris. These results corroborate the chemical profile observed in the literature data for the Asteraceae family, whose flavonoids and sesquiterpene lactones are considered chemotaxonomic markers. No phytochemical investigation has been previously reported for this species and an UPLC-MS/MS analysis verified the presence of phenolic acids and flavonoids. Thus, compounds 1, 3, 9, 10, 11, 12 were isolated, and compounds 13, 14, 15, 16, 19, 20, identified in the Piptocarpha genus for the first time. It is worth noting that the biological results presented herein for P. axillaris are new, while this study comprises the first cytotoxic activity evaluation concerning the histiocytic lymphoma U-937 and acute monocytic leukemia THP-1 cell lines for the flavonoid 5-hydroxy-3′,4′,7-trimethoxyflavanone (10). The dichloromethane extract displayed low activity, but the most significant result against the U-937 cell line, with an IC50 of 63.82 ± 1.06 μg/mL and a good selectivity index (SI) of 5.51. Acknowledgments The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for research fellowships (31033016013P0). Thanks are also due to the Universidade Federal Rural do Rio de Janeiro (UFRRJ) for the 1H (400 MHz) and 13C (100 MHz) NMR analysis. In addition, the authors also thank Aline 29
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I.d.S.P. Pereira, et al. nematicida. Cienc. E Agrotecnol 34, 1224–1231. https://doi.org/10.1590/S141370542010000500021. MassBank High Resolution Mass Spectral Database. accessed. https://massbank.eu/ MassBank, Accessed date: 12 September 2018. Mathur, S.B., Gonzalez, L., 1982. Identification of terpenoids from the leaves of Piptocarpha paradoxa and their biological activities. J. Nat. Prod. 45, 495–496. https://doi.org/10.1021/np50022a025. Mendes, N.M., Queiroz, R.O., Grandi, T.S., Anjos, A.M., Oliveira, A.B., Zani, C.L., 1999. Screening of Asteraceae (Compositae) plant extracts for molluscicidal activity. Mem. Inst. Oswaldo Cruz 94, 411–412. https://doi.org/10.1590/S007402761999000300023. Paes, M.M., Vega, M.R.G., Cortes, D., Cabedo, N., Kanashiro, M.M., 2015. Cytotoxic activities of acetogenins from annona dioica roots. Intermt. J. Sci. 4, 19–29. https://doi. org/10.18483/ijSci.875. Ribeiro, A.O., Silva, A.F., Castro, A.H.F., 2010. Identificação de espécies da família Asteraceae, revisão sobre usos e triagem fitoquímica do gênero Eremanthus da Reserva Boqueirão, Ingaí-MG. Rev. Bras. Plantas Med. 12, 456–465. https://doi.org/ 10.1590/S1516-05722010000400009. Robinson, H.E., 2002. Three new species of Piptocarpha (Asteraceae: vernonieae) from Ecuador and Peru. Novon 12, 393–398. https://doi.org/10.2307/3393087. Rodrigues, A.M.S., Paula, J.E., Degallier, N., Molez, J.F., Espíndola, L.S., 2006. Larvicidal activity of some cerrado plants extracts agains Aedes aegypti. J. Am. Mosq. Control Assoc. 22, 314–317. https://doi.org/10.2987/8756-971X. (2006)22[314]:LAOSCP] 2.0.CO;2. Scio, E., Mendes, R.F., Motta, E.V.S., Bellozi, P.M.Q., Aragão, D.M.O., Mello, J., Fabri, R.L., Moreira, J.R., Assis, I.V.L., Bouzada, M.L.M., 2012. Antimicrobial and antioxidant activities of some plant extracts. Phytochem. as Nutraceuticals - Glob. Approaches to Their Role Nutr. Heal 65–80. https://doi.org/10.5772/27308. Seaman, F.C., 1982. Sesquiterpene lactones as taxonomic characters in the Asteraceae. Bot. Rev. 48, 121–595. https://doi.org/10.1007/BF02919190. Silva, J.R.A., Rezende, C.M., Pinto, A.C., Pinheiro, M.L.B., Cordeiro, M.C., Tamborini, E.,
Young, C.M., Bolzani, V.S., 1998. Ésteres triterpênicos de Himatanthus sucuuba (Spruce) Woodson. Quim. Nova 21, 24–26. https://doi.org/10.1590/S010040421998000600005. Silva, C.A.M., Melo, R.O., Resck, I.S., Silveira, D., 2016. Triterpenes from Pouteria gardneri ( mart . & miq .) baehni extracts. J. Appl. Pharm. Sci. 6, 197–201. https://doi.org/10. 7324/JAPS.2016.601229. Silva, E.M., Valencia, A., Grossi-de-Sá, M.F., Rocha, T.L., Freire, É., Paula, J.E., Espindola, L.S., 2009. Inhibitory action of Cerrado plants against mammalian and insect αamylases. Pestic. Biochem. Physiol. 95, 141–146. https://doi.org/10.1016/j.pestbp. 2009.08.003. Smith, G.L., 1981. New taxa in Piptocarpha R . BR . (vernonieae: compositae). Ann. Mo. Bot. Gard. 68, 661–667. https://doi.org/10.2307/2398894. Smith, G.L., Coile, N.C., 2007. Piptocarpha (compositae: vernonieae). Flora Neotrop. 99, 1–94. https://www.jstor.org/stable/4393953. Sobrinho, D.C., Hauptli, M.B., Appolinario, E.V., Kollenz, C.L.M., Carvalho, M.G., BrazFilho, R., 1991. Triterpenoids isolated from Parahancornia amapa. J. Braz. Chem. Soc. 2, 15–20. https://doi.org/10.5935/0103-5053.19910004. Suffness, M., Douros, J., 1982. Current status of the NCl plant and animal product program. J. Nat. Prod. 45, 1–14. https://doi.org/10.1021/np50019a001. Suffness, M., Pezzutto, J.M., 1991. Assays for cytotoxicity and antitumor activity. In: Hostettmann, K. (Ed.), Methods in Plant Biochemistry. Academic Press, London, pp. 133. Suffness, M., Pezzutto, J.M., 1990. Assays related to cancer drug discovery. In: Hostettmann, K. (Ed.), Methods in Plant Biochemistry: Assays for Bioactivity. Academic Press, London, pp. 220. Zheleva-Dimitrova, D., Gevrenova, R., Zaharieva, M.M., Najdenski, H., Ruseva, S., Lozanov, V., Balabanova, V., Yagi, S., Momekov, G., Mitev, V., 2017. HPLC-UV and LC–MS analyses of acylquinic acids in geigeria alata (DC) oliv. & hiern. And their contribution to antioxidant and antimicrobial capacity. Phytochem. Anal. 28, 176–184. https://doi.org/10.1002/pca.2658.
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